ISEV2021 Abstract Book (2024)

Plenary 1: EVs neurodegenerative diseases‐ Andrew Hill, Professor, La Trobe University

FA01. Mitochondrial Dysfunction Alters the Number and Content of Mitovesicles, Newly Identified Mitochondria‐derived Extracellular Vesicles

Pasquale D'Acunzo, Nathan S. Kline Institute for Psychiatric Research

Efrat Levy, Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, New York 10962, USA

Introduction: Mitochondrial damage is a well‐established player of neurodegenerative diseases, including Alzheimer's disease and Down syndrome (DS). We previously showed that the extracellular matrix of the brain contains a newly identified population of metabolically active extracellular vesicles (EVs) of mitochondrial origin that we have named ‘mitovesicles’. We investigated the effect of mitochondrial dysfunction in vivo on the number and content of mitovesicles in DS brains as compared with diploid controls and in vitro on secretion of mitovesicles by primary fibroblasts.

Methods: EVs were isolated from murine and human DS and control brains using a high‐resolution density step‐gradient that fractionates subtypes of EVs. EVs were analyzed by nanoparticle tracking analysis, Western blotting, mass spectrometry, and qPCR. EVs were also isolated from media of human fibroblasts following treatment with the electron transport chain inhibitor antimycin‐A and analyzed by Western blotting.

Results: The in vitro study revealed that mitochondrial damage enhances mitovesicle release in a mitophagy‐independent fashion. Consistently with these data, human and murine DS brains showed higher number of mitovesicles when compared to controls. Additionally, DS mitovesicles displayed perturbation in cargo loading, given that the amount of several mitochondrial proteins and mRNAs were lower in DS compared to controls when equal number of vesicles were considered. Quite the reverse, the amount of mitochondrial DNA, which is a strong pro‐inflammatory agent, was higher in DS mitovesicles compared to controls, consistent with the reported neuroinflammatory phenotype in DS.

Summary/Conclusion: Brain mitovesicle levels and cargo are modified in DS, suggesting that mitovesicles may be a previously unrecognized player of mitochondria quality control and may have a yet undiscovered role in the response to oxidative stress, neuroinflammation and synaptic regulation.

Supported by NIH grants AG017617, AG057517, AG056732, DA044489

Plenary 2: EVS in Cardiovascular Disorders‐ Chantal Boulanger, Research Director at the French Biomedical Research Agency (INSERM)

FA02. Blood Flow Tunes Uptake and Fate of Extracellular Vesicles

Benjamin MARY, INSERM U1109 tumor biomechanis lab

Nandini ASOKAN, INSERM UMR_S1109, Tumor Biomechanics Lab; Université de Strasbourg, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg France

Olivier LEFEBVRE, INSERM UMR_S1109, Tumor Biomechanics Lab; Université de Strasbourg, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg France

Jacky GOETZ, INSERM UMR_S1109, Tumor Biomechanics Lab; Université de Strasbourg, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg France

Vincent Hyenne, INSERM / CNRS

Introduction: Circulating tumor EVs (ctEVs) are abundant in blood of cancer patients and favor metastasis by inducing the formation of pre‐metastatic niche in distant organs. Yet, how they react to the intravascular hemodynamic conditions remain poorly understood.

Methods: Here, we mimicked realistic bloodstream conditions in vitro and in vivo, using microfluidics and zebrafish respectively, to dissect the impact of blood flow parameters on the efficiency and endocytic route of ctEVs uptake.

Results: While moderate blood flow regimes (velocities around 400 μm/s) promote the uptake of ctEVs by the endothelium compared to a static condition, increasing shear and blood flow velocities cancel this positive effect. Adhesive properties of ctEVs is instrumental in their intravascular behavior notably via the adhesion molecule CD146 expressed on EV surface. Thus, it is likely that harsh hemodynamic constraints compete with their adhesive potential on the endothelium. Further and ongoing investigations will determine whether additional receptors (CD44, integrins) also contribute to ctEVs uptake.

Upon arrest, we identified clathrin‐independent endocytosis as a route of uptake of ctEVs and observed that hemodynamic forces affect the subcellular localization of ctEVs taken up by endothelial cells. We further observed that hemodynamic forces upregulate lysosomal pathways in endothelial cells. We are currently testing whether tuning blood flow impacts ctEVs cargo transfer and how this affects endothelial behavior using a combination of imaging and transcriptomic approaches.

Summary/Conclusion: Altogether, our work demonstrates that hemodynamics could tune the endothelial uptake of ctEVs. Since this is likely to control the function of ctEVs, we propose a novel route by which ctEVs can impact the establishment of pre‐metastatic niches.

Commensal gut bacteria‐derived extracellular vesicles: Mediators of gut microbe‐host crosstalk and vehicles for mucosal drug delivery‐ Simon Carding, Professor Mucosal Immunology

FA03. Melanoma‐secreted exosomes prepare the formation of pre‐metastatic niches in sentinel lymph nodes

Susana Garcia‐Silva, Spanish National Cancer Research Center (CNIO), Madrid, Spain.

Alberto Benito‐Martín, Weill Cornell Medicine, New York, USA

Laura Nogues, Spanish National Cancer Research Center (CNIO)

Alberto Hernández‐Barranco, Spanish National Cancer Research Center (CNIO)

Vanesa Santos, Spanish National Cancer Research Center (CNIO)

Marina Mazariegos, Spanish National Cancer Research Center (CNIO)

Marta Hergueta, Spanish National Cancer Research Center (CNIO)

Raghu P. Kataru, Memorial Sloan Kettering Cancer Center

Sara Sanchez‐Redondo, Spanish National Cancer Research Center (CNIO)

Osvaldo Graña‐Castro,Spanish National Cancer Research Center (CNIO)

Irina Matei, Weill Cornell Medicine

Sagrario OrtegaSpanish National Cancer Research Center (CNIO)

Raúl Torres‐Ruiz, Spanish National Cancer Research Center (CNIO)

Sandra Rodríguez‐Perales,Spanish National Cancer Research Center (CNIO)

Lola Martínez,Spanish National Cancer Research Center (CNIO)

Manuel Pérez,Spanish National Cancer Research Center (CNIO)

Diego Megías,Spanish National Cancer Research Center (CNIO)

Babak J. Mehrara,Memorial Sloan Kettering Cancer Center

David Lyden, Weill Cornell Medicine

Hector Peinado,Spanish National Cancer Center

Introduction: Secreted EVs built a network of communication around primary tumors and distant organs favoring metastasis. Our previous works have demonstrated the role of tumor‐secreted EVs in pre‐metastatic niche formation and metastatic organotropism in distal organs. In this work we have analyzed the dynamics of tumor‐derived small EVs (sEVs) in the lymphatic system and their role establishing the pre‐metastatic niche formation in sentinel lymph nodes.

Methods: We have analyzed melanoma‐derived sEV biodistribution in the lymphatic system by in vivo imaging, confocal microscopy and flow cytometry. We performed RNA‐seq in lymphatic endothelial cells after sEV uptake combined with proteomic analysis of sEVs. We have analyzed the effect of sEV‐shed neurotrophin receptor lymphangiogenesis both in vitro and in vivo. In addition, we performed experimental and spontaneous metastasis as well as survival assays after education with melanoma‐derived sEVs in preclinical models. Finally, we have evaluated the expression of neurotrophin receptors in primary tumors and sentinel lymph nodes correlating with disease outcome.

Results: We found that sEVs derived from metastatic melanoma cell lines spread through the lymphatic system. We observed that lymphatic endothelial cells are the main and first cell type incorporating tumor‐derived sEVs followed by macrophages in the lymph nodes. Melanoma‐derived sEVs induced lymphangiogenic gene expression (e.g. LYVE‐1, VEGF‐C), tumor cell adhesion, and the activation of ERK and NF‐kB pathways in lymphatic endothelial cells. Mechanistically, we observed that neurotrophin receptors are secreted in melanoma‐derived sEVs and shuttled to lymphatic endothelial cells concomitant with the acquisition of a pro‐lymphangiogenic phenotype. Blocking sEVs‐induced signals reduced melanoma metastasis and improved survival in pre‐clinical models. Analysis of human samples showed that the expression of specific neurotrophin receptors in metastatic pioneering cells within the sentinel lymph node is correlated with poor survival.

Summary/Conclusion: Our data shows for the first time that both tumor intrinsic and extrinsic factors such as tumor‐secreted sEVs are involved in lymphangiogenesis and pre‐metastatic niche formation in sentinel lymph nodes through an neurotrophin receptor‐dependent mechanism favoring metastatic spread in melanoma.

Biogenesis of the Multivesicular Endosome‐ Jean Gruenberg, Honorary Professor, Department of Biochemistry

FA04. Specificity determinants for RNA release into extracellular vesicles

Marie Mosbach, Institute of Biochemistry, Justus Liebig University of Gießen

Christina Pfafenrot, Institute of Biochemistry, Justus Liebig University of Gießen

Elke Pogge von Strandmann, Institute for Tumorimmunology, Center for Tumor Biology and Immunology, Philipps University Marburg

Albrecht Bindereif, Institute of Biochemistry, Justus Liebig University of Gießen

Christian Preußer, Institute for Tumorimmunology, Center for Tumor Biology and Immunology, Philipps University Marburg

Introduction: Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material such as various classes of coding and non‐coding RNAs, a few of which have been shown to be selectively targeted into vesicles. However, factors and mechanisms contributing to this specificity remain largely elusive, and only a few putative protein factors involved in packaging have been described.

Methods: Here we used reporter systems to decipher the loading efficacy of different RNA species into EVs, based on different expression constructs and transfection in mammalian cells: First, we studied RNA polymerase‐ (RNA pol) dependent effects at the 5' end of the RNA as well as different transcriptional terminators at the 3' end. Second, the size dependence of RNA loading was investigated, based on a series of reporter constructs. The relative and the absolute abundance of the different reporters as well as of some endogenous RNAs were determined.

Results: We could show that RNA pol III transcripts are more efficiently loaded into EVs than RNA pol II transcripts, and that even in these overexpression system, only relatively few RNA molecules per EV could be detected. This conclusion is further supported by the absolute quantification of endogenous EV‐associated RNAs. Regarding size distribution, we observed that shorter RNAs are more efficiently released into EVs than longer RNAs, indicating that size is an important determinant of RNA export.

Summary/Conclusion: Our results reflect the current debate on EV‐associated RNAs. The initial enthusiasm on the enrichment of various RNAs in EVs has been dampened by some reports and our own analysis: RNAs appear to be EV‐associated only at low copy numbers, and the question is still open whether this RNA association reflects internal EV encapsulation, or merely a less tightly bound state at the vesicle surface.

CC1.1. Mitochondrial‐lysosomal crosstalk induces mitochondrial‐derived vesicle generation in cisplatin chemoresistance

Flora Guerra, Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento

Sinforosa Gagliardi, Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento

Silvia Caterina Resta, Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento

Cecilia Bucci, Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento

Introduction: Different studies suggest a key role of the crosstalk between mitochondria and lysosomes in cellular physiology and its dysregulation is present in cancer. Indeed, it is known that mitochondrial impairment can influence lysosomal function and viceversa. RAB7 is a small GTPase with multiple key roles in cellular physiology. RAB7 controls transport to late endocytic compartments and regulates late endocytic organelle biogenesis, lysosomal positioning and functions, trafficking and degradation of several signaling receptors and extracellular vesicle (EV) secretion. In recent works, RAB7 was described also as regulator of mitophagy, mitochondrial‐lysosomal contacts and mitochondrial dynamics. Moreover, it is known that RAB7 may determine fusion between mitochondrial derived vesicles (MDVs) with late endosome, but its role in MDVs biogenesis is poorly understood.

Methods: In this context, we have purified trough ultracentrifugation and immunoisolation EVs from cisplatin chemosensitive and chemoresistant ovarian cancer cell lines. We verified their endosomal biogenesis and mitochondrial content through western blotting. Moreover, we performed PCR to analyze the presence of mitochondrial DNA (mtDNA).

Results: We found that RAB7 downregulation and impairment of late endocytic functions characterize all chemoresistant cells. Moreover, in chemoresistant cells we observed increase of EV secretion compared to matched chemosensitive cells. Interestingly, we found that purified EVs contain several mitochondrial proteins and mtDNA.

Summary/Conclusion: Here, we concluded that cisplatin chemoresistance is associated with alteration of late endocytic pathway and with consequent increase of EV secretion. It is known that cisplatin treatment induces mitochondrial dysfunction. In this context, RAB7 is not able to induce autophagic degradation of dysfunctional mitochondria which are secreted becoming potential markers of chemoresistance.

CC1.2. The role of Notch pathway in the pro‐tumorigenic activity of extracellular vesicles in multiple myeloma

Domenica Giannandrea, Università degli Studi di Milano

Michela Colombo, Università degli Studi di Milano

Natalia Platonova, Università degli Studi di Milano

Valentina Citro, Università degli Studi di Milano

Mara Mazzola, Università degli Studi di Milano

Anna Pistocchi, Università degli Studi di Milano

Raffaella Adami, Università degli Studi di Milano

Vincenza Dolo, Università degli Studi dell'Aquila

Ilaria Giusti, Università degli Studi dell'Aquila

Laura Cantone,Università degli Studi di Milano

Valentina Bollati, Università degli Studi di Milano

Mauro TurriniValduce Hospital

Raffaella Chiaramonte, Università degli Studi di Milano

Introduction: Multiple myeloma (MM) is characterized by the tight interaction between MM cells and bone barrow (BM) niche, resulting in tumor progression.

MM cells overexpressed Notch 2 and Jagged 1 and 2, triggering Notch pathway activation on BM population and their pro‐tumorigenic activity.

Extracellular vesicles (EV) represent novel pro‐tumorigenic players in the MM microenvironment.

In this work we assess the tumorigenic effect of MM‐derived EVs and the role played by the Notch pathway in EV‐mediated communication between MM cells and the BM cells.

Methods: EVs from MM cell lines (MM‐EVs) or MM cell lines constitutively inhibited for Jagged1/2 (MMJ1/2KD‐EVs) or Notch2 (MMN2KD‐EVs) were characterized for Notch2 and Jagged1 and 2 content by Western blot and for size and number by nanoparticle tracking analysis and electronic transmission microscopy. The transfer of HA‐tagged Notch2 via EVs was evaluated by an engineered system of HEK293 sending and receiving cells. Notch pathway activation was evaluated in vivo by injecting MM‐EVs in the duct of Cuvier of 2 days post fertilization Notch‐reporter Tg(T2KTp1bglob:hmgb1‐mCherry)jh transgenic zebrafish embryos. The pro‐tumorigenic effect of MM‐EVs, MMJ1/2KD‐EVs and MMN2KD‐EVs were assessed in vitro by measuring the osteoclastogenic potential, the ability to induce human endothelial cells to organize tubular structures and assessing changes in stromal cell‐mediated drug resistance.

Results: MM‐EVs carry Notch2 Jagged1 and 2 and transfer them to recipient cells; Notch members levels depend on their expression in MM cells.

The analysis of the functional effects indicates that MM‐EVs interact and activate Notch pathway in receiving cells in vitro and in vivo and display a pro‐tumorigenic effect. MM‐EVs show osteoclastogenic effect and angiogenic ability and boost drug resistance induced by the BM stromal cells HS5. All these effects are lost when EVs are produced by MMJ1/2KD and MMN2KD cells.

Summary/Conclusion: These results provide the first evidence that targeting the Notch pathway may be a valid therapeutic strategy to hamper the pro‐tumorigenic role of EV in MM progression.

CC1.3. Extracellular vesicles from triple negative breast cancer promote differentiation of pro‐inflammatory macrophages associated with better clinical outcome

Jessie Thalmensi, Institut Curie / INSERM U932

Eleonora Timperi, Institut Curie / INSERM U932

Paul Gueguen, Institut Curie / INSERM U932

Nathalie Névo, Institut Curie / INSERM U932

Eleonora Grisard, Institut Curie U932

Philemon Sirven, Institut Curie / INSERM U932

Federico Cocozza, Institut Curie / INSERM U932

Alizée Gouronnec, Institut Curie / INSERM U932

Lorena Martin‐Jaular,Institut Curie / INSERM U932

Mabel Jouve, Institut Curie / CNRS UMR 3215

Coralie GuérinInstitut Curie / FlowCytométrie Platform

Vassili Soumelis, Université de Paris / INSERM U976

Emanuela Romano,Institut Curie / INSERM U932

Elodie Segura,Institut Curie / INSERM U932

Clotilde Thery, MD PhD,Institut Curie / INSERM U932

Clotilde Thery, MD PhD,InstitutCurie / INSERM U932

Introduction: Tumor associated macrophages (TAMs) are highly abundant in human cancers, representing widely heterogeneous populations. The contribution of various tumor‐derived signals to differentiation of circulating monocytes into distinct TAM subsets is not well understood. In particular, tumors release both soluble factors and extracellular vesicles (EVs: exosomes, ectosomes and others) containing a complex set of signaling molecules, whose impact on TAM precursors may be different.

Methods: Here, we used Size‐exclusion chromatography (SEC) to separate EVs from soluble molecules in the secretome of triple negative breast cancer (TNBC) cell lines. We cultured human blood monocytes with EV‐rich or EV‐poor SEC fractions and analyzed the phenotype of differentiated monocytes in terms of cell surface marker expression and cytokine secretion, and by global transcriptomic analysis. We generated by CRISPR/Cas9 tumor cell lines KO for several genes that we identified as relevant to disclose the mechanisms mediating the observed effects (CSF1, Rab11, STING). We compared the in vitro obtained gene signatures with those of macrophages isolated from human breast tumor patients and analysed by single cell RNASeq.

Results: We show that both EVs and soluble secretome promote monocyte differentiation towards macrophages. However, EVs specifically promoted a subset of pro‐inflammatory macrophages bearing an IFN signature. CSF‐1 exposed on EVs was necessary for macrophage differentiation and the cGAS/STING axis was involved in the activation of the IFN‐response. Macrophages imprinted with an EV‐signature or with the soluble molecule signature were both found in patient's TAMs. Strikingly, EV‐induced macrophage signature positively correlated with T cell infiltration and patient survival.

Summary/Conclusion: Together these data suggest that TNBC‐released CSF‐1‐bearing EVs promote a tumor immune microenvironment associated with a favourable prognosis in TNBC patients.

CC1.4. Cancer cells shuttle extracellular vesicles containing oncogenic mutant p53 proteins to the tumor microenvironment

Bibek Bhatta, Ben‐Gurion University of the Negev

Ishai Luz, Ben‐Gurion University of the Negev

Christian Krueger, Division of Cellular & Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre Singapore

Fanny Xueting Teo, Division of Cellular & Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre Singapore

David Lane, p53 Laboratory (p53Lab), Agency for Science, Technology, and Research (A*STAR)

kanaga sabapathy, Division of Cellular & Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre Singapore

Tomer Cooks, Ben‐Gurion University of the Negev

Introduction: Mutations in the TP53 gene (encoding for the p53 tumor suppressor protein) are the most common molecular event in human cancer. Cancer cells harboring gain‐of‐function (GOF) mutant p53 are more aggressive than cancer cells harboring inactivating mutations or wild‐type (WT)‐p53. Notably, multiple studies have delineated the presence of GOF‐mutant p53 protein in untransformed cells or in stromal compartments of tumor microenvironment (TME). In recent years, the involvement of extracellular vesicles (EVs) in cell‐to‐cell communication has emerged as a major route by which cells can interact with each other. On the same note, cancer cells were shown to produce excessive amounts of EVs received by TME cells, thus recruiting the TME to become tumor‐supportive, therefore suggesting that EVs‐mediated communication between cancer cells and cells of TME imparts an aggressive trait to the underlying cancer. To this end, we hypothesize that mutant p53 protein can be shuttled via EVs to TME cells thus shedding light on a novel non‐cell autonomous role of mutant p53 cancers.

Methods: EVs were isolated from various cancer cell lines differing by their p53 status and the effect on neighboring cancer cells and TME cells was studied in vitro and in‐vivo. We used PANC‐1 pancreatic ductal adenocarcinoma cells, we knocked‐out the R273H endogenous mutant p53 using the CRISPR‐Cas9 system as well as with shRNA stable knockdown of the mutant p53 gene. In H358 lung carcinoma cells, we used Tetracycline‐controlled transcriptional activation cell system overexpressing several different p53 GOF mutants (V157F, R175H, R249S and R273H), simulating inactivated p53, compared with the WT form and an empty vector. We utilized the human colorectal Colo‐320DM cancer cell xenograft model, which expresses the R248W p53 mutant. FFPE sections of subcutaneous tumors derived from the Colo‐320DM xenografts were stained for p53 using the DO‐1 antibody that specifically recognizes human p53.

Results: Our data demonstrated that mutant p53 protein can be selectively sorted into EVs; that mutant p53 in EVs can be taken up by neighboring cancer cells and macrophages that do not harbor mutant p53 and that mutant p53 expression is found in non‐tumor cells in both human cancers, and in non‐human tissues in human xenografts.

Summary/Conclusion: In this report, we tested and corroborated the fundamental hypothesis suggesting the cancer cells that harbor GOF p53 mutants, can package these mutant proteins in EVs, and deliver them to neighboring cancer cells and to the TME.

CC1.5. A comprehensive comparative analysis of extracellular vesicle release in non‐small cell lung cancer and its potential to drive cancer hallmarks in non‐cancerous lung epithelial cells

Humna Hasan, Purdue University

Ikjot Singh Sohal, Purdue University

Zulaida M. Soto‐Vargas, zsotovar, Purdue University

Anjali Byappanhalli, Purdue University

Sean Humphry, Purdue University

Hana Kubo, Northwestern University

Sarunya Kitdumrongthum, Mahidol University, Thailand

Arthit Chairoungdua, Mahidol University, Thailand

Andrea Kasinski, Purdue University

Introduction: Cancer‐derived extracellular vesicles (EVs) play a pivotal role in cancer progression by mediating bi‐directional communication between cancer cells and their environment. While multiple lines of evidence have shown how non‐small cell lung cancer (NSCLC) EVs promote cancer progression by evaluating distinct aspects of cancer, it remains unclear how EVs from different NSCLC cells differ in their secretion profile and in their potential to promote various cancer hallmarks.

Methods: To address this need, we performed a comparative analysis of (i) EV release from non‐cancerous bronchial epithelial cells (HBEC/BEAS‐2B) and several NSCLC cells (A549, H460, H358, SKMES and Calu6) as well as (ii) the potential of NSCLC EVs including EV‐encapsulated RNA in driving cancer hallmarks in HBEC/BEAS‐2B cells.

Results: The isolated EVs were in the 100–150nm size range and were enriched in CD9 and CD81 tetraspanins. While literature indicates that cancer cells generally have higher secretion rate, our secretion analysis indicated that only two (Calu6 and H358) out of 5 NSCLC cells had higher secretion rate compared to HBEC/BEAS‐2B cells. We observed differential uptake of NSCLC EVs by non‐cancerous cells with A549 and SKMES EVs showing the highest uptake. We showed that EVs derived from high secretion rate cells (H358 and Calu6) were able to disrupt BEAS‐2B epithelial barrier and significantly increase permeability, which was further confirmed by downregulation of E‐cadherin and ZO‐1 junctional complex proteins. Similarly, only H358 and Calu6 EVs dramatically enhanced invasive phenotype in HBEC/BEAS‐2B cells. On the other hand, EVs derived from low secretion rate cells neither impaired epithelial barrier (except SKMES) nor induced invasive phenotype to a large extent. Furthermore, EV‐encapsulated RNA was attributed as a contributing factor in mediating the above‐mentioned phenotypes for H358 and Calu6 EVs.

Summary/Conclusion: More nuanced analysis suggested that the NSCLC subtypes can be correlated with the potential of their distinct EVs to drive cancer hallmarks. In conclusion, the study lays the groundwork which will guide future studies to detail the role of EVs and their cargo in modulating the microenvironment at various stages of lung cancer progression.

CC2.1. Genome‐wide shRNA screening identifies factors required for exosome secretion from microglia

ZHI RUAN, Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine

Kayo Takamatsu‐Yukawa, Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine

Yuzhi Wang, Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine

Adam T. Labadorf, Department of Neurology, Bioinformatics Program, Boston University

Seiko Ikezu, Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine

Tsuneya Ikezu, MD, PhD, Department of Pharmacology & Experimental Therapeutics, Center for Systems Neuroscience,Boston University School of Medicine; Department of Neuroscience, Mayo Clinic Florida

Introduction: Microglia are the principal immune cells in the central nervous system, serving for functional and metabolic homeostasis as well as innate immune response to pathogen invasion and neuronal damage. After rapidly responding to noxious stimuli, activated microglia could release exosomes, the smallest extracellular vesicles, carrying various pro‐inflammatory cytokines including IL‐1β. The molecules critical for regulating the exosome production from microglia are yet to be understood.

Methods: Tetraspanin protein CD63 was constructed with tdTomato and packaged by VSV‐G pseudotyped lentivirus to establish murine microglial BV2 cell line stably expressing CD63‐tdTomato. The pooled shRNA library was constructed with the Dharmacon SMARTvector Lentiviral shRNA library expressing short hairpin RNAs (shRNAs), which target genome‐wide 21,745 genes with 8 clones per target and expressing TurboGFP under the control of murine EF1α promoter. After sorting of TurboGFP+ CD63‐tdTomato+ BV2 cell library, cells were exposed to 5mM ATP stimulation for exocytosis of CD63‐tdTomato+ exosomes, and sorted for TurboGFP+ CD63‐tdTomatohigh and CD63‐tdTomatolow cells. The sorted cells were subjected to genomic DNA extraction, PCR amplification of barcoded shRNA region and next generation sequencing. The enrichment of shRNA clones in CD63‐tdTomatohigh and CD63‐tdTomatolow cells were ranked by the Z‐score to identify the “Hit” candidates.

Results: ATP‐induced exosome secretion from CD63‐tdTomato+ BV2 cells and reduction of the tdTomato signal in cells, showing that CD63‐tdTomatohigh and CD63‐tdTomatolow cells show suppression or enhancement of CD63‐tdTomato+ exosomes, respectively. By using a barcoded lentivirus‐based pooled short‐hairpin RNA (shRNA) library combined with next generation sequencing, we identified 1353 silenced host genes highly enriched in cells resistant to the ATP induced exosome secretion. The majority of these genes are the integral component of membrane and plasma membrane and participate in the cell‐to‐cell communication as determined by the DAVID gene ontology analysis and Metascape. Validation experiments were performed on several top hits, and determined essential for the exosome secretion from microglia as assessed by nanoparticle tracking analysis and CD63 ELISA. Finally, silencing of these three genes by siRNAs suppressed the lipopolysaccharide‐ and ATP‐induced IL‐1β release from murine primary cultured microglia.

Summary/Conclusion: These findings provided novel candidate genes for microglial exosome secretion, which is a therapeutic target of neuroinflammatory and neurodegenerative disorders.

CC2.2. Novel method for isolating extracellular vesicles from hippocampal interstitial fluid in Alzheimer's disease

Morgan Pait, Wake Forest School of Medicine

Sarah Kaye, Wake Forest School of Medicine

Yixin Su, Wake Forest School of Medicine

Andy Snipes, Wake Forest School of Medicine

Jingyun Lee, PhD, Wake Forest School of Medicine

Cristina Furdui, PhD, Wake Forest School of Medicine

Gagan Deep, PhD, Wake Forest School of Medicine

Shannon Macauley, PhD, Wake Forest School of Medicine

Introduction: Abnormal protein aggregation is a hallmark of Alzheimer's disease (AD). Exosomes, endosome‐derived, small extracellular vesicles (EVs), can transport AD‐related proteins amyloid‐beta (Aβ) and tau, making them potential AD blood‐based biomarkers. However, it is unclear how peripheral exosomes compare to exosomes in the brain's interstitial fluid (ISF) as AD pathology progresses in vivo. Here we describe a novel method for collecting exosome‐enriched EVs from hippocampal ISF using in vivo microdialysis in the presence and absence of Aβ pathology.

Methods: In vivo microdialysis was used to collect hippocampal ISF from 3‐and 9‐month‐old, unanaesthetized, unrestrained, APPswe/PS1″E9 (APP/PS1), a mouse model of Aβ overexpression, and B6C3 wildtype (WT) mice. Exosome‐enriched ISF EVs were isolated via ultracentrifugation then underwent nanoparticle tracking analysis and immunogold labeling. Mass spec and proteomic analysis were performed on both EV surface and cargo proteins.

Results: ISF EVs from APP/PS1 and WT mice were 40–150nm and CD63‐ and CD9‐positive. Of ExoCarta's Top 100 exosome proteins, 59 were found in the 3mo, WT ISF EVs. EV concentration increased with age in males, but not females, and was lower in APP/PS1 vs. WT. In 3mo APP/PS1 mice, fewer endothelial‐related proteins were on EV surface compared to WT. At 9mo, following Aβ plaque formation, astrocyte, microglia, neuronal and oligodendrocyte‐related proteins were altered on the surface and in the core of exosome‐enriched EVs. EV surface and core proteins changed with age, with 612 proteins unique to 3mo, 161 unique to 9mo, and 333 proteins shared across ages. At 3mo, WT and APP/PS1 shared 325 proteins, WT had 495 unique proteins and APP/PS1 had 125 unique proteins. At 9mo, WT and APP/PS1 shared 372 proteins, while 101 were specific to WT but only 21 to APP/PS1.

Summary/Conclusion: In vivo microdialysis is a novel method for brain ISF EV collection. Exosome‐enriched, ISF EVs are altered with age and Aβ pathology. Aβ plaques may impair release, clearance and/or uptake of EVs sex‐dependently. Furthermore, cell‐type specific (astrocyte, neuron, etc.) ISF EVs and their contents are altered in APP/PS1 mice. In vivo ISF exosome‐enriched EVs offer a unique opportunity to identify novel AD biomarkers and validate them in peripheral EVs.

CC2.3. Outreach of striatum derived EVs in the mouse brain

David Rufino‐Ramos, CNC ‐ Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

Koen Breyne, PhD, Molecular Neurogenetics Unit, Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA, USA

Killian O'Brien, PhD, Molecular Neurogenetics Unit, Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA, USA

Kevin Leandro, CNC ‐ Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

Thomas S Van Solinge, Molecular Neurogenetics Unit, Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA, USA

Shadi Mahjoum, Molecular Neurogenetics Unit, Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA, USA

Sevda Lule, Molecular Neurogenetics Unit, Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA, USA

Shilpa Prabhakar, Molecular Neurogenetics Unit, Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA, USA

Luís Pereira De Almeida, CNC ‐ Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal;

Xandra O Breakefield, Molecular Neurogenetics Unit, Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA, USA

Introduction: Extracellular vesicles (EVs) are known as mediators of intercellular communication, primarily due to their capacity to transfer functional cargo and ultimately modulate cellular function. Transfer of functional cargo in the brain can be demonstrated using the Cre‐LoxP reporter system.

The aim of this work is to evaluate the biodistribution of functional striatum‐derived EVs in a mouse brain over time by exploiting the genome modifications ilicited by EV‐encapsulated Cre‐recombinase (Cre) in the recipient cells of a floxed reporter mice.

Methods: HEK293T cells were transduced with lentivirus encoding Cre and Fluc under the PGK and UBC promoter, respectively. EVs were isolated by Size Exclusion Cromatography (SEC) and the presence of Cre encoding mRNA was demonstrated by qPCR. In vitro transfer was determined by direct co‐culture and incubation of CRE‐EVs with both Ai9 and Nanoluc reporter cell lines. Fluorescence and luminescence was measured and RT‐PCR was used to confirm editing at the DNA level.

The lentivirus was injected into the right striatum of Ai9 reporter mice. Mice were monitored by IVIS imaging and were sacrified at designated time‐points. Whole brain coronal sections were analyzed for tdTomato‐positive signal by immunohistochemistry and ddPCR. Moreover, the level of DNA editing in other brain regions was analysed by correlating floxed and unfloxed DNA.

Results: Stably transduced cells were able to produce EVs encapsulating functional Cre mRNA that was delivered to Ai9 and Nanoluc reporter cell lines, resulting in induction of tdTomato and luciferase in a time‐dependent manner. Ai9 mice intracranially injected with lentivirus encoding Cre and Fluc express functional Cre and tdTomato signal not only at the injection site, but also in other brain regions.

Summary/Conclusion: This study emphasizes the ability of striatum‐derived EVs to distribute throughout the brain and transfer functional cargo.

CC2.4. Interneuronal exchange and functional integration of synaptobrevin via extracellular vesicles

Natali L. Chanaday, Department of Pharmacology ‐ Vanderbilt University

Alejandro Vilcaes, CIQUIBIC‐CONICET Depto. Quimica Biologica Fac. de Ciencias Quimicas Pabellon Argentina Universidad Nacional de Cordoba

Ege T. Kavalai, Professor & Acting Chair, Department of Pharmacology, William Stokes Chair In Experimental Therapeutics, Vanderbilt University Department of Pharmacology

Introduction: In the past few years the role of extracellular vesicles (EVs) as short and long distance messengers has grown considerably. We now know that these secreted vesicles mediate the interchange of genomic materials, proteins and lipids in numerous tissues and can change the phenotype of the target cells. However, the functions of EVs in the nervous system and the underlying molecular mechanisms are only starting to be comprehended. The goal of the present work was to understand the putative role of EVs in neurotransmission from a molecular neuroscience perspective.

Methods: We isolated EVs from glia‐free dissociated rat and mouse hippocampal neurons. EVs were characterized by proteomics, electron microscopy and nanoparticle tracking analysis. We then studied the impact of EVs on neuron physiology using a combination of live fluorescence imaging and electrophysiology. Studies were complemented with Western blots, dot blots and localization analysis via confocal microscopy.

Results: We found that neuronal EVs contain several neuron‐specific proteins, including synaptic vesicle proteins that regulate the release of neurotransmitters, among them synaptobrevin‐2 (syb2). Syb2 from EVs can be functionally incorporated into synaptic vesicles in the target neurons, leading to a selective, calcium‐dependent increase in inhibitory neurotransmission. Syb2 recruitment to EVs is dependent on the tetraspanin CD81. Moreover, EVs containing syb2 can partially rescue spontaneous neurotransmission in syb2 knock‐out neurons. Conversely, EVs from hippocampal astrocytes, which lack syb2, have no effect on spontaneous neurotransmission.

Summary/Conclusion: These findings shed light on the molecular underpinnings of a novel form of interneuronal communication mediated by EVs and suggest the trafficking routes of EVs and synaptic vesicles may be interconnected.

CC2.5. Monitoring of the effect neutral sphingomyelinase 2 inhibition on neural extracellular vesicles release by surface plasmon resonance imaging

Silvia Picciolini, IRCCS Fondazione Don Carlo Gnocchi

Carolyn Tallon, PhD, Johns Hopkins University School of Medicine

Cristiano Carlomagno, IRCCS Fondazione Don Carlo Gnocchi

Alice Gualerzi, PhD, IRCCS Fondazione Don Carlo Gnocchi ONLUS

Seung‐Wan Yoo, Johns Hopkins University School of Medicine

Ajit G. Thomas, Johns Hopkins University School of Medicine

Arindom Pal, Johns Hopkins University School of Medicine

Jesse Alt, Johns Hopkins University School of Medicine

Norman J. Haughey, Johns Hopkins University School of Medicine

Rana Rais,Johns Hopkins University School of Medicine

Barbara S. Slusher, Johns Hopkins University School of Medicine

Marzia BedoniIRCCS Fondazione Don Carlo Gnocchi

Introduction: Considering the implication of EVs in the spread of pathogenic proteins, inhibitors of Neutral Sphingomyelinase 2 (nSMase2), an enzyme critical in EV biogenesis, have been studied as therapeutic agents for several diseases. We recently discovered phenyl(R)‐(1‐(3‐(3,4‐dimethoxyphenyl)‐2,6‐dimethylimidazo[1,2‐b]pyridazin‐8‐yl)pyrrolidin‐3‐yl)carbamate (PDDC), the first potent, selective, orally‐available, and brain penetrable nSMase2 inhibitor, capable of reducing EV release in vitro and in vivo. Herein, we used Surface Plasmon Resonance imaging (SPRi) to evaluate which specific brain cell‐derived EVs were affected by PDDC in response to a neuroinflammatory insult.

Methods: Mice fed vehicle or PDDC‐containing chow were administered an intra‐striatal IL‐1β injection and 2h later their plasma were collected. EVs were isolated from plasma by size‐exclusion chromatography and different brain‐derived EVs were detected on a SPRi‐based biosensor by probing specific EV membrane molecules.

Results: IL‐1β‐induced injury selectively increased the levels of plasma EVs derived from CD171+ neurons and PLP1+ oligodendroglia that were normalized by PDDC, while GLAST+ astrocyte‐derived EVs were unchanged. IL‐1β injection increased the amount of EVs released from activated, CD11b+ microglia compared to EVs released from non‐activated microglia in plasma. The increase in EVs from activated microglia was normalized with PDDC.

Summary/Conclusion: We found that PDDC was able to normalize the increased release of neuronal‐, oligodendrocyte‐ and activated microglial‐derived EVs into plasma following an acute brain injury. These data support nSMase inhibition as a therapeutic strategy for acute brain injury, the use of circulating brain‐derived EVs as indicators of neuroinflammation status and the use of SPRi to evaluate the efficacy of therapeutics.

CC3.1. Crosslinking mass spectrometry reveals the EV‐surface protein composition, structural terrain and interactome

Julia Bauzá‐Martinez, Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands

Gadi Armony, Utrecht University

Albert J.R. Heck, Utrecht University

Wei Wu, Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands

Introduction: EVs mediate key processes like intercellular communication and immune activation. EVs can directly trigger T‐cell activation by forming immunological synapses with the TCR, or indirectly after uptake by APCs. Although membrane proteins are involved in most of EV‐mediated processes, the study of such proteins remains challenged by technical limitations. In crosslinking mass spectrometry (XL‐MS), a chemical crosslinker is used to covalently link surface residues of proteins in close‐proximity, i.e., < 30Å. Yet, XL‐MS relies on using lots of material, a key limitation when working with EVs. Here, we overcome this limitation by using a highly efficient crosslinker (disuccinimidyl suberate; DSS) and a fractionation strategy to enrich for crosslinked peptides. We apply this to study the surface interactome of intact B‐cell derived EVs.

Methods: EVs from JY cells were isolated by ultracentrifugation (UC). Intact EVs were reconstituted in PBS and crosslinked with DSS for 10 min. Crosslinked EVs were lysed and proteins were trypsin‐digested. Crosslinked peptides were enriched by strong cationic exchange fractionation before LC‐MS analysis. Data was searched against a tailored database containing abundant proteins of the EV proteome. EV purity and composition were characterized by multiple techniques, including NTA, TEM and bottom‐up proteomics.

Results: Isolated EVs ranged between 30 ‐ 200 nm as shown by NTA and TEM, and appeared intact after UC. Compared to source cells, EVs were highly enriched in EV markers and membrane proteins, including CD81 and MHC‐I. By XL‐MS we identified ∼1,000 crosslinks from EVs, simultaneously mapping several membrane and EV‐specific protein interactions. The large matrix of distance restrains generated not only describes the EV‐surface terrain but can also be used to make structural models. Finally, we contrasted EV‐specific interactions to the JY cell surface interactome, with focus on supramolecular MHC complexes.

Summary/Conclusion: Here, we demonstrate the feasibility of using XL‐MS to study the EV‐interactome. By selecting a suitable crosslinker and enriching for crosslinked species, we boosted sensitivity overcoming one of the main limitations of working with EVs. We envision this approach will be highly suitable for EV‐surface interactome profiling, to aid in rationalizing membrane fusion and uptake processes in recipient cells.

CC3.2. A novel enzymatic surface functionalization approach for generating engineered extracellular vesicles for targeted drug delivery

Migara K. Jayasinghe, Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore

Tin C. Pham, Department of Biomedical Sciences, College of Veterinary Medicine and Life Sciences, City University of Hong Kong

Thach T. Pham, Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore

Yuqi Yang, Department of Biomedical Sciences, College of Veterinary Medicine and Life Sciences, City University of Hong Kong

Marco Pirisinu, Department of Biomedical Sciences, College of Veterinary Medicine and Life Sciences, City University of Hong Kong

Boya Peng, Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore

Jiahai Shi, Department of Biomedical Sciences, College of Veterinary Medicine and Life Sciences, City University of Hong Kong

Minh T. Le, Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore

Introduction: Given their propensity for intercellular communication and their excellent biocompatible profile, extracellular vesicles (EVs) have been viewed for many years as promising vectors for drug delivery. However, endogenous EVs are often lacking in their target specificity leading to the incorporation of artificial modifications that elevate their therapeutic potential. Thus far, most studies on EV surface engineering have relied on slight variations of four principle approaches ‐ genetic engineering of cells, lipid insertion, affinity interactions and chemical conjugation. While demonstrating impressive functionality in some cases, they tend to be tedious and inefficient due to their reliance on methods such as genetic manipulation, transient interactions and harsh chemical treatments. Here we describe a novel approach for the surface functionalization of EVs based on enzymatic ligation.

Methods: Via the use of protein ligases, we are able to efficiently and site‐specifically conjugate a desired targeting molecule of choice at high copy number onto pre‐existing EV membrane proteins via the formation of covalent peptide bonds. The approach is biocompatible, requires no genetic manipulation and has no effect on EV integrity or physicochemical characteristics.

Results: Conjugation of targeting peptides and antibodies onto the EV surface enables efficient and specific delivery of encapsulated drugs to target cells expressing corresponding receptors such as EGFR, EpCAM and CXCR4. Furthermore, we demonstrate tumor specific delivery of EV‐encapsulated chemotherapeutics, peptides and RNA drugs upon systemic administration in multiple mouse models of cancer, including a lung cancer xenograft, leukemia and breast cancer. Targeted delivery of encapsulated therapeutics leads to significantly better tumor suppression and improved treatment outcomes in vivo. We also demonstrate the ability to target other receptors such as SIRP‐α to enhance anti‐phagocytic properties of EVs.

Summary/Conclusion: We present here a novel method of surface functionalization that improves upon the efficiency and versatility of existing methods and preserves the endogenous biocompatible profile of EVs while concurrently utilizing a more clinically translatable approach by virtue of the non‐immunogenic, non‐mutagenic and stable nature of enzymatic ligation.

CC3.3. On‐chip magnetic immuno‐extraction of small extracellular vesicles from human plasma

Monica Araya‐Farias, Curie Institute

Dario Brambilla, CNR ‐ SCITEC

Lucile Alexandre, McGill University

Laura Trapiella‐Alfonso, Chimie Paris Tech‐PSL University

Giacomo Gropplero, Curie Institute

Marine Verhursel, Fluigent‐Smart Microfluidics

William Cesar, Fluigent‐Smart Microfluidics

Marcella Chiari, National Research Council of Italy ‐ Institute of Chemical Sciences and Technologies (CNR ‐ SCITEC)

Than‐Duc Mai, Université Paris‐Saclay

Marco Morani,Université Paris‐Saclay

Stéphanie Descroix, Curie Institute

Introduction: Extracellular vesicles (EVs) have emerged over the past years as the new biomarkers of the future for non‐invasive disease diagnostics, sometimes called as the new Hermes biomarkers as they will provide a large quantity of information. However, to exploit their potential in human diagnostics, a fast, high‐throughput and reproducible method is needed for their extraction and analysis. Here, we present an original microfluidic approach that combines immuno‐extraction and fluidized bed (FB) technology to isolate EVs directly from human plasma allowing the capture and release of EVs in a single device.

Methods: We have developed a miniaturized and microfluidic fluidized bed in which magnetic and drag forces are balanced to keep in suspension antibody‐functionalized magnetic beads in a triangular shaped‐chamber during sample perfusion. Beads were functionalized with EVs‐specific antibodies (AntiCD63 or AntiCD9) by using a novel DNA‐directed immobilization (DDI) strategy. A low"cost FB microfluidic platform was developed to carry out the experiments in a completely automated manner.

Results: We evaluated the effect of the flow rate ranging from 0.5 to 5μL/min to tune the residence time of the sample in the chip in order to achieve the highest efficiency. EVs were extracted from human plasma with around 80% of capture efficiency. We demonstrated thanks to Nano Tracking Analysis (NTA) that our microfluidic strategy allowed to capture and to release significantly a large number of EVs (40% higher than an experiment performed in tube) with a size between 100 to 200 nm. This integrated and automated approach enabled the release of intact vesicles which was successfully confirmed by imaging techniques such as Transmission Electron Microscopy (TEM) and Cryo‐Electron Microscopy as well as Capillary Electrophoresis Coupled Laser Induced Fluorescent Detection (CE‐LIF).

Summary/Conclusion: We demonstrated the feasibility of our fluidized bed approach to efficiently extract small EVs from a complex biological matrix. Further experiments will be performed to assess how these fluidized bed devices can be sequentially coupled to extract different EVs subpopulations.

CC3.4. AFM‐based high throughput mechanical characterization of single EVs from different natural sources

Andrea Ridolfi, Dipartimento di Chimica “Ugo Schiff”, Università degli Studi di Firenze, 50019 Firenze, Italy

Marco Brucale, Consiglio Nazionale delle Ricerche, Istituto per lo Studio dei Materiali Nanostrutturati, 40129 Bologna, Italy

Costanza Montis, Dipartimento di Chimica “Ugo Schiff”, Universitàdegli Studi di Firenze, 50019 Firenze, Italy

Lucrezia Caselli, Dipartimento di Chimica “Ugo Schiff”, Università degli Studi di Firenze, 50019 Firenze, Italy

Lucia Paolini, Dipartimento di Medicina Molecolare e Traslazionale, Università degli Studi di Brescia, 25123 Brescia, Italy

Anne Borup, Department of Clinical Medicine, Faculty of Health, Aarhus University, 8200 Aarhus, Denmark

Anders Toftegaard Boysen, Department of Clinical Medicine, Faculty of Health, Aarhus University, 8200 Aarhus, Denmark

Francesca Loria, HansaBioMed Life Sciences OÜ, Mäealuse 2/1, 12618 Tallinn, Estonia

Martijn van Herwijnen, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.

Marije Kleinjan,Department of Biomolecular Health Sciences, Utrecht University, The Netherlands

Peter Lindberg Nejsum, Department of Clinical Medicine, Faculty of Health, Aarhus University, 8200 Aarhus, Denmark

Natasa ZarovniHansaBiomed Life Sciences, 12618 Tallinn, Estonia

Marca H.M. H.M. Wauben, Department of Biomolecular Health Sciences, Utrecht University, The Netherlands

Debora Berti,Dipartimento di Chimica “Ugo Schiff”, Università degli Studi di Firenze, 50019 Firenze, Italy

Paolo Bergese,Dipartimento di Medicina Molecolare e Traslazionale, Università degli Studi di Brescia, 25123 Brescia, Italy

Francesco Valle,Consiglio Nazionale delle Ricerche, Istituto per lo Studio dei Materiali Nanostrutturati, 40129 Bologna, Italy

Francesco Valle,Consiglio Nazionale delle Ricerche, Istituto per lo Studio dei Materiali Nanostrutturati, 40129 Bologna, Italy

Introduction: The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function in terms of e.g. cellular adhesion, endo/ exocytosis, cellular uptake, and mechanosensing. Moreover, these characteristics provide a significant contribution to the heterogeneity that affects different EV subtypes, which is one of the main issues hindering the application of EVs in multiple biomedical fields. EVs nanomechanics can be studied by Atomic Force Microscopy‐based Force Spectroscopy (AFM‐FS); however, the low throughput and the need for dedicated instrumentation usually limit this approach to specialists, making it unable to address the demand of the EV community for a simple and rapid mechanical characterization.

Methods: In the attempt of providing a solution to this open issue, we herein present a simple AFM‐based high throughput characterization method that allows for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale (Ridolfi A. et al, 2020, Anal. Chem. 92:10274"10282). The procedure is based on the measurement of the contact angle displayed by each vesicle, upon adsorption on a surface.

Results: Results suggest that the contact angle can be regarded as a mechanical fingerprint of the typical “vesicle‐like” behavior, allowing for the detection of contaminants within an EV sample and for rapidly assessing the presence of EVs even in samples for which no established assays and/or commercial kits are available. Moreover, we show that a vesicle's contact angle is directly related to its stiffness as measured by AFM‐FS, and can hence be used to obtain a high throughput mechanical characterization of single EVs from different populations.

Summary/Conclusion: In this framework, we present our latest results regarding the mechanical characterization of multiple EV samples coming from distinct natural sources, thereby demonstrating the applicability of the method to a wide variety of EV subtypes and its ability to map EV samples’ heterogeneities, which remain inaccessible to most of the currently used bulk characterization techniques.

CC3.5. Novel strategies to increase efficacy and to overcome drug resistance in BRAF‐driven tumours

Adrián Varela. Varela‐Vázquez, CellCOM Research Group. Research Biomedical Institute of A Coruña (INIBIC)

Amanda Guitián‐Caamaño, CellCOM Group, Instituto de Investigación Biomédica de A Coruña (INIBIC), Servizo Galego de Saúde (SERGAS), Universidade da Coruña (UDC)

Paula Carpintero‐Fernández, CellCOM Group, Instituto de Investigación Biomédica de A Coruña (INIBIC), Servizo Galego de Saúde (SERGAS), Universidade da Coruña (UDC).

Marta Varela‐Eirín, European Research Institute for the Biology of Ageing (ERIBA);, University Medical Center Groningen (UMCG), University of Groningen.

Susana B. Bravo‐López, Instituto de Investigación Sanitaria de Santiago de Compostela IDIS, Proteomics laboratory.

Teresa Calleja‐Chuclá, Hospital Pharmacy Service. CH‐Universitario A Coruña (XXIAC). Servizo Galego de Saúde (SERGAS). Universidade da Coruña (UDC).

María Quindós‐Varela, Translational Cancer Research Group, Instituto de Investigación Biomédica de A Coruña (INIBIC). CH‐Universitario A Coruña (XXIAC). Servizo Galego de Saúde (SERGAS). Universidade da Coruña.

David Santamaría, University of Bordeaux, INSERM U1218, ACTION Laboratory, IECB.

Eduardo Fonseca, Dermatology Deparment; CellCOM Group, Instituto de Investigación Biomédica de A Coruña (INIBIC), Servizo Galego de Saúde (SERGAS), Universidade da Coruña (UDC).

María D. Mayán,CellCOM Group, Instituto de Investigación Biomédica de A Coruña (INIBIC), Servizo Galego de Saúde (SERGAS), Universidade da Coruña (UDC).

Introduction: Connexin43 (Cx43) has been described as a tumor suppressor in primary melanoma, but its role in disease progression is still under debate. Small extracellular vesicles (sEVs) containing Cx43 allow the exchange of small molecules such as small RNAs (sRNAs) via gap junction channels. BRAF/MEK inhibitors (BRAF/MEKi) have become the standard therapy in patients with BRAF‐mutated melanoma. However, resistance to therapy often develops within 12 months. Drug resistance continues to be a major problem for totally efficient to this therapy.

Methods: Expression vectors and sEVs‐enriched in Cx43 were used to increase Cx43 activity in tumor cells. sEVs were isolated by ultracentrifugation and characterized by NTA, electron microscopy and WB. IP was performed to study protein interactions and analyzed by LC‐MS/MS. RNA‐seq was used to identified sRNAs. Standard methods were used to study cellular senescence and apoptosis.

Results: Cx43 overactivity decreases proliferation and increases senescence and apoptosis in BRAF mutant cells in absence/presence of BRAF/MEKi. sEVs‐positive for Cx43 radically changes their function and the content of proteins and smallRNA, indicating that Cx43 may participate in the recruitment of proteins and sRNA. Also, restoration of Cx43 using sEVs, in BRAF‐mutated tumors, increases the efficacy of the BRAF/MEKi, and prevents drug resistance by reinforcing senescence and enhancing apoptosis alone and in combination with the senolytic drug navitoclax.

Summary/Conclusion: We have identified a new therapeutic target for the treatment of BRAF‐mutated tumors and to overcome drug resistance to BRAF/MEKi based on the combination of sEVs containing Cx43 along with BRAF/MEKi which increases in more than 80% inhibitors efficacy. We have demonstrated that sEVs can be used as drug carrier to transport transmembrane proteins such as Cx43. Our results could impact in the manage and treatment of metastatic tumors with a potential clinical benefit in patients with a metastatic disease.

CC4.1. Determining the Timing and Mechanisms of Cardiac Recovery by Extracellular Vesicles secreted by Induced Pluripotent Stem Cell Derived Cardiomyocytes

Bryan Z. Wang, M.S., Columbia University

Bohao Liu, Columbia University

Trevor Nash, Columbia University

Xiaokan Zhang, Columbia University

Lori luo, Columbia University

Manny Tamargo, Columbia University

Sharon Fleischer, Columbia University

Roberta Lock, Columbia University

Gordana Vunjak‐Novakovic, Columbia University

Introduction: Extracellular vesicles from induced pluripotent stem cell derived cardiomyocytes (iCM‐EV) have potential for treating myocardial infarction (MI). The efficacy of subacute treatment with iCM‐EV has not been studied. Also, the mechanisms of their action in human tissue are unknown.

Methods: Studies were done in a rat model of MI, and in human cell and tissue models of ischemia. To induce MI, rats underwent left anterior descending artery ligation. iCM‐EV laden patches with sustained release of EVs were implanted on the infarct zone immediately after MI induction, or 24 hours post‐MI. Echocardiography and histological analysis of hearts were performed after four weeks. In vitro, iCM were injured with hypoxia, treated with EVs, and evaluated by RNA sequencing. Western Blot and qPCR were used to evaluate iCMs. Physiologically matured human engineered cardiac tissues (ECT) were generated using our previously established methods, injured with hypoxia, treated with EVs, and evaluated by calcium imaging (for function) and qPCR.

Results: iCM‐EV significantly reduced the infarct size and increased cardiac function when implanted immediately after ligation. Delaying treatment by 24h reduced therapeutic benefit. RNA sequencing and protein analysis showed increased anti‐apoptotic and pro‐inflammatory signatures in EV‐treated cardiomyocytes. Activation of epidermal growth factor receptor (EGFR) signaling was identified as a key pathway affected by EV treatment. Analysis of iCM‐EV cargo showed an abundance of EGFR in EVs. iCM‐EVs upregulated EGFR phosphorylation, concomitant with increased pro‐survival ERK1/2 activity. In ECT, iCM‐EV significantly altered tissue calcium handling. qPCR confirmed increases in pro‐inflammatory and anti‐apoptotic gene expression.

Summary/Conclusion: Acute, but not subacute, treatment with iCM‐EVs was effective in treating MI, underscoring their proinflammatory and anti‐apoptotic effects which are potentially mediated through activation of EGFR signaling.

CC4.2. MicroRNA‐30 is a potential circulating biomarker for dysregulated microvascular endothelial cell function and metabolism in heart failure with preserved ejection fraction

Shawn C. Veitch, University Health Network / University of Toronto

Makon‐Sebastien Njock2Makon‐Sebastien Njock, University Health Network

Mark Chandy, University Health Network

M. Ahsan Siraj, University Health Network

Zhiqi Chen, University Health Network

Lijun Chi, The Hospital for Sick Children

Faisal Alibhai, University Health Network

Dakota D. Gustafson, Toronto General Hospital Research Institute

Dorrin Zarrin Khat, University Health Network / University of Toronto

Patrick Meagher,St. Michael's Hospital

Henry S. Cheng, University Health Network / University of Toronto

Kim ConnellySt. Michael's Hospital

Paul Delgado‐Olguin, The Hospital for Sick Children

Mansoor Husain,University Health Network

Jason E. Fish, PhD,Toronto General Hospital Research Institute

Introduction: Type 2 diabetes (T2D) is associated with cardiac microvascular dysfunction, which can contribute to the development of diastolic dysfunction and heart failure with preserved ejection fraction (HFpEF). The molecular mechanisms responsible for HFpEF remain unclear, and no effective diagnostics or treatments are available.

Methods: T2D mouse (db/db) and rat (Goto‐Kakizaki) models were used in this study. The presence of HFpEF was assessed using echocardiography and pressure‐volume loop analysis. The microRNA content of circulating extracellular vesicles (EVs) during the pathogenesis T2D‐associated HFpEF was measured using a RT‐qPCR microRNA array. EV concentration and size was measured using NanoSight NS300 Nanoparticle Tracking Analysis (NTA). Finally, RT‐qPCR was used to assess the role of miR‐30 in endothelial cells (ECs) at a molecular level. Seahorse and fluorometric assays were used to assess the role of miR‐30 in EC metabolism.

Results: We found that miR‐30d and miR‐30e were increased in EVs prior to echocardiographic evidence of diastolic dysfunction. These microRNAs may serve as biomarkers of microvascular dysfunction as they are upregulated in the endothelial cells (ECs) of the left ventricle of the heart, but not other organs. Furthermore, the induction of the miR‐30 family in ECs is regulated by senescence, a characteristic feature of diabetic ECs. Assessment of pathways regulated by miR‐30d/e revealed a large number of target genes involved in fatty acid metabolism. Importantly, over‐expression of miR‐30e in ECs increased exogenous fatty acid oxidation and the production of reactive oxygen species, while inhibiting the miR‐30 family had the opposite effect. Additionally, miR‐30e over‐expression synergized with fatty acid exposure to dramatically down‐regulate the expression of eNOS, an important regulator of microvascular function.

Summary/Conclusion: Circulating miR‐30d/e may represent early biomarkers of diastolic dysfunction that reflect altered fatty acid metabolism and microvascular dysfunction in the heart. Furthermore, the pathways regulated by miR‐30 may represent therapeutic targets for diabetes‐associated HFpEF.

CC4.3. Pluripotency‐associated miRNAs underlie the superior in vitro bioactivity of human induced pluripotent stem cell (hiPSC)‐derived extracellular vesicles in mediating cardiac repair

Ana Filipa F. Louro, iBET; ITQB‐NOVA

Marta Paiva, iBET; ITQB‐NOVA

Marta Oliveira, iBET

Patrícia Gomes‐Alves, iBET; ITQB‐NOVA

Paula Alves, iBET; ITQB‐NOVA

Margarida Serra, iBET; ITQB‐NOVA

Introduction: Studies on the cardiac repair potential of Extracellular Vesicles (EV) traditionally use mesenchymal or cardiac progenitor cell derived EV. In this study, we isolated EV from key stages of the hiPSC‐cardiomyocyte (hiPSC‐CM) differentiation and maturation, i.e. from hiPSC (hiPSC‐EV), cardiac progenitors (CPC‐EV), immature (CMi‐EV) and mature (CMm‐EV) cardiomyocytes, with the aim of identifying an efficient cell biofactory for therapeutic EV production, and selecting miRNA candidates for cardiac regeneration.

Methods: hiPSC were differentiated into hiPSC‐CM and matured in 3D culture, according to in‐house protocols. EV were separated by density, and characterized in terms of expression of specific EV‐associated markers, yield, particle size and particle size distribution. Bioactivity was assessed in human umbilical vein endothelial cells (HUVEC) and hiPSC‐CM based on EV uptake and its effect on angiogenesis, cell migration and proliferation. Small RNA‐Seq was performed to identify differentially expressed miRNA in the four EV groups.

Results: Bioactivity assays showed increased tube formation and migration in HUVEC treated with hiPSC‐EV compared to EV from committed cell populations (p < 0.0001). hiPSC‐EV also increased hiPSC‐CM proliferation (p < 0.01). Small RNA‐Seq analysis identified 15 miRNAs differentially expressed along hiPSC‐CM differentiation and maturation. A cluster involved in stemness maintenance was highly expressed in hiPSC, and gradually repressed along CM differentiation. These miRNAs were found to target the PI3/AKT pathway and were transfected into HUVEC and hiPSC‐CM to further investigate their role in cardiac repair.

Summary/Conclusion: Our findings suggest a superior in vitro bioactivity for hiPSC‐derived EV possibly mediated by pluripotency associated miRNA, intrinsically involved in cell proliferation processes.

CC4.4. Coronary artery disease ameliorates extracellular vesicle lncRNA PUNISHER regulates angiogenic response and endothelial cells function via NFkB‐dependent mechanism

Mohammed Rabiul Hosen, University of Bonn

Introduction: Augmenting evidence indicates that long noncoding RNAs are playing a crucial role in diverse cellular/pathological processes. Intercellular transfer of extracellular vesicles transmitted lncRNA regulates vascular health and diseases. However, whether lncRNA expression in EVs is regulated in patients with coronary artery disease, is unknown.

Methods: A PCR‐based lncRNA array analysis revealed that EV‐PUNISHER was significantly upregulated in patients with CAD (n = 221) compared to healthy subjects. To examine the specific role of PUNISHER in EC phenotypic regulation, siRNA‐mediated silencing in ECs revealed that depletion of PUNISHER suppresses the migration and proliferation of ECs. Depletion of PUNISHER decreases cell survival by reducing cell viability and proliferation through increased apoptosis and cytotoxicity. To investigate EC function in PUNISHER depleted cells, sprouting and tube formation assay revealed that PUNISHER is an important mediator EC functions. In vitro atherosclerotic stimuli (OxLDL, TNF‐α, IL‐6) increased PUNISHER expression in EV/EC in a dose‐dependent way. Microarray analysis identified a series of pro‐ and anti‐angiogenic genes as well genes directly involved in cell viability that are differentially regulated.

Results: We confirmed that PUNISHER is incorporated into endothelial microvesicles (EMVs) and transferred to recipient cells by using different experiments. By using lncRNA‐FISH and vesicle degradation assays, we showed that PUNISHER is incorporated into EMVs, augmented recipient EC function in vitro upon transfer via EMVs. To examine whether EMV‐PUNISHER promotes the EC function, different in vitro functional experiments with ECs (tube formation, angiogenic sprouting, migration, proliferation, apoptosis, etc.) confirmed that PUNISHER is an important regulator. Mass spectrometry analysis has revealed EMV contains numerous proteins, including RNA binding proteins such as hnRNPU, hnRNPK, hnRNPA2/B1, etc. By using RNA‐pulldown and RNA immunoprecipitation (RIP), we confirmed that PUNISHER interacts with hnRNPU, which facilitates packaging into EMV prior to transfer to recipient EC. The interaction acts as an important positive regulator of cell viability and survival of recipient cells, identified using functional assay (migration, viability, proliferation, and angiogenesis). The transcription factor array has shown that PUNISHER regulates NFkB to control cellular viability and apoptosis. A murine re‐endothelialization model after injection of EVs or ncRNAs revealed that EV‐PUNISHER promotes reendothelialization.

Summary/Conclusion: Our study unveiled that EV‐incorporated PUNISHER exerts its function in ECs which might be beneficial in cardiovascular pathologies

CC4.5. Engineering small extracellular vesicle‐like vehicles carrying pro‐reparative microRNA for cardiac repair after myocardial infarction

Sruti Bheri, Georgia Institute of Technology and Emory University

Michael E. Davis, Ph.D., Georgia Institute of Technology and Emory University

Introduction: Small extracellular vesicles (sEVs) are released by ckit+ progenitor cells (CPCs) for cardiac repair after myocardial infarction (MI). These sEVs carry microRNA (miR) cargo and have a complex lipid membrane which is associated with efficient uptake. However, as sEVs are secreted by cells, we have little control over sEV yield, the cargo loaded and its concentration. To address this, synthetic sEV mimics allowing custom cargo loading have been developed. However, they do not have the sEV's complex membrane, often having high toxicity and lower uptake. Here, we've designed sEV‐like vehicles (ELVs) to combine the benefits of both sEVs and mimics, by containing an sEV‐derived membrane and allowing tailored cargo. Further, to test ELV functionality, we investigated its ability to deliver pro‐reparative miR cargo to cardiac cells.

Methods: sEVs were isolated from CPC conditioned media using differential ultracentrifugation. They were ruptured using freeze‐thaw cycling and sonication to remove inherent cargo. Thin film hydration was then used to create a uniform lipid film. Finally, ELVs were formed by rehydrating the film in the presence of pro‐reparative miR‐126 or ‐133 cargo. ELVs were then post‐processed to remove unbound miR and form sEV‐sized vehicles. ELV size and concentration were assessed with nanoparticle tracking analysis. For testing ELV functional response, miR‐126 ELVs were dosed to cardiac endothelial cells (CECs) and angiogenesis and proliferation were quantified. Finally, the anti‐fibrotic effects of miR‐133 ELVs were studied on rat cardiac fibroblasts (RCFs).

Results: ELVs were successfully created with selective miR encapsulated, although there was some sample loss during synthesis. Treatment of CECs with miR‐126 ELVs increased angiogenesis and proliferation over 48hr treatment, compared to sEV alone. Similarly, ELVs containing anti‐fibrotic miR‐133 reduced RCF fibrotic response in a dose dependent manner.

Summary/Conclusion: This study provides the groundwork for a potent and tailored cardiac miR carrier for MI, which mitigates the challenges with sEVs and sEV mimics. Moreover, given the extensive role of miRs as therapeutics, ELVs have scope to be used for cargo delivery in other cardiac diseases and beyond.

CC5.1. Intracellular sorting and EV‐mediated release of Y‐RNA and Y‐RNA binding proteins

Tom Driedonks, PhD, Johns Hopkins Medical School / Utrecht University

Sarah Ressel, Institute of Immunology & Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom

Thi Tran, Dept. Biomolecular Health Sciences, Fact. Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Amy H. Buck, Institute of Immunology & Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom

Esther N.M Nolte – ‘t Hoen, Dr., Utrecht University

Introduction: Y‐RNA is a non‐coding RNA that is abundantly present in EV from various cell types and biofluids such as plasma. EV‐associated Y‐RNA has been associated with immune‐regulatory functions. We previously showed that Toll‐like receptor (TLR) activation of primary immune cells alters the abundance of Y‐RNA in EV, but not in the cytoplasm, suggesting that Y‐RNA shuttling is a regulated process. Y‐RNA interacts with various RNA‐binding proteins (RBP) in cells. Using subcellular fractionation of THP1 macrophages, we investigated how activation‐induced changes in intracellular sorting of Y‐RNA and their RBP relate to enhanced release of Y‐RNA in EV.

Methods: Post‐nuclear cytosol of TLR2/1‐stimulated and unstimulated THP1 was separated on Optiprep gradients into fractions enriched in ER, Golgi and endo/lysosomes. Y‐RNA and RBP in subcellular fractions and EV were analyzed in parallel by Western blot, RT‐qPCR and Northern blot.

Results: Full‐length Y‐RNA was mainly detected in subcellular fractions containing endo/lysosomal markers Lamp‐1, CD63 and Tsg101. Y‐RNA binding proteins Ro60, La, HuR and hnRNP K, but not YBX1, co‐localized with Y‐RNA in these fractions. This supported the idea that Y‐RNA can be sorted into EV at multivesicular endosomes (MVE). Of the tested RBP, only Ro60 was detected in Y‐RNA containing EV. While TLR stimulation did not affect cytoplasmic Y‐RNA levels, we observed an increase in the local Y‐RNA concentration in MVE. This was paralleled with a TLR stimulation‐induced increase in Y‐RNA and Ro60 incorporation into EV.

Summary/Conclusion: Our data exemplify how parallel analysis of subcellular compartments and EV can be used to study cell stimulation‐induced changes in the sorting of EV‐associated RNAs and their interacting proteins. We demonstrate that full‐length Y‐RNA and a subset of Y‐RNA binding proteins are enriched at EV biogenesis sites. Our results suggest that TLR‐activation changes the incorporation of Y‐RNA into EV by altering its local concentration at EV‐biogenesis sites.

CC5.2. Unconventional protein trafficking for extracellular vesicle packaging and function

Petra Parać, Metabolic Research Laboratories, Wellcome Trust‐Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, UK

Anna Albecka, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

Stephanie J. Popa, Metabolic Research Laboratories, Wellcome Trust‐Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, UK

Julien Villeneuve, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK

Suresh Mathivanan, Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3083, Australia

Sarah E. Stewart, Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3083, Australia

Australia

Introduction: The current model for EV biogenesis dictates that the subcellular location of proteins is reflected in the EV architecture, with cytosolic proteins in the lumen and cell surface proteins on the surface of EVs. This is irrespective of whether the EVs originate from the plasma membrane or are formed in the multivesicular body. However, myself and others have reported that cytosolic proteins, such as annexin A2, are reside on the surface of EVs. This is not entirely surprising, we have also shown that annexins are primarily secreted from cells via type I unconventional protein secretion, directly crossing the plasma membrane. This suggests that cytosolic proteins, including annexin A2, reside on the cell surface and hence the surface of EVs.

Methods: To build on these observations, we are investigating EV architecture and protein packaging using a proteomic approach to identify proteins known to be unconventionally secreted on the surface of EVs by tryptic digest. Candidates of interest are further investigated for roles in EV biogenesis and extracellular function using gene knockdown and CRISPR/Cas9 knockout, imaging and biochemical approaches.

Results: Several proteins known to be secreted by unconventional protein secretion are identified on the surface of EVs. Surprisingly other cytosolic proteins, not known to be secreted by cells are also detected on the surface of EVs. Proteins of interest were assessed for their role in EV function, such as binding and uptake in recipient cells. At least one EV surface protein was found potentially mediate EV uptake by recipient cells. Additionally, we investigated the role of several candidates in the regulation of EV biogenesis. Again, we identified one surface protein appears to regulate EV biogenesis or secretion.

Summary/Conclusion: The concept of cytosolic proteins residing on the surface of EVs is important for understanding the extracellular functions of EVs, including uptake and fusion which is likely to be mediated by proteins on the surface of EVs and cells. This data adds to the body of knowledge by which proteins are secreted through unconventional protein secretion and may identify new molecular pathways for packaging proteins onto the surface of EVs.

CC5.3. Biogenesis of RNA‐containing extracellular vesicles at VAP‐A directed endoplasmic reticulum membrane contact sites

Bahnisikha Barman, Vanderbilt University

Jie Ping, Vanderbilt University Medical Center

Evan Krystofiak, Vanderbilt University

Ryan Allen, Vanderbilt University Medical Center

Nripesh Prasad, HudsonAlpha Institute for Biotechnology

Kasey Vickers, Vanderbilt University Medical Center

James G. Patton, PhD, Vanderbilt University

Qi Liu, Vanderbilt University

Alissa Weaver, Department of Cell and Developmental Biology, Vanderbilt University School of Medicine

Introduction: Promising data suggest that extracellular RNAs (exRNAs) can affect gene expression, function, and phenotypes of recipient cells. While several RNA binding proteins (RBPs) are known to carry RNAs into extracellular vesicles (EVs), where and how in the cell this occurs is unclear. Here, we identify VAP‐A positioned endoplasmic reticulum membrane contact sites (ER MCS) as key locations for the biogenesis of RNA‐containing EVs.

Methods: Methods: We used bioinformatics, RNA‐sequencing, lipidomic, confocal and transmission electron microscopy, tumor xenograft and various biochemical techniques to analyze EV biogenesis and cargo content in colon cancer cell lines molecularly engineered for molecules that control ER MCS (VAP‐A‐KD, VAP‐A OE, CERT‐KD, ORP1L‐KD).

Results:RNA‐Seq analysis revealed a number of small RNAs that are altered in VAP‐A KD small and large EVs compared to control cells. Density gradient fractionation revealed that VAP‐A regulates a select subpopulation of small EVs that are enriched with RNA. Furthermore, this VAP‐A‐controlled small EV population is critical for transfer of miR‐100 to recipient cells and for growth of xenograft mouse tumors. Lipidomics analysis of small and large EVs revealed that VAP‐A controls levels of ceramide and cholesterol, two lipids involved in EV biogenesis. Furthermore, KD of the VAP‐A binding ceramide and cholesterol transporters CERT and ORP1L led to similar defects in EV biogenesis.

Summary/Conclusion: Summary/Conclusion: We uncovered a novel pathway of EV biogenesis that takes place at ER MCS. These data suggest a model in which lipid transfer at ER MCS drives biogenesis of a select subpopulation of EVs containing RNA‐RBP complexes. Beyond improving our understanding of EV biogenesis, we anticipate that these findings may be useful for future engineering of therapeutic EVs as well as exploring the functions of RNA‐containing EVs.

CC5.4. High‐throughput screening for the identification of drugs affecting secretion of two types of Extracellular Vesicles in breast cancer cells

Eleonora Grisard, Institut Curie U932

Aurianne Lescure, Istitut Curie Biophenics Laboratory

Maxime Corbé, Insitut Curie Biophenics Laboratory

Mercedes Tkach, Institut Curie / INSERM U932

Lorena Martin‐Jaular, Institut Curie / INSERM U932

Mathilde Mathieu, Insitut Curie U932

Grégory Lavieu, PhD, Université de Paris, inserm, umr7057/cnrs

Mabel Jouve, Institut Curie / CNRS UMR 3215

Elaine del Nery, Institut Curie Biophenics Laboratory

Clotilde Thery, MD PhD,Institut Curie / INSERM U932

Introduction: Eukaryotic cells, including cancer cells, secrete many different types of extracellular vesicles (EVs) as mediators of inter‐cellular communication. Secreted EVs can originate in different subcellular locations, resulting in populations highly heterogeneous in size, composition and function. Despite the most commonly used EV isolation techniques co‐isolate mixtures of these heterogeneous EVs, the specific mechanisms of secretion of the different EV subtypes are still largely unknown and tools to specifically modulate them are still lacking.

Methods: To analyze different EV subtypes, we first engineered MDA‐MB‐231 triple‐negative breast cancer cells to express constructs encoding for either CD63 or CD9 EV markers tagged with the Nanoluciferase (Nluc) enzyme. We then set up a high‐content screening assay to detect the secretion of Nluc‐CD63 or Nluc‐CD9 tagged EVs by quantifying Nluc activity in the supernatant of cells. We used a compound library of 1,280 FDA, EMA approved drugs (Prestwick Chemicals V3) to identify drugs able to modulate the secretion of Nluc‐CD63 or Nluc‐CD9 positive EVs. Finally, a panel of selected drugs depicted from the screening were used to further quantify EV release upon classical techniques of EV isolation and characterization (Size exclusion chromatography, Nanoparticle tracking analysis, Western blot, Electron microscopy).

Results: Our screening allowed the identification of several drugs modulating either Nluc‐CD63 cells or Nluc‐CD9 cells, or both. The hit validation step highlighted several potential misinterpretations due to unpredictable interaction between drugs and Nluc enzyme. After the establishment of stringent criteria to select drugs, we identified a new drug which robustly increases EV secretion in both Nluc‐CD63 and Nluc‐CD9 cells and we validated it also in the parental MDA‐MB‐231. Interestingly, this drug increases a EV subpopulation enriched in a novel combination of surface markers.

Summary/Conclusion: A novel EVs secretion multi‐drug high‐content screening allowed the identification of a never described drug able to increase the secretion of a specific EV subpopulation.

CC5.5. Identification of the Wnt Signal Peptide that directs secretion in Extracellular Vesicles

Uxia Gurriaran, Ottawa Hospital Research Institute

David Datzkiw, 1 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. 2 Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.

Leandro G. Radusky, Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Barcelona, Spain

Solomon Fisher, 1 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. 2 Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.

Fan Xiao, 1 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. 2 Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.

Hong Ming, 1 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. 2 Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.

Yves De Repentigny, 1 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. 2 Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.

Rashmy Kothary, 1 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. 2 Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.

Luis Serrano, 3 Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Barcelona, Spain 4 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

Michael Rudnicki,1 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. 2 Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.

Introduction: Wnt proteins are a secreted family of hydrophobic glycoproteins that govern essential developmental, growth, and regenerative processes, as well as pathological conditions. Recently, our group discovered that upon a muscle injury there is an upregulation of Wnt7a expression on new regenerating myofibers. Moreover, Wnt7a local‐muscle injection into dystrophic mice restores muscle function. However, Wnt7a is highly hydrophobic impairing its systemic delivery to treat all muscles. Despite their relative insolubility due to the palmitoylation required for specific Frizzled receptor biding, Wnt proteins actively participate in long‐range paracrine signaling between Wnt‐producing cells and distal recipient cells. These observations raise the unanswered controversial question of how long‐range Wnt signals are regulated. Recently, we discovered that Wnt7a is secreted in vitro through small Extracellular Vesicles (EVs). Our goal is to decipher the specific mechanism of Wnt7a‐EVs secretion to manufacture highly efficient Wnt7a‐EVs for systemic Duchene Muscular Dystrophy (DMD) treatment.

Methods: Using Tangential Flow Filtration we have standardized a Wnt purification protocol that allows the isolation of Wnt EVs without cross‐contamination with other sources of Wnt secretion. Structure‐function analysis, BioID experiments and in silico modeling interaction experiments were performed to elucidate the specific mechanism that regulates Wnt‐EVs secretion.

Results: We discovered that Wnt7a is secreted at high levels on exosomes following muscle injury to stimulate regeneration. Structure‐function analysis identified the signal sequence in Wnt7a, that we termed Extracellular Vesicle Signal Peptide (ESP), which directs EVs secretion, and revealed that palmitoylation is not required. This peptide binds specifically to Coatomer proteins through a positively charged motif to direct trafficking of Wnt to EVs. The positively charged motif and mechanism is conserved among Wnts.

Summary/Conclusion: Our studies identify a novel signal peptide that traffics Wnt cargo to EVs surface and elucidate the mechanisms that facilitate long‐range Wnt signaling. Our experiments suggest that systemic delivery of Wnt7a loaded on EVs represents a potential therapy for DMD. Moreover, the use of the ESP to direct the display of cargo proteins on the surface of EVs opens the door for multiple therapeutic applications. We anticipate that our discovery will be a starting point for more sophisticated delivery systems as well as establish the fundamental knowledge for Wnt secretion in pathological contexts.

CC6.1. Cardiotropic Mechanisms of EV‐Encapsulated AAVs for Gene Therapy in Heart Failure

Sabrina La Salvia, SL, Icahn School of Medicine at Mount Sinai

Yaxuan Liang, YL, Cardiovascular Research Center Icahn School of Medicine at Mount Sinai

Marta Adamiak, MA, Cardiovascular Research Center Icahn School of Medicine at Mount Sinai

Shweta Lodha, SL, Research Center Icahn School of Medicine at Mount Sinai

Samantha Osinki, SO, Research Center Icahn School of Medicine at Mount Sinai

Mihir Parikh, MP, Cardiovascular Research Center Icahn School of Medicine at Mount Sinai

Kimberly Okoli, KO, Icahn School of Medicine at Mount Sinai

Tzu‐Yi Chen, Icahn School of Medicine at Mount Sinai, Department of Department of Genetics and Genomic Sciences

Nicole C. Dubois, NCD, Cardiovascular Research Center Icahn School of Medicine at Mount Sinai

Navneet Dogra, PhD,Department of Genetics and Genomics Sciences, Icahn School of Medicine at Mount Sinai

Susmita Sahoo, SS, Department of Cardiology, Icahn School of Medicine at Mount Sinai

Introduction: Adeno‐associated viruses (AAVs) are one of the most commonly used viral vectors in cardiac gene therapy. However, preexisting neutralizing antibodies (NAb) bind to free to AAVs and impair their clinical effect. Here, we describe extracellular vesicles (EV)‐encapsulated AAVs (evAAVs) as a superior cardiac gene delivery system offering high NAb resistance.

Methods: We characterized the ultracentrifuge‐isolated evAAVs using TEM, qPCR, Western blot, DLS, qNano, imaging flow cytometry and ExoView technologies. We determined the gene delivery efficacy both in presence and absence of NAbs using human iPS‐derived cardiomyocytes (CM) and human left‐ventricular CM‐AC16 cell line in vitro, and in mice in vivo, via flow cytometry and IVIS bioluminescence imaging. We determined the therapeutic efficacy of SERCA2a (a gene that regulates cardiomyocyte contraction) delivery using either evAAVs or free AAVs in a mouse model of myocardial infarction, in presence of NAbs. We investigated the mechanisms of evAAV uptake and trafficking both in vitro and in vivo using fluorescence‐labeled evAAVs.

Results: evAAVs outperformed free AAV in delivering genes in the presence of NAbs, both in vitro and in vivo. Intramyocardially injected evAAVs carrying SERCA2a significantly improved cardiac function (ejection fraction, %EF, ∼60%) compared to free AAVs (∼40%) and saline control (∼20%), in mice with NAb. evAAVs colocoalized with endosomes (Rab5, 7) and delivered significantly higher amounts of Luciferase to human CMs compared to free AAVs. We postulate that evAAVs deliver higher amounts of viruses to the nucleus via their acidification in endosomal compartments, and Bafilomycin A1, which inhibits the proton pump in endosomes, will inhibit this process. In addition, fluorescence‐tagged evAAVs injected to mouse hearts were internalized into all cell types (CMs and non‐CMs). However, evAAV‐mCherry was primarily expressed in CMs (8.3%), but not in non‐CMs (1.2%) showing evAAV‐mediated gene delivery to the heart is cardiotropic

Summary/Conclusion: Our study demonstrated that evAAV‐mediated gene delivery can circumvent neutralizing antibody (NAb) issue for gene therapy. We show a complex mechanism of evAAV biology, trafficking and cardiotropism. evAAV is a superior gene delivery vector and a new platform to treat heart failure.

CC6.2. Nasal administration of MSC‐derived small extracellular vesicles reverses cisplatin‐induced cognitive impairments, changes in white matter and neuronal mitochondrial function in mice

Bojana Milutinovic, The University of Texas MD Anderson Cancer Center

Luis Arroyo, The University of Texas MD Anderson Cancer Center

Mayela Mendt, The University of Texas MD Anderson Cancer Center

Faiza Hancock, The University of Texas MD Anderson Cancer Center

Alex Seua, The University of Texas MD Anderson Cancer Center

Shruti Dharmaraj, The University of Texas MD Anderson Cancer Center

Elizabeth Shpall, The University of Texas MD Anderson Cancer Center

Annemieke Kavelaars, The University of Texas MD Anderson Cancer Center

Cobi j. Heijnen, The University of Texas MD Anderson Cancer Center

Introduction: Cognitive deficits including impaired attention, memory and executive functioning are common neurotoxic side effects of chemotherapy. Up to 75% of chemotherapy patients experience cognitive decline and in 30% of the patients it persists after treatment. There are no FDA‐approved therapeutic options. We recently showed that neuronal mitochondrial dysfunction is the underlying mechanism of cisplatin‐induced cognitive impairment (CICI) and is accompanied by changes in white matter organization and hippocampal synaptic loss. Based on our successful use of mesenchymal stem cells (MSCs) to reverse CICI, we proposed that hBM MSC‐derived small extracellular vesicles (sEVs) are also capable of restoring cognition.

Methods: sEVs were isolated by differential centrifugation of hBM MSC cell culture medium. Animals were injected with cisplatin according to Chiu et al, 2017. sEV were administered nasally 48 and 96 h after the last cisplatin injection. Cognition was tested in the Puzzle box and Novel Object Place Recognition Test (NOPRT). Distribution of sEVs was monitored using fluorescent DiR. Synaptosomal mitochondrial morphology was analyzed by transmission electron microscopy and mitochondrial oxygen consumption using Seahorse technology. Synaptophysin and Black Gold II staining were used to assess synaptic loss and white matter integrity.

Results: Nasal sEV administration reversed the deficits in working and spatial memory as well as executive functioning in a dose‐dependent manner. Damage to mitochondrial morphology and the decrease in mitochondrial oxygen consumption was restored as well as the cisplatin‐induced changes in white matter and synaptic integrity. sEV entered the brain within 30 minutes. Clearance began 24 h after treatment but sEV remained in the brain for at least 48 h.

Summary/Conclusion: Nasal administration of MSC‐derived sEV is a potentially effective therapeutic approach to reverse the debilitating effects of CICI.

CC6.3. Exploring the crosstalk between endothelial autophagy and extracellular vesicle biology: potential role in atherosclerosis

Pierre‐Michaël Coly, Université de Paris, INSERM U970, Paris Cardiovascular Research Centre, Paris, France

Shruti Chatterjee, Université de Paris, INSERM U970, Paris Cardiovascular Research Centre, Paris, France

Fariza Mezine, Université de Paris, INSERM U970, Paris Cardiovascular Research Centre, Paris, France

Florent Dingli, Curie Institute, PSL Research University, Laboratoire de Spectrométrie de masse Protéomique, Paris, France

Damarys Loew, Université de Paris, INSERM U970, Paris Cardiovascular Research Centre, Paris, France

Xavier LOYER, INSERM U970‐PARCC

Chantal Boulanger, Inserm U970‐ Paris Cardiovascular Research Center

Introduction: Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Our team has recently demonstrated that endothelial autophagy is a protective process, stimulated under conditions of high shear stress (HSS) when compared to LSS, and hampers the development of atherosclerotic lesions. Endothelial EVs have been shown to regulate inflammation, senescence and angiogenesis and might therefore play a crucial role in vascular homeostasis and disease. While previous studies have shown links between autophagy and extracellular vesicle formation, the exact role of the autophagic machinery in the release and uptake of EVs remains elusive. Our aim is therefore to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress.

Methods: Confluent human umbilical vein endothelial cells (HUVEC) were exposed to either LSS (2 dyn/cm2) or HSS (20 dyn/cm2) for 24 hours in culture medium containing 2% EV‐free FCS. Large (>200 nm) and small EVs were isolated from conditioned medium by sequential centrifugation and size exclusion chromatography. They were characterized by Western blot analysis of EV markers (CD9, CD63 and HSC70), tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using Membright or Vybrant‐DID‐labelled EVs and differences between groups were assessed by flow cytometry and confocal microscopy.

Results: Levels of large and small EVs were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions compared to HSS. Interestingly, silencing ATG5 in HUVECs increased EV uptake by cells exposed to HSS. Additionally, endothelial LSS‐EVs appeared to have a greater affinity for HUVECs than HSS‐EVs or EVs derived from platelets, red blood cells, granulocytes and peripheral blood mononuclear cells. Proteomic analysis revealed that LSS‐EVs were enriched in adhesion proteins such as PECAM1, MCAM and integrins. We found that neutralizing PECAM1 and MCAM on EVs significantly reduced their uptake by HUVECs. Finally, inhibiting autophagy using pharmacological or genetic approaches elevated levels of adhesion proteins in HSS‐EVs and caused them to be more readily taken up by HUVECs

Summary/Conclusion: These findings suggest that endothelial shear stress and autophagy may have an important function during EV biogenesis and uptake. Given the major role of EVs and autophagy in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction.

CC6.4. High performance of α‐galactosidase A lysosomal enzyme loaded in extracellular vesicles through recombinant protein overexpression

Joaquin Seras‐Franzoso, Drug Delivery & Targeting (DDT), CIBBIM‐Nanomedicine, Vall d'Hebron Institut de Recerca (VHIR)

Zamira V. Díaz‐Riascos, Drug Delivery & Targeting (DDT), Functional Validation & Preclinical Research, CIBBIM‐Nanomedicine, Vall d'Hebron Institut de Recerca (VHIR)/Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER‐BBN)

José Luis Corchero, Institut de Biotecnologia i de Biomedicina (IBB) and Department of Genetics and Microbiology, Universitat Autònoma de Barcelona (UAB)/Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER‐BBN)

Patricia González, Drug Delivery & Targeting (DDT), CIBBIM‐Nanomedicine, Vall d'Hebron Institut de Recerca (VHIR)

Natalia García‐Aranda, Drug Delivery & Targeting (DDT), Functional Validation & Preclinical Research, CIBBIM‐Nanomedicine, Vall d'Hebron Institut de Recerca (VHIR)/Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER‐BBN)

Mònica Mandaña, Drug Delivery & Targeting (DDT), Functional Validation & Preclinical Research, CIBBIM‐Nanomedicine, Vall d'Hebron Institut de Recerca (VHIR)/Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER‐BBN)

Roger Riera, Nanoscopy for Nanomedicine Group, Institute for Bioengineering of Catalonia (IBEC)

Ana Boullosa, Drug Delivery & Targeting (DDT), Functional Validation & Preclinical Research, CIBBIM‐Nanomedicine, Vall d'Hebron Institut de Recerca (VHIR)/Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER‐BBN)

Sandra Mancilla, Drug Delivery & Targeting (DDT), Functional Validation & Preclinical Research, CIBBIM‐Nanomedicine, Vall d'Hebron Institut de Recerca (VHIR)/Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER‐BBN)

Alba Grayston,Neurovascular Research Laboratory, Vall d'Hebron Institut de Recerca (VHIR)

Marc Moltó‐Abad, Drug Delivery & Targeting (DDT), CIBBIM‐Nanomedicine, Vall d'Hebron Institut de Recerca (VHIR)/Division of Rare Diseases, Reference Center for Hereditary Metabolic Disorders (CSUR, XUEC, MetabERN, and CIBER‐ER), Vall d'Hebron University Hospital

Elena Garcia‐FruitósInstitut de Biotecnologia i de Biomedicina (IBB) and Department of Genetics and Microbiology, Universitat Autònoma de Barcelona (UAB)/Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER‐BBN)

Rosa Mendoza, Institut de Biotecnologia i de Biomedicina (IBB) and Department of Genetics and Microbiology, Universitat Autònoma de Barcelona (UAB)/Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER‐BBN)

Guillem Pintos‐Morell,Drug Delivery & Targeting (DDT), CIBBIM‐Nanomedicine, Vall d'Hebron Institut de Recerca (VHIR)/Division of Rare Diseases, Reference Center for Hereditary Metabolic Disorders (CSUR, XUEC, MetabERN, and CIBER‐ER), Vall d'Hebron University Hospital

Lorenzo Albertazzi,Nanoscopy for Nanomedicine Group, Institute for Bioengineering of Catalonia (IBEC)

Anna Rosell,Neurovascular Research Laboratory, Vall d'Hebron Institut de Recerca (VHIR)

Josefina Casas,RUBAM, Biological Chemistry, Institute of Advanced Chemistry of Catalonia (IQAC‐CSIC) / Networking Research Center on Hepatic and Digestive Diseases (CIBEREHD)

Antonio Villaverde,Institut de Biotecnologia i de Biomedicina (IBB) and Department of Genetics and Microbiology, Universitat Autònoma de Barcelona (UAB)/Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER‐BBN)

Simó Schwartz JrDrug Delivery & Targeting (DDT), CIBBIM‐Nanomedicine, Vall d'Hebron Institut de Recerca (VHIR) / Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER‐BBN)

Ibane Abasolo,Drug Delivery & Targeting (DDT), Functional Validation & Preclinical Research, CIBBIM‐Nanomedicine, Vall d'Hebron Institut de Recerca (VHIR)/Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER‐BBN)

Introduction: Fabry disease (FD), is a lysosomal storage disorder (LSD) caused by the mutation of α‐galactosidase A (GLA) and enzymatic replacement therapy (ERT) is its preferred therapeutic option. However, ERT requires life‐long administration of large amounts of recombinant enzyme and depends highly on mannose‐6‐phospate (M6P) mediated cell internalization. We propose an alternative system to deliver lysosomal enzymes in EVs simplifying the enzyme downstream processing while protecting the enzyme's activity and improving its bioavailability.

Methods: EVs loaded with GLA (EV‐GLA) were produced by protein overexpression in CHO cells. EVs were routinely isolated by water exclusion precipitation and further validated by tangential flow filtration and iodixanol gradient ultracentrifugation. EV‐GLA activity in vitro was assessed by specific enzymatic activity (EA) assay, GLA substrate (Gb3) accumulation and MP6 competition assays. FD mice models were used to evaluate EV‐GLA biodistribution and therapeutic potential.

Results: EV‐GLA loaded significant amounts of GLA exhibiting 6 times higher EA than free enzyme. In FD in vitro cultures, EV‐GLA restored WT levels of Gb3 more efficiently than GLA. In vivo, DiR labeled EV‐GLA was detected in liver, lungs and kidneys by ex‐vivo imaging but also in difficult‐to‐target organs such as kidneys and brain. Consequently, single intravenous administration of EV‐GLA restored basal Gb3 levels in highly accessible organs like liver or lungs but also reduced Gb3 in kidneys and brain. Cellular uptake revealed that EV internalization was independent on the MP6 route, fact that could partially explain the higher efficacy and bioavailability of EV‐GLA compared to the free enzyme.

Summary/Conclusion: Our data picture EVs loaded by recombinant lysosomal enzymes as natural drug delivery systems increasing the stability, bioavailability and efficacy of the cargo

CC6.5. Pro‐inflammatory cytokines primed mesenchymal stromal cell‐derived exosomes contribute to tissue regeneration in experimental inflammatory bowel disease

Anna Maria Tolomeo, L.i.f.e.L.a.b. Program, Consorzio per la Ricerca Sanitaria (CORIS), Veneto Region, Padua, Italy

Ignazio Castagliuolo, Department of Molecular Medicine, University of Padova, Padua, Italy

Martina Piccoli, Laboratory of Tissue Engineering, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy

Michele Grassi, Department of Women's and Children's Health, University of Padova, Padua, Italy

Fabio Magarotto, Department of Women's and Children's Health, University of Padova, Padua, Italy

Giada De Lazzari, Department of Women's and Children's Health, University of Padova, Padua, Italy

Ricardo Malvicini, Instituto de Medicina Traslacional, Trasplante y Bioingenieria (IMeTTyB‐CONICET)

Federico Caicci, Department of Biology,University of Padua, Italy

Chiara Franzin, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy;

Melania Scarpa, Department of Women's and Children's Health, University of Padova, Padua, Italy

Antonella Viola, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy;

Andrea PorzionatoDepartment of Neurosciences, University of Padova, Padua, Italy

Michela Pozzobon, Department of Women's and Children's Health, University of Padova, Padua, Italy

Maurizio Muraca, Department of Women's and Children's Health, University of Padova, Padua, Italy

Introduction: Several reports have described a beneficial effect of Mesenchymal Stromal Cells (MSCs) and of their secreted extracellular vesicles (EVs) in mice with experimental colitis. However, the effects of the two treatments have not been thoroughly compared in this model.

Methods: Here, we compared the effects of MSCs and of MSC‐EV administration in mice with colitis induced by dextran sulfate sodium (DSS) treatment. Since cytokine conditioning was reported to enhance the immunomodulatory activity of MSCs, the cells were kept either under standard culture conditions (naïve, nMSCs) or primed with pro‐inflammatory cytokines (IL1b, IL6 and TNFa; induced, iMSCs). Colitis was induced in C57BL/6N mice by 3% dextran sulfate sodium (DSS) in drinking water for 6 days followed by 3 days on plain water. Healthy controls received plain water only. Mice with colitis received an intraperitoneal injection (IP) of MSCs (4.00E+6 nMSCs, 4.00E+6 iMSCs) on days 4 and 8 or of EVs (1.00E+9 nMSC‐EVs and 1.00E+9 iMSC‐EVs) on days 4, 6 and 8. Control mice received PBS only. Animals were sacrificed on day 10.

Results: In our experimental conditions, nMSCs and iMSCs administration resulted in both clinical and histological worsening and was associate with pro‐inflammatory polarization of intestinal macrophages. However, mice treated with iEVs showed clinico‐pathological improvement, decreased intestinal fibrosis and angiogenesis and a striking increase in intestinal expression of Mucin5ac, suggesting improved epithelial function. Moreover, treatment with iEVs resulted in the polarization of intestinal macrophages towards and anti‐inflammatory phenotype and in an increased Treg/Teff ratio at the level of the intestinal lymph node.

Summary/Conclusion: Collectively, these data support the concept that MSCs can behave either as anti‐ or as pro‐inflammatory agents depending on the host environment. In contrast, MSC‐EVs showed a beneficial effect, suggesting a more predictable behavior. Moreover, we show for the first time that MSC‐EV administration is associated with enhanced epithelial protection mediated by Mucin5ac hypersecretion.

CC7.1. Apoptotic bodies mediate mesenchymal stem cell‐based therapy for type 2 diabetes

Chenxi Zheng, Center for Tissue Engineering, The Fourth Military Medical University

Bingdong Sui, Center for Tissue Engineering, The Fourth Military Medical University

Jiachen Hu, Center for Tissue Engineering, The Fourth Military Medical University

Shiyu Liu, Center for Tissue Engineering, The Fourth Military Medical University

Yan Jin, Center for Tissue Engineering, The Fourth Military Medical University

Introduction: Mesenchymal stem cells (MSCs) have been identified as a promising cell source for translational application in multiple diseases, including type 2 diabetes (T2D). Specifically, MSCs undergo apoptosis and release apoptotic bodies (ABs) during therapeutic application. Nevertheless, the feature, fate and function of MSC‐derived ABs remain largely unknown.

Methods: We established high‐fat diet (HFD)‐induced and genetic Db/db T2D mouse models. We explored the therapeutic efficacy of MSCs with apoptosis inhibition. Then, we isolated MSC‐derived ABs and identified their features. The therapeutic potency of ABs was evaluated in two T2D models. Next, the in vivo fate of ABs and the effects of ABs on T2D liver macrophages were analyzed. Finally, the molecular mechanisms underlying the uptake of ABs were evaluated.

Results: We firstly found that inhibition of apoptosis impaired the therapeutic efficacy of MSCs in T2D. Next, we found that infusion of isolated ABs was able to alleviate both diet‐induced and genetic T2D models. We further revealed that ABs were efferocytosed by liver macrophages and functionally modulated them, leading to inhibition of macrophage infiltration and transformation of macrophages towards anti‐inflammation phenotype. At the molecular level, we showed that calreticulin was exposed on the surface of ABs which acted as a critical “eat me” signal mediating the functional efferocytosis and therapeutic potency of ABs.

Summary/Conclusion: This study revealed the therapeutic mechanism that MSCs undergo apoptosis and release ABs to treat T2D, clarified ABs’ in vivo fate of targeting macrophages and the underlying molecular mechanism, and verified the modulatory effects and therapeutic potential of ABs, thus promoting the establishment of novel T2D treatment strategies.

CC7.2. Unraveling EV‐mediated cardioprotection: EV‐dependent and ‐independent mechanisms?

Marieke T. T. Roefs, Department of Experimental Cardiology, University Medical Center Utrecht, Utrecht University, The Netherlands

Pieter Vader, CDL Research, University Medical Center Utrecht, The Netherlands

Joost P.G. Sluijter, J.P.G., Department of Experimental Cardiology, University Medical Center Utrecht, Utrecht University, The Netherlands

Joost P.G. Sluijter, J.P.G., Department of Experimental Cardiology, University Medical Center Utrecht, Utrecht University, The Netherlands

Introduction: Cardiac progenitor cell (CPC)‐derived extracellular vesicles (EVs) have been shown to protect the myocardium against ischemia/reperfusion injury. However, the underlying mechanisms for CPC‐EV‐mediated cardioprotection remain elusive. By exploring protein‐mediated effects of CPC‐EVs, we discovered that crude EV preparations activate recipient endothelial cells through EV‐dependent and "independent pathways.

Methods: CPCs were stimulated with calcium ionophore (ca ion‐EVs), previously shown to influence EV release, or vehicle (control‐EVs) for 24 hours and crude EVs were isolated using size exclusion chromatography (SEC). EV concentration and size was assessed using NTA and proteomic composition was profiled using mass spectrometry. Following SEC, Optiprep gradient ultracentrifugation was used to separate EVs from free proteins. EV‐ and protein fractions were functionally characterized based on endothelial cell activation assays.

Results: Endothelial cells displayed enhanced phosphorylation of ERK1/2 and AKT and increased wound closure after stimulation with control‐EVs, but not with ca ion‐EVs. Proteomic analysis identified multiple proteins uniquely expressed or enriched in control‐EVs compared with ca ion‐EVs. Surprisingly, when investigating the contribution of individual candidate proteins, the extent of endothelial cell activation was found to be influenced by the purity of the EV preparations. EVs isolated using Optiprep gradients lost part of their ability to activate endothelial cells compared to crude EV preparations. In addition, several candidate proteins were found to be present in the free protein fraction instead of the EV fraction. This hints towards a co‐stimulatory role of co‐isolated proteins in recipient cell activation.

Summary/Conclusion: A specific set of EV proteins is identified that may be functionally responsible for the activation of endothelial cells upon exposure to CPC‐EVs. It is important to identify if these proteins are EV‐associated or represent co‐isolated factors that contribute to endothelial cell activation. This may lead to a better mechanistic understanding of CPC‐EV‐mediated cell activation and translation of EV‐mediated therapeutics.

CC7.3. Administration of Extracellular Vesicles Derived from Amniotic Fluid Stem Cells Rescues Autophagy in Underdeveloped Fetal Lungs

Kasra Khalaj, PhD, MSc, The Hospital for Sick Children

Lina Antounians, MSc, The Hospital for Sick Children

Rebeca Figueira, PhD, MSc, The Hospital for Sick Children

Martin Post, PhD, The Hospital for Sick Children

Augusto Zani, MD, PhD, FACS, FAAP, The Hospital for Sick Children

Introduction: We previously showed that administration of extracellular vesicles derived from amniotic fluid stem cells (AFSC‐EVs) promote lung growth and maturation in fetuses with pulmonary hypoplasia (PH) via a miRNA‐mediated mechanism. It has been recently reported that autophagy is crucial for normal lung development. Herein, we aimed to assess whether PH lungs have abnormal autophagy and if AFSC‐EV treatment can restore normal levels of autophagy.

Methods: EVs were separated from rat AFSC conditioned medium by ultracentrifugation (100,000g/14hrs), and characterized for size (nanoparticle tracking analysis), morphology (transmission electron microscopy), and Western blot (WB; EV‐specific markers: CD63, CD81, TSG101). Following our established PH model in fetal rats (nitrofen administration to rat dams at E9.5), lungs were harvested at E14.5 and E17.5 and explants were established. PH explants were treated with AFSC‐EVs (10%v/v AFSC‐EVs; NA) or medium alone (N) for 72h. Fetal lungs from untreated dams served as control. Gene and protein levels of autophagy markers (activator: ATG5,BECN1; repressor: SQSTM1) were analyzed via RT‐qPCR and WB. Purified EV‐RNA was sequenced using NEB‐Next. We compared N with NA treated samples for miRNAs associated with autophagy. Statistics: One‐way (qPCR and WB) and Two‐way (RNA‐seq) ANOVAs.

Results: PH explants had lower Atg5 and Becn1 and higher levels of impaired autophagy marker, SQSTM1 at both time points. AFSC‐EV treatment to hypoplastic lungs restored expression levels of all markers back to normal control levels. RNA‐sequencing identified 3 miRNAs (miRs‐17,‐20a,‐93) known to be associated with autophagy (in relation to Sqstm1) and were enriched in the AFSC‐EV‐treated group.

Summary/Conclusion: This is the first study demonstrating impaired autophagy in PH, which can be rescued by the administration of AFSC‐EVs. miRNA cargo contained in AFSC‐EVs targets autophagy mechanism in developing lungs and represents a promising approach to treat PH.

CC7.4. Angio‐modulatory role of amniotic fluid derived extracellular vesicles in preeclampsia

Natalia Gebara, University of Turin

Renata Skovronova, University of Turin

Julia Scheel, Department of systems biology and bioinformatics

Luca Marozio, Department of Surgical Sciences,Obstetrics and Gynecology

Benedetta Bussolati, University of Turin, Department of Molecular Biotechnology and Health Sciences, Italy

Introduction: Preeclampsia is a hypertensive pregnancy disorder that affects up to 8% of pregnancies worldwide. Many metabolically active cell types of the placenta or fetus secrete within the amniotic fluid bio‐factors, such as VEGF, endoglin, PGF‐1 as well as extracellular vesicles (EVs). In pathological conditions, such as hypoxia, these factors may affect the physiology of pregnancy and cause multiple pathologies such as preeclampsia. This study aims to fully characterize, test, and compare the bio‐function of amniotic fluid‐derived EVs from normal and preeclamptic pregnancies.

Methods: The amniotic fluid was obtained from gestationally matched normal and preeclamptic pregnancies, during cesarean sections. Samples were immediately processed for EV isolation. The physical parameters were tested by Nanosight technology. EVs were further characterized by a super‐resolution microscopy. The identification of exosomal markers was tested by Macsplex exosome kit and NanoView. Angiogenic effects of EVs were tested using tube formation assay. A comparative bioinformatic analysis of 754 miRNAs, between, normal and pre‐eclamptic patients, was performed.

Results: We set up a method for EV isolation from the amniotic fluid using differential centrifugation and filtration steps. EVs from 22 normal pregnancies and 7 preeclamptic samples were analyzed. The EV size and concentration were 222.8 +/‐ 6nm and 2.8 × 1010 /ml. In a comparison of 171.5+/‐7nm and 2.7 × 10 9 /ml for 7 preeclamptic pregnancies samples. For miRNA and NanoView experiments, we further purified the EV samples using size exclusion columns. Size and tetraspanin expression were confirmed by dSTORM analysis using the superresolution NanoImager microscope. NanoView and Mascplex analysis showed differences between the angiogenic marker (CD105 (endoglin)), progenitor markers (CD24 and SSEA‐4), and the MHC‐1 (HLA‐ABC). Tube formation assay showed pre‐eclampsia derived EVs to significantly downregulate formation of tubules in comparison with controls and normal pregnancy derived‐EVs. Several miRNAs were detected to be differentially expressed in EVs derived from preeclamptic pregnancies

Summary/Conclusion: Our results show that pre‐eclamptic EVs differ in their content and angiogenic ability, as well as in their size in comparison with normal pregnancy amniotic fluid‐derived EVs. Our data support the importance of EV's role as cell‐cell communicators and their significant function in the development of pre‐eclampsia.

CC7.5. Pharmacokinetics and biodistribution of EV administered intravenously versus intranasally in mice and macaque models

Tom Driedonks, PhD, Johns Hopkins Medical School / Utrecht University

Bess Carlson, Dept. Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, MD

Suzanne Queen, Dept. Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, MD

Olesia Gololobova, Johns Hopkins University School of Medicine

Zheng Han, Russell H. Morgan Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Guanshu Liu, Russell H. Morgan Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Lyle Nyberg, Dept. Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, MD

Gabriela Lima, Dept. Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, MD

Kayla Schonvisky, Dept. Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, MD

Natalie Castell,Dept. Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, MD

Barbara Smith, Dept. Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD

Charles Lai, PhD, Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan

Jessica Izzi, Dept. Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, MD

Eric Hutchinson,Dept. Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, MD

Kelly Pate,Dept. Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, MD

Kenneth W. Witwer,Johns Hopkins University School of Medicine

Introduction: Extracellular vesicles (EVs) have potential to deliver therapeutic cargo to tissues including brain. It was previously shown in mice that intravenously administered EVs have a half‐life of minutes and are taken up mostly by the liver and spleen. Alternative administration routes may result in different distributions. Although mice are a valuable preclinical model, physiologic differences between mice and humans may limit the translational value of findings. Here, we compared the blood and cerebrospinal fluid (CSF) pharmacokinetics of EVs administered intravenously (i.v.), intranasally (i.n.), and intrathecally (ith.) to macaques. We also compared organ biodistribution of i.v. and i.n. EVs in mice and macaques.

Methods: EVs containing a palmitoylated GFP‐Nanoluciferase (palmBRET) dual reporter were produced in Expi293F cells, concentrated by TFF, labelled with MemGlow (700 nm) lipid dye, and purified by SEC. EVs were characterized by Western blot, NTA, electron microscopy, SP‐IRIS, and imaging flow cytometry. 3E10^10 " 7E10^10 EVs were administered i.v., i.n., or ith. into macaques, and plasma and CSF were sampled at regular intervals for 24h. 6E10^9 EVs were administered i.v. or i.n. into mice, and organs were harvested after 60 min. Presence of the EV reporter in tissue lysates and biofluids was measured by NanoGlo assay.

Results: PalmBRET bioluminescence was present in EV‐containing SEC fractions, colocalized with CD9, CD63 and CD81 by SP‐IRIS, and was protease resistant in the absence of detergent. PalmBRET EVs were detectable over a 10^5‐fold dilution series in macaque plasma. The route of administration affected EV levels in biofluids. After ith. administration, EVs were cleared rapidly (t1/2 = 2 min) from CSF. Intravenously injected EV were rapidly cleared from plasma (t1/2 = 2 min). At the administered dose, i.n. EVs were not detected in plasma. EVs administered i.n. and i.v. accumulated in CSF between 1h and 24h. In mice, the administration route affected EV biodistribution, with i.v. EVs accumulating predominantly in the liver, spleen, and kidney, and i.n. EVs accumulating in lung and brain.

Summary/Conclusion: Highly sensitive reporters such as palmBRET enable pharmacokinetic studies of EVs in large animal models. Our data reveal that EV administration route affects the uptake and biodistribution of EVs, which may inform future therapeutic applications of EVs in humans. Studies of higher EV doses and distribution in macaques are ongoing.

CC8.1. ACE2‐containing extracellular vesicles and exomeres bind the SARS‐CoV‐2 spike protein

Qin Zhang, Vanderbilt University Medical Center

Dennis Jeppesen, Vanderbilt University Medical Center

James Higginbotham, Vanderbilt University Medical Center

Jeffrey Franklin, Vanderbilt University Medical Center

James Crowe, Vanderbilt University Medical Center

Robert Coffey, Vanderbilt University Medical Center

Introduction: Small extracellular vesicles (sEVs) and exomeres (a recently discovered nanoparticle) are increasingly implicated in both physiological and pathophysiological conditions. The COVID‐19 pandemic is due to SARS‐CoV‐2 that via its spike (S) protein binds the host cell receptor angiotensin‐converting enzyme 2 (ACE2) to enter cells. Host serine protease TMPRSS2 primes the spike (S) protein of SARS‐CoV‐2 for cellular entry. Recently, human recombinant (r) soluble ACE2 has been shown to attenuate SARS‐CoV‐2 infection in vitro and in patients, presumably acting as a viral decoy. TMPRSS2 and the metalloprotease TACE cleave and release the ectodomain of ACE2, raising the possibility that extracellular ACE2 also may act as a viral decoy.

Methods: sEV pellets (sEV‐Ps) and exomeres were isolated by differential high‐speed ultracentrifugation from the conditioned medium of human colorectal cancer cell lines (DiFi, DKO‐1, LIM1215, Caco‐2, LS174T). The binding of ACE2 in sEVs and exomeres to the receptor binding domain (RBD) of the S1 subunit of S protein was examined by flow cytometry and co‐immunoprecipitation. α2,6‐sialylated proteins were pelleted with the SNA lectin. For cytokine treatment, CRC or Calu‐3 lung cancer cells were treated with 200 U/ml or 1000 U/ml of rhuman IFN‐γ and/or with 50 ng/ml of rhuman TNF‐α.

Results: Full‐length ACE2 is released in sEVs from CRC cells and two ectodomain fragments of ACE2 are enriched in exomeres. TMPRSS2 and TACE are expressed in CRC cells and secreted in sEVs. ACE2 in cells, sEVs and exomeres is α2,6‐sialylated by ST6Gal‐1. ACE2‐containing sEVs and exomeres bind the RBD of the SARS‐CoV‐2 S protein S1 subunit. Intriguingly, IFN‐γ induces inflammation and reduces ACE2 expression in these CRC cells and in Calu‐3 cells.

Summary/Conclusion: CRC cell‐derived sEVs contain full‐length ACE2 and TMPRSS2 that primes the S protein of SARS‐CoV‐2 for entry. Exomeres contain ectodomain‐shed ACE2. ACE2 in sEVs and exomeres undergoes 2,6 sialylation by ST6Gal‐1. Studies are underway to determine 1) the relative efficiency of ACE2‐containing sEVs and exomeres to bind S protein compared to recombinant human ACE2 and 2) if inflammatory modulation of cellular ACE2 expression results in altered secretion of ACE2 in sEVs and exomeres. Our findings support the hypothesis that ACE2‐containing sEVs and exomeres may act as viral decoys for SARS‐CoV‐2 and have potential important implications for the treatment COVID‐19.

CC8.2. Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release (EV‐CATCHER): A customizable purification assay designed for small‐RNA biomarker identification and functional evaluation of extracellular vesicles

Megan I. Mitchell, Center for Discovery and Innovation, Hackensack Meridian Health

Iddo Ben‐Dov, Hadassah‐Hebrew University Medical Center

Kenny Ye, Albert Einstein College of Medicine

Christina Liu, Center for Discovery and Innovation, Hackensack Meridian Health

Kar Chow, Hackensack University Medical Center

Yael Kramer, Hackensack University Medical Center

Anju Gangadharan, Hackensack University Medical Center

Steven Park, Center for Discovery and Innovation, Hackensack Meridian Health

Sean Fitzgerald, Center for Discovery and Innovation, Hackensack Meridian Health

Andrew Ramnauth,Weill Cornell Medicine

David Perlin, Center for Discovery and Innovation, Hackensack Meridian Health

Michele Donato,Hackensack University Medical Center

Emily Bhoy, Center for Discovery and Innovation, Hackensack Meridian Health

Ehsan Manouchehri Doulabi,Uppsala University

Masood Kamali‐Moghaddam,University of Uppsala

Olivier Loudig,Center for Discovery and Innovation, Hackensack Meridian Health

Introduction: Circulating nucleic acids in extracellular vesicles (EVs) provide a stable source of disease biomarkers. However, the selective isolation of disease‐associated EVs from whole biofluid is critical. Many available purification assays rely on the physical properties of extracellular vesicles, rather than inherent cellular characteristics. We established a highly selective purification assay, termed EV Capture by AnTibody of CHoice and Enzymatic Release: EV‐CATCHER, designed for high‐throughput analysis of low‐abundance miRNAs and the evaluation of EV functional characteristics.

Methods: Evaluation of EV‐CATCHER sensitivity by specific capture and small‐RNA sequencing of mouse EVs spiked into human plasma. Western blotting, nanoparticle tracking, and TEM were used to evaluate EV purification between EV‐CATCHER and commercial assays. miRNA sequencing was performed on EV‐CATCHER purified EVs from sera of consented, hospitalized patients with either mild or severe Covid‐19 disease. Finally, In vitro functional evaluation of Covid‐19 convalescent EVs isolated from sera using EV‐CATCHER was performed.

Results: Our miRNA data demonstrates the purity and sensitivity of EV‐CATCHER in isolating mouse EVs spiked in human plasma, using a mouse‐specific CD63 antibody. Small‐RNA sequencing of EVs isolated from sera of mildly and severely ill Covid‐19 patients identified hsa‐miR‐146a and hsa‐miR‐126‐3p to be significantly downregulated with severity, two miRNAs associated with inflammation and endothelial cell repair inhibition, not significantly detectable in whole sera. Finally, using EV‐CATCHER we identified neutralizing properties against SARS‐CoV‐2 for EVs isolated from high anti‐spike IgG convalescent sera, demonstrating that our assay allows for release of intact and functional EVs.

Summary/Conclusion: EV‐CATCHER represents a versatile molecular assay for the highly specific purification of EVs from all biofluids, with unique properties to identify circulating biomarkers and functionality associated with disease.

CC8.3. The progression of a Covid‐19 pneumonia to Sars‐CoV‐2 acute respiratory distress syndrome (ARDS) is regulated by serum cell‐free miRNAs in precipitated extracellular vesicles

Agnes S. Meidert, University Hospital, Ludwig‐Maximilians‐University Munich

Stefanie Hermann, Technical University of Munich

Florian Brandes, Ludwig‐Maximilians‐University Munich

Benedikt Kirchner, MSc, Technical University of Munich

Dominik Buschmann, Technical University of Munich

Anja Lindemann, University Hospital, Ludwig‐Maximilians‐Universität München

Marlene Reithmair, University Hospital, Ludwig‐Maximilians‐Universität München

Gustav Schelling, University Hospital, Ludwig‐Maximilians‐Universität München

Michael W. Pfaffl, PhD, Chair of Animal Physiology & Immunology

Introduction: Extracellular vesicles (EVs) and their biologically active molecules regulate the intercellular communication during inflammatory lung response. EVs and enveloped viruses share several molecular and structural characteristics, and some viruses can hijack EV biogenesis to promote their dissemination. We assume that serum circulating EVs released during Sars‐CoV‐2 infection support inflammatory processes and might be associated with the progression of Covid‐19 pneumonia (CoP) to severe Covid‐19 associated pulmonary failure (ARDS).

Methods: We studied 20 symptomatic patients with confirmed CoP (age = 63.5±14.8 years, CURB‐65 score = 1.2±1.2), 20 mechanically ventilated patients with Covid‐19 ARDS (age = 62.5±10.9, paO2/FiO2 ratio = 139.9±57.9) and 20 healthy controls (age = 40±12.9). EVs were purified from serum by precipitation, total RNA was isolated and small‐RNA was profiled by NGS. Differential gene expression (DGE) was performed, and differentially regulated miRNAs were analyzed by Ingenuity Pathway Analysis (IPA) to characterize signaling pathways in CoP and the progression to ARDS with healthy individuals serving as controls.

Results: DGE revealed 42 differentially expressed miRNAs in CoP compared to the healthy state, and 19 regulated miRNAs in patients with CoP in comparison to ARDS. IPA revealed differentially regulated signaling networks between CoP and healthy individuals with miR‐542‐3p targeting TNF, and miR‐3168 or miR‐338‐5p targeting IL‐6. miR‐197‐3p and miR‐338‐5p also targeted OR52N2, an olfactory receptor in the nose that triggers the perception of smell. The olfactory pathway is currently regarded as a major route for neuroinvasiveness of the Sars‐CoV‐2 virus. Comparing CoP to ARDS resulted in a fully activated network of cytokines and transcription factors controlled by downregulated miR‐4433b‐5p, miR‐4433b‐3p and miR‐3168.

Summary/Conclusion: Our data indicate a role for cell‐free miRNAs as mediators of pathophysiologic changes in CoP and the associated progression to ARDS. EVs could also serve as carrier vehicles for future RNA based vaccines against SARS‐CoV‐2.

CC8.4. Single‐particle detection and analysis of SARS‐CoV‐2 protein‐carrying extracellular vesicles

Linglei Jiang, Johns Hopkins University

Tom Driedonks, PhD, Johns Hopkins Medical School / Utrecht University

Wouter Jong, Abera Bioscience AB, Stockholm, Sweden

Zhaohao Liao, Johns Hopkins University

Olesia Gololobova, Johns Hopkins University School of Medicine

Fengying Li, Johns Hopkins University

Joen Luirink, Vrije Universiteit Amsterdam

Kenneth W. Witwer, Johns Hopkins University School of Medicine

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the causative agent of COVID‐19, with a death toll reaching two million in just over a year after the first reported case. Despite rapid development and deployment of vaccines, continuing spread, vaccine availability, unknown longevity of immune responses, and viral mutations suggest that SARS‐CoV‐2 may be a problem for years to come. Here, we used several single‐particle analyses to determine if viral proteins could be detected on the surface of extracellular vesicles as virus mimetics.

Methods: The receptor binding domain (RBD) of SARS‐CoV‐2 Spike protein was expressed in mammalian cell culture and purified. Extracellular vesicles were collected from an engineered bacterial strain. RBD was then conjugated to the EV surface using affinity tag technology. EVs were then characterized in bulk by Western blot, zeta potential measurement, and particle tracking. Single‐particle analyses were done with immunogold EM and single particle interferometric reflectance imaging sensing (SP‐IRIS).

Results: Highly efficient conjugation of Spike RBD to bacterial EVs was observed by densitometry and confirmed by immunogold labelling. The diameter range of conjugated particles was from about 30 nm to 200 nm. Particles were successfully detected with custom SP‐IRIS capture chips printed with affinity reagents to Spike epitopes and bacterial LPS. Furthermore, SP‐IRIS in fluorescence mode confirmed co‐localization of Spike and bacterial LPS.

Summary/Conclusion: Our results show proof of principle that SARS‐CoV‐2 protein produced in mammalian cell culture can be conjugated to bacterial EVs, and also that viral protein‐containing particles can be detected and characterized at the single‐particle level by technologies including SP‐IRIS. These findings may have implications for diagnosis and monitoring of infection as well as mobilization of highly tractable bacterial systems for future vaccines.

CC8.5. Deciphering the biogenesis mechanisms of JC polyomavirus associated extracellular vesicles

Jenna Morris‐Love, Brown University

Bethany O'Hara, MS, Brown University

Gretchen V. Gee, PhD, University of Massachusetts Medical School, MassBiologics

Aisling S. Dugan, PhD, Assumption University

Brandon Armstead, MS, Brown University

Ryan O'Rourke, Brown University

Benedetta Assetta, PhD, Brown University

Sheila A. Haley, PhD, Brown University

Walter J. Atwood, PhD, Brown University

Introduction: JC polyomavirus (JCPyV) is a small non‐enveloped virus that causes the neurodegenerative disease Progressive Multifocal Leukoencephalopathy (PML). JCPyV establishes productive infections using either receptor‐dependent or receptor‐independent mechanisms. In the receptor‐dependent mechanism naked JCPyV requires attachment to a sialic acid containing receptor followed by interaction with a member of the 5‐hydroxytryptamine type 2 serotonin receptor family that triggers clathrin‐mediated endocytosis. In the receptor‐independent mechanism JCPyV is associated with extracellular vesicles that are taken up by target cells via clathrin‐mediated endocytosis or macropinocytosis regardless of the presence of either attachment or entry receptors. The biogenesis of extracellular vesicles involves several non‐mutually exclusive pathways with a myriad of proteins. Here, we begin to characterize the general pathways and individual proteins crucial to the production of JCPyV associated extracellular vesicles (EVs).

Methods: Using chemical and/or genetic depletion techniques we targeted neutral sphingomyelinase 2 (nSMase2), endosomal sorting complexes required for transport (ESCRT) proteins, common EV associated tetraspanins, and factors involved in secretory autophagy.

Results: We found the use of drug treatments against nSMase2 decreased the spread of purified JCPyV over time. Genetic depletion of nSMase2 caused an increased in EVs produced per cell yet decreased the quantity of protected viral genomes associated with EVs and reduced infectious EV production. Knockdown of seven different ESCRT‐related proteins“HRS, ALIX, TSG101, VPS25, VPS20, CHMP4a, and VPS4a”did not significantly affect JCPyV‐EV infectivity or production, whereas knockdown of the tetraspanins CD9 and CD81 or the secretory autophagy related proteins Rab8a, Rab27a, and GRASP65 all significantly reduced the spread of purified JCPyV and decreased production of infectious EVs.

Summary/Conclusion: These findings point to a general role for exosomes and secretory autophagy in the release of JCPyV associated EVs with a specific role for nSMase2, CD9, CD81, Rab8a, Rab27a, and GRASP65 proteins.

OD01.01. The specific enrichment in lipids, proteins and miRNAs of exosomes released from skeletal muscle explants is altered in obese mice and modulated lipid storage in adipocytes

AUDREY JALABERT, INRAe

Laura Reininger, CeeD

EMMANUELLE BERGER, INRAe

YOHAN COUTE, Univ. Grenoble Alpes, CEA, INSERM, IRIG, BGE, Grenoble, France

EMMANUELLE MEUGNIER, INRAe

Alexis FORTERRE, DIATECH, UMR 7294, Centre européen d’étude du Diabète

ELIZABETH ERRAZURIZ‐CERDA, UNIVERSITY OF LYON

ALAIN GELOEN, CNRS

Karim Bouzakri, CEED

JENNIFER RIEUSSET, INSERM

SOPHIE ROME, INRAE

Introduction: We have determined for the first time, the specific enrichment in lipids, proteins and miRNAs of exosome‐like vesicles (EXO) released from skeletal muscle (SkM) explants from mice. Then we have determined the impact of obesity‐induced insulin resistance on SkM‐EXO composition and biological functions

Methods: Ob/ob and C57BL/6 WT mice were fed for 12 weeks (standard chow diet). At sacrifice quadricep (Quad) and gastrocnemus (Gast) were excised and cut into small pieces to remove all contaminant tissues and incubated for 24h in serum‐free DMEM. EXO were extracted by differential centrifugations/filtrations/ultracentrifugations. EXO were labeled with anti‐CD81and visualized by TEM. TSG101 and ALIX were detected by WB. EXO proteins were quantified by using a mass spectrometry‐based quantitative proteomic approach. Individual phospholipid classes and and cholesterol were quantified from Gast or from EXO, released from the same Gast muscle to calculate specific lipid enrichment. MiRNAs were quantified by TaqMan(R) Low Density Arrays and compared with their expressions in Quad explants.

Results: ob/ob‐Quad released significantly less EXO than WT‐Quad, in agreement with the decrease in RAB35 and with the increase in cholesterol concentration vs WT‐Quad. WT‐EXO contained 798 proteins involved in glucose metabolism, signalling pathways and inflammation. They were located in intracellular organelles (also mitochondria), but not in plasma membrane validating the endosomal origin of EXO. 49 proteins were differentially abundant between WT‐EXO and ob/ob‐EXO, involved in lipid oxidation and with catalytic activities. Compared with Gast, Gast‐EXO accumulated sphingomyelin and cholesterol. The levels of specific subspecies of ceramides, sphingomyelin and phosphatidylcholine were modified in ob/ob‐EXO vs WT‐EXO but these variations did not mirror those between ob/ob‐Gast and WT‐Gast. The WT‐EXO miRNA population was surprising as many miRNAs had a nuclear addressing sequence and computer predictions indicated that their target genes were enriched in genes encoding proteins with nuclear activities. On the 7 miRNAs differentially expressed between WT‐EXO and ob/ob‐EXO, 4 had been found expressed in nucleus of various cell types and 2 had a nuclear motif in their 3’ regions. Ob/ob‐EXO induced lipid storage whereas WT‐EXO prevent lipid accumulation in adipocytes, suggesting for the first time, the existence of a cross‐talk between SkM and adipose tissue in favor of the adipose tissue expansion in obesity.

Summary/Conclusion: During obesity, all SkM‐EXO components are altered and can individually participate in the spread of a deleterious endocrine signal from SkM to adipose tissue, by targeting different intracellular organelles in these recipient cells, including the nucleus.

OD01.02. Characterization of Brain Derived Extracellular Vesicle Lipids in Alzheimer's Disease

Huaqi Su, The University of Melbourne

Yepy Rustam, The University of Melbourne

Colin Masters, The University of Melbourne

Enes Makalic, The University of Melbourne

Catriona McLean, The Florey Institute of Neuroscience and Mental Health

Andrew Hill, La Trobe University

Kevin Barnham, The Florey Institute of Neuroscience and Mental Health

Gavin Reid, The University of Melbourne

Laura Vella, The Florey Institute of Neuroscience and Mental Health

Introduction: Lipid dysregulation is associated with Alzheimer's disease (AD) pathogenesis, however the application of lipids as blood‐based biomarkers of AD, has proven difficult. Most biomarker studies have focused on examining the entire lipidome, without focusing on brain‐specific lipids. However, the small extracellular vesicles (EV), can pass the blood‐brain barrier (BBB) and enter the periphery, while carrying a subset of lipids which make them uniquely suited for biomarker exploration.

Methods: To determine the potential of EV lipids as biomarkers, using the protocol developed for brain‐derived exosome isolation and characterization (Vella, etal. JEV. 2017) and quantitative mass spectrometry based lipidome analysis (Rustam and Reid. Anal. Chem. 2018), we revealed the lipidomic characterization of post‐mortem frontal cortices and the brain derived EV (BDEV) from AD subjects versus controls.

Results: Western blot result showed enrichment of syntenin and TSG101 in the isolated BDEV preparations with little calnexin, indicating minimum cell lysis. Transmission electron microscopy revealed the typical round and cup‐shaped EV morphology with vesicle size being 50–150nm. A total of 692 lipids were identified and quantified. Enrichment of glycerophosphatidylserine (PS) lipids, especially the ether PS lipids, alkyl‐ and alkenyl‐, were observed in BDEV. Remodeling in phosphatidylethanoamine (PE) lipid class and polyunsaturated fatty acyl containing lipids (PUFA‐) was observed in AD BDEV in comparison to control BDEV.

Summary/Conclusion: We, for the first time, characterized the lipid composition of EVs in human frontal cortex. BDEVs offered improved detection of dysregulated lipids in AD over global lipid profiling of the brain region. Many of these changes have previously been reported to play key roles in AD pathogenesis, suggesting BDEV provide a readout of lipid dysregulation in AD and highlighting the potential use of these lipids as disease biomarkers in the periphery.

OD01.03. Glycerophospholipid and sphingolipid composition of equine synovial fluid derived extracellular vesicles obtained pre‐ and post LPS‐induced acute synovitis

Laura Varela, Division of Equine Sciences, Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Chris H.A. van de Lest, Division of Equine Sciences, Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

René P.R. van Weeren, Division of Equine Sciences, Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Marca H.M. H.M. Wauben, Department of Biomolecular Health Sciences, Utrecht University, The Netherlands

Introduction: Inflammation is the hallmark of many diseases and most joint disorders have inflammation as their common denominator, though causes of inflammation may differ. Extracellular vesicles (EVs) are known to play a role in intercellular communication in both health and disease states. The aim of this study was to investigate the lipidome profile of synovial fluid‐derived EVs in healthy equine joints and joints with lipopolysaccharide (LPS)‐induced synovitis, a model of acute, however transient and fully reversible, synovitis, characterized by an acute inflammation phase 5–8h after injection with the height of the resolution phase at around 24h post‐injection. Focus was on non‐neutral lipids, including the glycerophospholipids and sphingolipids.

Methods: Equine synovial fluid (SF) was harvested prior to LPS‐injection at 0 hours, and at 5 hours and 24 hours post‐injection. SF was centrifugated at 3000g and stored at ‐80°C. Thawed cell‐free SF samples were treated with hyaluronidase, DNAse I, and sodium citrate. Extracellular vesicles were isolated by differential ultracentrifugation with final steps at 10,000g and 200,000g, followed by EV purification with sucrose density gradients. From each fraction, lipids were extracted by the Bligh & Dyer method and analyzed using a Fusion orbitrap mass spectrometer. The subsequent data processing, as well as data visualization and statistical analyses, were performed using R packages and/or GraphPad software.

Results: We identified more than 250 lipid species within 14 lipid classes. Total lipid levels were significantly higher (p < 0.001) in EVs from the 5h (7‐fold) and 24h (2.6‐fold) collected‐SF when comparing to the 0h samples. Moreover, there was an increase of hexosylceramide (HexCer), phosphatidylserine (PS), phosphatidylcholine (PC), and sphingomyelin (SM) in the composition of EVs from both the peak and the resolutions phases. On the other hand, the amount of phosphatidylethanolamine (PE) relatively decreased in the inflamed samples compared to healthy SF.

Summary/Conclusion: Our results show that inflammation has clearly a profound effect on the lipidome composition of SF‐derived EVs.

OD01.04. Extracellular vesicle ‐mediated crosstalk between adipose tissue and liver in the development of non‐alcoholic fatty liver disease

Johanna Matilainen, University of Eastern Finland, Institute of Biomedicine

Ville Männistö, University of Eastern Finland, Institute of Public Health and Clinical Nutrition; Kuopio University Hospital

Natalia Rosso, Fondazione Italiana Fegato ONLUS, The Italian Liver Foundation

Jussi Pihlajamäki, University of Eastern Finland, Institute of Public Health and Clinical Nutrition

Uma Arasu, University of Eastern Finland, A.I. Virtanen Institute for Molecular Sciences

Ashik Deen, University of Eastern Finland, A.I. Virtanen Institute for Molecular Sciences

Petteri Nieminen, University of Eastern Finland, Institute of Biomedicine

Anne‐Mari Mustonen, University of Eastern Finland, Institute of Biomedicine

Reijo Käkelä, University of Helsinki, Molecular and Integrative Biosciences Research Programme

Kai Härkönen,Finnish Red Cross Blood Service

Kirsi Rilla, University of Eastern Finland, Institute of Biomedicine

Introduction: Recent studies have produced evidence that extracellular vesicles (EV) released from adipocytes and visceral adipose tissue (VAT) could have important roles in inter‐organ communication leading to ectopic fat accumulation in the liver (i.e., non‐alcoholic fatty liver disease, NAFLD). However, despite the promising results based on the analyses of the contents of AT‐EV, the predicted EV‐mediated effects in the liver have been rarely studied in detail. In our experiments, we investigate how obesity affects the secretion and contents of AT‐EV. EV will be isolated from human adipocyte cell line and from ex vivo AT cultures of obese mice as well as bariatric surgery patients. In addition, we aim to provide the first, detailed mechanistic data of how AT‐EV promote NAFLD development and progression. To examine this, EV from patient AT ex vivo cultures and adipocyte cell line will be added into hepatocyte cultures in vitro, after which EV‐mediated changes, particularly in fatty acid metabolism, were studied.

Methods: EV were isolated from human Simpson Golabi Behmel Syndrome (SGBS) adipocyte cells and NAFLD patient ex vivo AT cultures by ultracentrifugation, and EV counts were measured by Nanoparticle Tracking Analysis. EV were transferred to hepatocyte (immortalized human hepatocyte (IHH), human hepatoma cell line HuH7) cultures. The effects of EV and VAT on hepatocyte fatty acid metabolism and signaling promoting NAFLD were studied by qPCR and RNAseq. The amount of fatty acids were analyzed by the fluorescent labeling of intracellular lipids and confocal microscopy. Fatty acid profiles of hepatocytes and EV were determined by mass spectrometry" gas chromatography.

Results: Our preliminary results suggest that patient VAT secretes more EV than subcutaneous AT. Furthermore, our analyses revealed that EV from mature SGBS cells contained high proportions of saturated fatty acids and, particularly, palmitic acid. SGBS‐EV interacted with IHH cells, and caused increased expression of sterol regulatory element‐binding protein 1c and acetyl coenzyme A carboxylase enzymes, together with increased number of lipid droplets.

Summary/Conclusion: Our results suggest that inflammation drives the EV secretion from AT, and that AT‐EV may induce NAFLD‐promoting effects.

OD01.05. Lipidomic analysis of adipose‐derived extracellular vesicles reveals their potential as lipid mediators of obesity‐associated metabolic complications

Alexia BLANDIN, Institut du Thorax INSERM 1087

Grégory Hilairet, University of Angers

Maharajah Ponnaiah, ICANalytics

Marie Lhomme, ICANalytics

Soazig Le Lay, Institut du Thorax INSERM 1087

Introduction: Obesity‐related disorders are commonly associated with perturbations of lipid metabolism. Recent data show the ability of adipose extracellular vesicles (AdEV) to transport lipids that could participate in the development of metabolic dysfunctions. We aim to characterize the lipid content of AdEV in an healthy or obesity context in order to define their lipid signature and predict their role as mediators of metabolic disorders.

Methods: AdEV were purified from conditioned media of visceral adipose tissue (VAT) from control (Ob/+) or obese (Ob/Ob) mice by differential centrifugation to isolate large AdEV (13K) and small AdEV (100K). An untargeted mass spectrometry lipidomic approach was conducted to identify the whole sphingolipidome and phospholipidome as well as neutral lipids of AdEV, in comparison to secretory VAT.

Results: Lipidomic analysis of VAT reveals that lean tissue significantly differs from obese one at the level of both membrane lipid classes and molecular species. The most consistent changes in the obese VAT were the decrease in Lysophosphatidylethanolamines (LPE) and in PE plasmalogens (PEP) and the increase of Phosphatidylinositols (PI) and mitochondrial‐derived Phosphatidylglycerols (PG) in comparison to lean VAT. The PCA using lipid content data of AdEV and VAT shows a significant separation between both groups. Comprehensive lipidome analysis indeed reveals a specific EV enrichment of Ceramides (Cer) and its derivatives as well as in PG. EV lipidome was nonetheless more dependent of cell origin than on EV subtype, since obesity impacted AdEV lipid content of large and small AdEV in similar ways. For instance, obesity drives AdEV subtype enrichment in Triacylglycerols (TG) suggesting AdEV neutral lipid sorting independent of the canonical lipolytic pathway, and sphingomyelin (SM) enrichment at the expense of Cer illustrating dysregulated sphingolipid synthesis with obesity. Of interest, obese AdEV are particularly enriched in PG and arachidonic subspecies which might, respectively, contribute to mitochondrial dysfunction and chronic inflammation associated with obesity.

Summary/Conclusion: This study establishs for the first time the lipid fingerprint of VAT and their derived AdEV in a healthy and obesity context and highlights the AdEV potential as lipid mediators of obesity‐associated metabolic complications.

OD01.06. Inhibition of neutral sphingomyelinase 2 reverses depression‐ and cognitive‐associated behaviors in EcoHIV‐infected mice

XIAOLEI ZHU, JHU

Kristen Hollinger, JHU

Yiyao Huang, MD, Johns Hopkins University School of Medicine

Tanina Arab, JHU

Alejandra Borjabad, Icahn School of Medicine at Mount Sinai

Boe‐Hyun Kim, Icahn School of Medicine at Mount Sinai

Ajit G. Thomas, Johns Hopkins University School of Medicine

Mohammed Moniruzzaman, JHU

Lyndah Lovell, JHU

Camilo Rojas,JHU

Atsushi Kamiya, JHU

Kenneth W. WitwerJohns Hopkins University School of Medicine

David Volsky, Icahn School of Medicine at Mount Sinai

Norman J. Haughey,Johns Hopkins University School of Medicine

Barbara S. Slusher,Johns Hopkins University School of Medicine

Introduction: To determine if depressive‐like behaviors are present in the EcoHIV mouse model of HIV‐induced neurocognitive disorders, and to evaluate the behavioral and biochemical effect of administering phenyl(R)‐(1‐(3‐(3,4‐dimethoxyphenyl)‐2,6‐dimethylimidazo[1,2‐b]pyridazin‐8‐yl)pyrrolidin‐3‐yl)‐carbamate (PDDC). PDDC is an orally bioavailable, selective and brain penetrable small molecule inhibitor of neutral sphingomyelinase 2 (nSMase2), an enzyme involved in the production of ceramide and extracellular vesicle (EV) biogenesis.

Methods: Mice were infected with EcoHIV and treated daily with either vehicle or PDDC starting at 3 weeks post‐infection. After two weeks of treatment, depressive‐like behaviors were evaluated using three‐chamber social approach test and forced swim testing. In parallel a separate cohort of mice was treated similarly and evaluated for cognitive function using the radial arm water maze. Subsequently, mice were sacrificed and brain tissues and plasma were collected to determine the effect of PDDC on nSMase2 enzymatic activity and sphingolipids. EVs were also isolated from brain tissue to determine the effect of PDDC on EV number and cargo.

Results: EcoHIV‐infected mice exhibited depressive‐like behaviors and cognitive impairment, both of which were reversed by PDDC. Brain nSMase2 activity was significantly elevated in EcoHIV infected mice, resulting in decreased sphingomyelin levels and increased ceramide levels. PDDC treatment restored these levels to baseline. EcoHIV infected mice also exhibited changes in brain‐derived EV levels, along with altered miRNA and protein cargo; all were normalized by PDDC.

Summary/Conclusion: Inhibition of nSMase2 represents a new therapeutic strategy for the treatment of HIV‐associated cognitive impairment and depression.

OD02.01. Allergic sensitization of the mother influences the miRNA cargo and T cell modulatory properties of milk‐derived extracellular vesicles

Alberta Giovanazzi, Department of Biomolecular Health Sciences, Utrecht University, The Netherlands

Martijn van Herwijnen, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.

Marijke I. Zonneveld, Postdoctoral researcher, GROW‐School for Oncology and Developmental Biology, Maastricht University, The Netherlands

Joaquín Jurado Maqueda, BIOINF2BIO, Porto, Portugal; i3S, Universidade do Porto, Portugal

Tom Driedonks, PhD, Johns Hopkins Medical School / Utrecht University

Marije Kleinjan, Department of Biomolecular Health Sciences, Utrecht University, The Netherlands

Gerbrich N. van der Meulen, Department of Paediatric Allergy, Martini Hospital, Groningen, The Netherlands

Johan Garssen, Division of Pharmacology, Department of Pharmaceutical Sciences, Utrecht University, The Netherlands

Ruurd M. van Elburg, Department of Pediatrics, Emma Children's Hospital/Academic Medical Center, Amsterdam, The Netherlands

Carla Oliveira,BIOINF2BIO, Porto, Portugal; i3S, Universidade do Porto, Portugal

Frank A. A. Redegeld, Division of Pharmacology, Department of Pharmaceutical Sciences, Utrecht University, The Netherlands

Esther N.M Nolte – ‘t Hoen, Dr.Utrecht University

Marca H.M. H.M. Wauben, Department of Biomolecular Health Sciences, Utrecht University, The Netherlands

Introduction: Allergic diseases can alter the physiological milk composition, but it is still unknown whether EV function and cargo are affected. Recently, we compared the functionality of EVs derived from non‐allergic and allergic mothers and we found a reduced T cell modulatory capacity in the latter group. In this study, we analyzed the miRNA cargo of milk EVs and used T cell signaling network analysis to predict possible relations between altered miRNA cargo and changed T cell modulatory activity.

Methods: EVs were purified from human milk of non‐allergic and allergic mothers (total serum IgE ‐ 50 kU/ml and/or positive Phadiatop assay for specific IgE) by differential centrifugation, density gradient floatation and size exclusion chromatography. Small RNA sequencing was performed to pinpoint qualitative and quantitative differences in milk EV miRNA cargo. A T cell signalling network model based on miRNA‐target interactions was built to predict hotspots of milk EV regulation.

Results: Transcriptomic analysis of milk EVs revealed that some miRNAs were differentially expressed among non‐allergic and allergic samples. Our prediction model shows that miRNAs overrepresented in non‐allergic samples favour the attenuation of T cell activation‐downstream processes such as cell cycle progression, STAT3/IL6 pathway and glucose metabolism, while miRNAs overrepresented in allergic samples target pro‐apoptotic pathway and negative regulators of cyclins.

Summary/Conclusion: Milk EVs from non‐allergic mothers are stronger inhibitors of T cell activation and this might be linked to quantitative differences in milk EV miRNA cargo between allergic and non‐allergic mothers.

OD02.02. Umbilical cord blood‐derived small extracellular vesicles: immune‐modulating properties and regenerative potential for psoriatic lesions

Patricia Freire, Exogenus Therapeutics, S.A.

Silvia Rodrigues, Exogenus Therapeutics, S.A.

Patricia Freire, Exogenus Therapeutics, S.A.

Renato Cardoso, Exogenus Therapeutics, S.A.

Cláudia Gomes, Exogenus Therapeutics, S.A.

Ricardo Neves, Exogenus Therapeutics, S.A.; Center for Neurosciences and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal; Institute for Interdisciplinary Research (3Is), University of Coimbra, Coimbra, Portugal

Joana Correia, Exogenus Therapeutics, S.A.; Center for Neurosciences and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal

Introduction: Umbilical cord blood (UCB) has long been seen as a rich source of naïve cells with strong regenerative potential, likely mediated by the secretion of small extracellular vesicles (sEV). More recently, the immune‐modulating properties of stem‐cell‐derived sEV have attracted attention as a possible treatment for auto‐immune conditions. Despite their widely accepted use for transplantation into patients with blood disorders, UCB mononuclear cells (MNC) are seldomly studied for their regenerative and anti‐inflammatory potential.

Methods: With this work, we aimed to characterize the effect of UCB‐MNC‐sEV in different immune populations and determine their therapeutic effect in in vitro and in vivo models of psoriasis.

Results: In vitro, sEV were capable of shifting the profile of THP‐1 derived macrophages towards an anti inflammatory M2 phenotype, including in the presence of a pro‐inflammatory stimulus (LPS). In fresh PBMC, incubation with sEV resulted in a significant inhibition of total CD4+ and CD8+ T cell proliferation and pro‐inflammatory cytokine release (IFNg, TNFa, CCL20). Moreover, sEV were shown to influence the expression of transcriptional regulators T‐bet, RORgt and Foxp3, thereby supporting the development of regulatory T‐cells (Treg), while hindering Th1 and Th17 differentiation. In a 3D model of psoriatic epidermis, sEV strongly decreased the expression of antimicrobial peptides S100A7 and DEFB4, as well as of pro‐inflammatory mediators IL‐6, IL‐8, CXCL10 and COX‐2. Furthermore, in vivo, sEV significantly prevented or reversed epidermal thickening (acanthosis) in a model of imiquimod‐induced psoriasis, and tendentially increased the number of Treg in affected skin, while having no overall effect in macroscopic disease score.

Summary/Conclusion: In conclusion, this work provides evidence for the immune‐modulating effect of UCB‐MNC‐sEV, opening the door to the exploitation of this largely discarded material which may prove effective in the treatment of inflammatory skin diseases with high unmet needs, such as psoriasis.

OD02.03. Molecular and functional signatures of distinct subpopulations of extracellular vesicles provide a rationale for beta‐cell mediated immune disease

Grégoire Mignot, IECM laboratory USC 1383 INRAE

Khem Giri, IECM, ONIRIS, INRAE, USC1383, Nantes, France

Laurence de Beaurepaire, IECM, ONIRIS, INRAE, USC1383, Nantes, France

Dominique Jegou, IECM, ONIRIS, INRAE, USC1383, Nantes, France

Margot Lavy, IECM, ONIRIS, INRAE, USC1383, Nantes, France

Mathilde Mosser, IECM, ONIRIS, INRAE, USC1383, Nantes, France

Aurélien Dupont, MRic, Biosit, UMS3480 CNRS, University of Rennes 1, Rennes, France

Romain Fleurisson, PAnTher, INRAE, Oniris, Université Bretagne Loire, Nantes, France

Laurence Dubreuil, PAnTher, INRAE, Oniris, Université Bretagne Loire, Nantes, France

Julien Pichon,PAnTher, INRAE, Oniris, Université Bretagne Loire, Nantes, France

mayeul Collot, Laboratoire de Biophotonique et Pharmacologie, UMR CNRS 7213, Université de Strasbourg, Illkirch, France

Peter van EndertINSERM, U1151, Institut Necker‐Enfants Malades, Paris, France

Jean‐Marie Bach, IECM, ONIRIS, INRAE, USC1383, Nantes, France

Steffi Bosch,IECM, ONIRIS, INRAE, USC1383, Nantes, France

Introduction: Evidences accumulates for an active role of the insulin‐producing pancreatic beta cell in diabetes development. As metabolically highly active cells, beta cells readily undergo cellular stress responses imposed by environmental changes. Beta cells release self‐antigens and microRNA inside extracellular vesicles (EV), fostering the idea that EV act as mediators in communication with immune effectors in diabetes development. While evidence accumulates on specific immune functions of subtypes of EV, the complete beta vesiculome has not been explored yet.

Methods: Here, we make quantitative and qualitative side‐by‐side comparisons of the phenotype and function of large apoptotic bodies (AB), microvesicles (MV) and small EV (sEV) isolated from an equal amount of insulin‐producing beta cells in normal and inflammatory settings.

Results: Under normal conditions, AB and MV represent 93% of the volume and 90% of the vesiculome's insulin load. None of the EV present detectable amounts of cytokines. Under inflammatory conditions, a consistently higher release of all EV subtypes is observed, commensurate with EV‐associated export of the autoantigen insulin, cytokines/chemokines and immune‐stimulatory microRNA. While the concentration of insulin inside the vesicles remains unchanged, the concentration of specific microRNA sequences raises specifically in sEV. Enrichment of MCP‐1 in all EV subtypes and of interleukin‐27 solely in AB suggests selective sorting inside EV subpopulations. Functional assays in mouse macrophage and dendritic cell cultures revealed differences in the aptitude of EV subtypes to modulate expression of cytokines and maturation markers.

Summary/Conclusion: Our findings highlight the different imprints of environmental changes in subpopulations of EV, whose relative contributions should provide new insights into the development of the global immune response.

OD02.04. Small Extracellular Vesicles Propagate the Inflammatory Response After Trauma

Tanja Seibold, University Hospital Ulm

Jonathan Schönfelder, University Hospital Ulm

Florian Weeber, University Hospital Ulm

Milena Armacki, University Hospital Ulm

Andre Lechel, University Hospital Ulm

Markus Huber‐Lang, University Hospital Ulm

Miriam Kalbitz, University Hospital Ulm

Thomas Seufferlein, University Hospital Ulm

Tim Eiseler, University Hospital Ulm

Introduction: Trauma is the leading cause of death in individuals under 44 years of age. Thorax trauma (TxT) is strongly associated with trauma‐related death, an unbalanced innate immune response, sepsis, acute‐respiratory‐distress‐syndrome (ARDS), and multiple‐organ‐dysfunction (MODS), but also marked endothelial activation and inflammation has been described.

Methods: To investigate sEV secretion in response to trauma we used nanoparticle tracking analysis to characterize serum sEVs from mice after TxT, polytrauma (PT) and hemorrhagic shock (HS). Effects of sEVs on endothelial inflammation were elucidated by an in‐vitro trauma model using an inflammatory polytrauma cocktail (PTC). We assessed the role of sEVs for the post‐traumatic response in mice by injecting the sEV‐biogenesis inhibitor GW4869 10 min after TxT. Vice versa, TxT‐plasma‐sEVs were injected into healthy animals to investigate regulation of inflammation and endothelial barrier stability. Results were validated by analyzing PT‐patient plasma‐sEV.

Results: We show that different in‐vivo traumata, or PTC in‐vitro trigger secretion of sEVs from endothelial cells with pro‐inflammatory cargo. These sEVs transfer transcripts for adhesion molecules and cytokines, but also miRNAs to both propagate inflammation and destabilize endothelial barriers, respectively. Inhibition of sEV‐release after TxT in mice ameliorated local as well as systemic inflammation, neutrophil infiltration, and secondary acute kidney injury (AKI). In turn, injection of TxT‐plasma‐sEVs into healthy animals was sufficient to trigger pulmonary and systemic inflammation as well as AKI. Moreover, increased sEV concentrations and transfer of similar cargos was observed in polytrauma patients.

Summary/Conclusion: We show that diverse traumatic insults significantly increase sEV secretion and identify endothelial cells as major source. In summary, we here described a so‐far neglected role for endothelial‐derived sEVs in the transmission of local and systemic post‐traumatic inflammation.

OD02.05. Cytosolic dsDNA and extracellular vesicles participate to antitumor immunity induced by external and targeted radiotherapy

Julie Constanzo, IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut régional du Cancer de Montpellier

Jihad Karam, Institut de Recherche en Cancérologie de Montpellier

Alexandre Pichard, IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut régional du Cancer de Montpellier

Julien Faget, IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut régional du Cancer de Montpellier

Frank Bruchertseifer, European Commission ‐ Joint Research Centre

Alfred Morgenstern, European Commission ‐ Joint Research Centre

Isabelle Villa, IGH, Institut de Génétique Humaine, CNRS, Université de Montpellier, Molecular Basis of Inflammation Laboratory

Nadine Laguette, IGH, Institut de Génétique Humaine, CNRS, Université de Montpellier, Molecular Basis of Inflammation Laboratory, Montpellier

Nathalie Bonnefoy, IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut régional du Cancer de Montpellier

Jean‐Pierre Pouget,IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut régional du Cancer de Montpellier

Introduction: Beside conventional external beam radiotherapy (EBRT) using X‐rays dedicated to localized tumors, targeted radionuclide therapy (TRT) allows to specifically irradiate diffuse and metastatic tumors. TRT consists of the administration of radiopharmaceuticals made of monoclonal antibodies or peptides coupled to a radionuclide emitting alpha, beta and Auger particles. Here, we investigate the role of X‐rays, alpha, Auger and beta particles in triggering systemic effects through the activation of cGAS‐STING pathway in tumor and host immune cells and assessed the role of extracellular vesicles (EVs) in intercellular communications.

Methods: B16F10 melanoma cells were subcutaneously injected in C57BL/6J and athymic mice. Mice received intraperitoneal injections of TA99 mAb targeting TYRP‐1/gp75 tumor antigen radiolabeled either with 225Ac (1‐ 9.25 kBq; 74MBq/mg, alpha‐TRT) or 125I (2‐ 27 MBq; 37MBq/mg, Auger‐TRT), or with EVs purified from non‐treated cells or cells exposed to 2MBq/ml 177Lu‐TA99 (200 MBq/mg, beta‐TRT).

Results: In vivo, alpha‐ and Auger‐TRT efficacy was shown to require T‐cells for adaptive immunity. Median survival was 16 days and 29 days for alpha and Auger TRT, respectively versus 11 and 15 days for controls (**p = 0.0035). No difference with controls was observed in athymic nude mice. In vitro, B16F10 cells exposed to Auger‐TRT, demonstrated a protracted accumulation of cytosolic dsDNA over 48h as compared with beta‐TRT or X‐rays. In addition, an early and persistent activation of cGAS‐STING pathway was observed, from 1h to 48h following the beginning of TRT. However, knocked‐out cGAS genes in host immune cells did not affect Auger‐TRT response in vivo, while knocked‐out STING genes did, suggesting that dsDNA is not the only extracellular messenger involved in activation of immune cells. We therefore focused on extracellular vesicles as a second messenger released by cancer cells that may activate an antitumor immune response through the STING pathway. EVs purified from B16F10 cells exposed to beta‐TRT contained 3.5.10‐3 fg of dsDNA. Compared to EVs purified from non‐treated cells, 4 intratumoral injections of EVs‐Beta demonstrated a strong tumor growth delay and survival (***p = 0.0007) in C57Bl/6J mice, while no difference were observed in athymic nude mice.

Summary/Conclusion: Radiation‐induced cytosolic dsDNA and EVs mediate radiation‐induced systemic response in vivo.

OD02.06. The effects of mesenchymal stromal cells‐derived small extracellular vesicles on dendritic cells via IL‐10 in patients with allergic rhinitis

Ya‐Qi Peng, The First Affiliated Hospital, Sun Yat‐sen University

Qing‐Ling Fu, The First Affiliated Hospital, Sun Yat‐sen University

Introduction: Mesenchymal stromal cells (MSCs) are well known as their immunoregulatory roles on allergic inflammation particularly by acting on T cells, B cells, and dendritic cells (DCs). MSC‐derived small extracellular vesicles (MSC‐sEV) are increasingly considered as one of the main factors for the effects of MSCs on immune responses. However, the effects of MSC‐sEV on DCs in allergic diseases remain unclear.

Methods: MSC‐sEV were prepared from the induced pluripotent stem cells (iPSC)‐MSCs by anion‐exchange chromatography, and were characterized with the size, morphology, and specific markers. Human monocyte‐derived DCs were generated and cultured in the presence of MSC‐sEV to differentiate the so‐called sEV‐immature DCs (sEV‐iDCs) and sEV‐mature DCs (sEV‐mDCs), respectively. The phenotypes and the phagocytic ability of sEV‐iDCs were analyzed by flow cytometry. sEV‐mDCs were co‐cultured with isolated CD4+T cells or peripheral blood mononuclear cells (PBMCs) from patients with allergic rhinitis (AR). The levels of Th1 and Th2 cytokines produced by T cells were examined by ELISA and intracellular flow staining, and the following mechanisms were further investigated.

Results: We demonstrated that MSC‐sEV inhibited the differentiation of human monocytes to iDCs with down‐regulation of the expression of CD40, CD80, CD86, and HLA‐DR, but had no effects on the surface markers of mDCs. However, sEV treatment enhanced the antigen uptake of mDC. In addition, sEV‐mDCs suppressed the Th2 immune response by reducing the production of IL‐4, IL‐9, and IL‐13. Moreover, the treatment of neutralizing anti‐IL‐10 antibodies led to a significant reversal in levels of IL‐13+CD4+T cells, IL‐9 and IL‐13 production from T cells compared to the sEV‐mDCs alone. Additionally, the administration of sEV‐mDCs upregulated the levels of IL‐10+CD4+T cells and CD4+CD25+Foxp3+ Treg cells.

Summary/Conclusion: Our study identified that mDCs treated with MSC‐sEV exhibited the inhibition of Th2 responses, providing a novel evidence of the potential cell‐free therapy of on DCs in allergic airway diseases.

OD02.07. Red blood cells‐derived extracellular vesicles decrease Neutrophils survival, phagocytosis, and ROS production

Getulio P. Oliveira, Ph.D., Beth Israel Deaconess Medical Center

Brandy Pinckney, Beth Israel Deaconess Medical Center

Shulin Lu, Beth Israel Deaconess Medical Center

Alan Zimmerman, Northeastern University

John Tigges, Beth Israel Deaconess Medical Center

Alexander Ivanov, Northeastern University

Ionita C. Ghiran, MD, Beth Israel Deaconess Medical Center

Introduction: Extracellular vesicles (EVs) are membrane‐bound entities released by cells and tissues into biofluids involved in cell‐cell communication. Circulating red blood cells (RBCs), the most numerous cell‐type in the body, generate large numbers of EVs daily. The fate of the RBC‐EVs is currently unknown.

Methods: RBC plasma membranes were labeled with a red fluorescent dye, and EVs were generated by either Ionomycin 10 μM or A23187 10 μM for 1h. RBC‐EVs were purified by size exclusion chromatography (SEC), and characterized using microscopy (fluorescence and electron (EM)), nanoflow cytometry, western blotting, tunable pulse sensing, and LC‐MS‐based proteomics. EV uptake was measured by flow cytometry, fluorescence microscopy and cryo‐EM. The biological function of RBC‐EVs on neutrophil was assessed by survival assay at 24h (Annexin 5‐PI staining), phagocytosis of fluorescent E. coli BioParticles after 2h, and ROS production after 4h.

Results: The purity of RBCs‐EVs was confirmed by western blots and immuno‐EM, using anti‐CD235a and anti‐Band3 antibodies. Quantitative proteomic profiling revealed the enrichment of sorcin, stomatin, annexin A7, and RAB proteins into RBC‐EVs. Immuno‐EM showed two distinct populations of RBC‐EVs (positive and negative) for CD235a. Furthermore, we detect two mechanisms of RBC‐EVs uptake by neutrophils: fusion of the plasma membrane and complete internalization. RBC‐EVs were rapidly uptaken by neutrophils, even after 10 min of incubation, increasing the number of late apoptotic cells (Annexin 5‐ and PI‐positive cells) after 24h. The neutrophils' ability to phagocyte E. coli was slightly decreased after incubating with RBC‐EVs for 2h. Furthermore, the neutrophils production of reactive oxygen species (ROS) was impaired after 4h of incubation.

Summary/Conclusion: This work brings new insights into the communication among RBC‐EVs to PBMCs, and neutrophils in circulation.

OD03.01. Astrocyte‐derived small extracellular vesicles promote synapse formation via fibulin‐2 mediated TGF‐beta signaling

Mikin Patel, Vanderbilt University

Alissa Weaver, Department of Cell and Developmental Biology, Vanderbilt University School of Medicine

Introduction: Synapses are specialized neuronal structures that are critical for neuronal communication. Dendritic spines are postsynaptic membrane specializations that are critical for synapse formation and function. Previous studies have shown that astrocyte conditioned media can enhance neuronal spine and synapse formation, suggesting the importance of molecules secreted by astrocytes. Here, we examine the role of astrocyte‐derived small extracellular vesicles in the formation of neuronal dendritic spines and synapses.

Methods: Small extracellular vesicles (SEVs) were isolated from primary cortical neurons (CNSEVs), primary astrocytes (ADSEVs) and C6 glioma cells (C6SEVs) using differential ultracentrifugation. Purified SEVs were characterized for their size, morphology and common markers using NTA, TEM and Western blot. Day in vitro 10 cortical neurons were treated for 48 h with increasing doses of purified SEVs to analyze their effect on dendritic spines and synapses. iTRAQ proteomics analysis was performed to identify unique synaptogenic cargo present in ADSEVs. Phosphorylated Smad2 (pSmad2) was measured to examine activation of TGF‐beta signaling.

Results: Here, we find that ADSEVs, but not CNSEVs or C6SEVs, enhance dendritic spine and synapse formation. iTRAQ proteomics analysis revealed that ADSEVs are enriched in proteins distinct from CNSEVs or C6SEVs, including fibulin‐2, an extracellular matrix protein known to activate TGF‐beta. We find that treatment with fibulin‐2‐knockdown ADSEVs does not induce synaptogenesis, whereas treatment with recombinant fibulin‐2 induces synaptogenesis. Treatment of neurons with recombinant fibulin‐2 or ADSEVs leads to an increase in pSmad2, suggesting activation of TGF‐beta signaling. Also, the synaptogenic effects of fibulin‐2 or ADSEVs are reversed by inhibiting TGF‐beta signaling. Finally, the increase in pSmad2 level by ADSEVs is only slightly diminished by the presence of the endocytosis inhibitor Dynasore, suggesting that activation of TGF‐beta signaling by ADSEVS is likely to occur at the cell surface.

Summary/Conclusion: These results show that fibulin‐2 carried by astrocyte‐derived SEVs promotes formation of neuronal dendritic spines and synapses by activating TGF‐beta signaling.

OD03.02. Phenotypic and functional analysis of nigrostriatal astrocyte‐derived extracellular vesicles reveals an intrinsic brain area‐dependent neuroprotective potential in Parkinson's disease models

Loredana Leggio, Department of Biomedical and Biotechnological Sciences, University of Catania, Italy

Francesca L'Episcopo, Neuropharmacology Section, OASI Research Institute‐IRCCS, 94018 Troina (EN), Italy

Andrea Magrì, Department of Biological, Geological and Environmental Sciences, University of Catania, Italy

María José Ulloa‐Navas, Institute Cavanilles, University of Valencia, Valencia, Spain

Greta Paternò, Department of Biomedical and Biotechnological Sciences, University of Catania, Italy

Silvia Vivarelli, Department of Biomedical and Biotechnological Sciences, University of Catania, Italy

Cataldo tirolo, Neuropharmacology Section, OASI Research Institute‐IRCCS, 94018 Troina (EN), Italy

Nunzio Testa, Neuropharmacology Section, OASI Research Institute‐IRCCS, 94018 Troina (EN), Italy

Salvatore Caniglia, Neuropharmacology Section, OASI Research Institute‐IRCCS, 94018 Troina (EN), Italy

Carlos Bastos, University of Cambridge, Department of Veterinary Medicine, Cambridge, United Kingdom

Pierpaolo Risiglione, Department of Biomedical and Biotechnological Sciences, University of Catania, Italy

Nuno FariaUniversity of Cambridge, Department of Veterinary Medicine, Cambridge, United Kingdom

Stefano Pluchino, University of Cambridge, Department of Clinical Neurosciences, Cambridge, United Kingdom

Jose Manuel Garcia‐Verdugo, Institute Cavanilles, University of Valencia, Valencia, Spain

Angela Messina, Department of Biological, Geological and Environmental Sciences, University of Catania, Italy

Bianca Marchetti, Department of Biomedical and Biotechnological Sciences, University of Catania, Italy

Nunzio Iraci, Department of Biomedical and Biotechnological Sciences, University of Catania, Italy

Introduction: Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neuronal cell bodies in the ventral midbrain (VMB) and their terminals the striatum (STR), with consequent dopamine depletion. In this context, astrocytes (AS) play either destructive or beneficial roles and, when activated by the chemokine CCL3, they exert a robust neuroreparative action both in cellular and pre‐clinical PD models. We herein isolated EVs derived from VMB‐ and STR‐ AS, both in basal and CCL3 conditions, to evaluate their possible involvement in the complex cross‐talk between AS and neurons.

Methods: EVs were purified from AS supernatants by ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold‐transmission electron microscopy and western blotting. AS‐EV functional effects were evaluated on differentiated SH‐SY5Y cells under neurodegenerative conditions using immunofluorescence and high resolution respirometry (HRR).

Results: AS secrete vesicles with a dimension of "100 nm, positive for CD63, CD9 and Alix markers. In basal conditions, VMB‐AS release more EVs than STR‐AS, and only VMB‐AS respond to CCL3 by producing more EVs. Following internalization by SH‐SY5Y cells, we tested AS‐EV effects under H2O2 treatment. We found that both basal but mostly CCL3 AS‐EVs induce a significant reduction of Caspase 3 activation in oxidative conditions. Moreover, we tested EV effects in the presence of MPP+ PD neurotoxin and measured the mitochondrial functionality by HRR. We observed an important recover of complex I activity in the presence of AS‐EVs. Interestingly, during ATP production, the reduction of O2 flux and ADP phosphorylation caused by MPP+ was specifically restored by VMB‐AS‐EVs.

Summary/Conclusion: For the first time, to our knowledge, our study shows the existence of specific brain region‐linked mechanism(s) for AS‐EV secretion with possible functional implications in the intercellular communication within the nigrostriatal area, in the context of PD.

OD03.03. Cell type‐specific extracellular vesicles define disease‐related protein networks associated with astrocyte activation in Alzheimer's disease

Yang YOU, Boston university

Satoshi Muraoka, Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine, Boston, MA, USA

Mark P. Jedrychowski, Department of Cell Biology, Harvard Medical School, Boston, MA, USA

Shuiqiao Hu, Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine, Boston, MA, USA

Amanda K. McQuade, Department of Neurobiology and Behavior, Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, CA, USA

Tracy Young‐Pearse, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA

Roshanak Aslebagh, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA

Mohammad Abdullah, Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine, Boston, MA, USA

Scott A. Shaffer, PhD, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA

Mathew Blurton‐Jones,Department of Neurobiology and Behavior, Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, CA, USA

Wayne W. Poon, Department of Neurobiology and Behavior, Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, CA, USA

Steven P. GygiDepartment of Cell Biology, Harvard Medical School, Boston, MA, USA

Tsuneya Ikezu, MD, PhD, Department of Pharmacology & Experimental Therapeutics, Center for Systems Neuroscience,Boston University School of Medicine; Department of Neuroscience, Mayo Clinic Florida

Introduction: Extracellular vesicles (EVs) have gathered great interest in studying neurodegenerative diseases including Alzheimer's disease (AD) with the capability of transferring pathogenic molecules. Almost every cell type in the central nervous system (CNS) including neurons and glia are known to shed EVs. Capturing cell type‐specific EVs from patient‐derived samples and profiling their contents by transcriptomic or proteomic analyses provide a useful method to study the pathophysiology of AD. Indeed, recent studies immunoprecipitated CNS‐specific EVs from AD samples via specific antibodies and obtained promising results. However, a consensus on cell type‐specific EV markers is lacking due to the limited evidence of cell type‐specific EV proteomic datasets from human samples. To address these concerns, we sought to define human CNS cell type‐specific EV protein signatures that could be employed for cell type EV isolation, and investigate their potential roles in AD pathology.

Methods: We performed combined label‐free and TMT‐labeling based quantitative mass‐spectrometry of EVs isolated from human induced pluripotent stem cells (hiPSCs) and AD brain tissues to conduct a comprehensive EV proteomics study. The weighted protein co‐expression network analysis (WGCNA) was used to generate AD‐associated and cell type‐specific EV protein modules. The disease‐related proteins were further validated by purifying cell type EVs from AD brain using an independent cohort.

Results: Novel cell type‐specific EV protein markers were identified from hiPSC‐derived excitatory neurons (e.g., NCAM1, ATP1A3), astrocytes (e.g., LRP1, ITGA6), microglia‐like cells (e.g., CD300A, ITGAM) and oligodendrocytes (e.g., LAMP2, FTH1). WGCNA of brain‐derived EV proteomics from 11 healthy controls, 8 mild cognitive impairment and 11 AD patients identified a protein module, which were enriched with astrocyte‐derived EV markers and plasma membrane molecules, was most significantly associated with AD pathology and cognitive function. These proteins are biased towards the EV profile of activated astrocytes and significantly involved in inflammatory processes. We validated the elevated expression of ITGB1, a hub protein within the module, in AD astrocyte‐specific EVs by using an independent cohort.

Summary/Conclusion: Our study presents novel human CNS cell type‐specific EV markers, highlights the key role of astrocyte‐derived EVs in AD pathogenesis, and provides a featured framework for future EV studies on neurodegenerative diseases.

OD03.04. Measuring biomarkers for Parkinson's Disease using neuronal‐origin extracellular vesicles

Joseph M. Blommer, National Institute on Aging

Toni Pitcher, New Zealand Brain Research Institute

Wassilios Meissner, Institute of Neurodegenerative Disorders, University Bordeaux

Maja Mustapic, PhD, National Institute on Aging

Tim Anderson, New Zealand Brain Research Institute

Dimitrios Kapogiannis, National Institute on Aging

Introduction: Parkinson's Disease (PD) pathogenesis involves intraneuronal a‐synuclein accumulation, but also impaired insulin sensitivity, which is characterized by imbalance in Tyr and Ser insulin receptor substrate‐1 (IRS‐1) phosphorylations. Besides motor symptoms, some PD patients develop mild cognitive impairment (PD‐MCI) or dementia (PD‐D), perhaps as a result of Alzheimer's disease (AD) pathology (amyloid‐beta plaques and hyperphosphorylated tau tangles) developing concurrently with a‐synuclein pathology. Given its importance for disease prognosis, there is a need to develop biomarkers for distinguishing PD with normal cognition (PD‐N) from PD‐MCI/D. Neuronal‐origin extracellular vesicles (NEVs) contain cell signaling and pathogenic proteins that may serve as biomarkers for Alzheimer's disease and PD.

Methods: From 104 PD‐N, 83 PD‐MCI, and 39 PD‐D patients and 48 age and sex‐matched Controls, we immunocaptured plasma NEVs using anti‐L1CAM antibody. We measured biomarkers by electrochemiluminescence immunoassays for: 1) PD and AD hallmark pathogenic proteins (a‐synuclein, amyloid‐beta42, total and p181‐Tau 2) pTyr20 and pSer312‐IRS‐1 and 3) synaptic proteins reflecting synaptic degeneration. Particle concentration was measured in all samples by Nanoparticle Tracking Analysis and immunoblots were used to characterize NEV preparations.

Results: A‐synuclein was lower in PD compared to Controls (p < 0.01) and stepwise in PD‐MCI and PD‐D compared to PD‐N (p < 0.01) and tended to decrease with increasing motor symptom severity by MDS‐UPDRS III (p = 0.06). Amyloid‐beta42 trended towards being higher in PD‐MCI and PD‐D groups compared to PD‐N (p = 0.06). pTau181 was higher in PD patients compared to Controls (p < 0.005) and in PD‐MCI compared to PD‐N (p < 0.05). Total Tau was not different between groups. pTyr20‐IRS‐1 was lower in PD compared to Controls and in PD‐MCI compared to PD‐N (p < 0.05) and decreased with increasing motor symptom severity by MDS‐UPDRS III (p < 0.01). The ratio pSer312/pTyr20 IRS‐1 (indicating insulin resistance) was higher in PD patients compared to Controls (p < 0.05) and in PD‐MCI and PD‐D compared to PD‐N (p < 0.05). Synaptophysin and synaptopodin were not different between groups.

Summary/Conclusion: PD patients with cognitive impairment exhibited lower NEV levels of a‐synuclein and pTyr20‐IRS‐1and higher levels of pTau181 than cognitively intact PD patients. Additionally, a‐synuclein and IRS‐1pTyr20 were associated with PD motor symptom severity. Plasma NEVs are a valuable tool for discovering biomarkers in PD and investigating aspects of disease progression.

OD03.05. Flow cytometry detection of membrane nanoparticles with size and cargo characteristics of spontaneously released EVs from brain

Carlos J. Nogueras‐Ortiz, NIH/NIA/LCI

Christopher Dunn, NIH/NIA/LCI

Ana P. Amorim Gomes, University of Minho

Ioannis Sotiropoulus, University of Minho

Dimitrios Kapogiannis, National Institute on Aging

Introduction: We sought to determine the relative abundance of tetraspanins known to be enriched in distinct EV subpopulations and hence used as EV markers, in intact spontaneously‐released EVs from perfused mouse brain tissue, using high sensitivity nanoscale flow cytometry analysis combining side scatter‐ and fluorescence‐based particle detection.

Methods: Side scatter of particles isolated via ultracentrifugation (3,000g, then 100,000g) was detected using a 405 nm (violet) wavelength laser (vSSC) which has been demonstrated to improve EV resolution and to be a better indicator of particle size compared to 488 nm (blue) wavelength lasers.

Results: Simultaneous labelling of EVs with APC‐tagged anti‐CD9, CD63 and CD81 antibodies resulted in the detection of APC+ events with a vSSC range within that of fluorescent beads and fluorescent EVs isolated from YFP+ HEK cells, with sizes ranging from 100–1300 nm, whereas negative controls, including water, antibody‐alone and EVs labelled with isotype controls, showed low electronic noise and no signs of particle detection. APC+ nanoparticles were not detected after treatment with NP40 detergent, thus confirming their membrane composition.

To explore the relative abundance of tetraspanins, we assessed EV subpopulations positive for either CD9 or CD81, and both. First, we confirmed the capacity of our flow cytometry analysis to distinguish between single‐ and double‐positive events by comparing mixed EVs individually labelled with APC‐CD9 and PE‐CD81, and simultaneously labelled EVs. APC/PE double‐positive events were only detected when simultaneously labelling EVs, and not when individually labelled EVs were mixed prior to analysis, indicating the detection of double‐positive single nanoparticles in the absence of coincidental events due to swarming. Further analysis of simultaneously labelled EVs showed that 98% of APC‐CD9+ events are PE‐CD81+, whereas 42% of PE‐CD81+ events are APC‐CD9+.

Summary/Conclusion: These results concur with previous observations suggesting that CD81 is a specific marker of small EVs, whereas CD9 and CD63 are present in multiple EV subpopulations and validate the isolation of membrane nanoparticles with size and cargo characteristics of spontaneously released EVs from mouse brain tissue.

OD03.06. Plasma extracellular vesicle‐associated mitochondrial DNA declines with age

Nicole Noren Hooten, National Institute on Aging, National Institutes of Health

Stephanie Lazo, National Institute on Aging, National Institutes of Health

Jamal Green, National Institute on Aging, National Institutes of Health

Erez Eitan, National Institute on Aging, National Institutes of Health

Nicolle Mode, National Institute on Aging, National Institutes of Health

Qing‐Rong Liu, National Institute on Aging, National Institutes of Health

Alan Zonderman, National Institute on Aging, National Institutes of Health

Ngozi Ezike, National Institute on Aging, National Institutes of Health

Mark Mattson, National Institute on Aging, National Institutes of Health

Paritosh Ghosh,National Institute on Aging, National Institutes of Health

Michele Evans, National Institute on Aging, National Institutes of Health

Introduction: Aging is associated with the progressive decline in organ and tissue function over the lifetime leading to age‐associated diseases. Mitochondrial dysfunction is a factor that drives the aging process. Cellular mitochondrial DNA (mtDNA) can be released outside of the cell as circulating cell‐free mtDNA (ccf‐mtDNA) and can be measured from blood. Higher levels of ccf‐mtDNA have been detected in cancer and inflammatory diseases, suggesting that they may be indicators of health and disease. Mitochondrial components may be encapsulated in EVs, yet little is known about whether ccf‐mtDNA can be detected in EVs from the circulation and whether there are changes in ccf‐mtDNA in the context of normal physiological processes such as aging.

Methods: Here we examined ccf‐mtDNA in plasma‐derived EVs from a cross‐sectional and longitudinal cohort of individuals across the lifespan. DNA was isolated from plasma EVs and the EV‐depleted fractions and analyzed by quantitative real‐time PCR using four primer sets that were designed against different regions of the mitochondrial genome. EVs from young and old individuals were used to treat cells in vitro to determine the effects on mitochondrial energetics.

Results: We report that plasma‐derived ccf‐mtDNA can be encapsulated in EVs. Furthermore, we examined EV‐mtDNA levels across the lifespan and found that EV‐mtDNA levels significantly decline with age in both our cross‐sectional and longitudinal analyses. We tested whether EV‐age altered mitochondrial function. Basal and maximal respiration were higher in cells treated with young EVs compared to old EVs.

Summary/Conclusion: Our results indicate that plasma EVs can carry ccf‐mtDNA as cargo. Furthermore, we found that plasma EV‐derived mtDNA declines with advancing age, which may impact cellular mitochondrial function. These data shed new light on the relationship between EVs, EV cargo and age that may help guide the usage of EVs and their content as biomarkers of health.

OD03.07. ESC‐derived sEVs Rejuvenate Aging Hippocampal NSCs by Transferring SMADs to Regulate the MYT1‐Egln3‐Sirt1 Axis

Yuguo Xia, Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Zhifeng Deng, Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Guowen Hu, Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Yuguo Xia, Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Qing Li, Institute of Microsurgery on Extremities, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Yang Wang, Institute of Microsurgery on Extremities, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Zhifeng Deng, Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Introduction: Tissue stem cell senescence leads to stem cell exhaustion, which results in tissue homeostasis imbalance and a decline in regeneration capacity. However, whether neural stem cell (NSC) senescence occurs and causes neurogenesis reduction during aging is unknown. In this study, we aimed to investigate NSC senescence during aging and the effect of embryonic stem cell‐derived small extracellular vesicles (ESC‐sEVs) on rejuvenating NSC senescence as well as the underlying mechanism.

Methods: In this study, mice at different ages were used to detect age‐related hippocampal NSC (H‐NSC) senescence, as well as the function and mechanism of ESC‐sEVs in rejuvenating H‐NSC senescence.

Results: We found a progressive cognitive impairment, as well as age‐related H‐NSC senescence, in mice. ESC‐sEVs treatment significantly alleviated H‐NSC senescence, recovered compromised self‐renewal and neurogenesis capacities, and reversed cognitive impairment. Transcriptome analysis revealed that myelin transcription factor 1 (MYT1) is downregulated in senescent H‐NSCs but upregulated by ESC‐sEVs treatment. In addition, knockdown of MYT1 in young H‐NSCs accelerated age‐related phenotypes and impaired proliferation and differentiation capacities. Mechanistically, ESC‐sEVs rejuvenated senescent H‐NSCs partly by transferring SMAD family members 4 (SMAD4) and 5 (SMAD5) to activate MYT1, which downregulated egl‐9 family hypoxia‐inducible factor 3 (Egln3), followed by activation of hypoxia‐inducible factor 2 subunit a (HIF‐2a), nicotinamide phosphoribosyltransferase (NAMPT), and sirtuin 1 (Sirt1) successively.

Summary/Conclusion: Taken together, our results indicated that H‐NSC senescence caused cellular exhaustion, neurogenesis reduction, and cognitive impairment during aging, which can be reversed by ESC‐sEVs. Thus, ESC‐sEVs may be promising therapeutic candidates for age‐related diseases.

OD04.01. Stress‐induced extracellular vesicles enriched in Rab11a‐exosomes promote cetuximab resistance in colorectal cancer cells through activation of EGFR signalling

John D. Mason, University of Oxford

Ewan Marks, Department of Physiology, Anatomy & Genetics, University of Oxford

Shih‐Jung Fan, MD PhD, Department of Physiology, Anatomy & Genetics, University of Oxford

Clive Wilson, Department of Physiology, Anatomy & Genetics, University of Oxford

Freddie Hamdy, Nuffield Department of Surgical Sciences, University of Oxford

Adrian Harris, Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford

Chris Cunningham, Nuffield Department of Surgical Sciences, University of Oxford

Deborah C I Goberdhan, PhD, Department of Physiology, Anatomy & Genetics, University of Oxford

Introduction: We recently demonstrated that metabolic stress leads to the preferential release of extracellular vesicles (EVs) containing exosomes made in Rab11a‐labelled recycling endosomes from a panel of colorectal cancer cell lines. These vesicles carry increased levels of Amphiregulin (AREG), which promote EGFR‐ERK dependent growth in recipient cells. Here, we test whether these properties are transferable to other colorectal cancer cell lines and their role in promoting drug resistance in KRAS‐wild type colorectal cancer cells.

Methods: EVs were isolated using size exclusion chromatography from HCT116 cells (mutant KRAS) grown under glutamine‐replete and ‐depleted conditions, and their effect on the growth of Caco2 recipient cells (KRAS¬‐wild type) was measured. In addition, we tested whether the inhibitory effects of the clinically used anti‐EGFR antibody, cetuximab, and an intracellular EGFR kinase inhibitor, could be reversed by co‐treatment with switched EVs.

Results: Switched EVs isolated from HCT116 secreting cells strongly promoted AREG‐dependent growth of Caco2 recipient cells. Caco2, but not HCT116, cell growth was inhibited by increasing doses of cetuximab which could be partially reversed by co‐treatment of switched EVs. Consistent, with this being an AREG‐dependent mechanism, this property was lost by treating EVs with an AREG neutralising antibody. Furthermore, switched EVs were unable to rescue Caco2 cells treated with downstream EGFR kinase domain inhibitors, providing evidence that the EV‐induced drug resistance is mediated by binding of EV‐associated AREG to the EGFR, despite the low levels of AREG on these vesicles.

Summary/Conclusion: We conclude that switched EVs are important mediators of colorectal cancer cell growth and also cetuximab resistance via a competition or displacement mechanism, and could serve as useful novel biomarkers to predict response to treatment.

OD04.02. Altered cargo of EVs from mTORC1‐driven tumours enhances pro‐tumoral signalling in recipient fibroblasts of the tumour microenvironment

Muireann Ní Bhaoighill, Cardiff University

Andrew R. Tee, Cardiff University

Jason P. Webber, Swansea University

Elaine Dunlop, Cardiff University

Introduction: Loss‐of‐function of TSC1/2 growth suppressor genes enables hyperactivation of mammalian Target Of Rapamycin Complex 1 (mTORC1), which causes tumour growth. Subsequent inhibition of mTORC1 in cancer has limited impact, suggesting that feedback loops or other signalling mechanisms must facilitate tumour growth. We have previously shown that tumour‐derived extracellular vesicles (EVs) can facilitate tumour growth by activating stromal cells in the prostate microenvironment. However, little is known about how mTORC1‐active cells interact with their microenvironment, and the pathogenic role of EVs from these cells is unclear. Therefore, we investigated the characteristics and tumour‐promoting capacity of EVs secreted from mTORC1‐active tumour cells.

Methods: We explored characteristics, cargo, and functionality of EVs from TSC2‐expressing (control) and TSC2‐deficient/mTORC1‐active (disease) cells. EVs were isolated by ultracentrifugation‐based floatation within 30% sucrose, and characterised according to MISEV guidelines. We conducted a comprehensive screen of mRNA and protein cargo, with selected targets validated by qPCR and ELISA. EV‐mediated signalling activation in stromal fibroblasts was assessed and functional assays were used to determine EV contribution to disease phenotypes.

Results: We show increased EV secretion from mTORC1‐active cells compared to control cells. EVs from mTORC1‐active cells have distinct transcriptomic and proteomic profiles, enriched for cancer‐ and mTORC1 signalling‐associated markers. We show that such EVs can mediate tumour‐promoting signalling downstream of mTORC1 in recipient fibroblasts.

Summary/Conclusion: EVs from mTORC1‐active cells have altered characteristics and cargo, enabling these EVs to mediate signalling in the microenvironment to promote tumour growth. This furthers our understanding of mTORC1‐driven tumour development and reveals candidate biomarkers.

OD04.03. Exploring the role of extracellular vesicles in the cross‐talk between adipocytes and prostate cancer cells

Fabrizio Fontana, University of Milan

Monica Marzagalli, University of Milan

Michela Raimondi, University of Milan

Emanuela Carollo, Department of Biological and Medical Sciences, Oxford Brookes University

Patrizia Sartori, University of Milan

Patrizia Procacci, University of Milan

David R F. Carter, Department of Biological and Medical Sciences, Oxford Brookes University

Patrizia Limonta, University of Milan

Introduction: It is known that an association exists between obesity and risk of prostate cancer (PCa). A crosstalk between adipocytes and PCa has been demonstrated; however, the study of this dialog has been limited to metabolites and adipokines, despite emerging evidence points to a key role of extracellular vesicles (EVs) in the control of tumor progression.

Methods: After isolation by SEC, EVs were characterized by NTA, TEM and Western blot analysis (TSG101, Hsc70, Alix, calnexin and cytochrome c). In PC3 and DU145 PCa cells, the effect of 3T3‐L1 adipocyte conditioned media and EVs on cell proliferation was evaluated by Trypan blue exclusion assay; AKT phosphorylation was analyzed by Western Blot. After adipocyte media/EV conditioning, PC3 and DU145 metastatic potential was assessed by scratch test and Boyden chamber assay, and matrix metalloproteinase and epithelial‐to‐mesenchymal transition marker levels were analyzed by Western Blot, while changes in mitochondrial activity, ATP synthesis, lipid content and glucose consumption were assessed by flowcytometry. The effect of adipocyte conditioned media/EVs on PCa cell resistance to docetaxel was evaluated by Trypan blue exclusion assay and annexin V/PI assay; Western Blot analysis of caspase 3 and PARP levels as well as of CD44 expression was also performed.

Results: We demonstrated that 3T3‐L1 adipocyte conditioned media can affect PC3 and DU145 cell features, inducing increased proliferation, associated with AKT phosphorylation, and invasion, correlated with MMP2 and 9 activation, E/N‐cadherin switch and Snail upregulation. Moreover, PCa cells were found to accumulate lipid droplets and, more importantly, to undergo a neuroendocrine differentiation, accompanied by CD44 enhanced expression and docetaxel resistance. Notably, these results were confirmed in 3T3‐L1 EV‐treated PC3 and DU145 cells, where an increase in the glucose consumption, mitochondrial activity, ATP production and ROS generation was also observed, suggesting that adipocyte EVs can reprogram PCa metabolism.

Summary/Conclusion: These results highlight that an EV‐mediated crosstalk exists between adipocytes and PCa, driving tumor aggressiveness. Further studies will be performed to identify the adipocyte EV molecular cargo responsible for the modulation of this dialog.

OD04.04. Intra‐tumoral microniche critically regulates extracellular vesicle release in lung adenocarcinoma

Gyöngyvér Orsolya Sándor, Semmelweis University, Department of Genetics, Cell and Immunobiology

András Áron Á Soós, Semmelweis University, Department of Genetics, Cell‐ and Immunobiology

Judit Moldvay, Department of Tumor Biology, National Korányi Institute of Pulmonology, Semmelweis University

Zoltán Wiener, Semmelweis University, Department of Genetics, Cell and Immunobiology, Budapest, Hungary

Introduction: Lung adenocarcinoma (LUAD), one subtype of lung cancer, is a frequent disease with a poor survival. Wnt production by some tumor cells establishes a special microenvironment for highly proliferating LUAD cells in mouse models and this micro‐niche may critically influence the clinical outcome. The 3D organoid technology maintains the heterogeneity of in vivo epithelial tissues and has proved to be so far the best ex vivo model of human cancers. In our studies we set out i) to prove the presence of this micro‐niche in human LUAD and ii) to study its importance in EV secretion.

Methods: We used organoids of different origin. The Medical Research Council of Hungary approved the experiments and informed consent was obtained from patients. EVs were detected by antibody‐coated beads, NTA and TEM. Intra‐tumor heterogeneity was studied by RT‐qPCR and immunostaining.

Results: We found that only a subpopulation of mouse lung, normal lung bronchiolar (NL) and LUAD organoid cells produced active Wnt with a partial overlap with proliferating cells, thus, providing evidence for the presence of a Wnt producing microniche both in the normal lung and in human LUAD. Inhibiting Wnt secretion in NL or LUAD organoids critically changed not only cell proliferation, but EV secretion as well. In addition, fibroblast‐derived EVs contributed to the establishment of this intra‐tumor microniche via EVs.

Summary/Conclusion: We show here the presence of lung cancer cell subpopulations with different EV release. Thus, our findings may be of high importance when considering EVs as diagnostic tools.

OD04.05. Extracellular vesicles from retinal pigment epithelial cells expressing misfolded proteins induce epithelial‐mesenchymal transition in recipient cells

Mi Zhou, Department of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, USA.

Yuanjun Zhao, Department of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, USA.

Sarah Weber, Department of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, USA.

Han Chen, TEM Core Facility, Penn State College of Medicine, Hershey, PA, USA.

Alistair Barber, Department of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, USA.

Stephanie Grillo, Department of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, USA.

Jeffrey Sundstrom, Department of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, USA.

Introduction: Previous studies in our lab found that the expression of the misfolded protein, R345W‐Fibulin‐3, induces retinal pigment epithelial (RPE) cells to undergo epithelial‐mesenchymal transition (EMT). The purpose of the current study was to investigate the size, cargo and function of extracellular vesicles (EVs) derived from RPE cells expressing the R345W‐Fibulin‐3 mutation, and to determine the role of these EVs in regulating RPE cell dysfunction.

Methods: Transmission electron microscopy (TEM) and cryogenic electron microscopy (cryo‐EM) were performed to study EV morphology. The amount and size distribution of EVs were determined by Nanoparticle Tracking Analysis (NTA). EV protein concentrations were quantified using the DCTM Protein Assay (Bio‐Rad). EV cargo were analyzed by unbiased proteomics using LC‐MS/MS with subsequent pathway analysis (Advaita). The EV‐associated transforming growth factor beta 1 (TGF‐β1) protein was measured by enzyme‐linked immunosorbent assay (ELISA). The migration ability of ARPE‐19 cells, in the absence and presence of mutant EVs, was evaluated by using scratch assays.

Results: TEM imaging revealed concave‐appearing vesicles, while cryo‐EM imaging showed spherical vesicles with two subpopulations of EVs: a small group with diameters around 30nm and a large group with diameters around 100nm. Imaging also revealed a greater number of small EVs (∼30 nm) in the mutant group compared to the WT group. This result was further confirmed by NTA showing that, in the mutant group, the particle size distributions were smaller than those of the WT EVs. The protein concentration per EV in the mutant group was not significantly different from that of the WT group. Proteomics identified critical members of sonic hedgehog (SHH) signaling and ciliary tip components in the EVs derived from WT ARPE‐19 cells, whereas EVs derived from mutant ARPE19 cells contained EMT mediators, including TGF‐β‐induced protein (TGFBI), vimentin, and mothers against decapentaplegic homolog 4 (SMAD4). ELISA confirmed the elevated TGF‐β1 associated with mutant EVs compared to WT EVs. Critically, EV transplant studies showed that treatment of recipient cells with EVs derived from mutant cells was sufficient to increase migration and elevate EMT markers in RPE cells after scratch‐injury.

Summary/Conclusion: The protein cargo of EVs is determined by the phenotype of their parental cells. Expression of R345W‐Fibulin‐3 mutation also alters the size and autocrine function of EVs. Notably, EVs derived from RPE cells expressing R345W‐Fibulin‐3 are sufficient to induce EMT in wild‐type RPE cells.

OD04.06. Tumors regulate their nutrient demand in response to hypoxia via autocrine EV signaling

Marijke I. Zonneveld, Postdoctoral researcher, GROW‐School for Oncology and Developmental Biology, Maastricht University, The Netherlands

Joël E.J. Beaumont, Department of Radiotherapy, GROW‐School for Oncology and Developmental Biology, Maastricht University

Tom G.H. Keulers, Department of Radiotherapy

Kim Savelkouls, Department of Radiotherapy, GROW‐School for Oncology and Developmental Biology, Maastricht University

Kasper W. Derks, Clinical genetics, Maastricht University medical center, The Netherlands

Marca H.M. H.M. Wauben, Department of Biomolecular Health Sciences, Utrecht University, The Netherlands

Kasper M.A. Rouschop, Department of Radiotherapy, GROW‐School for Oncology and Developmental Biology, Maastricht University

Introduction: Hypoxia is an important component of the tumor microenvironment (TME), associated with increased angiogenesis, migration and treatment resistance. In a process known as phenocopying, extracellular vesicles (EV) have been shown to transfer specific traits, such metastatic potential and drug resistance, from one cell to another. We were interested whether EV could phenocopy hypoxia‐tolerance between tumor cells.

Methods: HT‐29 and MDA‐MD‐231 cells were exposed to moderate (0.2% O2) or severe (0.02% O2) hypoxia. EV were isolated using a density gradient or size‐exclusion chromatography and characterized by high resolution flow cytometry (HR‐FC) and western blot. Autocrine effects of EV on hypoxia‐naïve tumor cells were assessed via next generation sequencing, qPCR, and glucose/lactate uptake assays. The effects of EV pre‐conditioning on cell viability was assessed through clonogenic survival assay.

Results: Hypoxia did not alter the total number of EV secreted, nor were gross protein and RNA content affected. However, HR‐FC showed different scatter patterns and immunoblotting showed distinct patterns for CD9, CD63 and Flotillin‐1, indicative of different subpopulations being secreted. Ingenuitiy pathway analysis of target cell transcriptomes indicated that their metabolism changed in response to EV. qPCR showed a slight increase in GLUT1 (glucose transporter) mRNA levels in target cells exposed to EV from normoxic and moderately hypoxic cells, but not from severely hypoxic cells. To assess whether this was functionally significant, glucose uptake and lactate secretion was assessed. Indeed, EV from normoxic and hypoxic EV stimulated additional uptake of glucose, while EV from severely hypoxic cells did not. Lactate secretion was elevated in all EV‐exposed cells compared to controls. The overall survival of these cells was not significantly impacted.

Summary/Conclusion: Hypoxia alters the composition of EV, leading to adaptations in GLUT1 transcription and glucose uptake in target cells. These results show that the tumor regulates nutrient demand in response to hypoxia via EV in an autocrine fashion.

OD04.07. Carcinoma‐associated fibroblasts secrete small extracellular vesicles carrying Periostin and LOX to promote collagen cross‐linking

Xue Liu, Department of Basic Science of Stomatology, Shanghai Stomatological Hospital, Fudan University, Shanghai, China

Tingjiao Liu, Department of Basic Science of Stomatology, Shanghai Stomatological Hospital, Fudan University, Shanghai, China

Introduction: Extracellular matrix (ECM) stiffening is an important feature of tumor stroma and is related to tumor invasion, metastasis, drug resistance and prognosis. Recently, small extracellular vesicles (sEVs) play an important role in mediating cell communication. However, the interaction between EVs and ECM is rarely reported. It attracts researchers’ attention whether EVs produced by carcinoma‐associated fibroblast (CAF) can mediate cell‐ECM communication. In this study, we investigated the role of the CAF sEVs promoting collagen cross‐linking.

Methods: sEVs were isolated from the conditioned medium of four CAFs and NF by differential ultracentrifugation. CAF sEVs was added to NF for 12 hours, ELISA determined the contents of Pyridinoline, dihydroxylysinonorleucine (DHLNL) and hydroxylysinonorleucine (HLNL) to evaluate collagen crosslinking in vitro.

Results: Transmission electron microscopy (TEM) photos showed CAF sEVs were typical cup‐shaped contains lipid bilayer membrane. Western blot (WB) showed that sEVs expressed typical exosome markers, such as CD63, CD9 and Hsp70. Nanoparticle tracking analysis (NTA) results showed that the average particle size distribution of CAF sEVs mostly diameter in 150 nm. Periostin (POSTN) expression in CAFs was significantly higher than NF, and OSCC cell lines were not expressed. Similar to the cellular results, POSTN was expressed in CAF sEVs, but not in NF and OSCC cell lines derived sEVs. POSTN was located on the surface of CAF sEVs. POSTN content increased with the increase of CAF sEVs concentration, and POSTN content decreased significantly after CAF sEVs was added with POSTN blocking antibody. In addition, POSTN combines with BMP1 and αLOX in CAF sEVs. POSTN and αLOX were co‐located on the surface of CAF sEVs membrane. Content of LOX in CAF sEVs increased with the concentration of CAF sEVs, and the content of LOX in CAF sEVs decreased significantly after blocking with LOX antibody. ELISA showed that the contents of Pyridinoline, DHLNL and HLNL in CAF sEVs was significantly higher than CAF sEVs + LOX Antibody and CAF sEVs + BAPN.

Summary/Conclusion: Our data indicate that POSTN may be a biomarker that distinguishes CAF sEVs from OSCC cell‐derived sEVs, POSTN is located on the membrane surface of CAF sEVs. CAF sEVs directly promote collagen crosslinking in vitro carrying POSTN‐BMP1‐αLOX with bioactive LOX.

OD04.08. The role of the autophagy adaptor protein PLEKHM1 and exosome release in the leukaemic bone marrow microenvironment

Christina Karantanou, Georg‐Speyer‐Haus, Institute for Tumor Biology and Experimental Therapy

Valentina R. Minciacchi, Georg‐Speyer‐Haus, Institute for Tumor Biology and Experimental Therapy

Rahul Kumar, Georg‐Speyer‐Haus, Institute for Tumor Biology and Experimental Therapy

Costanza Zanetti, Georg‐Speyer‐Haus, Institute for Tumor Biology and Experimental Therapy

Georg Tascher, Institute of Biochemistry II, Medical Faculty, Goethe‐University

Christian Münch, Institute of Biochemistry II, Medical Faculty, Goethe‐University

Tobias Tertel, Institute for Transfusion Medicine, University Hospital Essen, Germany

Bernd Giebel, Prof, Institute for Transfusion Medicine, University Hospital Essen, Germany

David G. McEwan, Cancer Research UK Beatson Institute

Ivan Dikic,Institute of Biochemistry II, Medical Faculty, Goethe‐University

Daniela S. Krause, Georg‐Speyer‐Haus, Institute for Tumor Biology and Experimental Therapy

Introduction: Bone marrow microenvironment (BMM)‐derived mesenchymal stromal cells (MSC) play a role in the maintenance of normal haematopoiesis and leukaemia progression. Pleckstrin homology domain‐containing family M member 1 (PLEKHM1) serves as a hub for the fusion of intracellular vesicles and their secretion. We hypothesized that PLEKHM1 may have a role in the regulation of extracellular vesicle (EV) biogenesis and that EV secreted by BMM‐derived MSC may modulate leukaemia progression.

Methods: Retroviral transduction/transplantation mouse model of B‐cell acute lymphoblastic leukaemia (B‐ALL); flow cytometry; imaging; differential centrifugation; AMNIS analysis; proteomic and Western blot (WB) analysis.

Results: Induction of B‐ALL in WT or Plekhm1 KO primary recipient or in WT secondary recipient mice revealed an increase of B‐ALL aggressiveness and B‐ALL‐initiating cell number and/or function, respectively. Small EV (sEV) from Plekhm1 KO MSC did not differ in number, but contained increased levels of syndecan‐binding protein 1 (syntenin) and its binding partner, syndecan‐1, compared to WT. Treatment of B‐ALL cells with KO sEV led to increased levels of syntenin and syndecan‐1 and higher pAkt, a sign of pro‐survival and growth in target cells. This effect was reversed upon inhibition of EV uptake. Furthermore, pretreatment of leukaemia cells with KO sEV increased their aggressiveness in vivo. TNFα secretion by leukaemia cells may lead to PLEKHM1 downregulation with simultaneous upregulation of syntenin and syndecan‐1 in MSC.

Summary/Conclusion: Our data suggest that reduced PLEKHM1 levels in MSC may affect the cargo of EV. Uptake of these EV by leukaemia cells may increase their aggressiveness, thereby, leading to the establishment of a novel mechanism for the delivery of pro‐survival signals to leukaemia cells, triggered by the release of TNFα from leukaemia cells.

OD05.01. Optimization of the pre‐treatment and evaluation of different separation techniques to isolate EV subpopulations from a complex biofluid like synovial fluid

Daniele D'Arrigo, Regenerative Medicine Technologies Lab, Ospedale Regionale di Lugano

Chiara Arrigoni, Regenerative Medicine Technologies Lab, Ospedale Regionale di Lugano, Lugano (CH)

Marco Vanoni, Department of Biotechnology and Bioscience, Università degli studi di Milano‐Bicocca, Piazza della Scienza 2, Milan (IT)

Christian Candrian, Regenerative Medicine Technologies Lab, Ospedale Regionale di Lugano, Lugano (CH)

Matteo Moretti, Regenerative Medicine Technologies Lab, Ospedale Regionale di Lugano, Lugano (CH)

Introduction: Extracellular vesicles (EVs) isolated with liquid biopsies from biofluids emerged as a promising diagnostic tool for several diseases, including the osteoarthritis. Most of the studies were focused on small size EVs, but evidences demonstrated that also larger EVs have a pathophysiological role. However, the development of effective isolation and separation methods to obtain different EV subpopulations is not straightforward. This is made even more difficult due to the complexity of biofluids like the synovial fluid (SF). Aiming at developing a protocol to isolate and separate EV subpopulations with different size from SF, we optimized its pre‐treatment and compared different separation protocols.

Methods: We collected SF from the knee joints of 5 end‐stage arthritic patients underwent joint replacement. To optimize the pretreatment, we evaluated the effect of hyaluronidase digestion. Then, to remove from the SF the cell contaminants without affecting the yield of the EVs, we evaluated the effect of different centrifugation regimens on the SF samples. Finally, we tested and compared the effectiveness of different methods, such as ultracentrifugation, size‐exclusion chromatography also with HPLC and others, to isolate and separate different‐sized EV subpopulations.

Results: The hyaluronidase digestion decreased SF viscosity, increasing the total number of EVs isolated with ultracentrifugation by about 40%. We also found the best centrifugation regimen to remove the highest quantity of cell contaminants while minimizing the loss of larger EVs. On the other hand, the isolation techniques differed in the capability to separate the different‐sized EV subpopulations. Finally, we characterized the EVs belonging to each subpopulation by different methods among which light scattering, western blot (e.g. CD63, TSG101, Albumin, and others) and protein content quantification.

Summary/Conclusion: We optimized the pre‐treatment of the SF to facilitate the EV isolation process. We also demonstrated that various separation methods have a different capability in isolating EV subpopulations with different size. This could be useful for future studies aiming to isolate different‐sized EV subpopulations from complex biofluids like SF.

OD05.02. DNA‐directed immobilization of antibodies and its application in extracellular vesicles purification and fabrication of EV‐based diagnostics

Dario Brambilla, CNR ‐ SCITEC

Laura Sola, National Research Council of Italy ‐ Institute of Chemical Sciences and Technologies (CNR ‐ SCITEC)

Marcella Chiari, National Research Council of Italy ‐ Institute of Chemical Sciences and Technologies (CNR ‐ SCITEC)

Introduction: Extracellular vesicles (EVs) are a powerful source of novel biomarkers in the diagnosis and prognosis of disease. In spite of their potential in human diagnostics, the choice of the most appropriate purification and characterization strategies is often troublesome. Immunoaffinity approaches offer and unmatched selectivity allowing the analysis of specific subpopulation of EVs. Unfortunately strategies involving antibodies suffer from disadvantages that limit their application. We present DNA‐directed immobilization (DDI) of antibodies to overcome some of the limitations. The use of DDI in immunoaffinity separation enhances the antibody affinity towards the target. We have exploited DDI in the immunoaffinity separation of EVs to enable the recovery of intact vesicles. In the proposed approach EVs are immunocaptured on magnetic beads and finally released using enzymatic cleavage of DNA linker mediated by DNase I.

Methods: Antibodies were tagged with different sequences of ssDNA exploting strain promoted azide‐alkyne cycloaddition. DNA‐antibody conjugates were used to generate antibody mciroarrays via DDI. The microarrays were used to characterize EVs derived by HEK‐293 cell culture media. Magnetic beads, coated with streptavidin, were also functionlized with DNA‐directed antibodies, incubated with EVs for 2.5 h at room temperature to capture EVs, and then treated with DNAse I for 1 h at 37°C. The released EVs were analyzed by Nanoparticle Tracking Analysis, nanoFlow Cytometry and TEM microscopy.

Results: Antibodies immobilized via DDI showed a greater affinity in comparison with antibodies conventionally immobilized on sensors, and this feature was exploited to develop an analytical platform and an immunoaffinity separation kit. The microarray sensor showed consistency with commercial kits, while DDI‐based immunoaffinity experiments allowed the separation of intact EVs, as confirmed by NTA, nanoFCM and TEM analysis.

Summary/Conclusion: We present an unprecedented application of DNA‐directed immobilization of antibodies for EVs separation and characterization. DDI‐based tools potentially pave the way towards the implementation of EV analysis in diagnostics.

OD05.03. Assessing the biomolecular landscape and dynamics of systemically circulating lipid‐carrying particles

Cláudio A. Pinheiro, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Cancer Research Institute Ghent, Ghent, Belgium

Glenn Vergauwen, Department of Gynecology, Ghent University Hospital, Ghent, Belgium

Joeri Tulkens, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium

Cláudio A. Pinheiro, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Cancer Research Institute Ghent, Ghent, Belgium

Francisco Avila Cobos, Cancer Research Institute Ghent, OncoRNALab, Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

Sándor Dedeyne, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Cancer Research Institute Ghent, Ghent, Belgium

Marie‐Angélique De Scheerder, Department of General Internal Medicine, Ghent University Hospital, Ghent, Belgium

Linos Vandekerckhove, Department of General Internal Medicine, Ghent University Hospital, Ghent, Belgium

Francis Impens, VIB Center for Medical Biotechnology, Department of Biomolecular Medicine, VIB Proteomics Core, Ghent, Belgium

Ilkka Miinalainen, Biocenter Oulu, University of Oulu, Oulu, Finland

Geert Braems, Cancer Research Institute Ghent, OncoRNALab, Department of Biomolecular Medicine, Ghent University, Department of Gynecology, Ghent University Hospital, Ghent, Belgium

Kris GevaertVIB Center for Medical Biotechnology, Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

Pieter Mestdagh, Cancer Research Institute Ghent, OncoRNALab, Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

Jo Vandesompele, Cancer Research Institute Ghent, OncoRNALab, Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

Hannelore Denys, Department of Medical Oncology, Ghent University Hospital, Ghent, Belgium

Olivier De Wever, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium

An Hendrix, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium

Introduction: Separating lipid‐carrying particles from blood is challenging and complicates the biological understanding and biomarker development of extracellular vesicles (EV) and lipoprotein particles (LPP). In this study, we fractionate blood plasma samples (n = 36) with the aim to assess the biomolecular composition and dynamics of EV and LPP in multiple disease conditions.

Methods: Size‐exclusion chromatography (SEC) followed by density gradient (DG) centrifugation fractionates blood plasma in two dimensions, namely size and density. EV and LPP extracts are characterized by EM, western blot, ELISA, NTA, LC‐MS/MS and small RNAseq. To validate the repeatability of the protocol, we analyze pooled blood plasma from healthy donors and breast cancer patients. We investigate longitudinal samples from ovarian cancer patients and HIV patients to test the variability of EV cargo composition during treatment.

Results: SEC prepares crude extracts from blood plasma retaining 1.4%, 9.67% and 73.0% of APOA1‐containing LPP, APOB‐containing LPP and CD9‐containing EV. DG centrifugation further fractionates crude extracts with high specificity into LPP and EV extracts. LPP extracts (1.04 " 1.07 g/mL) retain 85.2% and 95.4% of respectively APOA1 and APOB‐containing LPP while EV extracts (1.09‐1.10 g/mL) hold 30% of EV. Multi‐omics analysis of technical replicates confirms high methodological repeatability. Differential analysis of the protein composition of EV versus LPP extracts identifies the compositional nature of the EV protein corona and provides a catalog of proteins associated with systemically circulating EV versus LPP. Finally, we reveal that EV carry a unique, dynamic, context‐dependent protein composition, and miRNA and tRNA profile, which are not directly measurable in LPP extracts and total blood plasma, respectively.

Summary/Conclusion: The implementation of blood plasma fractionation substantially advances the biological understanding and biomarker development of systemically circulating lipid‐carrying particles.

OD05.04. Automated liquid handling for highly specific and reproducible density‐based separation of extracellular vesicles from human body fluids

Sofie Van Dorpe, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium

Lien Lippens, PhD studen, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium

Robin Boiy, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium

Hannelore Denys, Department of Medical Oncology, Ghent University Hospital, Ghent, Belgium

Olivier De Wever, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium

An Hendrix, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium

Introduction: Extracellular vesicles (EV) in body fluids are extensively studied as potential biomarkers for numerous diseases. Major impediments of EV‐based biomarker discovery include the specificity and reproducibility of EV sample preparation. To tackle this, we present an automated liquid handling workstation for the density‐based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers.

Methods: Variability in density‐based EV separation using manual versus automated liquid handling is first evaluated by spiking PBS with trackable recombinant extracellular vesicles (rEV) followed by the quantification of rEV recovery efficiency using fluorescent NTA and ELISA (Geeurickx etal., Nature Comm, 2019). Next, EV are separated from blood plasma and urine through the orthogonal implementation of size‐exclusion chromatography and manual or automated density‐gradient centrifugation as previously described (Dhondt etal., STAR Protoc, 2020; Tulkens etal., Nature Protoc, 2020). Variability, EV yield and purity are assessed by MS‐based proteomics and transmission electron microscopy.

Results: Automated versus manual liquid handling significantly reduces variability in rEV recovery after density‐based separation (CVauto 11.6% vs CVman 26.4%), and significantly decreases variability in EV preparations obtained from blood plasma and urine. Indeed, the median CVauto for protein group quantification are between 17.2% (plasma) and 20.0% (urine), CVman,exp between 38.5% (plasma) and 22.6% (urine), and CVman,inexp between 43.2% (plasma) and 27.6% (urine). While retaining an equal EV yield compared to manual liquid handling, automation significantly diminishes the presence of contaminating abundant proteins in EV preparations, including lipoproteins in plasma (2‐fold decrease in ApoB) and uromodulin in urine (2.6‐fold decrease in THP).

Summary/Conclusion: Automated liquid handling ensures EV separation from body fluids with high reproducibility and specificity.

OD05.05. Capillary‐channeled polymer (C‐CP) fiber solid‐phase extraction micropipette tips for the isolation of extracellular vesicles (EVs) from complex biofluid matrices

Kaylan K. Jackson, Clemson University

Rhonda R. Powell, Clemson University

Terri F. Bruce, MDPhD, Clemson University

R. Kenneth Marcus, FRSC, FAAAS, FSAS, FNAI, Clemson University

Introduction: Available extracellular vesicle (EV) isolation methods are time‐consuming, costly, and result in low recoveries and purity. Typically, EVs are contaminated with protein/lipoprotein aggregates. This creates difficulties in the quantification and characterization of recovered EVs, hindering fundamental research and downstream applications. A method capable of producing pure EVs, repeatedly, across diverse size scales is of critical importance. Capillary‐channeled polymer (C‐CP) fiber micropipette tips are employed here in a solid‐phase extraction (SPE) workflow for the efficient isolation of EVs.

Methods: Polyester (PET) C‐CP fibers are employed here in a spin‐down micropipette tip format for the SPE of EVs, allowing processing using simple table‐top centrifugation of sample volumes down to 10s of microliters. Demonstration matrices have included urine, saliva, cervical mucus, blood serum and plasma, milk, and cell culture media. EV purity is assessed based on the removal of free lipoproteins and using an ELISA to apolipoprotein B100 and tetraspanin proteins (CD81, CD63). Electron and confocal microscopy confirm the presence of EVs, with concentration and size distributions determined via absorbance (scattering) detection and nanoparticle tracking analysis.

Results: Quantitative recoveries of up to 1E10 EVs are readily obtained from 100 μL of biofluids using the C‐CP fiber tip method. TEM analysis confirms the retention of the characteristic EV cup‐shape following isolation. The HIC tip isolation workflow removes up to 89% of biofluid‐originating proteins. A >60% reduction of spiked lipoproteins is demonstrated by ELISA detection of the ApoB‐100 LDL‐marker in the EV fractions, with MS proteomics suggesting even higher purities.

Summary/Conclusion: The C‐CP fiber tip EV isolation technique is beneficial in terms of time (< 15 min), cost (< $1 per tip), ease of use, and clinical tailor‐ability in terms of capture and labeling. The C‐CP tip workflow produces clean and concentrated EV recoveries. This new isolation method introduces a simple capture mode based on vesicle hydrophobicity, with EV imaging techniques performed directly on the fiber surface. The long‐term goal is to create an efficient, practical EV isolation method for fundamental and clinical applications.

OD05.06. A defected metal‐organic framework featuring cleavable lipid probe for efficient and non‐destructive isolation of extracellular vesicles

Weilun Pan, Nanfang hostipital

Bo Li, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University

Feng Jun Jie, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University

Introduction: Extracellular vesicles (EVs) are phospholipid bilayer surrounded particles ranging from 30 nm‐1 μm released by all types of cells in an evolutionally conserved manner. Increasing evidence suggests that EVs have potential as clinical biomarkers and therapeutic agents. However, because EVs with heterogeneous sizes are present in biological fluids such as plasma, serum, urine, saliva, and cell supernatant, in which also non‐EV biomolecules are abundant. Thus, it is urgent to develop an efficient platform for the isolation of high pure EVs.

Methods: Herein, we constructed a defected nanoscale Metal‐organic frameworks (MOF) UiO‐66‐NH2 as a supporter, a cleavable lipid probe PO43–Spacer‐DNA‐Cholesterol (PSDC) connected to the MOF as cpaturer to isolate EV. With the strong affinity between PO43‐ on PSDC and Zr(IV) in the MOF, the MOF@PSDC platform was constructed easily. Next, due to the high binding efficacy between cholesterol and phospholipid bilayer of EVs, a system of MOF@PSDC@EVs was developed to separate EVs at 12 800 g within 10 minutes ascribe to the high specific gravity of MOF. After trapping of EVs, the MOF@PSDC@EVs was further incubated with deoxyribonuclease I (DNase I) to hydrolyze DNA, resulting in detachment of EVs from the MOF. With the second centrifugation to remove the MOF, isolated EVs were obtained rapidly and efficiently.

Results: The MOF@PSDC was constructed successfully by the confirmation of a series of characterization. Model EVs derived from breast cancer cells MDA‐MB‐231 were applied for identifying of the feasibility of the MOF@PSDC in isolating EVs with high efficiency and purity. This MOF@PSDC platform was further used to isoalted EVs from plasmas samples from patients with breast cancer in different clincal stages, benign lesions and healthy individuals and sujected to the detection of three biomarkers glycipan‐1 (GPC‐1), CD63 and human epidermalgrowth factor receptor‐2 (HER‐2). The resultes indicated that by using the established platform MOF@PDC, EVs from plasmas could be isolated in a quick and high quality maner for downstream analysis.

Summary/Conclusion: In summary, a universal EVs isolation platform based on a defected MOF featuring cleavable lipid probe (MOF@PSDC) has been constructed successfully. Such a novel defected MOF platform featuring the cleavable lipid probe shows merits over existing EVs isolation methods in terms of its advantages that include fast, efficient, non‐destructive, and contamination‐free without requirement of the expensive equipment. It could be further studied to apply for harvesting EVs from complex biological fluids to facilitate EVs‐based research in clinical theranostics. Furthermore, this study also provides new insights into the design of functional materials for isolating subcellular structures or biomacromolecules.

OD06.01. NK‐cell derived EVs target and kill resistant cancer cell‐derived spheroids

Miriam Aarsund Larsen, University of Oslo

Marit Inngjerdingen, Oslo University Hospital

Introduction: Natural killer (NK) cells are innate lymphocytes that recognize and kill cancer cells. NK cell‐based therapies has shown promise for hematological tumors, but their use for solid cancers is hampered by their poor ability to infiltrate the tumor. EVs secreted from NK cells (NK cell‐derived EVs; NK‐EVs) are shown to contain the cytolytic material necessary for targeting and killing cancer cells, indicating that NK‐EVs may be ideal therapeutic agents.

Methods: EVs were isolated from primary NK cells or the NK cell lines NK92 or KHYG‐1 upon culture in either IL‐15 (resting) or a cocktail of IL‐12, IL‐15, and IL‐18 (activation). EVs were precipitated, and successful isolation confirmed by Western blotting, NTA, and electron microscopy. Phenotypic characterization was done by Western blotting and mass spectrometry. Function were tested in cultures and in spheroids from a total of seven different solid cancer cell lines (HCT116 and HCT 15 (colon), DU145 (prostate), OVCAR (ovarian), SKRB3 and T4D7 (breast), WM9 (melanoma)). Tumor growth and kinetics of apoptosis upon treatment with NK‐EV was obtained by continuous monitoring for 7 days using InCuCyte technology. Evaluation of EV penetration into spheroids was evaluated by histology.

Results: Apoptosis and tumor shrinkage was observed upon treatment with primary NK cell‐ or NK92‐derived EVs in colon, melanoma, prostate and ovarian spheroids, but not breast cancer‐derived. We found that activating NK cells with IL‐12, IL‐15, and IL‐18 generated EVs with enhanced killing capacity, although they produced similar amounts of EVs as resting cells. The enhanced capacity of IL‐12/15/18‐activated NK cell‐derived EVs was linked to enhanced protein levels of cytolytic proteins. We further observed differential expression of ligands for the activating receptors NKG2D by the cancer cell lines, that matched their susceptibility to NK‐EVs. The killing activity was conversely abrogated using an anti‐NKG2D antibody. Finally, we found that NK‐EVs were able to penetrate into the spheroid core.

Summary/Conclusion: We have in this study systematically analyzed NK‐EV targeting of cancer cells derived from solid tumors, and demonstrated potent killing activity of spheroids.

OD06.02. Separating tumor EV subtypes by Asymmetric Flow field flow fractionation (AF4) unravels differential functional capacities

Federico Cocozza, Institut Curie / INSERM U932

Lien Lippens, PhD studen, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium

Mabel Jouve, Institut Curie / CNRS UMR 3215

Aurélie Di Cicco, Institut Curie/UMR CNRS168

Bruno G. De Geest, Department of Pharmaceutics, Ghent University, Ottergemsesteenweg 460, 9000, Ghent, Belgium

Daniel Levy, Institut Curie/UMR CNRS168

An Hendrix, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium

Clotilde Thery, MD PhD, Institut Curie / INSERM U932

Mercedes Tkach, Institut Curie / INSERM U932

Introduction: Extracellular vesicles (EVs) represent a heterogenous population of vesicles of different sizes, densities and compositions, whose common and distinct functional properties are still ill defined. The asymmetric flow field‐flow fractionation (AF4) technology has recently been shown to efficiently separate EV subtypes based on their size, and the use of this technology led to the identification of a distinct very small nanoparticle, named exomere (Zhang etal., 2018, Nat Cell Biol 20: 332). The goal of our study was to establish the AF4 technology to separate tumor‐derived EV subtypes, and compare their efficiency to be captured by target phagocytic immune cells (dendritic cells).

Methods: Mouse mammary tumor E0771 cells were transfected with a general membrane‐binding tag (myr/palm) fused to mCherry to target the fluorescent protein towards lipid membranes and therefore a broad set of secreted EVs.

EVs obtained by differential ultracentrifugation (dUC) and AF4 fractionation were characterized by Cryo Electron Microscopy and Western Blot. Flow cytometry and immuno‐fluorescence microscopy were used to assess the uptake of mCherry by mouse dendritic cells.

Results: A protocol with a good resolution to separate EVs from 40nm to 180nm from the myr/palm ‐mCherry expressing cells was set. Western blot and electron microscopy confirmed the presence of exomeres and at least two types of EVs including one with viral particle features. mCherry was present together with other EV markers, in all the types of particles. Exomere markers recovery was sensitive to an ultracentrifugation washing step.

Our results show that all types of particles can be uptaken but with different efficacies.

Summary/Conclusion: The size distributions of EVs secreted by E0771 cells show the existence of two populations of sEVs (below and above 100nm in diameter) plus exomeres (smaller than 50nm), which can all be uptaken by dendritic cells. The functional consequences of this uptake (i.e. degradation or delivery to cytosol) must now be evaluated.

OD06.03. The αvβ6 Integrin Regulates IFIT3 Protein Levels in PrCa Cells and their EVs

Nicole M. Naranjo, Thomas Jefferson University

Israa Salem, Thomas Jefferson University

Shiv R. Krishn, Thomas Jefferson University

Larry Harshyne, Thomas Jefferson University

D. Craig Hooper, Thomas Jefferson University

Lucia R. R. Languino, Thomas Jefferson University

Introduction: The αvβ6 integrin (αvβ6), a transmembrane cell adhesion receptor, is a known biomarker of distinct cancers, including prostate cancer (PrCa). We have shown that transfer of αvβ6 via sEVs to recipient cells, including monocytes, affects their phenotype and function. Therefore, we aimed to evaluate if αvβ6 regulates the expression of specific downstream effectors of the adaptive immune response, specifically the Interferon Induced Proteins with Tetratricopeptide Repeats family (IFIT) in PrCa and their EVs.

Methods: To investigate αvβ6 effect on the IFIT protein expression in cells and/or EVs, we have isolated LEVs and sEVs derived from PrCa cells treated with siRNA to downregulate αvβ6 expression. We have characterized LEVs and sEVs by using immunoblotting analysis as well as assessed their size and concentration via Nanoparticle tracking analysis (NTA). Moreover, to isolate and characterize LEVs and sEVs devoid of αvβ6 or IFIT3, we have generated homozygous αvβ6 or IFIT3 knock out PC3 PrCa cells using CRISPR‐Cas9 strategies.

Results: Our analysis shows that among other downstream effectors, the IFIT3 protein, which is an antiviral protein induced by type I and II interferon pathways, is upregulated in both LEVs and sEVs derived from αvβ6 siRNA treated PrCa cells. Furthermore, IFIT3 protein expression is increased in LEVs and sEVs derived from CRISPR‐αvβ6 deleted PrCa cells compared to PrCa cells containing αvβ6. NTA analysis shows that the presence or absence of αvβ6 does not influence the size or concentration of LEVs and sEVs released by PrCa cells. Moreover, αvβ6 protein levels are not affected in LEVs or sEVs derived from CRISPR‐IFIT3 deleted PrCa cells.

Summary/Conclusion: Overall, these results show that the IFIT3 protein expression in PrCa and their EVs is regulated by the αvβ6 integrin. Furthermore, they suggest that transfer of PrCa EVs, enriched in IFIT3 and devoid of αvβ6, may affect recipient cell phenotype and functions.

OD06.04. Release of Immunosuppressive Tumor‐Derived Extracellular Vesicles Impairs with CD8+‐T cell Activity in Response to Radiotherapy

Yohan Kim, Mayo Clinic

Roxane R Lavoie, Mayo Clinic

Haidong Dong, Mayo Clinic

Sean Park, Mayo Clinic

Fabrice Lucien, MD PhD, Mayo Clinic

Introduction: Stereotactic ablative radiotherapy (SABR) is the effective treatment for prostate cancer (PCa) patients with few metastatic lesions (oligometastasis). SABR induces immunogenic cell deaths and elicits a systemic antitumor immune response at non‐irradiated distant metastatic sites (abscopal effect). Despite clinical observations of abscopal effect, cases remain scarce as most patients develop wide‐spread metastasis. There is an unmet need to elucidate the underlying mechanisms that impair with SABR‐mediated abscopal effect and develop more effective combination therapies. Extracellular vesicles (EVs) have emerged as key players of antitumor immunity locally and systemically. However, their functional roles in radiotherapy‐associated antitumor immune response have not been fully investigated yet.

Methods: To examine the cross‐talk between PCa‐derived EVs (PCEVs) and CD8+‐T cell in response to radiotherapy, nanoscale flow cytometry was used to analyze plasma samples from oligometastatic castration‐resistant prostate cancer (omCRPC) patients treated with SABR. Co‐culture of human CD8+‐T cells with PCEVs was used to examine the molecular and cellular mechanisms involved in EV‐mediated CD8+‐T cell activity. Finally, integrated transcriptomic and proteomic profiling was employed to identify molecular determinants responsible for the immunomodulatory activity of PCEVs.

Results: By using clinical samples, we have observed that SABR induces release of PCEVs into bloodstream and high PCEV levels were associated with higher risk of recurrence. High levels of PCEVs negatively correlated with levels of effector memory CD8+‐T cells. We also observed that EVs released from irradiated PC3 and DU145 cells inhibit CD8+‐T cell proliferation and cytotoxic function in vitro. Profiling of immunomodulatory cell‐surface proteins on PCa cells identified PD‐L1 and B7‐H3 as key players of EV‐mediated immunosuppression in response to radiotherapy. Blockade of PD‐L1 and B7‐H3+ EV release led to improved CD8+‐T cell function.

Summary/Conclusion: This study reveals a novel cellular mechanism that compromises radiation‐mediated antitumor immune response through the release immunosuppressive tumor‐derived EVs. Targeting these EVs in combination with SABR may represent a promising therapeutic approach for the treatment of omCRPC patients.

OD06.05. Identification of miRNAs released in vivo by extracellular vesicles upon BRAFV600E induction in thyrocytes; implication for immune cell recruitment

Ophélie Delcorte, DDUV institute‐ Cell Unit

Catherine Spourquet, DDUV Institute‐CELL Unit

Pascale Lemoine, DDUV Institute‐CELL Unit

Christophe E. Pierreux, DDUV Institute‐CELL Unit

Introduction: Papillary thyroid carcinoma (PTC) carrying BRAFV600E mutation is the most frequent subtype of thyroid cancers. Despite a very good prognosis in most cases, postsurgery recurrences and metastases occur in 10–15% of patients. Moreover, differential diagnosis between benign and malignant nodules is still challenging. Knowledge about extracellular vesicles (EVs) in PTC is rather weak when it could rise a double interest: a better understanding of the biology of PTC and its clinical behavior, and an improvement of differential diagnosis between thyroid cancer types.

The goal of this project is to (i) identify miRNAs actors and markers, released via EVs by the tumor, and (ii) decipher the mechanisms by which they impact on thyroid cancer and its microenvironment.

Methods: Using a mouse model in which BRAFV600E is selectively expressed in thyrocytes upon doxycycline injections, we developed a protocol to isolate control and PTC‐EVs from dissociated tissue by differential ultracentrifugations. Vesicles in the high‐speed pellet displayed a size and some specific markers that were consistent with exosomal characteristics. Sequencing was performed to identify miRNAs with a differential abundance in EVs isolated from control and BRAFV600E PTC tissue. In silico and in vitro analyses were carried out to elucidate their role within tumor microenvironment.

Results: We identified miRNAs that were progessively deregulated during PTC development in tissue and EVs. To study the role of those EV‐miRNAs, we focused on upregulated candidates, as those were the first deregulated and the most susceptible to be involved in intercellular communications within the tumour microenvironment. The in silico analysis of upregulated EV‐miRNAs and downregulated tissue‐mRNAs identified by mRNA sequencing was performed. It revealed a network organized around 6 miRNAs with many common mRNA targets, that were enriched in immune‐related pathways.

Summary/Conclusion: We provide a gradual tissue‐ and EV‐miRNAs profiling along BRAFV600E‐driven PTC development and thyrocyte dedifferentiation, and identify miRNAs candidates which could have an impact on tumorigenesis and communication within the tumor microenvironment, especially on the immune microenvironment.

OD06.06. Tumor secreted extracellular vesicles regulate T‐cell costimulation and can be engineered to induce tumor specific T‐cell responses

Subbaya Subramanian, Department of Surgery, University of Minnesota

Ce Yuan, Department of Surgery, University of Minnesota

Dechen Wangmo, Department of Surgery, University of Minnesota

Xianda Zhao, Department of Surgery, University of Minnesota

Xianda Zhao, Department of Surgery, University of Minnesota

Introduction: Colorectal Cancer (CRC) is a major cause of cancer‐related deaths world‐wide. Immune checkpoint blockade therapy (ICBT) is effective in 30–60% of the microsatellite instable‐High (MSI‐H) subtype. Unfortunately, most CRC patients (>85%) have microsatellite stable (MSS) tumors, that do not respond to ICBT. In this study, we aim to decipher the underlying tumor intrinsic mechanisms critical for improving immunotherapy in colorectal cancer.

Methods: We used human and mouse tumor samples, cell lines, and various syngeneic orthotopic mouse models of late‐stage CRC to define the effects of tumor cell‐secreted extracellular vesicles (EVs) on the antitumor immune response. T cell priming and functional assays were carried out using primary human CRC organoids and cell lines and preclinical mouse models.

Results: Our analyses of human CRC immune profiles and tumor‐immune cell interactions revealed that TEVs containing microRNA miR‐424 suppressed the CD28‐CD80/86 costimulatory pathway in tumor‐infiltrating T cells and dendritic cells, leading to immune checkpoint blockade therapies (ICBT) resistance. Modified TEVs with miR‐424 knocked down enhanced T‐cell mediated antitumor immune response in CRC tumor models and increased the ICBT response. Intravenous injections of modified TEVs induced tumor antigen‐specific immune responses and boosted the ICBT efficacy in CRC models that mimic aggressively progressing late‐stage disease.

Summary/Conclusion: Collectively, we demonstrate a critical role for TEVs in antitumor immune regulation and immunotherapy response, which could be developed as a novel treatment for immune checkpoint blockade therapy resistant colorectal cancer.

OD06.07. Metabolomic, proteomic, transcriptomic and fatty acid profiling of glioblastoma‐stem cells and their small extracellular vesicles

Tolga Lokumcu, DKFZ Heidelberg

Lisa Schlicker, KDFZ

Klinke Glynis Fiona, Centre for Organismal Studies Heidelberg

Frederik Bethke Bethke, DKFZ

Kendra Maass, DKFZ

Karsten Richter, DKFZ

Almut Schulze, DKFZ

Violaine Goidts, DKFZ

Introduction: Glioblastoma is the most common and aggressive brain tumor with the median survival is about 12–15 months after diagnosis. It is an extremely aggressive tumor showing high degree of intra‐ and inter‐tumoral heterogeneity and the exceedingly heterogeneous nature of glioblastoma creates a major challenge to implement better treatment strategies. It is now known that small sub‐population of cancer cells, known as glioblastoma stem‐like cells (GSCs), are responsible for the therapy resistance of glioblastoma, resulting in low overall survival in patients. Different population of glioblastoma cells have the ability to communicate with each other and with various different cell types in the microenvironment by exchanging proteins, nucleic acids, lipids and metabolites through extracellular vesicles. The extracellular vesicles taken up by neighboring tumor cells and normal cells in the microenvironment can change the phenotype of recipient cells, which results in increased angiogenesis, immune suppression, promoted cell invasion and metabolic deregulation. The goal of our work was to identify the factors that might be responsible for the glioblastoma subtype plasticity.

Methods: Proneural (PN) GSCs were treated with the different fractions of conditioned medium of mesenchymal (MES) GSCs and the changes in the expression of MES/PN markers (CD44 and CD133, respectively) were determined by flow cytometry. The metabolites and fatty acids of GSCs/small extracellular vesicles were identified by GC‐MS and the proteomic profiling of GSCs/sEV was assessed by LC‐MS. Finally, the RNA content of GSCs/sEV was detected by total RNA seq.

Results: PN cells increased the expression of mesenchymal marker CD44 when they were treated with the conditioned medium of MES cells. Whereas PN cells treated with 2.000g and 10.000g depleted conditioned medium increased the expression of CD44, as shown with complete CM transfer, cells treated with 100.000g depleted CM lost their high CD44‐expressing phenotype. Given that exosomes are depleted by 100.000g centrifugation, our data suggest that exosomes secreted by MES cells change the expression/abundance of CD44 in recipient cells. Importantly, metabolomic, proteomic, transcriptomic and fatty acid profiling of glioblastoma‐stem cells and their small extracellular vesicles showed that GSC‐derived sEVs harbor a diverse repertoire of biomolecules, which makes them critical mediators of glioblastoma heterogeneity and cell‐to‐cell communication.

Summary/Conclusion: Our data show that GSC‐derived sEVs carry various biomolecules including metabolites, proteins, RNAs and fatty acids, making them crucial biological structures for the cell‐to‐cell communication and the maintenance of heterogeneity in glioblastoma.

OD07.01. Extracellular vesicles rejuvenate aged tissues by activating the glutathione pathway

Juan Antonio Fafian‐Labora, Queen Mary University of London

Jorge Pascual‐Guerra, Hospital Ramon y Cajal

Marta Posada, Hospital Ramon y Cajal

Jesus Alarcon, Hospital Ramon y Cajal

Jose Antonio Rodríguez‐Navarro, Hospital Ramon y Cajal

Ana O'Loghlen, Queen Mary University of London

Introduction: Ageing is a major risk factor for many human diseases. It is a complex process that progressively compromises most of the biological functions of the organisms, resulting in an increased susceptibility to disease and death. It is characterised by different hallmarks, one of which is cellular senescence. In fact, senescent cells accumulate in different organs during ageing. Senescence is a cellular phenotype characterized by a stable cell cycle arrest and a particular secretome denominated senescence‐associated secretory phenotype (SASP). The SASP is compromised by soluble factors and extracellular vesicles (EV) but the role the latter play are understudied.

Methods: Functionality experiments were performed with extracellular vesicles of small size (sEV). sEV were isolated by either serial ultracentrifugation and/or size exclusion chromatography. A variety of technique involved to identify the activation of senescence or the presence of ageing‐related markers were used. In addition, different techniques involved in determining the activation of the glutathione pathway were also used both using cell culture models and tissues from old and young mice.

Results: Here, we have evaluated the role that EV of small size (sEV) play in ageing and cellular senescence. Previous data from our lab show that sEV from senescent cells are mediators of senescence in a non‐cell autonomous fashion. We further identified proteins within the interferon pathway as partial mediators of sEV‐mediated paracrine senescence. However, as during ageing, the organs are formed by a mixture of proliferating and senescent cells here we determined the influence of proliferating cells on senescent and ageing cells. Interestingly, we found that sEV from proliferating cells ameliorated several senescence and ageing features both in vivo and in vivo. Furthermore, we identified that sEV from proliferating cells have intrinsic glutathione‐S‐transferase (GST) activity due to their enrichment in the GST‐related protein, GSTM2. Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their GST and antioxidant capacity. Thus, sEVs from proliferating cells increase the levels of reduced glutathione and decrease oxidative stress including the GSTM2‐specific downstream signalling, lipid peroxidation, both in vivo and in vitro.

Summary/Conclusion: In conclusion, we found that sEV have an important role in the intercellular communication mediated through sEV during cellular senescence, ageing and rejuvenation processes respectively.

OD07.02. Clinical grade MSC‐EVs promote human cartilage recovery in vitro

Maria Elisabetta Federica Palamà, Department of Experimental Medicine (DIMES), University of Genoa, Italy

Simona Coco, IRCCS Policlinico San Martino, Genoa, Italy

Daniele Reverberi, U.O. Molecular Pathology, IRCCS Policlinico San Martino, Genoa, Italy

Georgina Margaret Shaw, Regenerative Medicine Institute (REMEDI), National University of Ireland Galway (NUI Galway), Galway, Ireland,

Dario Pisignano, Nanoscience Institute CNR‐NANO (NEST), Pisa, Italy

Katia Cortese, Department of Experimental Medicine (DIMES), University of Genoa, Italy

Frances Peter Barry, Regenerative Medicine Institute (REMEDI), National University of Ireland Galway (NUI Galway), Galway, Ireland

Mary Murphy, Regenerative Medicine Institute (REMEDI), National University of Ireland Galway (NUI Galway), Galway, Ireland

Chiara Gentili, Department of Experimental Medicine (DIMES), University of Genoa, Italy

Introduction: Osteoarthritis (OA) is a disabling joint disorder causing articular cartilage degeneration. Currently, treatments are mainly pain‐ and symptom‐modifying, rather than disease‐modifying. Human bone marrow stromal cells (hBMSCs) have emerged as a promising paracrine mechanism‐based approach for the treatment of OA.

Methods: We cultured hBMSCs in a novel xeno‐free culture system (XFS). We characterized extracellular vesicles (EVs) derived from hBMSCs grown in XFS compared to a conventional fetal bovine serum (FBS) culture system, in normoxic and hypoxic culture setting. We investigated also the therapeutic potential of EVs in an in vitro model of OA. Also, miRNA content of EVs in different culture setting was investigated, to select putative miRNA that could be involved in a biological function.

Results: The biological effects of XFS‐ and FBS‐cultured hBMSCs was tested on IL‐1α treated human articular chondrocytes (hACs), in an experiment designed to mimic the OA environment. We observed that hBMSC‐derived EVs counteract the inflammatory state of hACs, promoting the homeostasis maintenance. This effect was streghtened by XFS culture, both in normoxia and hypoxia. Analysis of miRNA content showed the upregulation in XFS‐hBMSC‐derived EVs of miRNA known to have a chondroprotective role, such as miR‐17, miR‐140, miR‐145, miR‐30a, miR‐29a, miR‐130a, miR‐199a. Interestingly, most of the miRNA found in our preparations seem to be involved in cartilage homeostasis and they affect TGF‐beta signaling.

Summary/Conclusion: In conclusion, the XFS medium was found to be suitable for isolation and expansion of hBMSCs, which secrete EVs with high therapeutic function. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum‐containing media and makes ready‐to‐use clinical therapies more accessible.

OD07.03. Mesenchymal stem cell extracellular vesicles as therapy for acute and chronic lung diseases: A systematic review and meta‐analysis

Alvin Tieu, Ottawa Hospital Research Institute

Kevin Hu, Ottawa Hospital Research Institute

Catherine Gnyra, Ottawa Hospital Research Institute

Joshua Montroy, MSc, Ottawa Hospital Research Institute

Dean Fergusson, PhD, Ottawa Hospital Research Institute

Duncan Stewart, MD, Ottawa Hospital Research Institute

David Allan, MD, Ottawa Hospital Research Institute

Manoj Lalu, MD, MDPhD, Ottawa Hospital Research Institute

Introduction: Mesenchymal stem cell EVs (MSC‐EVs) are reported to reduce inflammation and improve organ function in preclinical lung diseases. Prior to translation, an objective analysis of all available data is needed. Moreover, identifying EV characteristics associated with greater efficacy may help refine EV therapy. This systematic review aims to determine the efficacy of MSC‐EVs for lung diseases.

Methods: A protocol was registered a priori (PROSPERO CRD42020145334). MEDLINE and Embase were searched for in vivo studies of MSC‐EVs as therapy for acute lung injury (ALI), bronchopulmonary dysplasia (BPD) and pulmonary arterial hypertension (PAH). A random effects meta‐analysis was conducted to measure efficacy. Subgroup analysis identified EV methods/characteristics associated with improved efficacy. Data is presented as standardized mean differences (SMD) or risk ratios (RR) with 95% confidence intervals (CI).

Results: After screening 1167 reports, 52 studies met our eligibility criteria. For ALI, MSC‐EVs markedly reduced lung injury (SMD 4.33, CI 2.92‐5.73), vascular permeability (SMD 2.43, CI 1.82‐3.05), and mortality (RR 0.39, CI 0.22‐0.68). No differences were seen between MSC tissue sources, immunocompatibility or isolation techniques. However, small EVs were more effective than large EVs. For BPD, alveolarization was improved by EVs (SMD 1.45, CI 0.82‐2.08) with small EVs being more consistently beneficial then small/large EVs. In PAH, right ventricular systolic pressure (SMD 4.16, CI 2.64‐5.68) and hypertrophy (SMD 2.80, CI 1.91‐3.68) were attenuated by EVs. Allogeneic EVs were more beneficial than xenogeneic. In both BPD and PAH studies, EVs from tangential flow filtration (TFF) displayed no efficacy, whereas ultracentrifugation alone or paired with other techniques resulted in improved therapeutic benefit.

Summary/Conclusion: All outcomes were significantly improved by MSC‐EVs demonstrating the potential of EV therapy for treating acute and chronic lung diseases. MSC tissue source or route of administration did not alter efficacy, whereas ultracentrifugation and small EVs were more consistently beneficial. More direct comparisons of isolation techniques and EV subtypes are needed to optimize EV therapy for clinical translation.

OD07.04. Honeybee Royal Jelly extracellular vesicles display promising antibacterial and pro‐regenerative properties for wound healing

Sebastian Aguayo, Pontificia Universidad Católica de Chile, Faculty of Medicine, School of Dentistry

Pamina Contreras, Clínica Alemana‐Universidad del Desarrollo, Facultad de Medicina, Centro de Medicina Regenerativa

Simón Alvarez, Clínica Alemana‐Universidad del Desarrollo, Facultad de Medicina, Centro de Medicina Regenerativa

Orlando Ramirez, Clínica Alemana‐Universidad del Desarrollo, Facultad de Medicina, Centro de Medicina Regenerativa

Christina M.A.P. Schuh, Clínica Alemana‐Universidad del Desarrollo, Facultad de Medicina, Centro de Medicina Regenerativa

Introduction: Honeybee Apis mellifera Royal Jelly (RJ), has been used in medicinal treatments by many cultures for centuries. RJ is a honeybee hypopharyngeal gland secretion known to be antibacterial and to exert beneficial effects on wound healing. However, the underlying mechanisms have not been fully elucidated yet. In recent years microvesicles have been identified as key players in cellular communication and are upcoming as promising therapeutic vehicles. Since glandular secretions present a rich source of active extracellular vesicles (EVs), we hypothesized that EVs are present in RJ and participate in its known antibacterial and pro‐regenerative effects.

Methods: EVs were isolated from RJ using ultracentrifugation and analyzed for size distribution by Nanoparticle Tracking Analysis (NTA) and Transmission Electron Microscopy (TEM). Furthermore, vesicles were tested for presence of exosomal markers as well as cargo proteins with Western Blot. Antibacterial effects were tested in microplate biofilm assays using wound‐associated strain Staphylococcus aureus ATCC 29213. The effect of RJ‐EVs on human mesenchymal stem cells (MSCs) and fibroblasts was assessed in cell cycle‐ and migration assays.

Results: Presence of EVs was verified with NTA and TEM, and exosomal origin was confirmed by Western Blot (Syntenin, CD63, due to apis origin). Major Royal Jelly Protein 1 (MRJP1), Defensin‐1 and Jellein‐3 were identified as relevant EV cargo. RJ‐EVs displayed strong bactericidal and biofilm‐disrupting effects on both bacterial strains (concentration ∼10:1 RJ‐EVS to CFU). Both MSCs and fibroblasts internalized bee‐derived RJ‐EVs resulting in a pro‐migratory effect for MSCs and increased fibroblast proliferation.

Summary/Conclusion: RJ‐EVs displayed promising properties for application in wound healing. On the one hand, they are strongly antibacterial and biofilm‐disruptive; on the other hand, RJ‐EVs are not limited to intra‐species communication: their internalization by mammalian cells promotes either migration or proliferation of cells associated to wound healing. The underlying mechanisms can be associated to the cargo proteins identified, as Defensin‐1, Jellein‐3 and MRJP1 are known to be antibacterial, and the latter to furthermore act as a growth factor for several cell types. Summarizing, we were the first to identify EVs in RJ and link known RJ proteins to active EV cargo. Being encased by vesicles, these proteins are delivered into mammalian cells in a directional manner, increasing their therapeutic potential. Thus, RJ‐EVs could be used as a novel vesicle‐based therapy for the treatment of chronic wounds, especially when associated to infection and biofilm development.

OD07.05. Enhanced angiogenesis and wound healing in vivo induced by extracellular vesicles from therapeutic grade allogeneic human placental stromal cells

Martin Wolf, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI‐TReCS), Paracelsus Medical University (PMU), Salzburg, Austria

Rodolphe Poupardin, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg

Constantin Blöchl, Dept. of Biosciences, Paris Lodron University Salzburg

André Cronemberger Andrade, Paracelsus Medical University

Fausto Gueths Gomes, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg

Patricia Ebner, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg

Balazs Vari, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg

Essi Eminger, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI‐TReCS), Paracelsus Medical University (PMU), Salzburg, Austria

Heide Marie Binder, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg

Anna M Raninger,Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg

Gabriele Brachtl, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg

Andreas SpittlerCore Facility Flow Cytometry & Surgical Research Laboratories, Medical University of Vienna

Thomas Heuser, Vienna Biocenter Core Facilities, Medical University, Vienna

Astrid Obermayer,Dept. of Biosciences, Paris Lodron University Salzburg

Christian G Huber,Dept. of Biosciences, Paris Lodron University Salzburg

Katarina Schallmoser,Department of Transfusion Medicine and SCI‐TReCS, PMU, Salzburg, Austria

Hans‐ Dieter Volf,BCRT & Institute of Medical Immunology, Charite ‐ Univeritätsmedizin Berlin, Germany

Strunk Dirk,Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI‐TReCS), Paracelsus Medical University (PMU), Salzburg, Austria

Introduction: Cell therapy approaches using of the shelf allogeneic products have shown surprising results despite lack of engraftment of the transplanted cells. Their efficacy was so far considered to be mostly mediated by secreted trophic factors. We tested if extracellular vesicles (EVs) contribute to their mode of action. Here we provide evidence that EVs derived from therapeutic placental‐expanded (PLX) stromal cells are potent inducers of angiogenesis and modulate immune cell proliferation in a dose‐dependent manner.

Methods: Crude EVs were enriched >100‐fold from large volume PLX conditioned media via tangential flow filtration (TFF) as determined by tuneable resistive pulse sensing (TRPS). Additional TFF purification was devised to separate EVs from cell‐secreted soluble factors. EV identity was confirmed by western blot, electron microscopy and super resolution microscopy. To identify the mode of action we compared isolated EV preparations with corresponding soluble factors via proteomic analysis, in vitro angiogenesis assay, stimulation capacity of Immune cells, and in vivo wound healing mouse model.

Results: Surface marker profiling of tetraspanin‐positive EVs identified expression of cell‐ and matrix‐interacting adhesion molecules. Differential tandem mass tag proteomics comparing PLX‐EVs to PLX‐derived soluble factors revealed significant differential enrichment of 258 proteins in purified PLX‐EVs involved in angiogenesis, cell movement and immune system signalling. At the functional level, PLX‐EVs and cells in contrast to soluble factors inhibited T cell mitogenesis and showed different activation patterns of proliferation pathways in Monocytes and T cells. PLX‐EVs and soluble factors displayed dose‐dependent proangiogenic potential by enhancing tube‐like structure formation in vitro as well as enhanced vessel density and wound healing in an in vivo mouse model.

Summary/Conclusion: Our findings indicate that the mode of PLX action involves an EV‐mediated proangiogenic function and immune response modulation that may help explaining clinical efficacy beyond presence of the transplanted allogeneic cells.

OD07.06. In vivo administration of extracellular vesicles derived from amniotic fluid stem cells improves lung development in experimental pulmonary hypoplasia

Rebeca Figueira, PhD, MSc, The Hospital for Sick Children

Noor Ramy, The Hospital for Sick Children

Lina Antounians, MSc, The Hospital for Sick Children

Kasra Khalaj, PhD, MSc, The Hospital for Sick Children

Sree Gandhi, The Hospital for Sick Children

Augusto Zani, MD, PhD, FACS, FAAP, The Hospital for Sick Children

Introduction: We previously reported that administration of extracellular vesicles derived from amniotic fluid stem cells (AFSC‐EVs) to in vitro rat lung models of pulmonary hypoplasia (PH) restores lung growth and maturation. We also reported that intra‐amniotically injected ExoGlow+ AFSC‐EVs reach the fetal lungs. Herein, we investigated whether AFSC‐EV administration could rescue normal lung growth also in an in vivo rat model of PH.

Methods: EVs were isolated from pre‐cleared conditioned medium of cKit+rat AFSCs using ultracentrifugation (100,000g, 14h), and characterized for size (nanoparticle tracking analysis), morphology (transmission electron microscopy), and expression of canonical EV protein markers CD63, Hsp70, Flo‐1, and TSG101 (Western blot). To reproduce fetal PH, nitrofen was administered to pregnant rats at embryonic day (E) 9.5. During the canalicular stage of lung development (E18.5‐19.5), dams were anaesthetized, uterine horns were exposed, and 100uL of either AFSC‐EVs (n = 9) or saline (n = 9) were injected into the amniotic sac. Fetal lungs were harvested at E21.5. Groups were compared for: 1) airway branch density (mean linear intercept with H&E); 2) number of vessels per mm2 (immunofluorescence: von Willebrand factor for endothelial cells + smooth muscle actin for muscle cells); 3) mean wall thickness of pulmonary arteries with 10–60um diameter (H&E). Statistics: t‐test.

Results: Compared to saline injection alone, intra‐amniotic administration of AFSC‐EVs to nitrofen‐exposed fetal lungs restored airway branch density (p = 0.0029), rescued the number of vessels per mm2 (p = 0.04), and decreased the mean wall thickness of pulmonary arterioles (p < 0.0001).

Summary/Conclusion: Antenatal in vivo administration of AFSC‐EVs improves fetal lung development by restoring airway branching and vascularization levels in the rat model of PH. AFSC‐EV antenatal administration represents a promising therapy for fetuses with PH.

OD07.07. Therapeutic Potential of Pericyte‐derived EVs for Skeletal Muscle Recovery Following Hindlimb Immobilization in Aged Mice

Yu‐Fu Wu, M.S., University of Illinois at Urbana‐Champaign

Noah Kim, University of Illinois at Urbana‐Champaign

Rebecca Jung, University of Illinois at Urbana‐Champaign

Marni D. Boppart, Sc.D., University of Illinois at Urbana‐Champaign

Introduction: Skeletal muscle disuse atrophy is the decline of muscle mass that results from physical unloading. Recovery via physical rehabilitation is often incomplete in older adults, which can lead to disability and loss of independence. Our laboratory previously demonstrated that perivascular stem cells (CD146+ pericyte) can effectively recover muscle mass in young adult mice following a period of immobilization (IM). The purpose of this study was to determine the extent to which pericytes and pericyte‐derived extracellular vesicles (EVs) could effectively improve muscle mass in aged mice.

Methods: Four‐month‐old and 24‐month‐old C57BL/6 mice were randomly separated into cell or EV treatment groups by age (n = 3‐5/group). One hindlimb was immobilized in full dorsiflexion via a surgical staple. After 2 weeks of IM, staples were removed, and either PBS (control), pericytes, or pericyte‐derived EVs (unprimed or hydrogen peroxide (H2O2) primed) were injected into the tibialis anterior muscle. Muscles were excised after 2 weeks of remobilization, and recovery was assessed by immunofluorescence. One‐way ANOVA was used to compare the extent of improvement between treatments within each age group.

Results: No significant improvement was observed with pericyte transplantation in aged mice, likely due to cell viability issues associated with the aged microenvironment. However, significant improvements in myofiber CSA and collagen remodeling were observed in both young (p = 0.011 and p = 0.002) and aged (p = 0.011 and p = 0.036) mice receiving primed EVs as compared to age‐matched PBS groups. Unprimed EVs resulted in variable responses in young and old compared to primed EVs.

Summary/Conclusion: Pericyte‐derived EVs represent an important new therapy that can potentially recover and rebuild muscle mass after a period of disuse in both young and older adults.

OD07.08. Pregnancy Resembles Heterochronic Parabiosis And Amniotic Fluid Extracellular Vesicles Transfer Regenerative Potential

Pascal Goldschmidt‐Clermont, M.D., Alzady International, LLC

Corinne Hubinont, M.D., Saint Luc University Hospital

Bruna Turnes, Ph.D., Harvard Medical School

Ian A. White, PhD, Neobiosis, LLC

Introduction: Aging is an evolutionary conserved mechanism driving genetic diversity in all eukaryotic organisms. This process of “timing out” individuals allows resources to be made available to “novel” genetic variants (offspring) potentially better suited to take advantage of local environments or to inhabit new. Aging progresses due to a gradual, but continuous, depletion of endogenous stem cells, which are capable of maintaining homeostasis through tissue repair. As a consequence, aged individuals experience progressively slower and less complete tissue repair. This process has been interrupted in murine laboratory experiments referred to as heterochronic parabiosis, where an old individual regains youthful repair potential when connected to a young individual's blood supply though a common dermal patch. This phenomenon is thought to happen by boosting the regenerative capacity of endogenous tissue stem cells. A natural counterpart exists and that is pregnancy.

Methods: Pregnancy is an unusual form of heterochronic parabiosis, as the placenta prevents most blood cells to be exchanged between the young and the older parabionts. Instead, plasma, including small extracellular vesicles (EVs), can readily cross the placental barrier. These nanosized EVs, which accumulate in the amniotic fluid, are essential for fetal organogenesis and growth and also impact the mother, as they are essential for maternal physiological changes in response to the stresses of pregnancy. Using an array‐based multiplex ELISA approach we have identified over 200 bioactive cytokines, proteases and soluble receptors related to inflammation and tissue repair from purified EVs derived from human amniotic fluid (hAFEVs). We tested the hypothesis that these EVs are capable of improving age‐related pathology by employing a murine model of chronic inflammatory mono‐arthritis of the knee.

Results: Through intra‐articular injection of hAFEVs we learned that hAFEVs were immunologically tolerated and reduced knee‐inflammation and promoted endogenous tissue repair in treated animals (n = 12) compared to control animals (n = 8) up to 21 days post injury.

Summary/Conclusion: These data suggest a potential strategy for accessing benefits of heterochronic parabiosis to boost the regenerative capacity of endogenous tissue stem cells in humans and regain the homeostatic repair potential of the young, using a readily available tissue source.

OD08.01. Immunophenotyping of single extracellular vesicles via nano‐flow cytometry for nasopharyngeal carcinoma diagnosis

Yunyun Hu, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University

Bin Hu, The First Affiliated Hospital of Xiamen University

Ye Tian, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University

Qin Lin, The First Affiliated Hospital of Xiamen University

Xiaomei Yan, PhD, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University

Introduction: Nasopharyngeal carcinoma (NPC) is one of the most common malignant epithelial tumors which presents a major public health problem worldwide, especially in South China. Epstein Barr Virus (EBV) infection is a vital factor that contributes to NPC pathogenesis. Although no viral particles are detected in the tumor, the EBV genome is present in virtually all NPC cells, encoding numbers of latent gene products including membrane proteins (LMP1 and LMP2). The traditional clinical diagnostics of NPC relies on invasive tissue biopsy and less‐sensitive medical imaging technology. Extracellular vesicles (EVs, ∼40'1000 nm) are emerging as a promising substitute of liquid biopsy for disease diagnosis. Herein, we performed protein analysis on single EVs from both the NPC cells and NPC patients by using a laboratory‐built nano‐flow cytometer (nFCM) that enables multiparameter analysis of single EVs as small as 40 nm.

Methods: EVs derived from NP69 (a normal nasopharyngeal epithelial cell line), C666‐1 (an NPC cell line consistently harboring EBV), CNE1 (an EBV‐negative NPC cell line) and NPC patient plasma were isolated by differential ultracentrifugation. Upon immunofluorescent staining, the expression levels of five markers (LMP2A, LMP1, EpCAM, PD‐L1, and EGFR) for isolated EVs were analyzed and quantified.

Results: Upon screening of a series of cancer biomarkers, the expression levels of LMP2A and LMP1 for EVs derived from C666‐1 were elevated compared to those of NP69 and CNE1, which indicated their potential for NPC diagnosis. When plasma EVs were analyzed by nFCM, the concentration of LMP2A+ EVs showed a great performance in distinguishing clinical patients with stage II‐IV NPC from healthy donors (AUC = 0.97). Of note, when concurrent analysis of other cancer biomarkers was conducted, the combined signature offered up to 100% sensitivity, specificity, and accuracy for early‐stage NPC detection.

Summary/Conclusion: nFCM provides a straightforward and non‐invasive approach for immunophenotyping of EVs derived from NPC patients. In particular, multiple protein profiling facilitates diagnosis of early‐stage NPC with 100% accuracy. To further expand the application of the identified marker set, we will enroll more clinical samples to evaluate the diagnostic potential in distinguishing NPC from other cancer types.

OD08.02. Characterisation of EVs separated from plasma and BALF of patients diagnosed with lung lesions including NSCLC

Magdalena Dlugolecka, Chair and Department of Biochemistry, Doctoral School, Medical University of Warsaw

Jacek Szymanski, Chair and Department of Biochemistry, Medical University of Warsaw

Lukasz Zareba, Chair and Department of Biochemistry, Medical University of Warsaw

Zuzanna Homoncik, Chair and Department of Biochemistry, Medical University of Warsaw

Joanna Domagala‐Kulawik, Department of Internal Diseases, Pneumonology and Allergology, Medical University of Warsaw

Malgorzata Polubiec‐Kownacka, Department of Surgery, Institute of Tuberculosis and Lung Diseases

Malgorzata Czystowska‐Kuzmicz, Chair and Department of Biochemistry, Medical University of Warsaw

Introduction: The molecular characterisation of tumor‐derived extracellular vesicles (EVs) can be beneficial for diagnostic and prognostic purposes. In lung diseases like non‐small cell lung cancer (NSCLC), bronchopulmonary lavage fluid (BALF) seems to be a respectable source of tumour‐derived EVs. Using modern analytic methods we phenotyped and compared EV populations from patients’ BALF and plasma.

Methods: Plasma EVs were separated using size‐exclusion chromatography (SEC). BALF from the lung affected by lung cancer or another lesion (cBALF) and BALF from the not affected lung (hBALF) were collected and EVs were separated by differential ultracentrifugation. EV enriched samples were characterised by Western blot, Cryo‐Transmission electron microscopy (Cryo‐TEM), bead‐assisted flow cytometry (using a mix of magnetic beads with antibodies against CD63, CD9 and CD81) and FL‐NTA, where EV enriched samples were labelled with a membrane dye (CMDR) and PE dyed antibodies against typical EV‐markers (CD9, CD81, CD63).

Results: Cryo‐TEM imaging showed that BALF EVs consists exclusively of double‐membrane vesicles, whereas plasma EVs have much more complex morphology than both BALF EV types, containing mostly, besides typical exosomal vesicles, single‐membrane liposomes. Exosomal markers were present in different amounts in all EV types analysed by Western blot. In contrast, flow cytometry showed that BALF EVs bound to magnetic beads were positive for CD63, CD9 and CD81 whereas in case of plasma EVs detectable was only CD63 in a small EV subpopulation. FL‐NTA showed a higher particle concentration in plasma than in both BALF types in respect to one ml of each biological fluid. The particles from plasma were only positive for CMDR, whereas both BALF EV types were positive not only for membrane‐labelling but also for exosomal markers.

Summary/Conclusion: Deep FL‐NTA analysis allowed us to highlight the significant differences between EVs from plasma and BALF. We have shown that there were no significant differences between cBALF and hBALF EVs.

OD08.03. In situ imaging of bacterial membranous extensions and their associated protein complexes using electron cryo‐tomography

Mohammed Kaplan, California Institute of technology

Grant Jensen, California Institute of technology

Introduction: The ability to produce membranous extensions (MEs) in the form of membrane vesicles and tubes is a widespread phenomenon amongst bacteria. Despite this, our knowledge of the molecular ultrastructure of these extensions and their associated protein complexes (PCs) in different species remains limited.

Methods: Here, we used electron cryo‐tomography (cryo‐ET) to survey the ultrastructure and formation of MEs and their associated PCs in numerous bacterial species.

Results: We describe the ultrastructure of MEs in the form of nanotubes with a uniform diameter (with or without an internal scaffold) or irregular diameter, pearling nanotubes, connected chains of vesicles (with or without neck‐like connectors), budding vesicles and nanopods. These forms were present either exclusively or combined in a species‐specific manner. Furthermore, we identified various PCs associated with the MEs and were located either randomly or exclusively at the tip of the MEs including a secretin like complex and a crown‐like complex associated with cell‐lysis.

Summary/Conclusion: In total, our results show that the molecular architecture of MEs and their associated PCs are variable even amongst closely‐related species.

OD08.04. Application of extracellular vesicle surface activity in the presence of gas‐liquid and liquid‐liquid interfaces for their characterization by using the dynamic surface/interfacial tension probe

Ekaterina Tsydenzhapova, Moscow Institute of Physics & Technology

Roman Chuprov‐Netochin, Moscow Institute of Physics & Technology

Sergei German, Skolkovo Institute of Science and Technology

Anastasiia Merdalimova, Skolkovo Institute of Science and Technology

Alexey Yashchenok, Skolkovo Institute of Science and Technology

Sergey Leonov, Moscow Institute of Physics & Technology

Dmitry Gorin, Skolkovo Institute of Science and Technology

Mikhail Skliar,University of Utah

Vasiliy S. Chernyshev, Skolkovo Institute of Science and Technology

Introduction: Surface activity is a dynamic phenomenon where molecules (e.g. proteins) or nanoparticles migrate towards and adsorb to a surface (liquid‐air) or interface (liquid‐liquid) to reach a more energetically favorable state, causing a change in surface tension (ST) or interfacial tension (IT). Despite the high potential of extracellular vesicles (EVs) in medicine, there is a lack of information about their potential surface activity at the liquid‐air and liquid‐liquid interfaces which are regularly encountered in research, development of diagnostic platforms and drugs. Since EVs are known to contain membrane macromolecules (e.g. proteins) that are exposed to surrounding fluid, we hypothesized their potential surface activity.

Methods: EVs were isolated from MCF7, MCF10a, MDA‐MB‐231 and SKOV3 cell culture media by ultrafiltration followed by SEC. SEC fractions containing EVs were analyzed by NTA, DLS, Scanning‐EM, BCA and WB. For surface activity analysis, dynamic ST and IT of each SEC fraction containing EVs in 1x PBS was measured for at least 4 hrs (n = 3) in the presence of air, isopropyl myristate, toluene and cyclohexane by using a custom‐made real‐time pendant‐drop tensiometer at room temperature. Statistical analysis was done by using Matlab software.

Results: SEC sample characterization confirmed presence of EVs with high purity in 3 fractions for each cell line which were used for ST and IT measurements. At early age of the pendant drop ST and IT values agreed with values reported for pure fluid‐air and fluid‐fluid systems. As time progressed the ST and IT values decreased and after 4 hours the value was 5–30% lower than at early age of the pendant drop (p < 0.05) due to EV diffusion to the surface/interface and adsorption. A distinct correlation between EV quantity and tension values was found at specific time points.

Summary/Conclusion: Dynamic ST and IT measurements allowed to determine that EVs are surface active, diffuse and adsorb at the liquid/air and liquid/liquid interfaces. This EV property not only can play a critical role in their biological function but also opened doors to a new approach for EV characterization, especially their quantitation by using the highly informative dynamic ST/IT probe.

OD08.05. Multi‐Objective Calibration and Standardization of EV Measurement Using Nanoscale Flow Cytometry

Edwin van der pol, Amsterdam University Medical Centers

Fabrice Lucien, MD PhD, Mayo Clinic

Introduction: Nanoscale flow cytometry (nFC) is a powerful method that combines fluorescence and light‐scattering detection to enumerate a large number of extracellular vesicles (EVs) within minutes. nFC also offers the opportunity to evaluate the clinical utility of EVs as prognostic markers in human diseases. However, single particle detection can be challenged by variances in pre‐analytical conditions and instrumentation settings. To overcome this, impressive collaborative efforts resulted in a framework for standardized reporting of EV flow cytometry (FC) experiments (MIFlowCyt‐EV). By building on previous milestones, we further evaluated the performance and limitations of nFC in EV enumeration from biofluids. We also provide novel insights on pre‐analytical conditions and acquisition parameters to reliably enumerate specific EV subpopulations.

Methods: Biofluid samples (blood and urine) of healthy donors and prostate cancer patients were prepared according to the recommendations of MISEV 2018. The Apogee A60‐MicroPlus nanoscale flow cytometer was used for all FC experiments. Gag‐GFP+HEK293T EVs, spiked in biofluids were used as biological reference. Different antibody labeling methods were also assessed for detection of EV subsets with differential abundance in biofluids. Light‐scatter and fluorescence detection of EVs was assessed with more than 12 different acquisition settings. By using Mie Theory, optical configurations, calibrations, and all EV data were converted into standardized units.

Results: The optimal concentration ranges of biofluid samples for reproducible EV enumeration after serial dilution were determined and most samples could meet median concentrations within the ranges. For EV immunophenotyping, the number of dyes per antibody and choice of fluorophores were critical factors for fluorescence detection of EVs. Multiplexing with up to 3 antibodies targeting different EV subpopulations did not affect concentration measurements. However, double staining of same EV subset may result in underestimation of EV concentrations from potential steric hindrance of antibodies on surface of EVs.

Summary/Conclusion: In conclusion, this study provides optimal settings and recommendations for reproducible EV enumeration using nFC, and ultimately help other research groups strive for developing EV‐based liquid biopsies.

OD08.06. Quantitative analysis of 2D and 3D cell culture‐isolated EV populations by ILM, TRPS and NTA technologies

Liliia Paniushkina, Medical Center Freiburg, Exosomes and Tumour biology group

Martin Wolf, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI‐TReCS), Paracelsus Medical University (PMU), Salzburg, Austria

Krisztina V. Vukman, Department of Genetics, Cell‐ and Immunobiology, Semmelweis University, Budapest, Hungary

Strunk Dirk, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI‐TReCS), Paracelsus Medical University (PMU), Salzburg, Austria

Edit I. Buzás, Department of Genetics, Cell‐ and Immunobiology, Semmelweis University, Budapest, Hungary

Irina Nazarenko, Medical Center Freiburg, Germany

Introduction: Different methods are used to measure particle number for characterisation of isolated EVs. These methods bring internal discrepancies when measuring the same EV sample. This study aimed to quantify four EV populations of different size isolated from 2D and 3D cell culture models by three technologies. We introduce an application of Interferometric Light Microscopy (ILM) in comparison with TRPS and NTA.

Methods: Noticed a growing interest in physiological models, we used 2D and 3D prostate cancer (22Rv1) cell cultures to analyse surface molecular signatures and EV cargo. We compared the particle number and size of four EV populations: EV5, EV12, EV120, sEV designated according to centrifugation speed and purified by density gradient centrifugation resulting in 10 fractions per population. The fractions were analysed using ILM, TRPS and NTA technologies following by the characterization of tissue and EV‐specific markers.

Results: We observed a highly significant difference in particle number measured by all three technologies in EV5. The TRPS gives the lowest concentration of particles (∼107 particles/ml), while ILM ‐ ∼109 particles/ml, and NTA ‐ ∼1011 particles/ml. When comparing the measured particle size, NTA was able to detect only small particles (30‐200nm), while TRPS using different membranes distinguished between small and large particles, showing two populations: 50–150nm and 200–600nm. Only ILM was able simultaneously estimated small and large particles in a size range: 80–720nm.

Summary/Conclusion: All three techniques ILM, TRPS and NTA detect and evaluate the particles at 80–200nm. The TRPS and ILM estimated large particles, while NTA did not. Our results show that ILM might simultaneously evaluate a polydisperse sample detecting small and large particles in one preparation. However, using TRPS, a broader size range of particles 50–2000 nm can be seen, while ILM is limited by 80nm for small. Our result demonstrated the importance of applying different methods of particle quantification to characterize different EV populations.

OD08.07. Association between infrared spectra and the lipidomic profile of human milk exosomes

Victoria Ramos‐Garcia, Neonatal Research Unit, Health Research Institute Hospital La Fe, Avda Fernando Abril Martorell 106, 46026 Valencia, Spain

Isabel Ten‐Domenech, Neonatal Research Unit, Health Research Institute Hospital La Fe, Avda Fernando Abril Martorell 106, 46026 Valencia, Spain

Abel Albiach‐Delgado, Neonatal Research Unit, Health Research Institute Hospital La Fe, Avda Fernando Abril Martorell 106, 46026 Valencia, Spain

Alba Moreno‐Giménez, Neonatal Research Unit, Health Research Institute Hospital La Fe, Avda Fernando Abril Martorell 106, 46026 Valencia, Spain

María Gormaz, Division of Neonatology, University & Polytechnic Hospital La Fe, Avda Fernando Abril Martorell 106, 46026 Valencia, Spain

Anna Parra‐Llorca, Division of Neonatology, University & Polytechnic Hospital La Fe, Avda Fernando Abril Martorell 106, 46026 Valencia, Spain

María Círia, Regenerative Medicine and Heart Transplantation Unit, Health Research Institute Hospital La Fe, Avda Fernando Abril Martorell 106, 46026 Valencia, Spain

Pilar Sepúlveda, Regenerative Medicine and Heart Transplantation Unit, Health Research Institute Hospital La Fe, Avda Fernando Abril Martorell 106, 46026 Valencia, Spain

David Pérez‐Guaita, Department of Analytical Chemistry, University of Valencia, 50 Dr. Moliner Street, research building, 46100 Burjassot, Valencia, Spain

Bernhard Lendl, Institute of Chemical Technologies and Analytics, Technische Universität Wien, Getreidemarkt 9/164. A 1060 Vienna, Austria

Guillermo Quintás, Health and Biomedicine, Leitat Technological Center, Carrer de la Innovació, 2, 08225 Terrassa, Spain

Julia Kuligowski, Neonatal Research Unit, Health Research Institute Hospital La Fe, Avda Fernando Abril Martorell 106, 46026 Valencia, Spain

Introduction: Exosomes are nanosized (50‐100 nm) membrane vesicles released by fusion of the multivesicular body with the plasma membrane. The main objective of this study was to determine the feasibility of a rapid characterization of the lipidomic profile of exosomes by infrared spectroscopy using exosomes isolated from human milk (HM) as a model example.

Methods: Exosomes were isolated employing a multi‐stage ultracentrifugation procedure. After a single‐phase extraction, lipidomic fingerprinting was carried out using ultra‐high performance liquid chromatography quadrupole‐time‐of‐flight mass spectrometry (UPLC"qTOF‐MS) operating in positive and negative ionization modes. Automated MSMS‐based annotation of metabolites was carried out using HMDB, METLIN, in silico LipidBlast and MSDIAL MS/MS databases. Then, dry films of 2 μL of HM exosomes suspended in PBS were directly analysed by Attenuated Total Reflectance ‐ Fourier Transform Infrared (ATR‐FTIR) spectroscopy.

Results: A total of 693 LC‐MS features detected in HM exosomes were successfully annotated. The classes with the most annotated features were glycerolipids (230), glycerophospholipids (217), sphingolipids (173), and fatty acyls (20). Multivariate analysis showed significant associations between specific regions of the ATR‐FTIR spectra and the concentrations of different lipid classes. Besides, the UPLC‐MS and ATR‐FTIR datasets were analysed by principal component analysis. Using the distances among samples in the PC1‐PC2 score space as criteria, the significance of the similarity between the trends observed in both scores plots was assessed by the Mantel test (p‐value < 0.005).

Summary/Conclusion: A correlation between the lipidomic profile of HM exosomes and their ATR‐FTIR spectra has been obtained, indicating that the latter technique can be used for a rapid evaluation of the composition of HM exosomes, thus supporting the development of a new tool for a direct and fast quality control of the exosome isolation procedure.

OD08.08. Determination of the kinetics of circulating small extracellular vesicles with an in situ membrane biotinylation strategy

Zili Yu, School and Hospital of Stomatology, Wuhan University

Yi Zhao, School and Hospital of Stomatology, Wuhan University

Gang Chen, School and Hospital of Stomatology, Wuhan University

Introduction: The in vivo kinetics of circulating small extracellular vesicles (sEVs), which possess great potential to serve as biomarkers for disease diagnosis or delivery vectors for personalized therapy, remain elusive.

Methods: The in vivo biosafety, kinetics and biodistribution of intravenously injected DSPE‐PEG‐Biotin were systematically evaluated in mice. Biotinylation of circulating sEVs were characterized by immuno‐electron microscopy using streptavidin‐conjugated gold nanoparticles. The level of biotinylated circulating sEVs at serial time points were monitored with flow cytometry. The in vivo kinetics of each subtype of circulating sEVs using in situ biotinylation strategy were evaluated with the antibodies against different cell markers.

Results: The concentration of biotin in the circulation of mice was rapidly decreased and almost undetectable at 12 h after injection. The in situ membrane biotinylation strategy is universal to label circulating sEVs with biotin biofriendly and efficiently in different animal models. The level of biotinylated circulating sEVs rapidly decreased with time, and finally fallen to the baseline level at 3 days after injection of DSPE‐PEG‐Biotin. The lifetime (halftime) was 1 (0.14), 2 (0.40), 2 (0.45), 2 (0.50), 2 (0.54) and 5 (0.95) days for endothelium‐derived sEVs, macrophage derived sEVs, platelet‐derived sEVs, leukocyte‐derived sEVs, lymphocyte‐derived sEVs and erythrocyte‐derived sEVs, respectively. The lifetime of tumor‐derived PD‐L1+ sEVs in circulation was around 2 days (halftime: 0.99 days). The circulating sEVs were biotinylated by the intravenously injected DSPE‐PEG‐Biotin mainly through the direct way other than donor cell‐assisted manner.

Summary/Conclusion: This study realized the first reliable in situ biotinylation strategy and revealed the dynamics kinetics of circulating sEVs, which will undoubtedly beneficial to the understanding of fundamental aspects of circulating sEVs physiology in vivo and have implications for the design and feasibility of circulating sEVs‐based therapeutics.

OD09.01. Drug‐induced lysosomal impairment drives the release of extracellular vesicles carrying autophagy‐ssociated markers

Lorena Urbanelli, Università di Perugia

Krizia Sagini, Department of Surgery, Division of Cancer Biology and Therapeutics, Cedars‐Sinai Medical Center

Sandra Buratta, Università di Perugia

Federica Delo, Università di Perugia

Roberto Maria Pellegrino, Università di Perugia

Carla Emiliani, Università di Perugia

Introduction: Amiodarone (AM) is a cationic amphiphilic drug used as antiarrhythmic agent. It is known to induce phospholipidosis (PLD), i.e. the accumulation of phospholipids into multilamellar structures within organelles of the endo‐lysosomal system. Extracellular vesicles (EVs) are now considered an additional manner to transmit intercellular signals, but they have been initially identified as a system to dispose extracellularly unnecessary cell material. Because of the important role played by the endolysosomal system in the biogenesis and secretion of EVs, we investigated their role in PLD.

Methods: AM‐treated HEK‐293 cells were engineered to produce fluorescently labelled vesicles by expression of the mCherry‐CD63 fusion protein. EVs were isolated from cell media by differential ultracentrifugation and both medium/large and small florescent EVs were retrieved in the 10K and 100K faction, respectively.

Results: AM induces the release of a higher number of EVs, mostly of medium/large size. Although EVs released upon AM treatment do not display significant morphological changes by EM or altered size distribution by NTA, they show a dose dependent increase of autophagy associated markers by IB. Proteolytic digestion shows that at least one of these markers, LC3B, is localized within EVs. Determination of EV phospholipid content by LC/MS showed that medium/large EVs released upon AM treatment contain more phospholipids than those released by untreated cells, indicating that AM‐treated cells release EVs enriched in phospholipids to possibly alleviate their intracellular accumulation. Drugs commonly used to block autophagy such as chloroquine or bafilomycin A also induce a higher release of EVs enriched in autophagic markers.

Summary/Conclusion: Our findings indicates that EVs enriched in autophagy markers may be related to lysosomal impairment in general and not to the specific type of accumulated substrate. Improving lysosomal function and autophagy by transfection of TFEB transcription factor, a master gene regulating lysosomal biogenesis, prevents AM‐induced EV release. This result confirms that the degradative cell capability is a key factor in determining undigested material fate (intracellularly degraded or extracellularly released) and suggests that this could be a feasible target to attenuate PLD‐induced abnormalities.

OD09.02. Drug repositioning screening for an inhibitor of EV secretion in ovarian cancer cells

Yusuke Yoshioka, Tokyo Medical University

Akira Yokoi, Nagoya University

Takahiro Ochiya, PhD, Department of Molecular and Cellular Medicine, Tokyo Medical University

Introduction: Cancer‐related EVs with their ability to act within the tumor microenvironment and distally and thus play a major role in tumor progression and metastasis. For example, we found that EVs derived from highly metastatic ovarian cancer cells promote peritoneal dissemination in vivo (Yokoi A etal., Nat Commun, 2017). Therefore, inhibition of EV secretion from cancer cells can serve as a novel therapeutic tool to inhibit cancer metastasis. This study focused on the screening of small‐molecule inhibitors for EV secretion in ovarian cancer cells.

Methods: We used an original screening system based on ExoScreen assay for monitoring CD9 positive EV secretion (Yoshioka Y etal., Nat Commun, 2014). After screening, we used ExoView to measure EV secretion as a validation of our screening results. To observe the influence of small molecules on cell growth, a proliferation assay was undertaken using IncuCyte. The EV secretion rate of cells was normalized to the cell growth rate. Using this screening system and a chemical compound library containing 1271 small molecules, inhibitors for EV secretion were identified in the ovarian cancer cell line ES‐2.

Results: Based on the first screening result, 45 small molecules were selected as putative inhibitors for EV secretion. These small molecules were further validated by ExoScreen. As a result of the validation, 8 small molecules were found to inhibit EV secretion in ES‐2 cells. To confirm the screening result, 4 cell lines, including 2 non‐cancer cell lines, were treated these 8 molecules and measured EV secretion by ExoView. Some molecules inhibited EV secretion in a cancer cell‐specific manner. These 8 molecules did not affect cell proliferation compared to 0.1% DMSO treated cells.

Summary/Conclusion: Here, we identify inhibitors for EV secretion in ovarian cancer cells. Based on these results, we are now analyzing the effects of these candidate molecules on gene expression in cancer cells, and we plan to test their therapeutic effects using a mouse model of ovarian cancer transplantation.

OD09.03. Controlling cell‐material interactions to tune therapeutic extracellular vesicle production

Stephen B. Lenzini, University of Illinois at Chicago

Singwan Wong, University of Illinois at Chicago

Angela Song, University of Illinois at Chicago

Raymond Bargi, University of Illinois at Chicago

Dolly Mehta, University of Illinois at Chicago

Jae‐Won Shin, University of Illinois at Chicago

Introduction: Success in biomanufacturing of therapeutic EVs from mesenchymal stromal cells (MSCs) depends on a fundamental understanding of how EVs are produced and transported in physiologically relevant environments. We recently show that both matrix stress relaxation properties and water transport through aquaporin‐1 enable EVs to deform and travel through the dense mesh of the extracellular matrix (Lenzini etal., Nat Nano 15: 217–223, 2020). However, it remains unclear how matrix properties impact EV production by MSCs. Since mechanical forces regulate membrane trafficking and mechanosensing, we hypothesized that substrate mechanics regulate EV production.

Methods: Alginate polymer was conjugated with an integrin binding peptide Arg‐Gly‐Asp (RGD). Alginate hydrogels with tunable stiffness were formed by adipic acid dihydrazide crosslinking. Human bone marrow MSCs (Lonza) were seeded on hydrogels followed by washing to remove unattached cells. To measure EV number, Nanoparticle Tracking Analysis (NTA) via NanoSight NS300 (Malvern) was used.

Results: MSCs produce ∼10 times more EVs on a soft hydrogel than on a rigid polystyrene surface, and ∼2.5 times more than on a stiffer hydrogel. Treatment with blebbistatin does not impact stiffness dependent EV production, suggesting this effect is independent of myosin‐II. EVs are produced more rapidly when MSCs are adhered to soft substrates for 4 hours versus 24 hours. Consistent with this result, activating integrins with Mn2+ decreases EV production, suggesting that soft substrates may enhance EV production by decreasing activation of integrins. Importantly, we show that EVs from MSCs on different substrates resolve a lipopolysaccharide‐induced model of acute lung injury in mice.

Summary/Conclusion: The results propose an optimal substrate stiffness and cell adhesion time to enhance EV production from MSCs. Production strategies designed based on these results will have a profound impact on advancing biomanufacturing of EVs from MSCs.

OD09.04. Extracellular vesicles containing I‐BAR proteins are released from the cell plasma membrane in an Arp2/3 dependent manner

Delphine M. Muriaux, CNRS & University of Montpellier

Introduction: Extracellular vesicles are nanometric membrane vesicles produced by cells and involved in cell‐cell communication. Extracellular vesicles (EVs) formation can occur in endosomal compartments (exosomes) or at the cell plasma membrane (microvesicles). How these cellular vesicles bud from the cell plasma membrane is not completely understood. I‐BAR proteins are cytosolic proteins, when activated, bind to the plasma membrane and are involved in plasma membrane protrusion formation including filopodia and lamellipodia. These proteins contain a conserved I‐BAR domain which sense and induce negative membrane curvatures at the plasma membrane. I‐BAR proteins also interact with actin co‐factors to induce membrane protrusions. Here we explore if ectopic I‐BAR proteins, such as IRSp53, IRTKS and Pinkbar, are in EVs.

Methods: To explore these hypotheses, we purified EVs from productive human 293T cells and characterize plasma membrane I‐BAR EVs using immunoblots, interferometric microscopy, Atomic Force microscopy and immuno‐fluorescence coupled to TIRF‐Microscopy on EVs, several exosomal markers, siRNA and actin drugs.

Results: We found that the I‐BAR EVs are small vesicles of 200nm diameter in average mainly associated with CD81, CD9, ALIX and less CD63 but a subtype of these I‐BAR EVs are CD81 negative, wich are produced from the cell plasma membrane in a TSG101 independent manner and in an Arp2/3 dependent manner.

Summary/Conclusion: Our results thus reveal that these previously undescribed I‐BAR EVs represent a subset of plasma membrane microvesicles whose production depends on branched actin.

OD09.05. Characterising treatment‐induced exosome release reveals novel insights into biogenesis pathways and the pathogenesis of ovarian cancer

Elise H. Padbury, Department of Biological and Medical Sciences, Oxford Brookes University

Štefan Bálint, Kennedy Institute of Rheumatology, University of Oxford

Emanuela Carollo, Department of Biological and Medical Sciences, Oxford Brookes University

Esther Becker, Department of Physiology, Anatomy and Genetics, University of Oxford

David R F. Carter, Department of Biological and Medical Sciences, Oxford Brookes University

Introduction: Due to the high level of heterogeneity within Extracellular Vesicle (EV) subtypes, key questions in EV biogenesis remain unanswered. By analysing how external cues modulate the release of multivesicular body (MVB) derived EVs, we can begin to classify exosome subpopulations and dissect the pathways which underly their biogenesis. Interestingly, we have identified the transient receptor potential channel 3 (TRPC3) as a potential facilitator of Ca2+‐mediated exosome release by ovarian cancer cells, but its role in pathogenesis is unclear.

Methods: Using a pH‐sensitive CD81 live‐cell reporter, we directly visualised MVBs fusing with the plasma membrane (PM) in SKOV3 ovarian cancer cells by total internal reflection fluorescence (TIRF) microscopy. Basal MVB‐PM fusion was characterised and compared with fusion following stimulation with histamine, ionomycin or the TRPC3 activator GSK 1702934A. Protein content of basal and induced EVs was determined using label‐free quantitative mass spectrometry. Proliferation, migration and invasion assays were used to evaluate the phenotypic effect of TRPC3 activation on SKOV3 cells.

Results: Stimulation with TRPC3 activator, histamine and ionomycin increased the rate of MVB‐PM fusion in SKOV3 cells. Treatment‐induced differences were also apparent in the size, fluorescence and duration of fusion events, and may be suggestive of distinct exosome biogenesis pathways. We also report novel fusion dynamics including localised, synchronised MVB‐PM fusion. Proteomic profiling of treatment‐induced EVs revealed distinct protein compositions which provide a mechanistic insight into their biogenesis pathways. Finally, modulating the activity of TRPC3 altered the growth and behaviour of SKOV3 cells.

Summary/Conclusion: Our work provides novel insights into the dynamics of exosome biogenesis and the molecular factors which facilitate the release of exosome subpopulations, potentially linking exosome biogenesis with the pathogenesis of ovarian cancer.

OD09.06. CD47 interactions with exportin‐1 regulate targeting of m7G‐capped RNAs to extracellular vesicles

Sukhbir Kaur, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, USA

Alejandra Cavazos Saldana, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, USA

Jennifer D. D. Petersen, PhD, Section on Integrative Biophysics, Division of Basic and Translational Biophysics, Eunice‐Kennedy‐Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA

Anush Arakelyan, Section on Intercellular Interactions, Division of Basic and Translational Biophysics, Eunice Kennedy‐Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, USA

Abdel G. Elkahloun, Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, USA.

Leonid Margolis, Section on Intercellular Interactions, Division of Basic and Translational Biophysics, Eunice Kennedy‐Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, USA

Joshua Zimmerberg, Section on Integrative Biophysics, Division of Basic and Translational Biophysics, Eunice‐Kennedy‐Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA

Lisa M Jenkins, Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, USA

David G Jordan, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, USA

Andy D Tran, Confocal Microscopy Core Facility, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, USA

David D. Roberts, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, USA

Introduction: CD47 is a ubiquitously expressed membrane protein that binds the ligands thrombospondin‐1 (TSP‐1) and signal‐regulatory protein alpha. Previously, we have shown that CD47+ EVs contain distinct non‐coding RNAs, including miRNAs, relative to CD63+ and MHC1+ EVs released by the same cells suggesting that the packaging of noncoding RNAs into specific subpopulations of EVs is directed by CD47 (PMID: 29416092), The mechanisms by which CD47 directly or indirectly regulates which RNAs are packaged into EV remain unknown.

Methods: EVs released from WT and CD47‐ T cells were evaluated using miRNA sequencing, real‐time PCR and RNA‐immunoprecipitation. Interactions between CD47 and exportin‐1/Ran complex was identified by mass spectrometry and confirmed by using co‐immunoprecipitation, subcellular localization, flow cytometry, and confocal and electron microscopy.

Results: EV released from human CD47‐ T cells and in cd47‐/‐ mouse plasma were enriched in 5’‐7‐methylguanosine (m7G)‐capped miRNAs and mRNAs that depend on the exportin‐1/RanGTP pathway. Globally, more precursor/seed miRNAs than mature miRNAs exhibited shared CD47‐dependence between EVs and cells or uniquely differed in WT versus CD47‐ EVs. Therefore, CD47 may preferentially regulate trafficking of precursor rather than mature miRNAs into EVs. Knockdown of CD47 in WT cells or TSP1‐1 treatment correspondingly enhanced levels of capped‐RNAs released in EV and re‐expressing CD47 in null cells decreased their levels. Mass spectrometry and co‐immunoprecipitation identified specific interactions of CD47 with components of the exportin‐1/Ran nuclear export complex and its known cargos and between the CD47 cytoplasmic adapter ubiquilin‐1 and the exportin‐1/Ran complex. Interaction of CD47 with exportin‐1 was inhibited by leptomycin B, which inactivates exportin‐1 and increased levels of cap‐dependent RNAs in EV released from wild type but not CD47‐ T cells. We have further identified CD47 in a subset of vesicles within MVBs visualized by electron microscopy with immunogold labeling in WT cells. Treatment with LMB did not alter the number of MVBs or the abundance of CD47‐expressing vesicles therein. Therefore, some release of CD47‐expressing EVs occurs via the MVB pathway, but exportin‐1 does not regulate the release of CD47‐expressing EVs at the level of MVB biogenesis. CD47 generally limits RNA export in EVs rather than their cellular expression.

Summary/Conclusion: These findings indicate that CD47‐dependent TSP1‐1 signaling regulates levels of cap‐dependent pre‐miRNAs and mRNAs released in EVs at least in part through ubiquilin‐1‐ and GTP‐dependent physical interactions of CD47 with the exportin‐1/Ran transport complex.

OD09.07. Single‐cell extracellular vesicle secretion detection with a home‐use scanner

YAO LU, Dalian Institute of Chemical Physics, Chinese Academy of Sciences

Fengjiao Zhu, Dalian Institute of Chemical Physics, Chinese Academy of Sciences

Introduction: We reported a high‐throughput single‐cell EV secretion analysis method based on a domestic home use scanner without cell counting, which combines gold nanoparticle enhanced silver staining and Poisson distribution.

Methods: The hard silicon mold was prepared by photolithography, and PDMS was poured on the mold to prepared a high throughput microwell array chip for capturing single cells. By optimizing the cell density, the Poisson distribution can be used to count the number of single cells in the microchip. To visualize EVs, standard ELISA procedures were implemented. A desktop scanner was used to record and read the results.

Results: By combining gold nanoparticle enhanced silver staining with Poisson distribution, overcoming the dependence on large and expensive instruments and making this platform an ideal choice for single‐cell EV secretion analysis in resource‐limited environments. The platform consists of an antibody‐coated poly‐L‐lysine glass slide used to capture EVs and a high‐throughput microwell array chip used to capture single cells. The distribution of cells on the microwell chip obeys Poisson distribution, and ∼1000 single cells were reliably captured (1213±354, n = 9). CD9+CD63+EV and CD63+EV secreted from OSCC cell line, and OSCC primary cells were analyzed, which revealed the cell heterogeneity at the single‐cell level.

Summary/Conclusion: We successfully developed a high‐throughput single‐cell EV secretion analysis method based on a domestic home use scanner without cell counting. OSCC cells have significant heterogeneity in the number and secretion rate of EV secretion.

OD09.08. Unravelling intercellular communication between keratinocytes and melanocytes in the skin: extracellular vesicles and their role in the regulation of melanocyte function

Cécile Giordano, Institut Curie CNRS UMR144 Structure and membrane compartments

Ilse Hurbain, Institut Curie, CNRS UMR144, Structure et Compartiments Membranaires, Université Paris Sciences et Lettres, Paris, France. Institut Curie, CNRS UMR144, Plateforme d'imagerie cellulaire et tissulaire (PICT‐IBiSA), Université Paris Sciences et Lettres, Pari

Graça Raposo, PhD, Institut Curie, CNRS UMR144, Structure et Compartiments Membranaires, Université Paris Sciences et Lettres, Paris, France. Institut Curie, CNRS UMR144, Plateforme d'imagerie cellulaire et tissulaire (PICT‐IBiSA), Université Paris Sciences et Lettres, Pari

Gisela d'Angelo, Institut Curie, CNRS UMR144, Structure et Compartiments Membranaires, Université Paris Sciences et Lettres, Paris, France

Introduction: Extracellular vesicles (EVs), which facilitate the transfer of proteins, lipids and genetic material molecules between cells, are recognized as an additional mechanism for intercellular communication. In the epidermis, the communication between melanocytes and keratinocytes is tightly regulated to maintain skin homeostasis. Melanocytes synthetize the melanin pigment in melanosomes that are transferred to keratinocytes via an intricate dendritic network in order to color and photo‐protect the skin against UV‐B radiations. We have recently shown that EVs secreted by keratinocytes modulate pigment synthesis in melanocytes, the first step of melanogenesis. However, whether keratinocyte‐EVs play additional roles in melanogenesis is not known. We hypothesize that keratinocyte‐EV contribute to melanogenesis by promoting melanocyte dendricity for an efficient pigment transfer.

Methods: We combined here cell biology, optical and electron microscopy, biochemical, and molecular biology approaches.

Results: We show that keratinocyte‐EVs induce morphological changes of melanocytes, and increase their dendricity. Exploiting our previously identified keratinocyte‐EV content, we also show that keratinocyte‐EV depleted for Rac1 protein, a member of the Rho family of GTPases, do not affect melanocyte morphology, and fail to promote melanocyte dendricity. Finally, by electron microscopy, we observe an accumulation of mature melanosomes at the tip of the dendrites. We are currently investigating the mechanisms underlying such an effect.

Summary/Conclusion: Altogether our results support the view that Rac1 containing‐EVs released by keratinocytes provide a means for modulating melanocyte dendricity and morphology. They also put forward a new function of keratinocyte‐EVs in programing and instructing melanocytes so that they coordinate their functions: melanosome biogenesis, dendricity, and accumulation of mature melanosomes that will ultimately by transfer by melanocytes to keratinocytes. Importantly, dysregulation of these pathways could underlie pigmentary disorders like melanoma and skin carcinoma.

OD10.01. Extracellular vesicles from mesenchymal stromal cells combined with tissue engineering improve cardiac function, reduce fibrosis and modulate immune response in acute myocardial infarcted pigs

Marta Monguió‐Tortajada, Health Science Research Institute Germans Trias i Pujol (IGTP), Can Ruti Campus, Badalona, Spain

Cristina Prat‐Vidal, ICREC, Health Science Research Institute Germans Trias i Pujol (IGTP), Can Ruti Campus, Badalona, Institut d'Investigació Biomèdica de Bellvitge‐IDIBELL, CIBERCV, Instituto de Salud Carlos III, Spain

Daina Martínez‐Falguera, ICREC, Health Science Research Institute Germans Trias i Pujol (IGTP), Can Ruti Campus, Badalona, Department of Medicine, Universitat de Barcelona (UB),CIBERCV, Instituto de Salud Carlos III, Spain

Micaela Munizaga‐Larroudé, ICREC, Health Science Research Institute Germans Trias i Pujol (IGTP), Can Ruti Campus, Badalona, Department of Medicine, Universitat Autònoma de Barcelona (UAB), CIBERCV, Instituto de Salud Carlos III, Spain

Carolina Soler‐Botija, ICREC, Health Science Research Institute Germans Trias i Pujol (IGTP), Can Ruti Campus, Badalona, CIBERCV, Instituto de Salud Carlos III, Spain

Miriam Moron‐Font, REMAR‐IVECAT Group, Health Science Research Institute Germans Trias i Pujol (IGTP), Can Ruti Campus, Badalona, Spain

Adriana Cserkoova, ICREC, Health Science Research Institute Germans Trias i Pujol (IGTP), Can Ruti Campus, Badalona, Spain

Antoni Bayes‐Genis, ICREC, Health Science Research Institute Germans Trias i Pujol (IGTP), Can Ruti Campus, Badalona, CIBERCV, Instituto de Salud Carlos III; Cardiology Service, Germans Trias i Pujol University Hospital; Department of Medicine, UAB, Spain

Francesc E. Borràs, REMAR‐IVECAT Group, Health Science Research Institute Germans Trias i Pujol (IGTP), Can Ruti Campus; Nephrology Service, Germans Trias i Pujol University Hospital, Badalona, Spain

Santiago Roura,ICREC, Health Science Research Institute Germans Trias i Pujol (IGTP), Can Ruti Campus, Badalona, CIBERCV, Instituto de Salud Carlos III, Spain

Carolina Gálvez‐Montón, ICREC, Health Science Research Institute Germans Trias i Pujol (IGTP), Can Ruti Campus, Badalona, CIBERCV, Instituto de Salud Carlos III, Spain

Introduction: Accumulating evidence supports the potential of extracellular vesicles (EVs) from mesenchymal stromal cell (MSC) as a therapy for cardiac healing after myocardial infarction (MI). Nevertheless, neither their efficient administration nor their therapeutic mechanisms are fully elucidated. Here, we evaluate the preclinical efficacy of a tissue engineering approach to locally deliver porcine cardiac adipose tissue MSCs (cATMSC‐EV) in an acute MI pig model.

Methods: Pigs (n = 24) were subjected to permanent ligation of the coronary artery. After 30 min, animals were randomized to Untreated or treated groups with a tissue engineered graft composed of a decellularized pericardial scaffold filled with peptide hydrogel and cATMSC‐EV purified by size exclusion chromatography (EV‐treated group) or buffer (Control group) placed over the post‐MI myocardium. Cardiac troponin levels and cardiac MRI revealed consistent myocardial damage and infarct size in all animals.

Results: After 30 days, cardiac function was significantly improved with less ventricle dilatation in the EV‐treated group, indicating less myocardial remodelling. MRI showed reduced scar size in EV‐treated animals, correlating with a decrease of fibrosis in the distal zone and increased vascular density in the infarct core. Less macrophage infiltration and more anti‐inflammatory phenotype (CD163+CD73+) were found in the infarct of treated animals. Surprisingly, local delivery of cATMSC‐EV also triggered a systemic effect, reducing PBMC increase 2‐days post‐MI and modulating systemic CD73+ and CCR2+ monocytes, related to immunomodulation and fibrosis modulation.

Summary/Conclusion: These results highlight the clinical potential of cATMSC‐EV in modulating key features of ischemic injury and promoting cardiac repair after MI.

OD10.03. MSC exosomes promote cartilage and subchondral bone repair in a porcine osteochondral defect model

Wei Seong Toh, PhD, Faculty of Dentistry, National University of Singapore

Shipin Zhang, PhD, Faculty of Dentistry, National University of Singapore

Keng Lin Wong, MD, Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore

Xiafei Ren, MD, Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore

Ruenn Chai Lai, PhD, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore

Sai Kiang Lim, PhD, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore

James Hoi Po Hui, MD, Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore

Introduction: We had previously reported the efficacy of human mesenchymal stromal cell (MSC) exosomes in repair of critical‐size osteochondral defects in both rats and rabbits. However, small animals unlike humans have inherent tendency for spontaneous healing of cartilage defects in addition to differences in size and biomechanics. To enable clinical translation of MSC exosomes, we therefore proposed a validation of the efficacy of MSC exosomes in a large animal model.

Methods: Bilateral osteochondral defects measuring 6mm diameter and 1mm depth were surgically created on the weight‐bearing area of the medial femoral condyles of 24 knees in 12 micropigs. Immediately after surgery and at days 8 and 15 post‐surgery, 6 micropigs in exosome/HA group received sequential administration of 1mg exosomes in 1ml phosphate‐buffered saline (PBS) followed by 1ml hyaluronic acid (HA; Synvisc(R)) in both knees, whereas the other 6 micropigs in the HA group received 1ml of PBS followed by 1ml HA in both knees. Except for magnetic resonance imaging (MRI) performed on day 15, 2 and 4 months, macroscopic, histological and micro‐computed tomography (micro‐CT) assessments were performed at 4 months.

Results: As early as day 15 post‐surgery, exosome/HA treated defects had a better MRI score of 4.46 than the score of 3.63 by HA treated defects (P = 0.017). The MRI scores for exosome/HA treated defects continued to improve, and were consistently higher than that for HA treated defects at both 2 months (7.83 vs 5.79; P = 0.023) and 4 months (9.25 vs 6.71; P = 0.024). At 4 months, exosome/HA treated defects had significantly better ICRS macroscopic score (9.22 vs 7.25; P = 0.008) and histological score (79.71 vs 65.10; P = 0.032) than HA treated defects. Micro‐CT analysis further revealed structural improvements in the subchondral bone with significantly higher BV/TV (49.38% vs 39.73%; P = 0.046) and Tb.Th (0.18mm vs 0.13mm; P = 0.009), but not Tb.N and Tb.Sp in exosome/HA treated defects, compared to HA treated defects.

Summary/Conclusion: Our results show that MSC exosomes and HA combination administered at a clinically acceptable frequency of three intra‐articular injections can promote osteochondral repair with significantly improved morphological and histological outcomes in a clinically relevant porcine model. Our study highlights a clinically translatable protocol utilizing MSC exosomes as an off‐the‐shelf and cell‐free therapeutic for patients with osteochondral injuries and potentially osteoarthritis.

OD10.04. Anti‐fibrotic effects of Membrane Particles from mesenchymal stromal cells in renal ischemia reperfusion injury mouse model

Ana Merino, Nephrology and Transplantation, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, The Netherlands

Zhaoyu Du, Internal medicine department, Erasmus MC

Anusha Shankar, Internal Medicine department, Erasmus MC

Sander Korevaar, Internal Medicine department, Erasmus MC

Derek Reijerkerk, Internal Medicine department, Erasmus MC

Carla Baan, Nephrology and Transplantation, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, The Netherlands

Marlies Reinders, Internal Medicine Department, Erasmus MC

Martin Hoogduijn, Nephrology and Transplantation, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, The Netherlands

Introduction: Membrane particles (MP) are nanovesicles artificially generated by extrusion of the mesenchymal stromal cell (MSC) membranes. MP were designed to circumvent the risks of MSC therapy such as a poor biodistribution due to their large size and unknown mechanistic behaviour after infusion, while keeping the reparative and immunomodulatory properties of MSC. We have demonstrated earlier that MP have immunomodulatory properties, endothelial regenerative capacity, and antifibrotic effect on lung fibroblasts in vitro. In this study, the aim is to demonstrate the efficacy of MP as an antifibrotic treatment on a renal ischemia reperfusion injury (IRI) mouse model.

Methods: Ischemia injury was performed by clamping the right kidney of the mice for 37 minutes. The MP were intravenously infused 3–5 hours after ischemia. Animals were sacrificed and the kidney harvested 3 days after renal IRI. Four groups of mice were analysed: Sham, IRI, IRI+MP derived from 1 million of MSC, and IRI+MP derived from half million of MSC. Gene expression of proinflammatory cytokines was measured in the kidneys such as IL6, and TNFa; kidney injury marker KIM1, infiltration of monocytes and lymphocytes; and profibrotic markers such as TGFb, PAI‐1, fibronectin, tenascin C, collagen I, and III.

Results: We found no difference between IRI mice treated with MP and IRI untreated mice respect to the proinflammatory markers IL6, TNFa, injury marker KIM‐1 or infiltration of monocytes and lymphocytes. IRI induced an upregulation of the gene expression of profibrotic markers such as TGFb and PAI‐1, and proteins from the extracellular matrix. Interestingly, both doses of MP significantly decreased the expression of the TGFb, PAI‐1 and the main extracellular matrix proteins involved in fibrogenesis.

Summary/Conclusion: Our findings show that MP have antifibrotic effects on renal IRI, opening a new potential avenue for treatment of organ fibrosis.

OD11.01. Extracellular vesicles are involved in circulation of Hepatitis B Virus RNA in infected cells’ supernatant and patients’ serum

Delphine Bousquet, INSERM U1052‐ Cancer Research Center of Lyon (CRCL), Lyon, France

Doohyun Kim, INSERM U1052‐ Cancer Research Center of Lyon (CRCL), Lyon, France

Annie Adrait, Univ. Grenoble Alpes, CEA, INSERM, IRIG, BGE, Grenoble, France

YOHAN COUTE, Univ. Grenoble Alpes, CEA, INSERM, IRIG, BGE, Grenoble, France

Maria Guadalupe Martinez, INSERM U1052‐ Cancer Research Center of Lyon (CRCL), Lyon, France

Alexia Paturel, INSERM U1052‐ Cancer Research Center of Lyon (CRCL), Lyon, France

Aaron Hamilton, Roche Molecular Diagnostics, Pleasanton, CA

Marintha Heil, Roche Molecular Diagnostics, Pleasanton, CA

Massimo Levrero, INSERM U1052‐ Cancer Research Center of Lyon (CRCL), Lyon, France

Barbara Testoni,INSERM U1052‐ Cancer Research Center of Lyon (CRCL), Lyon, France

Fabien Zoulim, INSERM U1052‐ Cancer Research Center of Lyon (CRCL), Lyon, France

Introduction: Despite the availability of effective vaccine, chronic Hepatitis B virus (HBV) infection remains a global health burden. Current antiviral strategies (nucleos(t)ide analogues, NUCs) are unable to eliminate the virus from infected hepatocytes and, thus, achieve a complete cure. Relevant and non‐invasive biomarkers are necessary to ameliorate patients’ management and the evaluation of new therapies. In this study, we aim at better characterizing the compartments containing extracellular HBV RNAs, which were recently proposed as a new surrogate marker of intrahepatic viral activity.

Methods: Supernatant from HBV‐infected HepG2‐NTCP cells, treated or not with NUCs, was collected and processed through sucrose/iodixanol gradient separation, to allow physical separation of extracellular vesicles (EVs) and viral particles according to their buoyant density. Viral and EVs‐associated proteins were analyzed by Western Blotting and Elisa, while HBV RNAs were detected by specific digital droplet (dd)PCR. NTA and mass spectrometry‐based proteomic analyses were used to further characterize the EVs components.

Results: Elisa assays for viral surface proteins after gradient separation showed that virions were found in fractions corresponding to a density of 1,21‐1,25 g/ml. Western Blotting for CD9 and CD63, markers of exosomes/EVs were detected only in lower density fractions (1,13"1,19 g/m), which were deprived of viral proteins. Interestingly, HBV RNAs were detected not only in virion‐like particles but also in lighter gradient fractions, suggesting that EVs could contribute to carry the circulating HBV RNA pool. No significant difference was found in NUC‐treated vs untreated samples. To further investigate the nature of EVs detected in light density fractions, NTA analysis was performed, showing that these fractions were indeed containing EVs in size spanning from 30 to 150 nm. Finally, proteomic analyses of the same fractions revealed the presence of specific markers of exosomes (CD63, CD9, TSG101 or HSC70).

Summary/Conclusion: Our study will shed light on the molecular biology of serum HBV RNA secretion and will aid the development of serum HBV RNA as a novel biomarker for chronic HBV infection.

OD11.02. Human cytomegalovirus infection modifies trophoblastic small extracellular vesicles secretion and composition, facilitating viral dissemination in recipient cells

Mathilde Bergamelli, Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), Université de Toulouse, INSERM, CNRS, UPS, Toulouse, France

Hélène Martin, Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), Université de Toulouse, INSERM, CNRS, UPS, Toulouse, France

Jean‐Michel Mansuy, CHU Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, France

Ilse Hurbain, Institut Curie, CNRS UMR144, Structure et Compartiments Membranaires, Université Paris Sciences et Lettres, Paris, France. Institut Curie, CNRS UMR144, Plateforme d'imagerie cellulaire et tissulaire (PICT‐IBiSA), Université Paris Sciences et Lettres, Pari

Jacques Izopet, CHU Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, France.

Graça Raposo, PhD, Institut Curie, CNRS UMR144, Structure et Compartiments Membranaires, Université Paris Sciences et Lettres, Paris, France. Institut Curie, CNRS UMR144, Plateforme d'imagerie cellulaire et tissulaire (PICT‐IBiSA), Université Paris Sciences et Lettres, Pari

Daniel Gonzalez‐Dunia, Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), Université de Toulouse, INSERM, CNRS, UPS, Toulouse, France

Gisela d'Angelo, Institut Curie, CNRS UMR144, Structure et Compartiments Membranaires, Université Paris Sciences et Lettres, Paris, France

Cécile Malnou, Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), Université de Toulouse, INSERM, CNRS, UPS, Toulouse, France

Introduction: Congenital infection by human Cytomegalovirus (hCMV) is a major public health issue because of its high incidence and the variety of induced neurological sequelae in neonates but despite intense research, pathophysiology of hCMV infection is not yet fully understood. As they participate in mother‐fetus communication, we examined the hypothesis that placental small extracellular vesicles (sEVs) could contribute to placental and fetal injury.

Methods: sEV from trophoblastic cells infected or not by hCMV were purified by differential ultracentrifugation and density gradient. sEV structure and composition were further analyzed by flow cytometry, nanoparticle tracking analysis, immune‐electron microscopy and proteomics. Finally, impact of trophoblastic sEV on hCMV permissiveness in recipient cells was examined.

Results: We observed that hCMV infection increased secretion of trophoblastic sEV that presented smaller size compared to non‐infected condition. Protein content of trophoblastic sEV was modified by hCMV infection, with the presence of viral proteins and with modification in cellular protein composition, suggesting that they could play a role in “priming” infection in recipient cells and thus facilitate further viral infection. Trophoblastic sEV were internalized in fetal cells with time and dose effect. Incubation of fetal cells with sEV from trophoblastic cells infected by hCMV increased significantly the infection rate compared to fetal cell incubated with sEV prepared from non‐infected cells.

Summary/Conclusion: In conclusion, we showed that hCMV infection modifies both trophoblastic sEV secretion and protein content, therefore priming fetal recipient cells for a future infection. Hence sEVs may be crucial mediators that could play an important role in maternal‐fetal transmission of hCMV by facilitating viral dissemination towards the fetus.

OD11.03. Administration of Amniotic Fluid derived Extracellular Vesicles in COVID‐19 Long Hauler Patients

Maria Ines Mitrani, M.D., Ph.D., Organicell Regenerative Medicine

Michael A. Bellio, Ph.D., Organicell Regenerative Medicine

Gwendolyn Haskell, Pharma D, Organicell Regenerative Medicine

George C. Shapiro, M.D., Organicell Regenerative Medicine

Introduction: Post‐COVID‐19 infection symptoms such as mental fog, tachycardia, and extreme fatigue are just a few of the symptoms wreaking havoc on patients’ lives. Patients with long‐term sequelae following COVID‐19 are being called long‐haulers. To date, long‐haulers are receiving little to no guidance from physicians on their lingering COVID‐19 symptoms with no treatment options available. Zofin is an acellular biologic that contains the extracellular vesicle (EV) fraction of human amniotic fluid and is under investigation for use as a COVID‐19 therapeutic. We have recently completed 4 single patient emergency/compassionate use eINDs investigating amniotic‐fluid derived EVs in COVID‐19 long haulers under our approved parent IND 19881 to demonstrate safety and feasibility.

Methods: FDA and IRB approval were obtained for these single patient cases investigating Zofin treatment in an outpatient setting. IND approval numbers were: eIND 25888, eIND 26560, eIND 26561, IND 26821. The therapeutic intervention, Zofin, is an allogenic, acellular biologic derived from human amniotic fluid containing 2.3 × 10^11 particles/mL with 70–80% positive expression of exosome markers CD63 and CD81. Zofin was administered intravenous as 1mL doses on baseline, day 4 and day 8 (3 doses). The approved clinical protocol included patient follow up with biomarker testing and chest X Rays (CXR) on Day 0, 4, 8, 14, 21, 28, and 60. The primary objective of these studies was to demonstrate the safety of Zofin. All patients tested positive for COVID‐19 a minimum of 2 months prior to treatment.

Results: Administration of the EV product was shown to be safe in all patients. One patient had detectable bilateral pneumonia at baseline treatment that was present 2 months after discharge from the hospital. On Day 14, repeated CXR showed improvement of the patchy peripheral pulmonary opacities. Then, the CXR report on Day 21 noted that the patient's lungs were clear. Furthermore, this patient experienced extreme shortness of breath prior to treatment with baseline pulse oximetry readings were 95% when seated and 93–94% when supine on room air. Improvements in fatigue were noted soon after the second dose and the patient was able to exercise to fatigue, at which time, he did not desaturate and remained at 97–98% on room air.

Summary/Conclusion: The single patient IND studies were completed without any reported adverse events or safety concerns. Furthermore, these completed studies demonstrate the feasibility and a therapeutic potential of amniotic fluid‐derived EVs for COVID‐19 long hauler intervention.

OD12.01. Tunability of platelet‐derived extracellular vesicles

Mari Palviainen, PhD, University of Helsinki

Puutio Johanna, University of Helsinki

Johannes A. Eble, University of Munster

Masood Kamali‐Moghaddam, University of Uppsala

Pia Siljander, University of Helsinki

Introduction: The proteome of anuclear platelets comprises >5000 proteins and is impacted e.g. by age and disease. The platelet secretome of over 300 proteins contains e.g. growth factors and immunomodulatory proteins and therefore, platelet products, such as platelet rich plasma are used in regenerative medicine. Platelets also release extracellular vesicles (EVs), both constitutively and upon activation. We studied 1) the tunability of platelet EVs by agonists engaging different critical platelet signaling pathways, 2) the characteristics of these EVs, and 3) macrophage responses to these EVs.

Methods: Isolated human platelets were activated by CRP (engaging GPVI), rhodocytin (CLEC‐2), and by thrombin and collagen co‐stimulus (all thrombin and collagen receptors). Agonist concentrations and the time of activation were optimized for maximal EV yield in the shortest possible time. EVs were isolated by ultracentrifugation using cushioned density‐gradient, and then characterized by particle concentration, size distribution (NTA) and marker protein expression (Exoview R100). The inflammation‐linked proteome of EVs was analyzed in a targeted array using Olink technology. Macrophages differentiated from THP‐1 cells were treated with equal numbers of EVs for 6 and 24 hours, and the secretome of macrophages was analyzed with Luminex technology targeted for cytokines and chemokines

Results: Although more CD63+/CD9+ EVs were generated from activated platelets when compared to non‐activated platelets, activation by rhodocytin resulted in a markedly lower EV yield compared to CRP or TC co‐stimulus. The signaling pathways engaged during activation significantly impacted on the inflammation‐related molecular cargo. These findings were further supported by the variability of the agonist‐, but also time‐dependent changes in the secretome of the EV‐treated macrophages reflecting the tunability of platelet‐derived EVs

Summary/Conclusion: Upon activating platelets, agonists have distinct impacts on the characteristics of secreted EVs, their proteome and functionality. These results imply that by differential activation it is possible to create tunable EVs e.g. for immunomodulatory purposes.

OD12.02. microRNA Enrichment of Extracellular Vesicle Content for Diabetic Wound Treatment

Ricardo C. de Abreu, Department of Molecular Genetics, Faculty of Sciences and Engineering, Maastricht University, Maastricht, Netherlands

Cristiana Ramos, Coimbra Chemistry Centre, Chemistry Department, Faculty of Science and Technology, University of Coimbra, Portugal

Clarissa Becher, Biomaterials and Stem‐Cell Based Therapeutics group, CNC‐ Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Miguel Lino, Biomaterials and Stem‐Cell Based Therapeutics group, CNC‐ Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Carlos Jesus, Biomaterials and Stem‐Cell Based Therapeutics group, CNC‐ Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Patrícia Martins, Biomaterials and Stem‐Cell Based Therapeutics group, CNC‐ Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Inês Albino, Biomaterials and Stem‐Cell Based Therapeutics group, CNC‐ Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Marta Barão, Biomaterials and Stem‐Cell Based Therapeutics group, CNC‐ Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Maria João Moreno, Coimbra Chemistry Centre, Chemistry Department, Faculty of Science and Technology, University of Coimbra, Portugal

Hugo Fernandes,Biomaterials and Stem‐Cell Based Therapeutics group, CNC‐ Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Paula da Costa martins, Department of Molecular Genetics, Faculty of Sciences and Engineering, Maastricht University, Maastricht, Netherlands

Lino FerreiraBiomaterials and Stem‐Cell Based Therapeutics group, CNC‐ Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Introduction: Extracellular vesicles (EVs) have been used for tissue regeneration but their native cargo may be insufficient to elicit a therapeutic effect. Here we describe a method to load small EVs with therapeutically active miRNAs and their application in a diabetic wound healing mouse model.

Methods: Electroporation, saponin, cholesterol, freeze‐thaw and Exo‐Fect™ were tested for their capacity to load EVs with a fluorescently‐labelled miRNA. Loading efficiency was calculated based on the percentage of total fluorescence in the EV fraction and confirmed by quantitative PCR. To test the functionality of miRNA‐transfected EVs we used a HEK‐293T cell line was transfected with miRNA‐loaded EVs. This line constitutively expresses mCherry, which is inhibited by the presence of functional miRNA. The internalization and intracellular kinetics of these EVs were analysed in endothelial cells. Finally, EVs were loaded with a pro‐survival miRNA and topically administered (2x/day for 10 days) on wounds of diabetic mice.

Results: Our data shows that Exo‐Fect™ is the most effective strategy to load EVs with >50% loading efficiency. This was then shown to be functional in a reporter cell line, where transfection with microRNA‐loaded EVs decreased reporter signal by 30%. Furthermore, modulated EVs were shown to be less signalled for lysosomal degradation than their native counterparts. In vivo, miRNA‐loaded EVs were able to improve wound healing in diabetic mice, partially by enhancing wound vascularization.

Summary/Conclusion: Our results show that Exo‐Fect™ is an efficient and functional way of loading microRNAs into EVs. Further, these modulated EVs can be used in vitro and in vivo as effective delivery agents.

OD12.03. Exploration of blood‐cerebrospinal fluid barrier targeted extracellular vesicles in brain drug delivery

Marie Pauwels, VIB‐UGent

Adam Ceroi, VIB‐UGent

Nele Plehiers, VIB‐UGent

Caroline Van Cauwenberghe, VIB‐UGent

Sophie Steeland, VIB‐UGent

Elien Van wonterghem, VIB‐UGent

Griet Van Imschoot, VIB‐UGent

Sriram Balusu, PhD, VIB‐UGent

Florencia Linero, VIB‐UGent

Imre Mäger,Department of Paediatrics, University of Oxford, Oxford, United Kingdom.

Lien Van Hoecke, VIB‐UGent

Roosmarijn E. VandenbrouckeVIB ‐ Ghent University

Introduction: Successful treatment of neurological diseases is hampered by the presence of tightly regulated nervous system (CNS) barriers that restrict drug delivery to the brain. Up until now, most delivery strategies have focused on the blood‐brain barrier, while targeting the blood‐cerebrospinal fluid barrier (BCSFB) remains largely unexplored. However, the presence of transporting mechanisms, extensive secretory activity and strong vesicular trafficking indicate that BCSFB targeting strategies might have great potential. Interestingly, an increasing amount of data suggest that extracellular vesicles (EVs) (i.e. membrane derived vesicles that transfer biological cargoes between cells) naturally cross the CNS barriers. The low immunogenicity, the relatively high stability; and the cell targeting capacities of EVs, urged us to explore the BCSFB crossing capacity of EVs and their potential as brain delivery system.

Methods: Here, we genetically decorated HEK293T cell‐derived EVs with new BSCFB targeting ligands. Additionally, BCSFB epithelial cell‐derived EV characteristics as well as their homing capacity and protein content are currently under investigation using biodistribution assay, flow cytometry analyses, and in vitro setups.

Results: Our in vivo biodistribution studies indicate that BSCFB targeted EVs are a promising strategy for EV mediated brain delivery upon systemic injection. Moreover, specific EV enrichment at the BCSFB was observed by flow cytometry and in vitro BCSFB assays were used to further study the barrier crossing capacity.

Summary/Conclusion: In conclusion, our results indicate that BSCFB targeted EVs have great potential as brain drug delivery system. Further research is ongoing to characterize the underlying molecular mechanisms and possible loading strategies to fully exploit the potential of EVs as brain drug delivery vehicle.

OD12.04. Exosome Topical Therapy Delivered In Bioinspired Synthetic Protein Hydrogel Enhances Cutaneous Healing Of Diabetic Wounds

Juan F. Cortes, PhD, NIDCR, NIH

Joseph Kuhn, MD, Hansjorg Wyss Department of Plastic Surgery, NYU Langone Health, New York, NY

Priya Katyal, MDPhD, Chemical and Biomolecular Engineering, NYU Tandon School of Engineering

Michael Meleties, Chemical and Biomolecular Engineering, NYU Tandon School of Engineering

Iraines De La Cruz, Hansjorg Wyss Department of Plastic Surgery, NYU Langone Health

Bibi Subhan, Hansjorg Wyss Department of Plastic Surgery, NYU Langone Health

Jin Montclare, Chemical and Biomolecular Engineering, NYU Tandon School of Engineering

Piul Rabbani, New York University School of Medicine

Introduction: We have used human bone marrow multipotent stromal cells (hBMSCs) and their secreted exosomes (Exo) to successfully promote wound closure in diabetic animal models of delayed healing. However, safe and easy delivery platforms that maintain exosome efficacy are necessary to for clinical translation. Here, we describe the development of Exo‐Q, a thermoresponsive soft protein matrix loaded with hBMSC‐Exo, that accelerates wound closure in a Type II diabetes model.

Methods: We isolated exosome preps by differential ultracentrifugation of conditioned media from hBMSCs, prior to detailed characterization. We synthesized Q, an engineered variant of the coiled‐coil domain of cartilage oligomeric matrix protein, and incorporated 3 × 109 exosomes during gelation to yield Exo‐Q hydrogels. We analyzed Exo‐Q using transmission electron microscopy (TEM) and rheology. using excisional stented wounds on 16 weeks old LepRdb/db mice, we either topically pipetted exosome preps or applied Exo‐Q hydrogels. We observed wounds for closure and collected intermediate time point tissues for biomolecular analysis and histology.

Results: Q self‐assembles into a fibrous matrix at low temperatures and exhibits an upper critical solution temperature phase behavior. Exo‐Q entangled protein fibers uniformly interspersed with Exo in TEM and increased Q storage modulus, indicating increased elasticity of a pliable hydrogel. Exo‐Q solubilizes at skin wound temperature (∼31°C) for sustained delivery of Exo into wounds, without invasive/painful routes. Pipetted single doses 1 × 109 or 3 × 109 Exo at post‐operative day 1 (POD1) demonstrated non‐significant changes in closure time (30.25±1.5 and 27±2 days, respectively, n = 4) vs PBS ‐treated diabetic wounds. Exo‐Q application at POD1 decreased time to closure of diabetic wounds to 17±1.4 days vs 28±1.5 days for Q vehicle alone (p < 0.01, n = 3), and correspondingly reduced wound burden relative to that with Q alone. Exo‐Q administration generated extensive CD31+ neovascularization in large areas of granulation tissue in the Leprdb/db diabetic wound bed by POD10. Exo‐Q does not interfere with wound healing progression. A single Exo‐Q dose resulted in upregulated gene expression of angiogenic and wound healing associated factors VEGF, SDF1 and PDGF in diabetic wound beds, compared to Q vehicle‐only treated wounds (all p <.05, n‐3).

Summary/Conclusion: Exo‐Q is an efficacious, translatable therapy that can reverse pathologic healing of diabetic wounds. Future iterations can include drugs for compound therapeutic hydrogels

OD12.05. Extracellular vesicles derived from human liver‐stem cells improve fibrosis and inflammation associated with non‐alcoholic steatohepatitis and modified lncRNA expression profile

Stefania Bruno, sbruno, Department of Medical Sciences

Giulia Chiabotto, Department of Medical Sciences, University of Torino

Elena Ceccotti, Department fo Medical Sciences, University of Torino

Chiara Pasquino, Molecular Biothecnology Center

maria Beatriz Herrera Sanchez, Molecular Biothecnology Center

Marta Tapparo, Molecular Biothecnology Center

Cristina Grange, Department of Medical Sciences, University of Turin

Massimo Cedrino, Molecular Biothecnology Center

Giovanni Camussi, University of Turin

Introduction: We recently demonstrated that non‐alcoholic steatohepatitis (NASH) is improved by treatment with human liver stem cells (HLSCs). The aim of the present study was to evaluate whether EVs released by HLSCs can influence the progression of NASH and modify the hepatic lncRNA expression profile.

Methods: EVs were obtained by ultracentrifugation and characterized in accordance with ISEV guidelines. NASH has been induced through a methionine‐choline‐deficient diet. EV‐treatment started at week 2 (2.5 × 109 EVs twice a week) and ended at week 4, when mice were sacrificed. Liver fibrosis and inflammation have been evaluated by histological and molecular analyses, using specific histological staining, array and real time PCR analyses. LncRNAs, known to be involved in inflammatory response, have been evaluated by PCR array.

Results: EVs significantly improved liver function and reduced liver fibrosis and inflammation, at both morphological and molecular levels. In particular, we observed that 28 out of 29 fibrosis‐associated genes up‐regulated in livers of NASH mice were significantly downregulated by EV‐treatment. Moreover, the anti‐inflammatory effect of EV‐treatment was demonstrated by the reduction of inflammatory cells accumulated in the liver as seen by immunofluorescence. Whereas inflammatory infiltrates were present in the liver of NASH mice, almost no leukocytes were observed in mice treated with EVs. The increase of IL‐10 expression level in livers of EV‐treated NASH mice confirmed the anti‐inflammatory effect of EVs. Evaluation of lncRNA expression profile indicated that 15 lncRNA were modulated by EV‐treatment.

Summary/Conclusion: HLSC‐derived EVs display anti‐fibrotic and anti‐inflammatory effects in a model of chronic liver disease, leading to an improvement of liver function. In addition, EV‐treatment induces changing in the expression of lncRNAs known to be involved in inflammation, indicating their possible contribution to the anti‐inflammatory EV‐effect.

OD12.06. Small Extracellular Vesicles Derived from Bone Marrow Stromal Cells Enhance Proliferation of Intestinal Stem Cells in Mice after Radiation

Lalitha S Y Nanduri, Department of Radiation Oncology, Albert Einstein College of Medicine

Shobhit Bhansali, Department of Radiation Oncology, Albert Einstein College of Medicine

Phaneendra K. Duddempudi, Department of Biochemistry, Albert Einstein College of Medicine

Shahin Shajahan, Department of Radiation Oncology, Albert Einstein College of Medicine

Brett Bell, Department of Radiation Oncology, Albert Einstein College of Medicine

Tatyana L. Tchaikovskaya, Department of Radiation Oncology, Albert Einstein College of Medicine

Weng‐Lang Yang, Department of Radiation Oncology, Albert Einstein College of Medicine

Shilpa Kulkarni, Department of Radiation Oncology, Albert Einstein College of Medicine

Chandan Guha, Department of Radiation Oncology, Albert Einstein College of Medicine

Introduction: Radiation damage to the intestine leads to acute and delayed toxicities after abdomino‐pelvic radiation therapy. We have previously demonstrated that bone marrow stromal cells (BMSCs) rescue intestine injury in mice after exposure to lethal‐dose radiation. This beneficial effect of BMSCs may be through secreting paracrine factors. Here, we investigated whether small extracellular vesicles derived from BMSC (BMSC‐sEVs) could promote proliferation and differentiation of intestinal stem cells in mice after radiation.

Methods: BMSC's culture supernatant was subjected to sequential ultracentrifugation at 300g for 10 min, 16,500g for 20 min, and 120,00g for 70 min to obtain sEVs. Enteroids were generated by isolating small intestinal crypts from C57BL/6 mice and seeding them into matrigel for culturing 5–7 days with the supplement of EGF, R‐spondin 1, and noggin. For the in vivo study, C57BL/6 mice were irradiated at 11 Gy with 2.5% bone marrow shielding. On days 1 and 3 after irradiation, vehicle and BMSC‐sEVs (100 μg/mouse) were injected intraperitoneally to the mouse (n = 3‐4/group). On day 4, the intestine was harvested for histologic analyses.

Results: An average of 1.6 × 10^10 sEV particles were obtained from 1 million BMSCs, which were < 200 nm in size and positive for CD9 and CD63. When enteroids were irradiated at 5 Gy, there was 34,9% survival after 4 days. However, addition of BMSC‐sEVs at 1, 10, and 100 μg/ml after irradiation increased the survival to 46.3%, 80.4%, and 76.8%, respectively (p < 0.001, unpaired t‐test). In the histologic analysis of intestines in the irradiated mice, BMSC‐sEV treatment significantly increased the numbers of crypts (7.3 vs. 4.7 crypts/mm, p < 0.007), EdU‐positive proliferation cells (2.56 vs. 0.93 crypts/mm, p < 0.001), Olfm4‐positive stem cells (11.8 vs. 6.4 crypts/mm, p < 0.001), and Lysozyme 1‐positive Paneth cells (10.1 vs. 3.5 crypts/mm, p < 0.001) compared to vehicle‐treated mice.

Summary/Conclusion: BMSC‐sEVs can rescue the survival of cultured enteroids and improve the intestinal stem cell proliferation and differentiation in the mice after exposure to irradiation. BMSC‐sEVs can be a potential mitigator for treating the radiation‐induced intestine injury.

OD13.01. PSMA, STIP1 and PPIA Detection in Urine‐Derived Extracellular Vesicles in Multiple Cancers

Lyssa Dimapanat, Columbia University Irving Medical Center

Alex J. Rai, PhD, DABCC, FAACC, Columbia University Irving Medical Center

Introduction: Biofluids are a favorable sample type for the identification of novel cancer biomarkers. In particular, urine is convenient, non‐invasively obtained, and available in large sample volumes. It can recapitulate biological information typically obtained from tissue biopsies. We are interested in exploring protein biomarkers in urine extracellular vesicles (EV). Recent studies have revealed the critical role of EV‐mediated intercellular communication and in regulating cancer processes including tumorigenesis and metastasis.

Methods: We isolated urine EVs using an optimized workflow based on serial centrifugation. The presence of EVs was confirmed by detection of canonical EV markers (ALIX, flotillin, and ACTN4) using western blotting. Nanoparticle tracking analysis and transmission electron microscopy revealed particle distribution and EV size. We surveyed 117 cancer samples and 23 control samples for the presence of three candidate protein markers identified in our earlier studies: PSMA, STIP and PPIA. Cancer samples were categorized into five different groups: genitourinary (GU), gastrointestinal (GI), brain, lymphomas and leukemias.

Results: EVs were isolated from urine samples. PSMA was detected in 62% (73/117) of samples across the various cancer types, and only 2 of 23 controls. Highest PSMA signals were obtained from GU cancers (prostate and bladder), with higher levels in metastatic disease. STIP1 was detectable in 29% (35/117), and PPIA in 5% (6/117) of the cancer samples. STIP1 was detected at highest levels in GI cancers (colorectal and liver), while PPIA was restricted to few samples from patients with higher tumor burden.

Summary/Conclusion: Our candidate biomarkers are detectable in urine EVs and can fulfill different unmet clinical needs for patient management. We propose that PSMA has potential application for monitoring therapeutic response in GU cancers, STIP1 may be useful for GI cancer subtyping, and PPIA's restricted detection may define a subset of aggressive cancers.

OD13.02. Clinical significance of circulating exosomal PD‐L1 in solid and hematological cancers

Valentin Vautrot, CGFL/ INSERM1231

marine Cordonnier, INSERM1231

Charlée Nardin, CHU Besançon

François Aubin, CHU Besançon

Cedric ROSSI, CHU Dijon/ INSERM Equipe Epi2THM

RENE‐OLIVIER casasnovas, CHU Dijon/ INSERM1231

Carmen Garrido, INSERM1231

jessica gobbo, CGFL/ INserm 1231

Introduction: Nowadays, immunotherapy is a real therapeutic revolution, especially thanks to inhibitors of the PD‐1/PD‐L1 pathway. Nevertheless, this field still lacks predictive biomarkers. Various circulating markers have been evaluated. Exosomal PD‐L1 is reported to be associated with immunosuppression in various cancers. However, its clinical significance has not been defined yet. We conducted a prospective study to demonstrate the clinical significance of ExoPD‐L1 in cancer patients.

Methods: A total of 146 patients with skin cancer (melanoma, squamous cell carcinoma (SCC), and Merkel cell carcinoma (MCC)) and lymphoma were included. Plasma exosomes were isolated by ultracentrifugation and evaluated by nanoparticle tracking analysis (NTA technology) and TEM. Isolated exosomes were tested for the expression of exosomal markers (TSG101, CD9, CD63, Alix). PD‐L1 expression in plasma‐derived exosomes (ExoPD‐L1) was measured using an ELISA.

Results: First, ExoPD‐L1 was detected in all patients independently of the type of cancer. In melanoma patients, ExoPD‐L1 was significantly higher than in other skin cancers (MCC, SCC) and lymphoma (respectively 64.26, 37.6, 42.5 and 6.64 pg/mL). Furthermore, we demonstrated that ExoPD‐L1 variations between baseline and after treatment varied over the course of the disease treatment. These variations (decreased level) were correlated with tumor response and survival. Moreover, we showed the capacity of ExoPD‐L1 to differentiate pseudoprogression in patients treated with immunotherapy from a true progression for which, to date in clinical practice no referential exists to guide clinicians.

Summary/Conclusion: PD‐L1 in circulating exosome was detected in all patients. Monitoring exosomal PD‐L1 is a potential biomarker of tumor response to immunotherapies and could represent a powerful tool to help clinicians to manage treatments in patients. The ability to capture Exo‐PD‐L1 directly from blood using a microchip requiring only a few microliters of blood demands urgent investigation.

OD13.03. EV‐associated proteins but not DNA are sensitive and specific biomarkers in metastatic breast cancer

Mercedes Tkach, Institut Curie / INSERM U932

Caroline Hego, Institut Curie, Circulating Tumor Biomarkers laboratory, INSERM CIC BT‐1428

Marc Michel, Institut Curie, INSERM U934/CNRS UMR3215, PSL Research University

Jean‐Yves Pierga, Institut Curie, Department of Medical Oncology, Circulating Tumor Biomarkers laboratory, INSERM CIC BT‐1428

François‐Clément Bidard, Institut Curie, Department of Medical Oncology, Circulating Tumor Biomarkers laboratory, INSERM CIC BT‐1428

Clotilde Thery, MD PhD, Institut Curie / INSERM U932

Charlotte Proudhon, Institut Curie

Introduction: Detection of cell‐free circulating tumor DNA (ctDNA) and cancer‐specific extracellular vesicles (EVs) in patient blood have been widely explored as non‐invasive biomarkers for cancer detection and disease follow up. However, most of the protocols used to isolate EVs co‐isolate other components and the actual value of EV‐associated markers remain unclear.

Methods: To determine the optimal source of clinically‐relevant biomarkers in breast cancer, we assessed the specificity and sensitivity of nucleic acids and surface proteins from various fractions of blood plasma. We applied a size exclusion chromatography procedure to isolate molecules associated to EV‐enriched or EV‐depleted fractions, in comparison to total plasma.

Results: Both nuclear and mitochondrial DNA (gDNA and mtDNA) were detected in EV fractions. Tumor‐specific nuclear alleles, targeting known mutations identified from tumor tissues, were detected in EV‐associated gDNA. However, we observed that ctDNA from total plasma shows equal specificity and better sensitivity. In contrast, mtDNA was preferentially enriched in EV fractions. Yet, EV‐associated mtDNA displayed similar levels in healthy and cancer subjects.

Next, using an EV capture test which allows to analyze the presence of 37 protein markers by flow cytometry (MacsPlex), we have identified 3 EV surface proteins which are enriched in metastatic breast cancer. These preliminary results suggest that a small set of these surface molecules could provide a disease signature and could allow enrichment of cancer‐specific vesicles.

Summary/Conclusion: Our findings provide evidence that the detection of DNA within total circulating EVs does not provide added value as compared to the whole plasma. However, analysis of a subtype of EV‐associated proteins may reliably identify cancer patients. These non‐invasive biomarkers represent a promising tool for cancer diagnosis and real‐time monitoring of treatment efficacy and our results will impact the development of therapeutic approaches using EVs as targets or biomarkers of cancer.

OD13.04. Klotho inside uEVs and its potential role as biomarker for kidney physiopathology in single ventricle pediatric patients

Cristina Grange, Department of Medical Sciences, University of Turin

Dario Tomanin, Department of Molecular Biotechnology and Health Sciences, University of Turin

Luca Deorsola, Regina Margherita Pediatric Hospital

Carlo Pace Napoleone, Regina Margherita Pediatric Hospital

Benedetta Bussolati, University of Turin, Department of Molecular Biotechnology and Health Sciences, Italy

Introduction: Extracellular vesicles (EVs) from urine are considered a promising biomarker of the overall state of the kidneys. We recently described that uEV also carry Klotho, an anti‐aging hormone released by the kidney with protective activity on the cardiovascular and neurological systems. We here aimed to characterize the uEVs of a population of pediatric patients affected by single ventricle congenital heart disease. In particular, we aimed to evaluate Klotho levels as a potential biomarker of renal damage.

Methods: In this study, we isolated uEVs from first morning urine of pediatric patients affect by single ventricle defects undergone to Fontan procedure (n = 15), and from aged match young healthy subjects. We subsequently characterized uEVs, combining the cytofluorimetric analysis of uEV surface markers, obtained with MACSplex Exosome Kit, with the quantification of uEVs‐Klotho measured by ELISA. Moreover, Klotho and classical exosomal and renal markers were confirmed by single‐molecule super resolution microscopy. Renal function was evaluated by creatinine and NGAL levels.

Results: We isolated a comparable number of uEVs from pediatric patients and healthy subjects. We observed the presence of renal progenitor markers (SSEA4, CD133, CD24) and of the epithelial marker CD326 in both experimental conditions. However, the expression of renal uEVs markers was decreased in single ventricle patients. Of interest, the quantification of Klotho levels within uEVs revealed the presence of Klotho only in uEVs from healthy subjects, and not in uEVs from single ventricle patients. No differences in renal functional parameters were observed.

Summary/Conclusion: The results suggest that in patients affected by single ventricle congenital heart disease, uEV characterization and Klotho expression might predict pre‐clinical damage to the renal tissue. Klotho loss could be involved in the systemic alterations observed in these patients.

OD13.05. Expression of PD‐L1 on extracellular vesicles in renal cell carcinoma patients

Philip Zeuschner, Department of Urology and Pediatric Urology, Saarland University

Dirk Himbert, Saarland University

Jadzia Sonnleithner, Department of Urology and Pediatric Urology, Saarland University

Greta Jaschkowitz, Department of Urology and Pediatric Urology, Saarland University

Angela Zaccagnino, Department of Urology and Pediatric Urology, Saarland University

Michael Stöckle, Department of Urology and Pediatric Urology, Saarland University

Kerstin Junker, Department of Urology and Pediatric Urology, Saarland University

Introduction: The PD‐1/PD‐L1 axis has revolutionized advanced renal cell carcinoma (RCC) therapy. Although the PD‐L1 expression in EVs can have diagnostic, prognostic or predictive potential in various cancers, not much is known about its role in RCC. For the first time, this study examined the PD L1 expression in primary tumor and healthy kidney tissue and their EVs, including the corresponding blood probes.

Methods: Nine patient samples with clear cell RCC were analyzed, in two cases, adjacent normal kidney tissue was available. EVs from fresh frozen tissue were isolated by tissue digestion and sequentially centrifugation with a saccharose gradient. In 8/9 patients, EVs from blood plasma were isolated with an EV isolation kit. The PD‐L1 expression was measured by Western Blot and the results were compared with immunohistochemical (IHC) stainings. Quality and quantity of EV isolation was proven by transmission electron microscopy, Nano Tracking Analysis and Western Blot.

Results: 8/9 tumors samples were PD L1 positive, but 7/8 had a very weak staining intensity as defined by IHC on tissue sections. All tumors were infiltrated by PD‐L1 positive immune cells and two tumors had very prominent PD‐L1 positive immune cell infiltrates.

From all primary tumor tissues, EVs were successfully isolated. PD‐L1 was detectable in 8/9 tumors and their EVs, but differed between the patients. In 5/9 cases, PD‐L1 was enriched in tumor EVs compared to the tumor tissue. In 3/9 cases, PD‐L1 was almost not detectable in primary tumor tissues, but highly enriched in the corresponding EVs. The PD‐L1 expression was lower in adjacent normal kidney tissues and their EVs. One patient had PD‐L1 positive tumor EVs in Western Blot, but was negative on IHC.

All patients had PD‐L1 positive EVs in plasma samples. PD‐L1 was enriched in the EVs compared to whole plasma samples in 7/8 patients. There was no clear association with the IHC staining.

Summary/Conclusion: For the first time, the PD‐L1 expression in EVs from primary RCC tissues was analyzed. PD‐L1 was enriched in the EVs compared to tumor cells, and was higher in the plasma EVs compared to soluble PD‐L1. Hence, the PD L1 status on EVs could have a higher a diagnostic, prognostic or predictive power in RCC than previously thought, which will further be elucidated in this ongoing study.

OD13.06. Extracellular Vesicle Subtypes Enable Precision Cancer Diagnosis

MEI HE, University of Florida

Introduction: Although extracellular vesicles (EVs) are emerging biomarker sources for developing liquid biopsy‐based cancer diagnosis, the large heterogeneity and significant size overlap between vesicle populations pose extraordinary challenges in developing the clinical utility. Defining EV subtypes could particularly improve the precision for cancer diagnosis. However, currently there is no standardized purification methods yet for obtaining pure EV subtypes that are specific to their cellular origin and molecular information.

Methods: Herein, we introduced a novel nanographene‐based immunomagnetic bead with unique 3D nano‐wing structures, which allows the specific marker‐defined capture and release of intact EV subtypes from nearly all types of biological fluids, including human blood, urine, cow milk, and cell culture medium, etc. The nanographene sheet layer with surface conjugated immune photo‐click chemistry enables on demand release of intact, captured EVs for ensuring subtype specificity and improving the sensitivity in downstream analysis.

Results: We demonstrated such isolated EV subtypes utilized in diagnosing bladder cancer via detecting DNA mutations and miRNA profiles from urinary EVs. Via the next generation sequencing (NGS) analysis, the specific mutations such as the KRAS, PIK3CA, and ERBB2 are abundantly identified from bladder cancer patient in comparison to healthy control group. The droplet digital PCR analysis also identified heterozygous mutation point in EGFR from bladder cancer patient derived urinary EV subtypes and validated by sanger sequence analysis. Compared to ultracentrifugation approach which isolates a mixture of vesicle populations, the purified EV subtypes defined by their surface markers exhibit much better specificity and sensitivity to detect bladder cancer tumor associated biomarkers.

Summary/Conclusion: The developed Nano‐Wing EV preparation approach can offer much purer EV subtypes with good specificity to pathogenesis for developing non‐invasive, liquid biopsy diagnosis of bladder cancer.

OD13.07. Integrated analysis of sequenced miRNAs from circulating extracellular vesicles and spatial profiling of epithelial ovarian cancer

Priyakshi Kalita‐de Croft, University of Queensland Centre for Clinical Research

Dominic Guanzon, PhD, Exosome Biology Laboratory, Centre for Clinical Diagnostics, UQ Centre for Clinical Research, The University of Queensland, Australia

Shayna Sharma, Exosome Biology Laboratory, UQ Centre for Clinical Research, Royal Brisbane and Women's Hospital, The University of Queensland, St Lucia, QLD, Australia

Margaret Cummings, Pathology Queensland, Royal Brisbane and Women's Hospital, Herston, QLD, Australia

Terry K. Morgan, MD, PHD, Oregon Health and Science University

Lewis Perrin, Mater Research Institute, University of Queensland, Translational Research Institute, Woolloongabba, QLD, Australia

John Hooper, PhD, Mater Research Institute

Kenneth O'Byrne, Queensland University of Technology, Centre for Genomics and Personalized Health, School of Biomedical Sciences, Faculty of Health, Woolloongabba, QLD, Australia

Sunil Lakhani, UQ Centre for Clinical Research, Royal Brisbane and Women's Hospital, The University of Queensland, St Lucia, QLD, Australia

Arutha Kulasinghe,Queensland University of Technology

Carlos Salomon, MSc, DMedSc, PhD, The University of Queensland

Introduction: A significant proportion of patients with epithelial ovarian cancer (EOC) often present with advanced stage disease, where treatment options are limited. Recent evidence suggests that miRNAs associated with small extracellular vesicles (sEVs) participate in the progression of EOC. Moreover, these sEV cargos may reprogram the tumour microenvironment (TME). Here, we studied the miRNA content of circulating sEVs in patients with EOC, and we integrated this data with spatial information of the tumour microenvironment.

Methods: Informed consent was obtained and approved by the human research ethics committee of the University of Queensland. sEVs were isolated and characterized from plasma of 48 patients with different clinical outcomes (recurrence, deceased and disease‐free). sEV‐associated miRNAs were then sequenced and specific miRNAs were validated using qPCR. Nanostring GeoMX Digital Spatial Profiler (DSP) platform was used to ascertain the TME contexture on formalin‐fixed EOCs for protein markers of various immune and tumour‐related modules. This was integrated with the sequencing results using Ingenuity Pathway Analysis.

Results: A total of 1218 sEV‐associated miRNAs were identified, out of which 49 were significantly different in disease‐free vs recurrence. Recurrent patients had a >20‐fold increase in miRNAs: miR‐3202‐1, miR‐3202‐2, miR‐4516, miR‐139‐5p, and miR‐6865‐5p. In deceased patients a 17‐fold increase in miR‐4740‐3p, and a 5‐fold increase in miR‐106b‐3p and miR‐144‐3p was observed compared to disease‐free and recurrence, respectively. Spatial profiling revealed 54 differentially expressed proteins between the tumour and its microenvironment. Within EOC, 32 proteins were dysregulated in deceased compared to disease‐free. In addition, the recurrent group displayed 26 proteins as highly expressed compared to the deceased group. Namely B7‐H3, Beta‐2‐microglobulin, CD14, CD34, CD44, and CD45RO were found to be most dysregulated in the TME. We also obtained a regulatory network of 10 miRNAs that could be modulating the expression of the proteins associated with ovarian cancer progression

Summary/Conclusion: We propose that sEVs present in the circulation of EOC patients transfer oncogenic miRNAs to cells within the TME to promote cancer progression

OD13.08. Integrating Machine Learning with exoRNA for Early Cancer Diagnosis

Tzu‐Yi Chen, Icahn School of Medicine at Mount Sinai, Department of Department of Genetics and Genomic Sciences

Edgar Gonzalez‐Kozlova, Department of Genetics and Genomics Sciences, Icahn School of Medicine at Mount Sinai

Taliah Soleymani, Department of Genetics and Genomics Sciences, Icahn School of Medicine at Mount Sinai

Susmita Sahoo, SS, Department of Cardiology, Icahn School of Medicine at Mount Sinai

Ash Tewari, Department of Urology, Icahn School of Medicine at Mount Sinai

Gustavo Stolovitzky, IBM T. J. Watson Research Center

Carlos Cordon‐Cardo, Department of Oncology Sciences and Pathology, Icahn School of Medicine at Mount Sinai

Navneet Dogra, PhD, Department of Genetics and Genomics Sciences, Icahn School of Medicine at Mount Sinai

Introduction: One of the greatest challenges in urology is not how to treat prostate cancer (PCa), but rather, whether to treat it at all. The current PCa clinical diagnosis tool, serum prostate‐specific antigen, can only suggest abnormality in prostate glands but cannot ascertain cancer. Furthermore, novel RNA‐based technologies are being developed for real‐time monitoring of various diseases, including cancer. However, a machine‐learning model could facilitate efficient disease prediction and diagnosis from large‐scale data. Tumor cell‐derived EVs, including microvesicles and exosomes, have sparked great interest in their potential as a liquid biopsy‐based, non‐invasive diagnosis, and detection method for cancer. Despite the tremendous clinical outlook of tumor‐derived EVs, we have yet to fully uncover their transcriptomic lineages before and after tumor resection.

Methods: EVs were isolated from aggressive PCa serum samples at the time of proctectomy and from post‐prostatectomy subjects with undetectable cancer. This was followed by quality assessment via electron microscopy, immunoblotting, nanoview, TRPS, nanoparticle tracking, and qPCR. Genome‐wide small exoRNA sequencing was performed to identify unique small RNA markers specific to tumor EVs each before and after tumor‐resection. Finally, 15 exoRNA signatures for PCa were successfully identified and implemented for machine learning model training and validation in independent cohorts.

Results: We have established ∼60 total small RNA‐sequencing profiles, and hundreds of qPCR from 17 aggressive prostate cancer (PCa) patient's tumor and adjacent normal tissue, and EVs isolated from serum, and cancer cell culture media. The 15 candidate exoRNA signatures are significantly upregulated in tumor EVs of PCa patients in contrast to controls with no detectable cancer. Novel small RNA biomarkers were orthogonally validated for their differential expression in the ‘biomarker discovery’ cohort using RT‐qPCR. Therefore, our ongoing work is to identify cancer specificity and sensitivity for PCa detection from cohorts and scaling it up to big data solutions.

Summary/Conclusion: We have established 15 exoRNA biomarkers to distinguish cancer from non‐cancer patients. Our ongoing work is to train and validate the selected cancer‐specific exoRNA in a large cohort of patients, leading to a minimally‐invasive, blood‐only, operator‐independent surveillance biomarker.

OD14.01. VEGF binding to HSPG on the surface of extracellular vesicles secreted by carcinoma‐associated fibroblasts promotes angiogenesis in a bevacizumab‐resistant manner

Jiao Li, Department of Orthodontics, Shanghai Stomatological Hospital, Fudan University, Shanghai, China

Xue Liu, Department of Basic Science of Stomatology, Shanghai Stomatological Hospital, Fudan University, Shanghai, China

Tingjiao Liu, Department of Basic Science of Stomatology, Shanghai Stomatological Hospital, Fudan University, Shanghai, China

Introduction: The blood vessel growth inhibitor bevacizumab targets vascular endothelial growth factor (VEGF), a crucial regulator of angiogenesis. Recently, small extracellular vesicles (sEVs) have been demonstrated to be important vehicles in the transport of growth factors to target cells. It attracts researchers’ attention whether VEGF can bind to EVs. In this study, we investigated the role of EV‐bound VEGF secreted by carcinoma‐associated fibroblast (CAF) on angiogenesis and its sensitivity to bevacizumab.

Methods: Four primary CAFs were isolated from human oral squamous cell carcinoma (OSCC) tissues. sEVs were separated from the conditioned medium of four CAFs, respectively. Tube formation of human umbilical vein endothelial cells (HUVEC) and Matrigel plug assay were performed to evaluate angiogenesis in vitro and in vivo.

Results: Compared with other non‐extracellular vesicle components, CAF‐derived sEVs were found to be the main regulators of angiogenesis. The ability of CAF sEVs to activate VEGF receptor 2 (VEGFR2) signaling in HUVEC was dependent on the association between sEVs and VEGF. In addition, sEV‐bound VEGF secreted by CAFs further activated VEGFR2 signaling in HUVEC in a bevacizumab‐resistant manner. We also found that CAF sEVs may stimulate angiogenesis via interaction with the receptors on endothelial cells independently of uptake. Besides VEGF was found to interact with heparan sulfate proteoglycans (HSPG) on the CAF sEV surface and could be released by heparinase I/III. The bioactivity of the dissociated VEGF was retained in vitro and in vivo and could be neutralized by bevacizumab.

Summary/Conclusion: Our data indicated that CAF‐secreted VEGF binds to the heparin chain of HSPG on the sEV surface, which might be responsible for the resistance to Bevacizumab. Our findings that heparinase releases VEGF from sEVs and regains sensitivity to Bevacizumab raise the possibility that combination of bevacizumab and heparinase might improve the anti‐angiogenic therapy effects in patients with high sEV‐VEGF levels.

OD14.02. Extracellular vesicles shed by follicular lymphoma B cells promote the polarization of bone marrow stromal cell niche

Erwan Dumontet, CHU de Rennes

Céline pangault, CHU de Rennes

David Roulois, INSERM U1236

Matthis Desoteux, INSERM U1242

Simon Leonard, INSERM U1236

Tony Marchand, CHU de Rennes

Maelle Latour, CHU de Rennes

Patricia Legoix, Institut Curie

Damarys Loew, Université de Paris, INSERM U970, Paris Cardiovascular Research Centre, Paris, France

Florent Dingli,Curie Institute, PSL Research University, Laboratoire de Spectrométrie de masse Protéomique, Paris, France

Joelle Dulong, CHU de Rennes

Erwan FlecherCHU de Rennes

Cédric Coulouarn, INSERM U1242

Guillaume cartron,CHU de Montpellier

Thierry Fest,CHU de Rennes

Karin Tarte,CHU de Rennes

Introduction: Follicular Lymphoma (FL) originates in the lymph nodes (LN) and infiltrates bone marrow (BM) early in the course of the disease. BM FL B cells are characterized by a lower cytological grade, a decreased proliferation, and a specific phenotypic and subclonal profile. Mesenchymal stromal cells (MSC) obtained from FL BM display a specific gene expression profile (GEP), including enrichment for a lymphoid‐stromal cell signature, and an increased capacity to sustain FL B‐cell growth. However, the mechanisms triggering the formation of the medullar FL permissive stromal niche have not been yet identified.

Methods: Extracellular vesicles (EVs) from malignant B cells or human bone marrow plasma were gather by differential ultracentrifugation and were characterized by TRPS, TEM and proteomic analysis. Their ability to modify the GEP of BM‐MSC were analyzed by RNAseq and compared to the TNF/LT stimulation. mRNA upregulation were confirmed by qPCR, verified at protein level by luminex assays and test‐ for cell differentiation on prolonged cultures. EV‐induced signalization patways in BM‐MSC were studied by a sensor cell line, flow cytometry and immunofluorescence by confocal microcopy. Finally, after comparing the GEP of LN and BM FL B cells by Affymetrix analysis, we used NicheNet algorithm to describe the putative interactome between BM FL B cells and EV‐primed stromal cells.

Results: In the current work, we demonstrated that FL B cells produced EVs that could be internalized by BM‐MSC, making them more efficient to support FL B‐cell survival and quiescence. Accordingly, EVs purified from FL BM plasma activated TGF‐b dependent and independent pathways in BM‐MSC, modified their GEP, triggering an upregulation of factors classically associated with hematopoietic stem cell niche, including CXCL12 or angiopoietin‐1. Moreover, we provided the first characterization of BM FL B‐cell GEP, allowing the definition of the landscape of molecular interactions they could engage with EV‐primed BM‐MSC.

Summary/Conclusion: This work identified FL‐derived EVs as putative mediators of BM stroma polarization and supported further investigation of their clinical interest for targeting the crosstalk between BM‐MSC and malignant B cells.

OD14 EVs in Cancer Pathogenesis II

OD14.03. RAB5a‐GTPase regulates prostate tumor growth and progression via targeting exosome biogenesis and cargo loading in hypoxic microenvironment

Ashish Kumar, Wake Forest Baptist Health

Susy Kim, Wake Forest Baptist MEdical Center

Mitu Sharma, Wake Forest Baptist Medical Center, Department of Cancer Biology

Pawan Kumar, Wake Forest Baptist Medical Center, Department of Cancer Biolo

Yixin Su, Wake Forest School of Medicine

Sangeeta Singh, Wake Forest Baptist Medical Center, Department of Cancer Biology

Gagan Deep, PhD, Wake Forest School of Medicine

Introduction: RAB5a‐GTPase is a known regulator of early endosome formation, and here we assessed its role in exosome biogenesis, prostate cancer growth and progression under normoxic and hypoxic conditions.

Methods: RAB5a knock‐down (KD) was achieved by siRNA or lentivirus. Exosomes concentration and cargos were assessed by nanoparticle tracking analysis, miRNA‐sequencing and mass spectrometry analysis. To mimic hypoxic environment cells were cultured under 1% O2. Effect of RAB5a KD was assessed on prostate cancer (PCa) cells and in vivo in nude and syngeneic mice models.

Results: Hypoxia promoted exosomes secretion by PCa cells while RAB5a knock‐down significantly reduced exosomes release, cell growth, migration and invasion. Orthotropic administration of RBA5a‐KD PC3 cells in the prostate of male nude mice showed both reduced primary tumor growth and micro‐metastasis to distant organs compared to vector control cells. Supplementing exosomes derived from PC3 cells under normoxia and hypoxia conditions restored the growth of RBA5a‐KD PC3 tumors; more strongly with hypoxic exosomes, confirming exosomes‐mediated regulation of tumor growth and metastasis. Moreover, in a syngeneic mouse model, injection of lentiviral particles with RAB5a‐shRNA in mice with established RM1 tumors reduced the tumor growth. Mass spectrometry and miRNA‐seq analyses revealed differential exosomal cargo loading under normoxia and hypoxia following RAB5a KD, with changes mainly associated with tumor growth and cancer progression. Notably, stable RAB5a KD was associated with higher carbonic anhydrase (CA9) expression; and simultaneous CA9 inhibition by specific siRNAs or chemical inhibitor, U‐104, almost completely inhibited PCa cells growth.

Summary/Conclusion: Present study demonstrate the critical role of RAB5a‐GTPase in the regulation of exosome biogenesis and cargo loading under both normoxia and hypoxia, and suggest RAB5a as a potential therapeutic target for the treatment of advanced PCa.

OD14.04. Stromal Heat Shock Factor 1 promotes gastric cancer via exosome mediated secretion of Thrombospondin 2 and Inhibin Subunit Beta A

Meirav Pevsner‐Fischer, Department of Bimolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel

Nil Grunberg, Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel

Tal Goshen‐Lago, Division of Oncology, Rambam Health Care Campus, Haifa, Israel

Judith Diment, Department of Pathology, Kaplan medical center. Rehovot, Israel

Yaniv Stein, Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel

Hagar Lavon, Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel

Yifat Ofir‐Birin, Wiezmann institute for science

Li‐Jyun Syu, Department of Dermatology, Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA

Salomon M Stemmer, Institute of Oncology, Davidoff Cancer Center, Rabin Medical Center, Beilinson Hospital, Petah Tikva, Israel

Baruch Brenner,Institute of Oncology, Davidoff Cancer Center, Rabin Medical Center, Beilinson Hospital, Petah Tikva, Israel

Andrzej A. Dlugosz, Department of Dermatology, Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA

David LydenWeill Cornell Medicine

Neta Regev‐Rudzki, Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel

Irit Ben‐Aharon,Division of Oncology, Rambam Health Care Campus, Haifa, Israel

Ruth Scherz‐Shouval,Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel

Introduction: Gastric cancer is the 3rd most lethal cancer worldwide, accounting for 3% to 10% of all cancer‐related deaths. The mutational landscape of gastric cancer cells has not translated into effective prognostic or therapeutic strategies, and the standard of care is surgical intervention combined with chemotherapy. We hypothesized that outcomes may depend on the tumor microenvironment (TME), and in particular cancer‐associated fibroblasts (CAFs). However, very little is known about the role of CAFs in gastric cancer.

Methods: We mapped the transcriptional landscape of human gastric cancer stroma by laser capture microdissection and RNA sequencing of CAFs from gastric cancer patients to identify signature genes associated with aggressive gastric cancer phenotype. EVs were isolated from Hsf1 null fibroblasts and used in mouse models and cell co‐cultures, to explore their functions.

Results: We discovered a stromal gene signature associated with aggressive disease and regulated by the transcription factor Heat Shock Factor 1 (HSF1). Among signature genes, we identified Inhibin Subunit Beta A (INHBA) and Thrombospondin 2 (THBS2) to be regulated by the HSF1and to be delivered to cancer cells via EVs to support cancer growth.

Using mouse models and cell co‐cultures, we found that EVs from Hsf1 null fibroblasts showed similar size, quantity, biogenesis and uptake into cancer cells as their WT counterparts, however their protein content was different. Specifically, INHBA and THBS2 were more abundant in WT versus Hsf1 null MEFs EVs. Furthermore, co‐injection of cancer cells with EVs derived from WT fibroblasts caused a significant increase in the growth of MC38‐injected tumors, and this effect was completely abolished when EVs from Hsf1 null MEFs were co‐injected with MC38 cells.

Summary/Conclusion: These findings suggest that HSF1 regulates the expression of INHBA and THBS1/2 in stromal cells. INHBA and THBS2 are then packaged into EVs in a HSF1‐dependent manner and secreted to the TME, where they are taken up by cancer cells and promote a more aggressive disease phenotype.

*Grunberg etal. Cancer‐associated fibroblasts promote aggressive gastric cancer phenotypes via heat shock factor 1‐mediated secretion of extracellular vesicles. Cancer Reseach 2021 Feb 5;canres.2756.2020. https://doi.org/10.1158/0008‐5472.CAN‐20‐2756. Online ahead of print.

OD14.05. IMP1 Enhances Extracellular Vesicle Secretion in a Transformation‐Dependent Manner

Sarah Andres, PhD, OHSU

Madeline Kuhn, OHSU

Ranjan Preet, University of Kansas Medical Center

Aurora Blucher, OHSU

John Favate, Rutgers University

Sukanya Das, Rutgers University

Shun Liang, Rutgers University

Louis R. Parham, University of Pennsylvania Perelman School of Medicine

Priya Chatterji, University of Pennsylvania Perelman School of Medicine

Kathy N. Williams,University of Pennsylvania Perelman School of Medicine

Sudheer Molugu, University of Pennsylvania Perelman School of Medicine

Wei ZhangUniversity of Pennsylvania

Jiegang Yang, University of Pennsylvania

Kathryn E. Hamilton,Children's Hospital of Philadelphia

Premal Shah,Rutgers University

Gordon Mills,OHSU

Dan A. Dixon,University of Kansas

Anil K. Rustgi,Herbert Irving Comprehensive Cancer Center, Columbia University

Introduction: The endosomal, extracellular vesicle (EV), and autophagy pathways are interrelated and influenced by their environment and cellular context. The RNA binding protein insulin‐like growth factor 2 mRNA binding protein 1 (IGF2BP1/IMP1) displays tissue‐specific roles in colorectal cancer (CRC) and can regulate autophagy following damage; however, a role for IMP1 in coordinating the endosomal, EV, and autophagy pathways in the gastrointestinal (GI) tract is not known.

Hypothesis: We hypothesized that IMP1 is a mechanistic link between the endosome and autophagy pathways in CRC.

Methods: Ribosomal profiling and RNA sequencing compared transcript and translational efficiency profiles between IMP1 null and IMP1‐overexpressing CRC cell lines. EVs were assessed by nanoparticle tracking analysis, western blot, and electron microscopy in CRC cells and non‐transformed, mouse intestinal enteroids. Effects of IMP1 on endosome, EV, and autophagy pathways were assessed by electron microscopy, immunofluorescence, and western blot.

Results: EV, endocytic, and exosome‐related pathways were the most significant differentially regulated pathways by gene ontogeny analysis of RNA sequencing data in IMP1 null vs IMP1‐overpressing CRC cells (p = 1.78 × 10‐10, 2.43 × 10‐10). We found that IMP1 increased EV secretion in HT‐29 (4483±403 vs 1934±414, p = 0.006) and SW480 (3604±399 vs 2293±464, p = 0.049) CRC cells vs null controls. IMP1 modulated translational efficiency or protein levels of early endosome proteins, including EEA1, but did not alter LC3II or p62 levels. When autophagic flux was inhibited (BafilomycinA1), the ability of IMP1 to enhance EV release was magnified. By contrast, Imp1 overexpression in non‐transformed intestinal enteroids did not enhance EV secretion.

Summary/Conclusion: Our novel findings suggest that IMP1 has transformation‐dependent effects on the endosomal pathway and EV secretion. Our findings have implications for the development of novel early detection and therapeutic approaches in CRC where IMP1 is overexpressed.

OD14.06. Molecular Imaging of the Distinct Characteristics of Exosomes and Microvesicles in a Mouse Breast Cancer Model

Masamitsu Kanada, Ph. D., Michigan State University

Ahmed Zarea, MSU

Gloria Perez, MSU

Benedikt Dolgikh, MSU

Matthew Bernard, MSU

Amelia McGill, MSU

Victoria Toomajian, MSU

Alicia Withrow, MSU

Lukose Thampy, MSU

Michael Bachmann,MSU

Introduction: Exosomes are produced as components of multivesicular bodies (MVBs) and are released from cells via the fusion of MVBs with the plasma membrane. Microvesicles (MVs) are formed by the outward budding of the plasma membrane. We have previously characterized the physical properties and the functional transfer of cargo molecules packaged in exosomes and MVs. To further understand the roles of exosomes and MVs in cancer, we developed a new EV reporter system that enables comparative analysis of distinct EV classes both in vitro and in vivo.

Methods: A new EV imaging reporter was created using a highly sensitive red‐shifted bioluminescence resonance energy transfer (BRET) protein called red enhanced Nano‐lantern (ReNL; a fusion of tdTomato with NanoLuc) with a palmitoylation (Palm) signal peptide. Since palmitoylated reporter proteins have been presumed to label all EV classes, we assessed their labeling efficiency in the fractions of exosomes and MVs derived from 4T1 murine mammary carcinoma cells stably ex‐pressing PalmReNL. Cellular uptake of the reporter exosomes and MVs by several recipient cell types were analyzed. Furthermore, we used in vivo bioluminescence imaging (BLI) to determine the biodistributions of the reporter exosomes and MVs in healthy and mammary tumor‐bearing mice.

Results: (1) Exosomes and MVs carrying PalmReNL exhibited red‐shifted emission spectra upon the addition of a luminescent substrate. (2) Both exosomes and MVs contained PalmReNL. (3) Culturing the reporter exosomes or MVs with various cell types for 2 h showed the highest cellular uptake efficiency in the recipient cells. (4) Both reporter exosomes and MVs administered intravenously into BALB/c mice showed similar biodistributions. Both EV classes preferentially accumulated in the lung, followed by liver, spleen, heart, bone, lymph nodes, and brain. (5) Unexpectedly, 4T1 cell‐derived MVs with PalmReNL significantly suppressed primary tumor growth in the syngeneic mouse breast cancer model, while MVs without the reporter did not affect the tumor growth. (6) 4T1 cell‐derived MVs induced severe pulmonary hemorrhage in the tumor‐bearing mice.

Summary/Conclusion: Using PalmReNL, we demonstrated the successful tracking of distinct classes of EVs both in vitro and in vivo. Both reporter exosomes and MVs derived from 4T1 cells showed similar biodistributions and organotropisms in healthy mice. The reporter MVs showed a negative effect on the primary tumor growth, possibly through host immune activation by antigenic PalmReNL. Our data suggest that tumor cell‐derived MVs do not support primary tumor growth but cause pathological changes in healthy lung tissues.

OD14.07. Aptamers for selective targeting of breast cancer exosomes in early diagnosis and therapy

Carla Lucia Esposito, 1 Istituto di Endocrinologia ed Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (CNR), 80100, Naples, Italy

Cristina Quintavalle, Istituto di Endocrinologia e Oncologia Sperimentale IEOS‐CNR, Via Pansini 5, IT80131, Napoli.

Francesco Ingenito, Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via Pansini 5, IT80131, Napoli.

Deborah Rotoli, Istituto di Endocrinologia e Oncologia Sperimentale IEOS‐CNR, Via Pansini 5, IT80131, Napoli.

Giuseppina Roscigno, Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via Pansini 5, IT80131, Napoli.

Silvia Nuzzo, IRCCS SDN SpA, Via Gianturco 113, IT80143, Naples Italy.

Renato Thomas, Mediterranea Cardiocentro, 80100, Naples, Italy

Silvia Catuogno,Istituto di Endocrinologia e Oncologia Sperimentale IEOS‐CNR, Via Pansini 5, IT80131, Napoli.

Vittorio De Franciscis, Istituto di Endocrinologia e Oncologia Sperimentale IEOS‐CNR, Via Pansini 5, IT80131, Napoli.

Gerolama Condorelli, Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via Pansini 5, IT80131, Napoli.

Introduction: Extracellular vesicles named exosomes have attracted growing interest as early diagnostic and prognostic biomarkers and therapeutic targets in several cancers including breast cancer (BC). However, tools to easily and specifically distinguish cancer cell‐derived exosomes are mostly unknown and are required to realize their clinical utility. In this context, nucleic‐acid aptamers are a promising class of three‐dimensional single stranded oligonucleotides that serve as high affinity ligands of disease‐associated proteins.

Methods: In order to isolate aptamers specific for BC‐derived exosomes, we developed a novel SELEX strategy by using exosomes purified from primary BC cells as positive selection target and exosomes derived from primary normal epithelial breast cell lines in the negative selection. The individual sequences were isolated, optimized and characterized as tools to specifically recognize BC exosomes and to alter exosome uptake.

Results: We isolated nuclease resistant RNA aptamers able to specifically discriminate BC‐derived exosomes from those produced by normal cells. The best sequences were optimized identifying short molecules (about 30–35 mer) that resulted effective in specifically detect BC exosomes. Further, they inhibited exosome cellular uptake antagonizing cancer exosome‐induced cell migration

Summary/Conclusion: We developed a SELEX strategy with wide applicability to exosome targeting in different tumor types and/or clinical problems. The isolated aptamers show great potential as tools for BC exosome targeting in early diagnosis and therapies. The identification of the aptamer targets is on‐going and can allow to find out new specific markers of BC exosomes.

OD15.01. Environmental Plasticity Of Staphylococcus aureus Extracellular Vesicles RNA Content

Brenda Silva Rosa da Luz, INRAE, Institut Agro, STLO, F‐35000, Rennes, France

Aurélie Nicolas, INRAE, Institut Agro, STLO, F‐35000, Rennes, France

Svetlana Chabelskaya, University of Rennes, Inserm, BRM [Bacterial Regulatory RNAs and Medicine] UMR_S 1230, Rennes, France

Vinícius Rodovalho, Laboratory of Cellular and Molecular Genetics, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil

Yves Le Loir, INRAE, Institut Agro, STLO, F‐35000, Rennes, France

Vasco Azevedo, Laboratory of Cellular and Molecular Genetics, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil

Brice Felden, University of Rennes, Inserm, BRM [Bacterial Regulatory RNAs and Medicine] UMR_S 1230, Rennes, France

Eric Guédon, INRAe, Institut Agro, STLO, Rennes, France

Introduction: Bacterial extracellular vesicles (EVs) carry various macromolecules able to affect host‐pathogen interactions, such as RNAs. Staphylococcus aureus, an important human and animal pathogen, releases EVs whose RNA content is still unkown. Here, we adress what classes of RNAs compose S. aureus EVs.

Methods: S. aureus strain HG003 was cultured in Brain Heart Infusion medium under different in vitro conditions: early‐ and late‐stationary phases, in the presence or absence of a sublethal concentration of vancomycin (0.5 μg/mL). EVs were purified from cell‐free culture supernatants using density gradient ultracentrifugation. Bacterial and EV samples were submitted to phenol‐chloroform RNA extraction, DNAse treatment, and library preparation (Ovation Prokaryotic RNA‐Seq, Nugen, rRNA depletion). Sequencing was performed using Illumina, NextSeq500, 75 cycles, single reading, High Output.

Results: Particle yields were similar between conditions, however, EVs from late‐stationary phases were ∼55% larger. On average, 78.0% of HG003 annotated genes were identified in EVs, while only ∼5% presented ‐ 90% read coverage. Highly covered EV RNAs included mRNAs coding for virulence‐factors (hld, agrBCD, psmB1, sbi, spa, isaB), ribosomal proteins, transcriptional regulators, and metabolic enzymes. sRNAs were also detected, including the bona fide rsaC. Interestingly, several of these RNAs were shown to belong to the same transcriptional units in S. aureus. Both nature and abundance of the RNAs in EVs were dramatically affected by growth conditions, whereas much less in the parent cells. Finally, the RNA abundance pattern differed between EVs and parent cells.

Summary/Conclusion: To our knowledge, this is the first work characterizing the RNA cargo of S. aureus EVs. Our findings show that EV RNAs are shaped by the environment, and suggest the selective packaging of RNAs into EVs. Finally, this study also shedds light to the possible roles of potentially functional RNAs in S. aureus EVs, notably in host‐pathogen interactions.

OD15.02. From root to fruit: tomato EVs

Ramesh Bokka, EVs&MS Laboratory, Institute of Biosciences and BioResources, National Research Council of Italy

Giorgia Adamo, Institute for Biomedical Research and Innovation, National Research Council of Italy

Alfredo Ambrosone, Department of Pharmacy, University of Salerno

Antonella Bongiovanni, PhD, Institute for Biomedical Research and Innovation, National Research Council of Italy

Darja Božič, Faculty of Health Sciences, Laboratory of Clinical Biophysics, University of Ljubljana

Monica De Palma, Institute of Biosciences and BioResources, Research Division Portici, National Research Council

Immacolata Fiume, EVs&MS Laboratory, Institute of Biosciences and BioResources, National Research Council of Italy

Michele Guescini, University of Urbino Carlo Bo

Matej Hočevar, Department of Physics and Chemistry of Materials, Institute of Metals and Technology

Matic Kisovec,Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry

Veronika Kralj‐Iglič, Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana

Mauro MannoInstitute of Biophysics, National Research Council of Italy

Ramila Mammadova, EVs&MS Laboratory, Institute of Biosciences and BioResources, National Research Council of Italy

Samuele Raccosta,Institute of Biophysics, National Research Council of Italy

Michelina Ruocco,Institute for Sustainable Plant Protection, National Research Council of Italy

Simon Sugar,MS Proteomics Research Group, Hungarian Academy of Sciences, Research Centre for Natural Sciences

Marina Tucci,Institute of Biosciences and BioResources, Research Division Portici, National Research Council

Lilla Turiák,MS Proteomics Research Group, Hungarian Academy of Sciences, Research Centre for Natural Sciences

Marjetka PodobnikNational Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology

Gabriella Pocsfalvi, EVs&MS Laboratory, Institute of Biosciences and BioResources, National Research Council of Italy

Introduction: Similar to a mammalian cell, intra and extracellular vesicles of a plant cell are involved in many physiological processes, including transport, unconventional protein secretion, defense and symbiosis. There are several studies published on the successful isolation and characterization of apoplastic vesicles. Moreover, EV‐like structures from edible plants are under intense investigation because of their utility as delivery vectors in nutra‐cosmeceutical applications. The presentation will focus on the comparison of different vesicle isolates from three different parts of tomato (Solanum lycopersicum L.) plant: root, leaf and fruit.

Methods: Vesicles were isolated by differential ultracentrifugation method from hydroponic washing fluid, leaf apoplastic washing fluid and tomato fruit juice. Isolates from the fruit were further separated by gradient ultracentrifugation or purified by size exclusion chromatography. Physical, molecular and biological characterization was performed with established methods to determine size distribution, concentration, density and morphological characteristics. Proteomics, metabolomics, lipidomics and in vitro bioassays were performed for biocargo analysis.

Results: Vesicles isolates from root, leaf and fruit showed different physiochemical characteristics and protein profiles. Root‐derived EVs express a set of proteins associated to biotic stress signaling and plant defense mechanisms. Moreover, they were active against fungal pathogens in vitro. The most abundant proteins identified in tomato apoplastic vesicles were key enzymes of the nitrogen and carbohydrate metabolism, photosynthesis and ripening. Tomato fruit‐derived nanovesicles are complex mixture of intra‐ and extra cellular vesicles. Top‐ranking proteins in the nanovesicles isolated from the fruit include lypoxigenase involved in plant growth and development, several proteins related to ripening and defense mechanisms and to chromoplast differentiation.

Summary/Conclusion: Tomato is the most highly consumed vegetable in the word, particularly important for the Mediterranean diet because it is the major dietary source of the antioxidant lycopene. The high yield of tomato fruit‐derived nanovesicles and their anti‐inflammatory effects makes them promising for future innovative applications. The comparative biocargo analysis helps to deepen our understanding on the roles of the vesicles in different organs of a plant.

OD15.03. Extracellular vesicles released by Mycobacterium tuberculosis: Lipid composition and their interactions with macrophages

Pierre Boyer, Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, Université Paul Sabatier

Jérôme Nigou, CNRS

Emilie Layre, Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, Université Paul Sabatier

Introduction: Tuberculosis is one of the top ten causes of death worldwide. M. tuberculosis (Mtb) is an intracellular pathogen of alveolar macrophages. In most cases, Mtb infection of macrophages is followed by a fine equilibrium between the bacillus mechanisms of evasion and host immune responses, leading to the formation of a granuloma that controls the bacillus dissemination but not its eradication. Understanding the molecular crosstalk that conditions this equilibrium is key to develop new anti‐TB tools. In this context, vesicles released by the bacillus, which shuttle bacterial factors between cells, are of key interest because they have the potential to significantly modulate the microenvironment at the site of infection. However, still in its early stages, the characterization of mycobacteria membrane vesicles composition and properties on macrophages, key actors of innate immunity, remain incomplete.

Methods: An optimized multi‐step purification of mycobacterial vesicles has been required prior to their molecular and functional characterization. The content of the purified vesicles in immunomodulatory lipidic components was comprehensively characterized using global mass spectrometry approach and by western blot. Their uptake by and properties on macrophages were characterized by combining the use of reporter cell lines, high‐resolution microscopy and in vitro bioassays.

Results: Our work has allowed defining the content of purified vesicles in (glyco)lipids and lipoglycans, including virulence‐associated molecules. Our functional studies highlighted that vesicles interact with several PRR, beyond the previously described activation of TLR2, such as with lectins. We also describe their rapid uptake by macrophages, their intracellular trafficking and the subsequent impact on macrophages functionalities involved in the control of Mtb infection, like autophagy and inflammatory response.

Summary/Conclusion: The current characterization of mycobacterial vesicles composition and immunomodulatory properties is still incomplete. Our work shows that their contribution in virulence effects and in host immune responses regulation is likely more complex than described so far.

OD15.04. Evaluation of parasite burden and cytokine production in mice immunized with extracellular vesicles released by Leishmania (Leishmania) amazonensis

Talita V. Vieira Dupin, Universidade Federal de São Paulo campus Diadema

Natasha FC. Ferraz de Campos Reis, Universidade Federal de São Paulo campus Diadema

Elizabeth Cristina Perez, Pós Graduação em Patologia Ambiental e Experimental, Universidade Paulista, São Paulo, Brazil

Rodrigo Pedro Soares, Laboratory of Cellular and Molecular Parasitology, René Rachou Institute, Oswaldo Cruz Foundation, Belo Horizonte, Minas Gerais, Brazil

Ana Claudia Claudia. Torrecilhas, Universidade Federal de São Paulo campus Diadema

Patricia X. Xander, Universidade Federal de São Paulo campus Diadema

Introduction: Leishmaniasis is a group of neglected diseases caused by Leishmania. The disease is endemic in 97 countries, with Brazil presenting 90% of the cases reported in the Americas. Studies have already shown that some species of Leishmania are capable of releasing extracellular vesicles (EVs) containing antigens, virulence factors, RNA, DNA and lipids from the parasite. It is known that changes in the cargo of EVs can have an impact on the immune response and disease progression, therefore EVs derived from parasites with different virulence profiles (attenuated and virulent parasites) can present relevant differences in the activation of the immune response. This study aimed to evaluate the modulation of the immune system in mice immunized with EVs of virulent and attenuated L. (L.) amazonensis by analysing the parasite burden and the production of cytokines.

Methods: Virulent promastigotes of L. (L.) amazonensis were obtained by consecutive and successive recovery of lesions in infected animals and the attenuated parasites were derived after a long period in culture (100 passages in culture). All EVs were obtained by several centrifugations and used in immunization protocols with 15 days of interval. After 2 or 3 immunizations, animals were challenged with the parasite.

Results: We observed a significant decrease in the parasite burden in animals immunized with L. (L.) amazonensis EVs emulsified in Alum adjuvant, compared to the non‐immunized animals and the control group (immunized with Alum adjuvant). Interestingly, more reduction in the parasite burden were observed in animals immunized twice as compared with animals that received 3 doses of EVs. The specific IgG1 and IgG2a levels were analyzed and animals that received 2 or 3 immunizations with EVs showed an increase in both Ig subtypes. The cytokine production analyzed in the supernatant of spleen cells re‐stimulated in vitro with total antigens or EVs showed a Th2 profile with high levels of IL‐10 and IL‐4 in animals immunized with 3 doses of EVs, compared with non‐immunized mice.

Summary/Conclusion: Thus, our data suggest a potential protective role for EVs in an experimental immunization model for the prevention of leishmaniasis. Furthermore, it seems that the protective role seems to be related to the better development and polarization of the Th1 response. Further studies involving the dose amount and different types of adjuvants may help to improve the potential of using Leishmania EVs in immunization protocols.

OD15.05. Extracellular vesicles in Cryptoccocus neoformans: from structural insights to vaccine strategy

Juliana Rizzo, Unité Biologie des ARN des Pathogènes Fongiques, Département de Mycologie, Institut Pasteur

Sarah Sze Wah WONG, Unité Mycologie Moléculaire, CNRS UMR2000, Département de Mycologie, Institut Pasteur

Anastasia D. GAZI, Ultrastructural Bio‐Imaging, UTechS UBI, Département de Biologie cellulaire et infection, UMR 3528 CNRS, Institut Pasteur

Frédérique MOYRAND, Unité Biologie des ARN des Pathogènes Fongiques, Département de Mycologie, Institut Pasteur

Thibault CHAZE, Plateforme Protéomique, Unité de Spectrométrie de Masse pour la Biologie (MSBio), Centre de Ressources et Recherches Technologiques (C2RT), UMR 2000 CNRS, Institut Pasteur

Pierre‐Henri COMMERE, Cytometry and Biomarkers, Centre de Ressources et Recherches Technologiques (C2RT), Institut Pasteur

Sophie NOVAULT, Cytometry and Biomarkers, Centre de Ressources et Recherches Technologiques (C2RT), Institut Pasteur

Mariette MATONDO, Plateforme Protéomique, Unité de Spectrométrie de Masse pour la Biologie (MSBio), Centre de Ressources et Recherches Technologiques (C2RT), UMR 2000 CNRS, Institut Pasteur

Gerard PEHAU‐ARNAUDET, Ultrastructural Bio‐Imaging, UTechS UBI, Département de Biologie cellulaire et infection, UMR 3528 CNRS, Institut Pasteur

Flavia C. G. REIS,Instituto Carlos Chagas, Fundação Oswaldo Cruz (FIOCRUZ)

Matthijn VOS, NanoImaging Core Facility, Centre de Ressources et Recherches Technologiques (C2RT), Institut Pasteur

Lysangela R. ALVESInstituto Carlos Chagas, Fundação Oswaldo Cruz (FIOCRUZ)

Robin C. MAY, Institute of Microbiology and Infection and School of Biosciences, University of Birmingham

Leonardo NIMRICHTER,Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro

Marcio L. RODRIGUES,Instituto Carlos Chagas, Fundação Oswaldo Cruz (FIOCRUZ)

Vishukumar AIMANIANDA,Unité Mycologie Moléculaire, CNRS UMR2000, Département de Mycologie, Institut Pasteur

Guilhem JANBON,Unité Biologie des ARN des Pathogènes Fongiques, Département de Mycologie, Institut Pasteur

Introduction: The encapsulated yeast Cryptococcus neoformans is the primary agent of cryptococcal meningitis, resulting in nearly 180,000 deaths worldwide each year. This pathogen has been reported to release EV that might regulate host response during infection; even so, the field still lacks fundamental information regarding EV biogenesis, structure, composition, and function.

Methods: Using cutting‐edge methodological approaches including cryogenic electron microscopy (cryo‐EM) and cryogenic electron tomography imaging techniques, proteomics, flow cytometry, and in vivo vaccination model, we provided a robust set of data regarding the structural and compositional aspects of EVs from the C. neoformans and explored their potential use as vaccine platforms.

Results: Our analysis suggested a new EV structural model, in which the vesicular lipid bilayer is covered by a mannoprotein‐based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. The analysis of EV structural diversity showed a polymorphic population, composed mostly of regular (round‐bilayered) vesicles (81.4%), but also irregular vesicles (not rounded " bilayer or multilayered). Notably, most of the EV analyzed by cryo‐EM was smaller than 100nm, contrasting with the diameter size values obtained by NTA. By analyzing EV protein cargo from three different cryptococcal species, we provided a list of 17 conserved proteins, most of them predicted to be membrane‐associated proteins, including tetraspanin (Tsh proteins) bearing a SUR7/PalI motif. Additionally, most of the Cryptococcus EVs conserved proteins are known to be protective antigens, suggesting their potential use as a vaccine. Indeed, our data showed that mice immunized with EVs obtained from an acapsular C. neoformans mutant rendered a strong antibody response and significantly prolonged survival of mice upon fungal infection.

Summary/Conclusion: This study described a new structure model of cryptococcal EV and identified the first EV protein markers. Additionally, the mice model of infection suggested that cryptococcal EVs can be used as an efficient vaccination strategy. Altogether, the data represent tools for future studies on EV biogenesis, cargo loading, and vaccination development approaches for cryptococcosis and potentially other fungal diseases.

OD15.06. Set‐up of multi‐parametric analytical assays for quantification, quality control and surrogate potency testing of helminth EVs

Francesca Loria, HansaBioMed Life Sciences OÜ, Mäealuse 2/1, 12618 Tallinn, Estonia

Anne Borup, Department of Clinical Medicine, Faculty of Health, Aarhus University, 8200 Aarhus, Denmark

Anders Toftegaard Boysen, Department of Clinical Medicine, Faculty of Health, Aarhus University, 8200 Aarhus, Denmark

Andres Lõhmus, HansaBioMed Life Sciences OÜ, Mäealuse 2/1, 12618 Tallinn, Estonia

Kadi‐Liis Veiman, HansaBioMed Life Sciences OÜ, Mäealuse 2/1, 12618 Tallinn, Estonia

Paolo Bergese, Dipartimento di Medicina Molecolare e Traslazionale, Università degli Studi di Brescia, 25123 Brescia, Italy

Peter Lindberg Nejsum, Department of Clinical Medicine, Faculty of Health, Aarhus University, 8200 Aarhus, Denmark

Nataša Zarovni, Exosomics / HansaBioMed Life Sciences OÜ

Introduction: Extracellular vesicles (EVs) have shown to be key players in parasite‐to‐parasite and parasite‐to‐host cross‐talks and have displayed important immunomodulatory activity. Ascaris suum is a prevalent porcine parasite commonly used to model helminth infection in general and, specifically, A. lumbricoides infection, which to date infects more than one billion people. Our objective was to develop an essential analytical package for A. suum EV quantification and characterization to be employed as a quality control (QC) and/or surrogate potency testing tool throughout EV purification and assessment.

Methods: EVs from A. suum incubation media were purified by size exclusion chromatography ‐ only or in combination with ultracentrifugation. EV quantification and characterization were performed by (fluorescence) nanoparticle tracking analysis, protein quantification assay, phospholipid quantification assay, enzymatic activity and fluorometric (lectin‐based) microplate assays.

Results: Different A. suum EV batches were analyzed in independent experimental rounds providing multi‐modal quantification, generic or source‐specific content determination, purity and bioactivity evaluation. Our results revealed promising robustness of the used methods, as well as structural (i.e. particle number, content and integrity of EV constituents) and functional parameter (i.e. enzymatic activity and ultimate immunomodulatory effects) intercorrelation. Gathered quantitative and qualitative outputs showed to reflect batch‐to‐batch variability and the variance introduced by the adoption of different methods for EV preparation.

Summary/Conclusion: We have implemented several user‐friendly and time‐saving analytical options, combining well‐established and ad‐hoc developed source‐tailored benchtop assays, in line with EV purification that result highly informative in correlating bulk and single‐particle parameters in digital and/or analog mode for comprehensive QC assessment and surrogate functional testing.

OD15.07. Extracellular vesicle formation in Lactococcus lactis is stimulated by prophage‐encoded holin‐lysin system

Yue Liu, Food Microbiology, Wageningen University

Svetlana Alexeeva, Food Microbiology, Wageningen University

Eline van Ophem, Food Microbiology, Wageningen University

Eddy J. Smid, Food Microbiology, Wageningen University

Tjakko Abee, Food Microbiology, Wageningen University

Introduction: Mechanistic insights are lacking on how extracellular vesicles (EVs) are released through the thick cell walls in Gram‐positive bacteria, as the peptidoglycan layer had been historically presumed to be a strong physical barrier preventing such event. In this study, we characterized underlying mechanisms of EV production and provide evidence for a role of prophage activation in EV release using Gram‐positive Lactococcus lactis as a model.

Methods: We applied a standard EV isolation procedure to the supernatant of (i) a lysogenic Lactococcus lactis strain FM‐YL11, (ii) its prophage‐cured mutant (“prophi) and (iii) its prophage‐encoded holin‐lysin knock‐out mutant (”holin‐lysin). All strains were cultivated under a non‐stressed condition or a prophage‐inducing condition (addition of mitomycin C). Isolated EVs were quantified by membrane‐specific fluorescent dyes, visualized by electron microscopy and characterized by flow cytometry and proteomics analysis.

Results: In FM‐YL11, the prophage‐inducing condition led to an over 10‐fold increase in EV production compared to the non‐stressed condition, whereas EV quantities from the two mutants “prophi and ”holin‐lysin remained at very low level under both conditions. Under prophage‐inducing condition, FM‐YL11 did not show massive cell lysis. Defective tailless phage particles were found to be released in and associated with EVs from FM‐YL11. The proteome profile of prophage holin‐lysin induced EVs has been obtained.

Summary/Conclusion: We demonstrated that 1) Gram‐positive L. lactis produces EVs; 2) Stress‐induced prophage activation stimulates EV production; 3) Prophage‐encoded holin‐lysin system mediates massive release of EVs. Findings from this study may provide leads to the discovery of EV producing events and underlying mechanisms of EV release from other Gram‐positive probiotic and pathogenic bacteria with roles in health and disease.

OD16.01. Quantitative characterization of Extracellular Vesicle Uptake and Content Delivery within Mammalian Cells

Émeline Bonsergent, Université de Paris, Inserm, UMR7057/CNRS

Eleonora Grisard, Institut Curie U932

Julian Buchrieser, Virus & Immunity Unit, Department of Virology, Institut Pasteur

Olivier Schwartz, Virus & Immunity Unit, Department of Virology, Institut Pasteur

Clotilde Thery, MD PhD, Institut Curie / INSERM U932

Grégory Lavieu, PhD, Université de Paris, inserm, umr7057/cnrs

Introduction: Extracellular Vesicles (EVs) are an emerging vector of communication between tissues and cells and seem to be a promising vector for targeted therapeutic delivery. However much remains to be done at the basic research level to hit translational success.

The delivery process within the acceptor cells need a final EV‐content release that is likely to require fusion between the EV membranes and target membranes of the acceptor cell. Such a fusion reaction is used by viruses and intracellular vesicles to release their content within the target compartments. For EV‐content delivery, we still don't know 1) where the final content mixing occurs within the acceptor cells, 2) how efficient is this process, and 3) what molecules are involved in this final delivery process. We developed a cell‐free assay, which suggested that EV‐content delivery occurs within endosomal compartment though a process that resembles viruses’ delivery.

We aimed here to validate our results within live cells and to acutely quantify this delivery process.

Methods: We developed a cell‐based assay, to first identify the entry and delivery points within the acceptor cells and then quantify the content delivery efficiency. Briefly, we engineered donor cells releasing EV‐loaded with luciferase‐ or GFP‐tagged Hsp70, a generic marker of EVs. After EV purification by sequential ultracentrifugation, EVs were incubated with acceptor cells. The fate of the EV content was followed by microscopy (GFP‐tag) or by biochemistry (Luciferase‐tag). Cell fractionation of acceptor cells enabled separation of membrane and cytosolic fractions, which were tested for luciferase activity, to quantify the fraction of EV‐cargo that has been released into the cytosol of the acceptor cells.

Results: Confocal imaging colocalization showed that at a fraction of EVs were internalized within endocytic compartment and reached lysosomes. Luciferase‐based assay revealed that 1% of the EVs is associated with acceptor cells after 1h of incubation, and that 30% of this fraction is indeed delivered within the cytosol of acceptor cells. Cytosolic delivery is inhibited when acceptor cells are treated with Bafilomycin, an inhibitor of endosome acidification and maturation. In addition, IFITM proteins, known as inhibitor of viral entry and fusion, decreased EV‐content delivery.

Summary/Conclusion: Our results suggest that the EV‐content release occurs at the level of acidic‐endosomes/lysosomes through a process that may require membrane fusion.

OD16.02. Reporter gene assay for membrane fusion and cytoplasmic cargo delivery of extracellular vesicles

Shun'ichi Kuroda, Osaka University

Masaharu Somiya, Osaka University

Introduction: Extracellular vesicles (EVs) are expected to mediate intercellular communication by delivering their cargo to recipient cells. However, the mechanisms of cytoplasmic cargo release remain largely unknown. In this study, we have established a novel reporter gene assay that can quantify the efficiency of EV membrane fusion and subsequent cargo release in recipient cells.

Methods: The transcription activator (TA) gene was fused with the EV marker protein tetraspanin gene. Forty‐eight to 96 hours after transfection of 293T cells (donor cells), modified EVs were recovered from the supernatant by PEG precipitation. When modified EVs fuse with the cellular membrane of recipient cells, TA is released into the cells and induces reporter gene expression. The expression of the reporter gene was measured after 24 hours.

Results: We confirmed that EVs containing TA‐tetraspanin fusion protein were secreted from donor 293T cells into the supernatant. However, the TA‐containing EVs derived from 293T cells did not induce reporter gene expression in recipient reporter cells. In contrast, EVs containing the viral membrane fusion protein VSV‐G strongly induced reporter gene expression. Mutation of VSV‐G resulted in complete loss of EV‐mediated membrane fusion, suggesting that the assay readout reflects the membrane fusion and cargo release of EVs. Besides, the treatment of drugs that inhibit endocytosis and membrane fusion blocked VSV‐G‐mediated EV entry and fusion. These data indicated that the reporter assay is useful in validating the cargo delivery mechanism of EVs.

Summary/Conclusion: Membrane fusion is a key event in EV‐mediated cargo delivery. This novel reporter gene assay is useful to decipher the membrane fusion and intercellular signaling of EVs. In the absence of VSV‐G, we couldn't observe membrane fusion in 293T‐EVs and 293T‐recipient cells indicated that unmodified EVs have no or very weak fusion activity. It is worth verifying whether other combinations of donor/recipient cells are more susceptible to EV‐mediated cargo delivery.

OD16.03. Quantum dot‐based single particle tracking reveals the spatiotemporal dynamics of tumor extracellular vesicle internalization and miRNA‐delivery into endothelial cells

Houfu Xia, School and Hospital of Stomatology,Wuhan University

Zili Yu, School and Hospital of Stomatology, Wuhan University

Lijuan Zhang, College of Chemistry and Molecular Sciences, Wuhan University

Gang Chen, School and Hospital of Stomatology, Wuhan University

Introduction: Developing visualization and tracking methods to elucidate the spatiotemporal dynamics of extracellular vesicles’ (EVs) behaviors will facilitate understanding their physiopathological functions and developing therapeutic strategies.

Methods: Quantum dots (QDs) were used to label oral cancer EVs (OCEVs) and the carried miRNA respectively based on biotinylation strategy and electroporation method. A universal single particle tracking (SPT) platform was developed based on a spinning disk confocal microscope. The multi‐color and multi‐component labeled OCEVs were co‐cultured with endothelial cells (ECs), and the dynamic interaction between them was visualized and recorded in situ and in real‐time through SPT. The characteristics of the internalization and transportation of EVs by ECs, and the specific steps of subsequent miRNA‐release were analyzed by trajectory reconstruction and motion analysis.

Results: By optimizing the culture concentration and period with DSPE‐PEG‐Biotin, we successfully achieved efficient biotinylation of different subtypes of EVs regardless of their size distribution or cell origin. The site‐specific labeling of EVs with QDs was realized through further adjustment of the incubation concentration. Fluorescently labeled miRNA were efficiently loaded into the EVs, achieving simultaneous multi‐color labeling of different components of EVs. ECs efficiently internalized OCEVs through clathrin‐mediated endocytosis in a time‐dependent manner, and subsequently transported them to the perinuclear region through a typical “slow‐fast‐slow” three‐stage pattern. Finally, the internalized EVs rapidly released their miRNA possibly through their interaction with acidic endosomes. In addition, targeting acidification effectively blocked the miRNA‐delivery by EVs, thereby exerting an inhibitory effect on tumor angiogenesis.

Summary/Conclusion: This study, for the first time, reported the entire process and detailed dynamics of OCEV transportation and cargo‐release in ECs, leading to better understanding of their pro‐angiogenic functions. Additionally, the QD‐based single particle tracking technique would help uncover more secrets of EV‐mediated cell‐cell communication.

OD16.04. PDZ networking regulates exosome production, composition, and uptake

Monica Castro‐Cruz, KU Leuven

Raphael Leblanc, Centre de Recherche en Cancérologie de Marseille

Sofie Meeussen, KU Leuven

Lore Van Herck, KU Leuven

Frederique Lembo, Centre de Recherche en Cancérologie de Marseille

Guido David, KU Leuven

Pascale Zimmermann, KU Leuven

Introduction: Our poor understanding of the molecular mechanisms governing exosome biogenesis, uptake, and heterogeneity limits the rational use of these nano‐sized extracellular organelles in health and disease. Because tetraspanins and syndecans, major components of exosomes, contain a binding site for PDZ proteins and because PDZ proteins co‐evolved with multicellularity, we hypothesized that PDZ proteins might represent important players in exosome biology.

Methods: To test for tetraspanin and syndecan PDZ interactions, we performed a comprehensive yeast two‐hybrid screen (ca. 150 PDZ proteins in the human genome). To collect exosome‐enriched fraction, we used differential ultracentrifugation of conditioned medium from MCF‐7 cells. Exosome number and composition were analyzed by nanoparticle tracking analysis and immunoblotting, respectively. Subcellular protein localizations, syndecan endosomal budding, HS localization at the plasma membrane, and uptake of CD63 positive particles were analyzed by microscopy.

Results: We revealed an unsuspected broad number of direct interactions between the PDZ proteome and syndecans, while the number of direct PDZ‐CD9, CD63, and CD81 interactions appears more discrete. We show that PDZ proteins fine‐tune exosome composition and regulate positively or negatively the number of exosomes. We observe that on the contrary to tetraspanins, syndecan c‐terminal fragments are reliable indicators of exosome number. We document that PDZ proteins impact on the steady‐state distribution of syndecans and tetraspanins, and regulate syndecan endosomal budding and processing. Moreover, PDZ proteins impact on the uptake of exosomes and control heparan sulfate levels at the cell surface.

Summary/Conclusion: In conclusion, our study establishes that tetraspanin‐ and SDC‐PDZ networking contributes to exosome heterogeneity and turnover, and highlights an important piece of the molecular framework governing intracellular trafficking and intercellular communication.

OD16.05. Intratumoral cellular heterogeneity as a critical factor for extracellular vesicle uptake in colorectal cancer

Andrea Kelemen, Semmelweis University

Idan Carmi, Semmelweis University, Department of Genetics, Cell‐ and Immunobiology, Molecular Cancer Biology Research Group

Zoltán Wiener, Semmelweis University, Department of Genetics, Cell and Immunobiology, Budapest, Hungary

Introduction: Colorectal cancer (CRC) is one of the most common cancers. The functionality of the intratumoral genetic and phenotypic heterogeneity of cancer cells are not yet fully understood. Patient‐derived 3D organoids are one of the most modern methods to study in vitro this heterogeneity. IFITM1 plays a critical role in virus uptake. Since our bioinformatical analysis showed a correlation between IFITM1 expression and patient survival in CRC, we aimed at i) determining the cellular heterogeneity with respect to IFITM1 expression and ii) characterizing the EV uptake of different CRC cell populations.

Methods: The Ethics Committee of the Medical Research Council of Hungary approved our experiments and informed consent was obtained from patients. We sorted patient‐derived organoid cells, we measured the ratio of proliferating cells, organoid diameter and analyzed gene expression profiles. We detected EVs by antibody‐coated beads and flow cytometry, NTA, Western‐blotting and TEM. The uptake of labelled large EVs was visualized by confocal microscopy and the functional importance of the uptake was assessed by immunocytochemistry.

Results: We observed a higher expression of IFITM1 in adenomas and CRCs than in the normal colon. IFITM1high CRC cell‐derived organoids were larger and they contained more Ki67+ proliferating cells compared to IFITM1low organoids. IFITM1low CRC cells took up more EVs, however, we could not detect this difference for syntetic liposomes. Furthermore, exposure of IFITM1low organoids to fibroblast‐derived EVs resulted in a more pronounced increase in Ki67+ cell number compared to IFITM1high cells, leading to the disappearance of the difference in organoid size.

Summary/Conclusion: Our data indicate that intratumoral heterogeneity leads to tumor cell subpopulations with different ability to take up EVs, leading to different proliferation potential. Thus, we propose that intratumoral heterogeneity should be considered as a critical factor when designing targeted EV‐based therapy in CRC.

OD17.01. YAP/TAZ facilitates extracellular vesicle release upon mechanical stimulation

Felix Nägele, Department of Cardiac Surgery, Medical University of Innsbruck

René Weiss, Danube University Krems

Tanja Eichhorn, Center for Biomedical Technology, Danube University Krems

Viktoria Weber, Center for Biomedical Technology, Danube University Krems

Johannes Holfeld, Department of Cardiac Surgery, Medical University of Innsbruck

Introduction: The physical stimulus of shockwave therapy (SWT) has a pro‐angiogenic impact on ischemic tissue, representing a promising regenerative approach. The Hippo signaling pathway YAP/TAZ plays a key role in angiogenesis and can be regulated by mechanical signals. Both SWT stimulation and YAP/TAZ activation cause a secretory release of extracellular vesicles (EVs). We aim to substantiate the mechanotransduction of SWT via YAP/TAZ facilitated EV release and subsequent angiogenic response.

Methods: In order to investigate the detailed underlying mechanisms, human umbilical vein endothelial cells were stimulated with 300 impulses at a frequency of 3 Hz and an energy flux density of 0,1mJ/mm2. Four hours thereafter, mRNA expression of YAP/TAZ target genes (ANKRD1, CYR61) was measured and the nuclear localization of YAP/TAZ was examined by immunofluorescence. The cell culture supernatant was collected. The release of EVs was characterized by flow cytometry to detect bigger EVs using annexin V (Anx5) as a marker of phosphatidylserine (PS) expressing EVs. Furthermore, EVs were analyzed by a bead‐based flow cytometry assay to detect smaller EVs by using CD63‐coupled magnetic beads. The particle concentration was measured by nanoparticle tracking analysis.

Results: SWT of HUVECs resulted in a higher concentration of Anx5+ EVs (12,675±2,863 vs.8,650±1614 EVs/μl) in the culture supernatant as compared to the untreated control. This observation was confirmed by a higher percentage of EV‐decorated beads after SWT. This was accompanied by higher mRNA expression of YAP/TAZ target genes ANKRD1 (p = 0.0005, respectively) and CYR61 (p = 0.0006, respectively). Immunofluorescence staining showed a nuclear translocation of YAP/TAZ upon SWT compared to untreated controls. These effects were abolished upon pharmacological inhibition of YAP/TAZ nuclear translocation.

Summary/Conclusion: EV release via SWT could represent a regenerative approach for ischemic heart disease.

OD17.02. Role of extracellular vesicles in the propagation of inflammation and oxidative stress during vascular calcification

Linda Yaker, MP3CV‐UR7517, CURS‐Université de Picardie Jules Verne, Avenue de la Croix Jourdain F‐80054 Amiens, France

Saïd Kamel, MP3CV‐UR7517, CURS‐Université de Picardie Jules Verne, Avenue de la Croix Jourdain F‐80054 Amiens, France

Jérôme Ausseil, INSERM UMR1043, CNRS UMR5282, University of Toulouse III, F‐31024 Toulouse, France

Agnès Boullier, MP3CV‐UR7517, CURS‐Université de Picardie Jules Verne, Avenue de la Croix Jourdain F‐80054 Amiens, France

Introduction: Vascular calcification (VC) is a cardiovascular complication in patients with diseases such as atherosclerosis or chronic kidney disease. During VC, vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and secrete a heterogeneous population of extracellular vesicles (EVs). Recent studies have shown the involvement of EVs in inflammation and oxidative stress observed in the VC. The objective of our study is to determine the role and the mechanism of action of EVs in the propagation of inflammation and oxidative stress during the VC process.

Methods: Inflammation (IL6, IL1β, TNF‐α, NLRP3), oxidative stress (iNOS, SOD‐1, Nrf2, Keap1), and expression of EVs biogenesis markers (SMPD3, TNAP, phospho‐1) were analyzed by RT‐PCR in a macrophage murine cell line (RAW 264.7) treated with lipopolysaccharide (LPS EK). EVs secreted by these macrophages were then collected by ultracentrifugation and characterized by analyzing the acetylcholinesterase (AChE) activity as well as CD9 and CD81 protein expression by Western blotting. A murine VSMCs cell line (MOVAS‐1) was incubated with these EVs in calcifying conditions (Pi 4 mM " 14 days) and calcification was assessed by the o‐cresolphthalein calcium assay.

Results: An increase of oxidative stress and inflammation via the inflammasome activation was observed in LPS‐EK“treated macrophages. The expression of the EVs biogenesis’ markers in these macrophages was significantly decreased as well as the AChE activity in EV‐derived from LPS‐EK” treated macrophages. In calcifying conditions, these EVs significantly increase the calcification of VSMCs. A 24h‐treatment of VSMCs with these EVs induces an inflammatory as well as an oxidative response.

Summary/Conclusion: EVs derived from LPS‐EK"treated‐macrophages are themselves able to induce a pro‐inflammatory and pro‐oxidative response in surrounding cells, such as VSMCs, thus aggravating the VC process. These EVs could therefore be a therapeutic target to limit VC in patients.

OD17.03. miRNAs transferred within extracellular vesicles as key mediators of antifibrotic MSC effects in vitro and in vivo

Nataliya Basalova, Institute for regenerative medicine, Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russia

Ivan Zaitcev, Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russia

Mikhail Arbatskiy, Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russia

Olga Grigorieva, Institute for regenerative medicine, Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russia

Maksim Vigovskii, Institute for regenerative medicine, Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russia

Uliana Dyachkova, Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russia

Anastasiya Tolstoluzhinskaya, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia

Vladimir Popov, Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russia

Natalia Kalinina, Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russia

Zhanna Akopyan,Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russia

Anastasia Efimenko, Institute for regenerative medicine, Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russia

Introduction: Applying extracellular vesicles of mesenchymal stromal/stem cells (EV‐MSC) is an effective approach for the treatment of fibrotic diseases. However, the exact mechanism of this effect remains unclear. It is known that the suppression of fibrosis is based on inhibiting the activity of myofibroblasts and stimulating their dedifferentiation in normal tissue‐specific stromal cells. The key molecules regulating these processes are miRNAs, which are transported mainly as a cargo of EVs. Therefore, we investigated the effects of selected miRNAs within EV‐MSC on myofibroblasts dedifferentiation in vitro and in vivo.

Methods: EVs were isolated from the conditioned medium of human MSC and characterized by standard methods. Inhibition and overexpression of the studied miRNAs were obtained by EVs transfection with synthetic oligonucleotides. The effects were assessed using in vitro model of dedifferentiation of human myofibroblasts (ICC, western blot, RT‐PCR, collagen contraction test) and in vivo model of bleomycin‐induced pulmonary fibrosis in C57Bl/6 mice.

Results: We showed that EV‐MSC stimulated the dedifferentiation of myofibroblasts at the morphological and functional levels. RNA sequencing of EVs revealed more than 50 miRNAs associated with the regulation of myofibroblasts dedifferentiation and fibrosis. We demonstrated that not a single miRNA, but a complex consisting of miRNA‐129 and ‐29c had a critical impact into the observed effects in vitro. Suppression of miRNA‐129 and ‐29c significantly decreased the in vivo antifibrotic effect of EV‐MSC leading to the increased number of activated myofibroblasts and fibrotic foci in the lungs.

Summary/Conclusion: A complex of specific miRNAs transferred within EV‐MSC are able to stimulate myofibroblast dedifferentiation in vitro and in vivo, thereby influencing the development of fibrosis. Our findings may provide a basis for a novel antifibrotic EV‐based therapy development.

OD17.04. Endothelial‐Derived Extracellular Vesicles: A New Paradigm in Cancer Therapy‐Related Cardiac Dysfunction

Crizza Ching, Institute of Medical Science, University of Toronto

Dakota D. Gustafson, Toronto General Hospital Research Institute

Paaladinesh Thavendiranathan, Peter Munk Cardiac Centre, University Health Network

Jason E. Fish, PhD, Toronto General Hospital Research Institute

Introduction: Cancer‐therapy related cardiac dysfunction (CTRCD) is a devastating cardiovascular sequalae often associated with anthracycline therapy. The lack of mechanistic understanding remains a barrier to improving cardiovascular prognosis for cancer patients. While most studies have focused primarily on toxicity in cardiomyocytes, the role of the endothelium in CTRCD has remained largely unexplored. Recently, extracellular vesicles (EVs) have been shown to mediate intercellular communication and induce phenotypic changes in recipient cells. As EVs are a component of the endothelial secretome, we aimed to elucidate the effect of cancer treatment on endothelial‐derived EVs.

Methods: The extent of endothelial activation and permeability following epirubicin treatment was evaluated through qRT‐PCR and VE‐Cadherin staining, respectively. Endothelial permeability was further assessed in real‐time using the xCELLigence platform. Human umbilical vein endothelial cell (HUVEC)‐derived EVs were isolated by ultracentrifugation following exposure to epirubicin (n = 4). Initially, EVs were characterized by immunoblotting and cryo‐electron microscopy. CytoFLEX was also utilized to further characterize specific EV subsets. Total particle count and mean particle size were determined using nanoparticle tracking analysis. To gauge their effect on endothelial homeostasis, HUVECs were exposed to EVs and permeability was assessed.

Results: Treatment of HUVECs with epirubicin led to increased expression of endothelial activation genes and increased permeability, with stronger effects observed at higher doses. Morphological assessment of EVs showed an increase in size and abundance post‐anthracycline treatment (p < 0.05). Changes in EV surface markers were also observed, with increased CD31+ EVs following treatment. Exposure of naïve HUVECs to EVs derived from epirubicin‐treated HUVECs showed increased endothelial permeability immediately after treatment compared to untreated HUVEC‐derived EVs.

Summary/Conclusion: Our results demonstrate that anthracycline treatment perturbs endothelial homeostasis, which could lead to the release of effector EVs capable of mediating further endothelial damage. As endothelial‐derived effectors can affect cardiac function, deciphering how these EVs affect cardiomyocyte function could have implications in understanding CTRCD pathogenesis.

OD17.05. Functional differences of small extracellular vesicle subpopulations

Simonides Immanuel van de van de Wakker, Department of Experimental Cardiology, University Medical Center Utrecht, Utrecht University, The Netherlands

Julia Bauzá‐Martinez, Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands

Carla Rios Arceo, Department of Experimental Cardiology, University Medical Center Utrecht, Utrecht University, The Netherlands

Eduard Willms, Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia

Olivier Gerrit G. de Jong, PhD, CDL Research, University Medical Center Utrecht and Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, (UIPS), Faculty of Science, Utrecht University, The Netherlands

Wei Wu, Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands

Joost P.G. Sluijter, J.P.G., Department of Experimental Cardiology, University Medical Center Utrecht, Utrecht University, The Netherlands

Pieter Vader, CDL Research, University Medical Center Utrecht, The Netherlands

Introduction: The use of cardiac progenitor cell (CPC)‐derived small extracellular vesicles (sEVs) has shown potential to stimulate cardiac repair. sEVs are released by cells and play a role in intercellular communication through transfer of their bioactive content. Increasing evidence indicates that sEVs present a heterogeneous population of vesicles. In the context of cardiac regeneration, studying sEV heterogeneity could provide new insights into mechanisms underlying sEV‐mediated cardiac repair properties and help to improve the therapeutic application of CPC‐derived sEVs.

Methods: A two‐step size‐exclusion chromatography (SEC) approach was employed to isolate and fractionate different EV‐subfractions derived from CPCs. Western blot analyses were performed on individual fractions to determine protein expression patterns and define sEV subpopulations. Characterization of particle size and number, as well as protein‐, lipid‐ and RNA content of the different sEV subpopulations was performed. Full proteomic composition was studied using mass spectrometry (MS). Functional effects on recipient endothelial cells (ECs) were studied using an ERK/AKT phosphorylation assay, a wound healing assay, and a sprouting assay and on recipient cardiomyocytes using a survival assay.

Results: Size‐based fractionation and subsequent characterization of CPC‐derived sEVs revealed that sEVs comprise heterogeneous subpopulations that differ in size and proteomic composition. Based on the differential expression of common EV markers between individual SEC fractions, three distinct sEV subpopulations were identified. MS analysis confirmed the differences in the expression levels of classical EV marker proteins, as well as annexins, rab proteins, integrins, histones and proteasomal proteins. Exposure of recipient cells to sEV subpopulations demonstrated their differential functional effects, including on recipient cell ERK/AKT phosphorylation status and migration properties.

Summary/Conclusion: Two‐step SEC allows for the identification of sEV subpopulations and in‐depth study of the functional heterogeneity of sEVs. Increased knowledge of sEV heterogeneity will contribute to a better understanding of the mechanisms of action of sEVs in cardiac regeneration. Usage of specific functional CPC‐derived sEV subpopulations to stimulate cardiac repair will allow an off‐the‐shelf approach to stimulate cardiac regeneration.

OD17.06. Systemic infusion of regeneration‐associated cells‐derived extracellular vesicles improved ischemia‐injured heart function

Hospital

Ainur Salybekova, MD, Advanced Medical Science, Tokai University School of Medicine

Sheng Ying, MD, PhD, Advanced Medical Science, Tokai University School of Medicine

Yoshiko Shinozaki, Teaching and Research Support Core Center, Tokai University School of Medicine

Keiko Yokoyama, Teaching and Research Support Core Center, Tokai University School of Medicine

Shuzo Kobayashi, MD, PhD, Kidney Disease and Transplant Center, Shonan Kamakura General Hospital

Takayuki Asahara, MD, PhD, Advanced Medical Science, Tokai University School of Medicine

Amankeldi A. Salybekov, MD., Ph.D, Clinical Research Center, Regeneration and Translational Science Department, Shonan Kamakura General Hospital

Introduction: In an earlier study, we showed that under vasculogenic conditioning, pro‐inflammatory cell subsets of peripheral blood mononuclear cells (PBMCs) such as macrophages type 1 (M1ɸ), T cells, and primitive EPC cells beneficially shifted their phenotype to pro‐regenerative cells such as vasculogenic EPCs, M2 macrophages, and regulatory T cells, collectively designated as regeneration‐associated cells (RACs). Furthermore, systemic transplantation of the low number of RACs (1 × 105) beneficially improved cardiac functions. Here, we evaluated therapeutic efficacy of RAC‐derived extracellular vesicles (RACev) in comparison with MSC‐derived EVs (MSCev) in the context of rat myocardial ischemia‐reperfusion injury (IRI).

Methods: Human PBMCs were cultured with defined growth factors for 7‐days to harvest RACs. RACev and MSCev were isolated via ultracentrifugation. EVs quantity and quality were characterized by NTA, surface protein (CD9, CD63, Alix) expression, and TEM. EVs miR was sequenced and regenerative responsible miRs were validated using TargetScan and miRBase. Allogeneic immune responses to the RACev and MSCev were evaluated. The function of RACev and MSCev were evaluated using HUVECs proliferation and cell‐cycle assays in vitro and repetitive (30min, d1, and d3 after IRI) systemic infusion with either RACev or MSCev in a myocardial IRI model.

Results: We observed significant differences in EV‐specific surface markers (CD9 and CD63) expression as well as in quantity of EVs, secreted from RAC than MSC (P>0.03). In vitro assay, RAVev markedly enhanced cell viability, proliferation, and migration of HUVECs in a dose‐dependent manner compared to MSCev. Notably, systemic injection of RAC (5 × 105) derived EVs beneficially ameliorated cardiac functions at four weeks such as fractional shortening (P>0.005), and preserved from mitral regurgitation (P>0.03) than MSCev treated group. In histology, RACev transplanted group showed less interstitial fibrosis and enhanced capillary densities compared to MSCev. These beneficial effects are coupled with significant expression of angiomiRs (miR‐195‐5p, miR‐200b‐5p, miR‐126‐3p/5p) and anti‐fibrosis miR‐133 family in RACev but not in MSCev. In vivo bioluminescence analysis depicted preferential accumulation of RACev into the IR‐injured myocardium (P>0.01) while MSCev is in a modest manner. Immune phenotyping analysis confirmed immunomodulatory effect of MSCev and RACev by inhibiting antigen‐presenting cells to escape from T cell recognition.

Summary/Conclusion: Taking together, repetitive systemic transplantation of RACev is superior to MSCev in terms of cardiac function enhancements via crucial angiomiRs, anti‐fibrosis miRs, and anti‐apoptosis miRs delivery to the ischemic tissue.

OD17.07. Characterisation of Intracardiac Extracellular Vesicles in the Context of Myocardial Infarction and Glucose Intolerance

Stephane M.I. MAZLAN, Paris Cardiovascular Research Centre

Vincent Duval, Université de Paris, INSERM U970, Paris Cardiovascular Research Centre

Cecile Devue, Inserm U970‐ Paris Cardiovascular Research Center

Michael Robillard, Inserm U970‐ Paris Cardiovascular Research Center

Fariza Mezine, Université de Paris, INSERM U970, Paris Cardiovascular Research Centre, Paris, France

Pierre‐Michaël Coly, Université de Paris, INSERM U970, Paris Cardiovascular Research Centre, Paris, France

Shruti Chatterjee, Université de Paris, INSERM U970, Paris Cardiovascular Research Centre, Paris, France

Stephane Camus, Université de Paris, INSERM U970, Paris Cardiovascular Research Centre

Ke Xiao,Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM)

Jan Fiedler, Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM)

Thomas ThumInstitute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School & Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM)

Philippe Menasché, Université de Paris, INSERM U970, Paris Cardiovascular Research Centre

Chantal Boulanger,Inserm U970‐ Paris Cardiovascular Research Center

Jean‐Sébastien Silvestre,Université de Paris, INSERM U970, Paris Cardiovascular Research Centre

Xavier LOYER, INSERM U970‐PARCC

Introduction: In response to myocardial infarction (MI), extracellular vesicles (EVs), including large (lEVs) and small (sEVs), are released within and from the heart to facilitate intercellular communication and maintain cardiac homeostasis by transferring miRNA content to recipient cells. As diabetes increases the risk of CVD, we investigated how glucose intolerance influences the release of intracardiac EVs post‐MI and their miRNA content.

Methods: B6J mice were fed chow diet or high‐fat diet (HFD) for 3 months. MI was induced by coronary artery permanent ligation. Left ventricles were harvested at different timepoints post‐surgery and processed for EV isolation. EVs were quantified by Tunable Resistive Pulse Sensing technology. Parental cell origin and EV characterisation were determined by flow cytometry and Western blot. EV miRNA content was determined by RNAseq and validated by qPCR. Using cardiomyocyte specific GFP+ mice, circulating EVs were analysed by flow cytometry to validate whether GFP+ cardiomyocyte EVs (CMEVs) are released into the circulation. As MI and diabetes involve persistent inflammatory responses, APC+ labelled hypoxic cardiomyocyte cell line (HL‐1) lEVs were injected in HFD/control mice post‐MI to determine preferred target cells in tissues by flow cytometry.

Results: In control mice, release of lEVs and sEVs was significantly increased at 24h post‐MI when compared to shams. lEV levels in HFD mice were significantly higher compared to sham and control mice post‐MI. There was no difference in sEV release between sham and MI HFD mice. Most intracardiac lEVs expressed cardiomyocyte marker caveolin‐3 and harbour EV markers. Global qPCR analysis revealed multiple EV miRNAs that were dysregulated post‐MI. Among these, there is a downregulation of miRs 146a‐5p and 503–5p expression in HFD lEVs compared to control and upregulation of miR‐378a‐5p expression post‐MI in both EV subsets. Levels of intracardiac GFP+Cav‐3+ lEVs were lower in HFD than in control mice, whereas levels of circulating GFP+Cav‐3+ lEVs were higher in HFD than in control mice. In vivo biodistribution studies revealed a preferential uptake of hypoxic HL‐1 lEVs by splenic myeloid cells, with an increase in uptake in HFD spleens than in control spleens post‐MI. Further investigations are needed to define specific targeted splenic myeloid subpopulations.

Summary/Conclusion: Our results show that glucose intolerance modulates the release of intracardiac EVs post‐MI as well as their miRNA cargo. Furthermore, the release of CMEVs into the circulation is increased as well as their uptake by splenic myeloid cells. Further work is warranted to fully investigate the functional impact of the miRNA of interest in intracardiac EVs in the diabetic heart post‐MI.

OD18.01. The influence of 3D printed scaffold architecture on osteoblast‐derived extracellular vesicles therapeutic efficacy for bone repair

Kenny Man, University Of Birmingham

Sophie Louth, School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom.

Mathieu Brunet, School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom.

David Hoey, Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland.

Sophie C. Cox, School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom.

Introduction: Extracellular vesicles (EVs) are considered promising nanoscale therapeutics for bone tissue engineering. To date, EVs are harvested from cells cultured on tissue culture plastic, limiting the surface area for cell growth as well as not replicating conditions in situ. Numerous studies have demonstrated the impact of 3D culture on promoting osteogenic differentiation. Additive manufacturing techniques such as 3D printing has allowed for the fabrication of biomimetic in vivo environments. Therefore, this study investigated the influence of different 3D printed scaffold architectures on the yield and therapeutic potency of osteoblast‐derived EVs for bone repair.

Methods: 3D printed titanium (Ti6Al4V) scaffolds with different pore sizes (500 and 1000 μm) and shapes (square and triangle) were fabricated by selective laser melting. EVs were harvested from osteoblasts cultured on different 3D printed scaffolds in osteogenic conditions for 2 weeks. Relative EV size and concentration were defined using transmission electron microscopy and nanoparticle tracking analysis. The osteogenic differentiation of human bone marrow stromal cells (hBMSCs) cultured with scaffold‐derived osteoblast EVs was evaluated by qPCR, biochemistry and histological analysis.

Results: Titanium scaffolds promoted the mineralisation of osteoblasts when compared to 2D culture. Moreover, osteoblasts cultured on 3D printed scaffolds secreted significantly enhanced EV quantity when compared to 2D, with scaffolds exhibiting larger pore sizes (1000 μm) and permeabilities (triangle) promoting EV yield. Osteoblast‐derived EVs isolated from scaffolds exhibited a triangle pore conformation significantly promoted hBMSCs osteogenic differentiation and extracellular matrix mineralisation when compared to EVs derived from scaffolds exhibiting square pore confirmation and 2D culture.

Summary/Conclusion: Taken together, these findings demonstrate the influence of 3D printed scaffold architecture on osteoblast‐derived EV yield and efficacy, indicating the potential use of these culture platforms to enhance the production of therapeutically potency EVs for bone repair.

OD18.02. Controlled release of osteoblast‐derived extracellular vesicles from an injectable chitosan‐collagen composite hydrogel to promote bone regeneration

Kenny Man, University Of Birmingham

Mathieu Brunet, School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom.

Sophie C. Cox, School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom.

Introduction: For bone tissue engineering, the use of extracellular vesicles (EVs) is emerging as a promising acellular approach compared to cell‐based therapies. Despite their promise, the short half‐life of these cell‐derived nanoparticles following systemic administration hinders their therapeutic potential. Therefore, this study aimed to develop an osteoinductive chitosan‐collagen composite hydrogel capable of controlling the release of osteoblast‐derived EVs to promote bone regeneration.

Methods: Chitosan‐collagen composites were fabricated at ratios of 100/0, 65/35, 25/75 and 0/100 wt%. The hydrogels gelation time, compressive modulus and pore size were characterised. Osteoblast‐derived EVs were incorporated within the composites and their release kinetics were determined using the CD63 ELISA. The size, morphology and concentration of released EVs were assessed via nanoparticle tracking analysis and transmission electron microscopy. The osteogenic differentiation of human bone marrow stromal cells (hBMSCs) within the EV‐functionalised hydrogel was evaluated by qPCR, biochemistry and histological analysis.

Results: The presence of collagen within the composite hydrogel significantly enhanced compressive modulus and reduced gelation times and pore size. Increasing collagen content within the hydrogel, lead to a dose‐dependent reduction in EV release kinetics. In monolayer, the functional activity of hydrogel released EVs was confirmed with enhanced hBMSCs proliferation and migration. Importantly, EV‐functionalised composite hydrogels significantly promoted encapsulated hBMSCs osteogenic differentiation and extracellular matrix mineralisation when compared to the EV‐free gel during osteogenic culture.

Summary/Conclusion: Together, these findings demonstrate the development of an osteoinductive chitosan‐collagen composite hydrogel capable of enhancing the therapeutic delivery of osteoblast‐derived EVs as an acellular tool to promote bone regeneration.

OD18.03. Senescence did not alter the chondroprotective effect of extracellular vesicles from adipose mesenchymal stem cells in osteoarthritis

Jérémy Boulestreau, Inserm

Marie Maumus, Inserm

Pauline Rozier, Inserm

Christian Jorgensen, University Montpellier

Daniele Noël, Inserm

Introduction: Age is the most important risk factor in degenerative osteoarthritis (OA) and is associated with the accumulation of senescent cells that contribute to functional decline of joint. We previously demonstrated that extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) largely mediate the therapeutic effect of parental cells in OA. Here, we assessed the impact of senescence on the characteristics of EVs from adipose tissue‐derived MSCs (ASC‐EVs) and their properties in an in vitro model of OA.

Methods: ASCs were induced to senescence using 25μM etoposide for 24 hours. Senescence was assessed by quantifying proliferation rate, SA‐βGal activity, nuclear γH2AX foci number, phalloidin staining and expression of cyclin dependent kinase inhibitors (CDKI) (RT‐qPCR). ASC‐EVs were isolated by differential ultracentrifugation and characterized by size, concentration, total protein content, structure (cryo‐TEM) and immunophenotype. In vitro OA model used chondrocytes isolated from OA patients, which were stimulated with IL1b for 48h before culture with ASCs or ASC‐EVs for 7 days. Expression of chondrocytic and inflammatory markers was quantified by RT‐qPCR and SASP factors were quantified by ELISA in supernatants.

Results: Senescence‐induced ASCs experienced growth arrest and increase of SA‐βGal staining, of p21 CDKI expression, of nuclear γH2AX foci, of stress fibers and of several SASP factors (IL6, IL8, MMP3) confirming the expression of main senescence features. Senescent ASCs produced 4‐fold more EVs than healthy ASCs and senescent ASC‐EVs were larger. In vitro, both healthy and senescent ASCs decreased fibrotic markers (type III COLLAGEN), catabolic and hypertrophic markers (MMP3, MMP13, AP) and increased COX2 expression in OA chondrocytes. By contrast, healthy ASCs decreased the expression of IL6 while senescent ASCs highly increased IL6. Looking at the role of ASC‐EVs on OA chondrocytes, we found out that both healthy and senescent ASC‐EVs were able to increase the expression of AGG and type II COLLAGEN while they decreased the expression of MMP13, AP, type X COLLAGEN, HMOX1 and IL6. Finally, healthy and senescent ASC‐EVs decreased the number of SA‐bGal positive chondrocytes but did not impact the expression of p21 in IL1b‐induced chondrocytes.

Summary/Conclusion: Our results indicated a chondroprotective effect of ASC‐EVs, independently of the senescent state of parental cells and suggested that EVs might act through different mechanisms than ASCs, which warrants further investigation

OD18.04. Exosomes derived from osteogenic tumor activate osteoclast differentiation and concurrently inhibit osteogenesis to promote bone metastasis

Lijuan Yu, Department of Laboratory Medicine

Xiaoke Hao, Clinical Laboratory Department

Introduction: In patients with prostate cancer (PCa), bone lesions appear osteoblastic in radiographs; however, pathological fractures frequently occur in PCa patients, and bone resorption is observed in all metastatic lesions under histopathologic assessment. The mechanisms that balance the activities of osteoblasts and osteoclasts in PCa patients remain unclear. Intercellular and interorgan communication, mediated by exosomes, is a novel and powerful means of communication. However, until now, the role of PCa exosomes in PCa bone metastasis is still unknown.

Methods: Exosomes were isolated using ultracentrifuge and characterized. The biodistribution was accessed by in vivo imaging, ex vivo imaging and immunolabeling. Trap staining, Trap activity and the mRNA expression were performed to assess the osteoclast differentiation. ARS S staining, ALP staining, ALP activity and the mRNA expression were performed to assess the osteoblast differentiation. The tibia for microCT, HE staining, Trap staining and OCN immunolabeling were harvested to examine the bone homeostasis in vivo. After 4 weeks education, MDA PCa 2b‐luc cells were injected into the right tibia of BALB/C nude mice, and then tumor growth in bone was detected.

Results: The bone targeting of PCa exosomes was verified. Exosomes derived from osteoblastic (MDA PCa 2b), osteoclastic (PC3), and mixed (C4‐2) PCa cells were found here to promote osteoclast differentiation and concurrently inhibit osteoblastogenesis in vitro and in vivo, are responsible for osteolytic lesions, bone ECM remodeling, and the aggressive growth of PCa cells in bone.

Summary/Conclusion: Our findings not only offer a novel perspective on tumor bone metastasis, where“contrary to our initial hypothesis” exosomes derived from an osteoblastic tumor induce osteoclast differentiation, but also suggest potential therapeutic targets for PCa bone metastasis.

OD18.05. The LC3‐conjugated extracellular vesicles originated from secretory autophagy initiate pathological calcification in osteoarthritis and regulated by sympathetic tone

Jianfei Yan, The Fourth Military Medical University

Minjuan Shen, The Fourth Military Medical University

Weicheng Lu, The Fourth Military Medical University

Xiaoxiao Han, The Fourth Military Medical University

Wenpin Qin, The Fourth Military Medical University

Kai Jiao, The Fourth Military Medical University

Introduction: Pathological cartilage calcification plays a very important role in osteoarthritis (OA) progression, but its initiating factor is still unclear. Secretory autophagy has been reported to facilitate calcified precursors secretion within the extracellular vesicles (EVs). Nevertheless, the relationship between secretory autophagy and EVs and their effects in pathological cartilage calcification of OA remain unknown.

Methods: The LC3‐mCherry/EGFP mice and α2 adrenergic receptor (Adra2) knockout mice and their wide types were used to establish the temporomandibular joint (TMJ) OA model, and the LC3‐conjugated EVs and the cartilage calcification were examined dynamically. LC3‐positive EVs from OA cartilage were collected and added to the mimic cartilage extracellular matrix to evaluate their effect on calcification. The histone deacetylase 6 (HDAC6) activity, acetyl‐α‐tubulin level and microtubule stabilization were also tested.

Results: LC3‐positive EVs were increased in OA cartilage comparing to the controls, and were tightly related to the OA cartilage calcification by a location‐ and time‐dependent manner. The calcified EVs within OA cartilage were produced by LC3‐positive secretory autophagy, resulting from the decreased fusion of the autophagosomes with lysosomes. In addition, the LC3‐conjugated EVs aggregated to produce calcifying nodules with high concentrations of calcium and phosphate, resembling those observed in OA calcified cartilage. Mechanism wisely, increased levels of HDAC6, but decreased level of acetylated α‐tubulin and aggravated microtubule destabilization were observed in OA cartilage comparing to the controls. The deletion of Adra2 in TMJ‐OA mice efficiently decreased the HDAC6 level, reversed α‐tubulin deacetylation and microtubule destabilization, decreased the secretion of the LC3‐conjugatied EVs, eventually ameliorating cartilage calcification and degradation.

Summary/Conclusion: Secretory autophagy is the origin of LC3‐conjugated EVs and plays an important role in initiating pathological cartilage calcification in OA. The activation of Adra2 by sympathetic tone plays an important role in LC3‐conjugated EVs‐induced cartilage calcification in OA, through regulating HDAC6‐mediated α‐tubulin deacetylation and microtubule destabilization.

OD19.01. Extracellular vesicles from skeletal muscle cells isolated from extremely obese patients with and without type 2‐diabetes show different protein‐ and miRNA‐pattern

Kari Bente Foss Haug, The Blood Cell Research Group, Section for Research, Development and Innovation, Department of Medical Biochemistry, Oslo University Hospital, Ullevål

Vigdis Aas, Department of Life Sciences and Health, Oslo Metropolitan University (OsloMet), Norway

Abdille Hussein, Department of Life Sciences and Health, Oslo Metropolitan University (OsloMet), Norway

Misbah Hussain, Department of Life Sciences and Health, Oslo Metropolitan University (OsloMet), Norway

Hans Christian D. Aass, The Blood Cell Research Group, Section for Research, Development and Innovation, Department of Medical Biochemistry, Oslo University Hospital, Ullevål, Norway

Berit S. Brusletto, The Blood Cell Research Group, Section for Research, Development and Innovation, Department of Medical Biochemistry, Oslo University Hospital, Ullevål, Norway

Ole Kristoffer Olstad, The Blood Cell Research Group, Section for Research, Development and Innovation, Department of Medical Biochemistry, Oslo University Hospital, Ullevål, Norway

Anne‐Marie Siebke Trøseid, The Blood Cell Research Group, Section for Research, Development and Innovation, Department of Medical Biochemistry, Oslo University Hospital, Ullevål, Norway

Trude Aspelin, Oslo University Hospital, Ullevaal

Tuula Anneli Nyman,Proteomics Core Facility, Oslo University Hospital, Rikshospitalet, Norway

Reidun Øvstebø, The Blood Cell Research Group, Section for Research, Development and Innovation, Department of Medical Biochemistry, Oslo University Hospital, Ullevål, Norway

Introduction: Skeletal muscle (SkM), the largest and highly adaptable organ in human, is responsible for locomotion and energy metabolism. SkM plays an important role as secretory organ, producing modulating factors like myokines and metabolites. Recently, SkM‐derived extracellular vesicles (EV), containing bioactive components, have been described. The aim of this project was to characterize myotube‐secreted EV from extremely obese patients with type 2 diabetes mellitus (T2DM) or normal glucose tolerance (NGT) and to search for component specific profiles.

Methods: Muscle biopsies from bariatric surgery (T2DM: n = 6, NGT: n = 6) were used to isolate, in vitro cultivate and differentiate satellite cells into mature myotubes. EV were isolated, separated and concentrated into microvesicles (MV) and exosomes using centrifugation and filtration. Size and concentration were revealed by NTA, presence of tetraspanins analysed by flowcytometry and Hsc70/Hsp70 and calnexin with Western blot. Proteomics and RNA‐content in exosomes were analysed by LC‐MS/MS and Affymetrix microarray, and bioinformatics examined by Ingenuity Pathway Analysis.

Results: Concentration, size and presence of CD63‐ and CD81‐positive MV and exosomes were similar between T2DM and NGT. A total of 494 exosome proteins from both groups were detected, where 204 proteins showed significant higher levels and 144 had significant lower levels when comparing the two groups, suggesting an association to energy homeostasis and metabolism, ubiquitination, and the cardiovascular system. Results from pilot microarray analysis showed series of miRNA with significant different amounts between the groups.

Summary/Conclusion: In vitro cultured myotubes from extremely obese patients with T2DM or NGT synthesize similar amounts of CD63‐ and CD81‐positive MV and exosomes. In contrast, large differences in protein and microRNA exosome pattern were observed between the groups, indicating that the exosome load reflect the state of the SkM.

OD19.03. Placental small extracellular vesicles regulate insulin sensitivity during pregnancy and induce metabolic changes in gestational diabetes mellitus

Soumyalekshmi Nair, University of Queensland

Katherin Scholz Romero, BSc, Exosome Biology Laboratory, Centre for Clinical Diagnostics, UQ Centre for Clinical Research, The University of Queensland, Australia

Dominic Guanzon, PhD, Exosome Biology Laboratory, Centre for Clinical Diagnostics, UQ Centre for Clinical Research, The University of Queensland, Australia

Andrew Lai, PhD, Exosome Biology Laboratory, Centre for Clinical Diagnostics, UQ Centre for Clinical Research, The University of Queensland, Australia

Harold David McIntyre, Mater Research Institute‐University of Queensland, Translational Research Institute, Woolloongabba, Australia

Martha Lappas, Obstetrics, Nutrition and Endocrinology Group, Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia and Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria, Australia.

Carlos Salomon, MSc, DMedSc, PhD, The University of Queensland

Introduction: Growing evidence shows that extracellular vesicles play important roles in the regulation of metabolic functions. The aim of this study was to elucidate the role of placenta‐derived small extracellular vesicles (sEVs) in the regulation of maternal insulin sensitivity in Gestational Diabetes Mellitus (GDM). GDM is the fastest‐growing type of diabetes and is the most common medical complication of pregnancy.

Methods: Primary Human Trophoblast (PHT) cultures from Normal Glucose Tolerant (NGT) and GDM pregnancies were developed and sEVs were isolated from cell‐conditioned media using density gradient centrifugation. The protein content of PHT cells and sEVs analyzed by data‐independent acquisition (DIA) mass spectrometry. Using Illumina TrueSeq Small RNA kit, a small RNA library was constructed and miRNA profile of PHT cells and sEVs were determined.

Results: We identified that PHT cells express a differential profile of miRNAs and proteins in GDM compared to NGT. Interestingly, the miRNA profile of sEVs showed a significant correlation (p‐value < 0.0001) to PHT cells, indicating that sEVs are “fingerprints” of the releasing cells and their metabolic status. However, we identified a set of miRNAs and proteins differentially expressed in sEVs compared to PHT cells in GDM and NGT. This indicates the presence of mechanisms by which molecular cargo are specifically sorted to sEVs.The expression of certain miRNAs (miR‐1260b, miR‐181‐5p, miR‐589‐5p, and miR‐660‐5p) varied in a consistent pattern in PTH and their secreted sEVs in GDM compared with NGT. Using IPA analysis, miRNA and proteomic data were integrated and miRNA‐gene interactions were identified. IPA core analysis showed that the top canonical pathway associated with these miRNAs were PI3/AKT signaling and glucose metabolism/insulin resistance, respectively.

Summary/Conclusion: sEVs released from placenta can modify the phenotype of target cells and regulate metabolic changes in GDM contributing to changes in insulin sensitivity during pregnancy.

OD19.04. Impact of lipid accumulation in hepatocytes in the release of COMT associated to extracellular vesicles

Maria Azparren‐Angulo, CICbioGUNE

Edward Milbank, Department of Physiology, CIMUS, University of Santiago de Compostela‐Instituto de Investigación Sanitaria de Santiago de Compostela, CIBERobn, Santiago de Compostela, Spain

Félix Royo, Exosomes laboratory, CIC biogune‐BRTA, CIBERehd, Derio, Bizkaia, Spain

Miguel López, Department of Physiology, CiMUS, University of Santiago de Compostela‐Instituto de Investigación Sanitaria

Juan Manuel Falcón‐Pérez, CIC bioGUNE

Introduction: Hepatocytes secrete extracellular vesicles (EVs), and we have published previously that liver damage induce important changes in the composition and amount of secreted EVs. Those circulating EVs are loaded with specific liver proteins, including active enzymes, such as, arginase 1, CYP450 and Catechol‐O‐methyl transferase (COMT). Indeed, COMT (EC 2.1.1.6) is an enzyme that metabolizes catecholamines catalysing the transfer of a methyl group from S‐adenosylmethionine to catecholamines as dopamine, epinephrine and norepinephrine. The activity of COMT has been implicated in different liver processes, such as glucose homeostasis and establishment and progression of obesity and diabetes.

Methods: We have studied the changes in COMT in models that mimic fatty liver and lipid accumulation in hepatocytes. As models, we have employed hepatic and non‐hepatic cell lines and compared their COMT expression. Regarding to animal models, we have employed obesity models of rats and mice. To mimic the liver damage caused by obesity, we have treated cells with a mix of oleic and palmitic acid (2:1 ratio, 1mM final) generating a lipid accumulation like the observed in vivo. Apart from in vitro treatment, we also have look at in vivo obesity models using animals with a spontaneous mutations of the Leptin gene (ob/ob) mouse and Zucker rat. For EVs extraction, the cells are cultured after the hepatic perfusion and, 48 hours later, the EVs are obtained by ultracentrifugation. The analyses of the vesicles have been done through western blot and immunofluorescence. Finally, to see the effect that EVs‐associated COMT can have in different organs and knowing that COMT has been linked to eating and neuropsychiatric behaviour, we have also studied the effect of COMT loaded EVs in rat brain.

Results: Our work shows differences in the abundance of COMT in extracellular vesicles depending on the cellular model and the metabolic conditions assayed in this study. In addition, the administration of EVs from metabolically‐altered hepatocytes into rat brain shows limited effect on food intake and body weight.

Summary/Conclusion: The lipid accumulation produce changes in the release of COMT loaded EVs that could influence food intake of animals.

OD19.05. Feces‐derived extracellular vesicles disorganize gut‐liver axis homeostasis and contribute in the development of liver diseases

Jérôme Boursier,Centre Hospitalo‐Universitaire Angers

Alexandre Villard, INSERM U1063 SOPAM

Nadia Benabbou, INSERM U1063 SOPAM

Raffaella Soleti, INSERM U1063

Lionel Fizanne, HIFIH

Erwan Delage, LS2N

Mireille Wertheimer, INSERM U1063 SOPAM

Thibauld Oullier, INSERM U1235 TENS

Samuel Chaffron, LS2N

Michel Neunlist, INSERM U1235 TENS

Ramaroson Andriantsitohaina, INSERM U1063 SOPAM

Introduction: Non‐alcoholic fatty liver disease (NAFLD) is currently considered as the main chronic liver disease, with a worldwide prevalence of 25%. NAFLD may lead to the apparition of the inflammatory form of the disease, the non‐alcoholic steatohepatitis (NASH) often associated with fibrogenesis. The gut microbiota participates in the progression of NAFLD towards NASH and fibrogenesis, especially by triggering hepatic inflammatory pathways via the toll‐like receptor4 (TLR4)/lipopolysaccharide (LPS)‐mediated response. The gut microbiota is also a provider of bacterial extracellular vesicles (EVs), which are found in feces samples. In this regard, EVs derived from mouse feces have been demonstrated to induce systemic inflammation via TLR4 in a mouse model. However, no real translational study has been performed on the direct involvement of feces‐derived EVs (fEVs) in NAFLD/NASH pathophysiology.

Methods: fEVs were isolated from feces samples and small circulating EVs (cEVs) were isolated from blood samples from NAFLD/NASH biopsy‐confirmed patients and from non‐NAFLD/non‐NASH donors.

Results: fEVs and small cEVs are derived from prokaryotic and eukaryotic origins including intestinal epithelial cells. Only fEVs from NASH patients exerted deleterious effects. NASH fEVs increased intestinal permeability associated with reduced expression of tight junction proteins by a mechanism sensitive to non‐muscular myosin light chain kinase inhibition in vitro or deletion in vivo. NASH fEVs increased endothelial cell permeability and production of inflammatory cytokines and chemokines by a TLR4/LPS‐mediated pathway. NASH‐fEVs, as well as NASH‐cEVs, increased profibrotic and proinflammatory proteins expression in hepatic stellate cells. Bacterial origins of fEVs were different between stages of the liver disease and 16 amplicon sequence variants were identified as differentially abundant.

Summary/Conclusion: Together, our translational results support the potential role of fEVs as key players in NAFLD progression towards NASH by acting on different stages of the gut liver‐axis and warrant further preclinical and clinical studies to confirm the key targets nmMLCK and TLR4 for NASH resolution and fibrosis.

OD19.06. Exosomes Secreted By Umbilical Cord Blood‐Derived Mesenchymal Stem Cell Promote Pancreatic Regeneration And Insulin Secretion In Mouse Model Of Type 1 Diabetes

manju kumari, Sanjay Gandhi Postgraduate Institute of Medical Sciences

SUMAN MISHRA, Sanjay Gandhi Postgraduate Institute of Medical Sciences

Dharmendra K. Chaudhary, Sanjay Gandhi Postgraduate Institute of Medical Sciences

Alok Kumar, Sanjay Gandhi Postgraduate Institute of Medical Sciences

Avni Batia, Batia Avni MD, Head of outpatient service Department of Bone Marrow Transplantation and Cancer Immunotherapy Hadassah Medical Organization, POB 12000 Jerusalem 91120 Israel

Swasti Tiwari, Sanjay Gandhi Postgraduate Institute of Medical Sciences

Introduction: Mesenchymal stem cells (MSCs) therapy is a recent innovative approach in diabetes due to its capacity to modulate tissue microenvironment and regeneration of glucose‐responsive insulin‐producing cells. In this study, we investigated how MSC‐derived exosomes affect the severity of diabetes and their mechanism of action.

Methods: Diabetes was induced in male C57Bl/6 mice by five consecutive doses of streptozotocin (STZ; 40 mg/kg body weight, i.p). The diabetic mice were administered (i.v) with MSC (1‐105 umbilical cord blood MSCs cells/day), their derived exosomes (MSC‐Exo group; that received exosomes derived from 1‐105 MSCs cells/day), or the same volume of PBS. Before administration, the potency of MSCs and their exosomes in immune cell modulation were evaluated in vitro T‐cell activation experiments. After day 7 of the treatments, blood samples, and pancreatic tissues were analyzed.

Results: The results revealed reduced pancreatic tissue damage with an improved histological structure in mice treated with MSCs or MSC‐Exo compared to PBS‐treated mice. Hyperglycemia was also attenuated in these mice with a concomitant increase in insulin production compared to mice in the PBS‐treated group. We found increased expression of genes associated with tissue regeneration pathways in the pancreatic tissue of mice treated with MSC or MSC‐Exo relative to PBS‐treated mice. miRNA profiling of MSCs derived exosomes showed the presence of miRs that may facilitate pancreatic regeneration.

Summary/Conclusion: These results demonstrate a potential therapeutic role of umbilical cord blood MSC ‐derived exosomes in diabetes by activating pancreatic islets' intrinsic regenerative abilities.

OD20.01. LncRNA‐encoded Peptides: a New Form of Cargo in Cell‐derived and Circulating Extracellular Vesicles

Tanxi Cai, Institute of Biophysics, Chinese Academy of Science

Qing Zhang, Institute of Biophysics, Chinese Academy of Science

Bowen Wu, Institute of Biophysics, Chinese Academy of Science

Jifeng Wang, Institute of Biophysics, Chinese Academy of Science

Na Li, Institute of Biophysics, Chinese Academy of Science

Tingting Zhang, Institute of Biophysics, Chinese Academy of Science

Zhipeng Wang, Institute of Biophysics, Chinese Academy of Science

Jianjun Luo, Institute of Biophysics, Chinese Academy of Science

Xiaojing Guo, Institute of Biophysics, Chinese Academy of Science

Xiang Ding,Institute of Biophysics, Chinese Academy of Science

Zhensheng Xie, Institute of Biophysics, Chinese Academy of Science

Lili NiuInstitute of Biophysics, Chinese Academy of Science

Weihai Ning, Department of Neurosurgery, Sanbo Brain Hospital, Capital Medical University, Beijing, China

Xiangqian Guo,Henan Provincial Engineering Centre for Tumor Molecular Medicine, School of Basic Medical Sciences, Henan University

Runsheng Chen,Institute of Biophysics, Chinese Academy of Science

Hongwei Zhang,Department of Neurosurgery, Sanbo Brain Hospital, Capital Medical University, Beijing, China

Fuquan Yang,Key Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

Introduction: Advancements in technology over the past few years have led to the discovery of numerous biologically relevant peptides encoded by small open reading frames (smORFs) embedded in long noncoding RNA transcripts (referred to as lncRNA‐SEPs here) in a variety of species. However, the mechanisms and modes of action that underlie the roles of lncRNA‐SEPs have yet to be fully characterized. Of particular interest is whether lncRNA‐SEPs are taking part in intercellular communication between tumor cells and stromal cells in either the local or distant microenvironment.

Methods: In order to discover the novel lncRNA‐SEPs from cell‐derived and circulating EVs, we developed an MS‐based lncRNA‐SEPs analytical workflow, including the development of a lncRNA‐SEPs database that could entirely cover the putative lncRNA‐SEPs in humans but avoid generating a dataset as excessively large as the six‐frame translation of the entire genome. The human lncRNA transcripts deposited in the NONCODE (http://www.noncode.org/) database were scanned by ORFfinder and six‐frame translation mode to obtain all possible smORFs, and then theoretically translated into SEPs. Purified EVs of Glioma cancer cells or circulating EVs from health donors and Glioma cancer patients were then subjected to MS‐based lncRNA‐SEPs analysis.

Results: We provide the first experimental evidence of 29 lncRNA‐SEPs in EVs derived from Glioma cancer cells, indicating that the EV‐mediated transfer of lncRNA‐SEP may represent a novel mechanism for intercellular communication. Intriguingly, when examining human plasma, 48, 11 and 3 lncRNA‐SEPs were identified from purified EVs, whole plasma and EV‐free plasma, respectively, suggesting that circulating lncRNA‐SEPs are primarily enriched in EVs. Most importantly, our preliminary data demonstrate that lncRNA‐SEPs in EVs can be used to distinguish Glioma cancer patients from healthy controls, indicating circulating lncRNA‐SEPs in EVs has potential diagnostic application in identifying patients with glioma.

Summary/Conclusion: Our results suggest that EV‐mediated transfer of lncRNA‐SEPs represent a novel mechanism of intercellular communication between cells in close proximity as well as those at a distance. This communication method could provide an opportunity to establish entirely new paradigms of intercellular and inter‐species information exchange based on the EV‐mediated release, transport, uptake, and regulatory roles of lncRNA‐SEPs.

OD20.02. Characterization of size‐based isolated EV populations from a metastatic melanoma cell line by lipid analysis and proteomics

Felice Accattatis, Università Degli Studi di Milano

Sara Mazza, Dipartimento Di scienze Farmacologiche e Biomolecolari ‐ Università Degli Studi di Milano

Agnese Granata, Dipartimento Di scienze Farmacologiche e Biomolecolari ‐ Università Degli Studi di Milano

Elisabetta Vergani, Laboratorio di Immunoterapia dei Tumori Umani IRCCS Istituto Nazionale dei Tumori Milano

Monica Rodolfo, Laboratorio di Immunoterapia dei Tumori Umani IRCCS Istituto Nazionale dei Tumori Milano

Sara Baroni, Dipartimento Di scienze Farmacologiche e Biomolecolari ‐ Università Degli Studi di Milano

Alberto Corsini, Dipartimento Di scienze Farmacologiche e Biomolecolari ‐ Università Degli Studi di Milano

Lidia Merlo, Dipartimento Di scienze Farmacologiche e Biomolecolari ‐ Università Degli Studi di Milano

Lorenzo Arnaboldi, Dipartimento Di scienze Farmacologiche e Biomolecolari ‐ Università Degli Studi di Milano

Introduction: New insights into size, protein and lipid composition may help in characterizing functionality od Extracellular Vesicles (EVs) and infer with their fast development as delivery tools. Unfortunately, despite latest size‐based classifications which divide EVs in small (50‐80nm) or large (80‐120nm) exosomes, microvesicles (< 1000nm) and the new smallest (< 50nm) population (exomeres), overlapping of different EV populations and unproper separation methods impair the comprehension of their biological role.

Methods: To overcome this problem, we set up a reproducible ultracentrifugation (UC) method for a size‐based separation of different EV populations by 5 UC steps, in which physical and dynamic parameters are determined by an algorithm developed by Livshts etal. In the 5 fractions isolated from the culture medium of a metastatic melanoma cell line, EVs size was analyzed by transmission electron microscopy (TEM) and dynamic light scattering (Zetasizer), lipid content by gas chromatography, and protein profile by mass spectrometry and Ingenuity Pathway Analysis (IPA).

Results: Zetasizer and TEM analysis documented the existence of 5 different EV populations, whose relative % in saturated fatty acids gradually and continuously increased from larger to smaller EV (from 37.21±0.21 to 64.79±9.47). Proteomics identified a total of 2003 proteins differentially distributed (or even unique) among the 5 EV populations (n = 697, 819, 1079, 1621 and 1654, respectively). IPA analysis of these distributions revealed different characteristics signaling pathways.

Summary/Conclusion: Melanoma‐released EVs include vesicles of different size, fatty acid and protein composition. These differences may translate into distinct behaviors and functions in biological fluids and help to define the role of specific EV populations in physiological and pathological processes. Finally, these results pave the road to new pharmacological treatments to modulate EVs functions or to use EVs as pharmaceutical tools.

OD20.03. Biofunctional peptide‐modified extracellular vesicles with encapsulation of boron compounds for boron neutron capture therapy (BNCT)

Shiori Hirase, Graduate School of Science, Osaka Prefecture University

Ayako Aoki, Graduate School of Science, Osaka Prefecture University

Kenta Morimoto, Graduate School of Science, Osaka Prefecture University

Kosuke Noguchi, Graduate School of Science, Osaka Prefecture University

Yoshihide Hattori, Research Center for Boron Neutron Capture Therapy, Osaka Prefecture University

Mitsunori Kirihata, Research Center for Boron Neutron Capture Therapy, Osaka Prefecture University

Ikuhiko Nakase, Graduate School of Science, Osaka Prefecture University

Introduction: Boron neutron capture therapy (BNCT) is a radiation therapeutic method for cancer therapy. In the BNCT, internalization of boron‐10 (10B) atoms by cancer cells induces the cell death by the generation of alpha particles and recoiling lithium‐7 (7Li) nuclei when irradiated with low‐energy thermal neutrons [1]. In this research, we aimed to develop extracellular vesicles (exosomes, EVs)‐based drug delivery technology for BNCT. EVs have been highly expected to be a next‐generation drug delivery carrier, because of their pharmaceutical advantages, including controlled immune responses, effective usage of cell‐to‐cell communication, and brain‐targeting. In our research, we successfully achieved for modification of hexadeca oligoarginine (R16) [2, 3] on the EV membrane to effectively induce the macropinocytotic cellular uptake (accompanied by actin reorganization, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid), leading to enhanced biological activity in BNCT.

Methods: Fluorescently‐labeled dodecaborate (FITC‐BSH) was encapsulated in the EVs (derived from HeLa cells) by electroporation (FITC‐BSH‐EVs). We synthesized the R16 peptides with a succinimide linker (R16‐EMCS)). The R16‐EMCS can covalently bind to amino‐group of FITC‐BSH‐EVs membrane proteins by their simple mixing. Internalization of FITC‐BSH‐EVs by C6‐glioma cells was analyzed using a confocal laser scanning microscope and a flow cytometer. Actin reorganization (lamellipodia) was assessed by phalloidin‐staining.

Results: Modification of the R16‐EMCS (20 μM) on the EV membrane significantly increased cellular uptake of FITC‐BSH (230 nM) into C6‐glioma cells (approximately 25‐fold increase by the peptide modification (24 hr treatment)). Macropinocytosis induction by the EVs modified with R16‐EMCS was confirmed by enhanced macropinocytosis marker and lamellipodia formations. In the thermal neutron irradiation experiments, the C6‐glioma cell‐killing effect of BSH was enhanced through macropinocytosis induction by encapsulation in the EVs and modification with R16‐EMCS.

Summary/Conclusion: These results provide fundamental knowledge for the further development of EV‐based intracellular delivery system in BNCT.

[1] Nakase, I. etal. ACS Omega, 5, 22731 (2020)

[2] Nakase, I. etal. Sci. Rep., 7, 1991 (2017)

[3] Nakase, I. Processes, 9, 224 (2021)

OD20.04. Electrical and Sensitive Quantification of Extracellular Vesicles with a Reduced Graphene Oxide Field Effect Transistor Biosensor

Yi Yu, Hubei university of chinese medicine

Guo‐Jun Zhang, Hubei university of chinese medicine

Introduction: Microvesicles (MVs) and exosomes have received extensive attention in recent years because they are closely related to the development of tumors. Therefore, it is very important to establish a high‐sensitivity and high‐specificity detection method to detect low‐concentration exosomes and MVs.Field effect transistor (FET) biosensor is one of the most promising biosensors in recent years. Through field effect transistors, microelectrical signals caused by the interactions between biomolecules on the sensing interface are transformed into readable electrical signals and amplified, with high sensitivity and good specificity.However, the detection of exosomes and MVs using FET biosensors has not yet been reported.

Methods: We first fixed reduced graphene oxide (RGO) on the surface of the sensor by drip coating, and realized the label‐free detection of exosomes by modifying the CD63 antibody. Secondly, we deposited gold particles on the surface of RGO by the chloroauric acid deposition method, and then modified the double aptamer (TLS 11a, EpCAM) on it to identify the microvesicles derived from liver cancer cells(HepG2‐MVs), through the Dirac point in the electrical signal The deviation of exosomes and microvesicles were detected separately.

Results: The exosomes and MVs were purified by ultracentrifugation.The functionalization process was proved by the Id‐Vg electrical signal characteristic curve. The detection sensitivity is 33 particles/ul and 84 parbicles/ul, respectively. The detection method is higher than that of most biosensors, and it can detect the analyte under a complex system with high specificity. Finally, the constructed functional sensor was used to detect the levels of exosomes and MVs in the plasma of prostate cancer patients and liver cancer patients. The results show that the constructed sensor can effectively distinguish cancer patients from healthy volunteers, and can be used repeatedly and still has high detection sensitivity, indicating that the method has the potential for clinical application and is expected to be used for early diagnosis of tumors.

Summary/Conclusion: 1. A functional RGO FET biosensor was established for the first time to detect exosomes and MVs. The detection sensitivity is 33 particles/ul and 84 particles/ul, respectively, which is higher than most biosensors. 2. This method can effectively distinguish tumor patients from healthy volunteers and has high specificity. 3. This method can complete the label‐free detection within 30 minutes, and the sensor can be used repeatedly, and the cost is low. 4. We have established a universal method to detect exosomes or MVs derived from specific tumor cells by modifying different antibodies or aptamers, providing a new detection idea and platform for clinical detection.

OD20.05. Development of a novel serum exosomal miRNA nomogram for the preoperative prediction of lymph node metastasis in esophageal squamous cell carcinoma

Tong Liu, Second Hospital, Shandong University

Chuanxin Wang, Second Hospital, Shandong University

Introduction: Preoperative prediction of lymph node (LN) metastasis is accepted as a crucial independent risk factor for treatment decision‐making for esophageal squamous cell carcinoma (ESCC) patients. Our study aimed to establish a non‐invasive nomogram to identify LN metastasis preoperatively in ESCC patients.

Methods: Construction of the nomogram involved 3 sequential phases with independent patient cohorts. In discovery phase (N = 20), LN metastasis‐associated miRNAs were selected from next‐generation sequencing (NGS) assay of human ESCC serum exosome samples. In training phase (N = 178), a nomogram which incorporated exosomal miRNA model and clinicopathologic was developed by multivariate logistic regression analysis to preoperatively predict LN status. In the validation phase (n = 188), we validated the predicted nomogram's calibration, discrimination and clinical usefulness.

Results: Four differently expressed miRNAs (chr 8‐23234‐3p, chr 1‐17695‐5p, chr 8‐2743‐5p and miR‐432‐5p) were tested and selected in the serum exosome samples from ESCC patients who have or do not have LN metastasis. Subsequently, an optimized 4‐exosomal miRNA model was constructed and validated in the clinical samples, which could effectively identify ESCC patients with LN metastasis, and was significantly superior to preoperative computed tomography (CT) report. In addition, a clinical nomogram consisting of the 4‐exosomal miRNA model and CT‐report was established in training cohort, which showed high predictive value in both training and validation cohorts (AUC: 0.880 and 0.869, respectively). Hosmer‐Lemeshow test and decision curve analysis implied the nomogram's clinical applicability.

Summary/Conclusion: Our novel non‐invasive nomogram is a robust prediction tool with promising clinical potential for preoperative LN metastasis prediction of ESCC patients, especially in T1 stage.

OD20.06. Exosomes loaded with Palladium nanosheets: targeted bioothogonal catalysts against cancer

María Sancho‐Albero, Institute of Nanocience and Materials of Aragon (INMA)

María Sancho‐Albero, Institute of Nanocience and Materials of Aragon (INMA)

Belén Rubio‐Ruiz, GENYO

Ana M Pérez‐López, Technische Universität Berlin

Victor Sebastián, Institute of Nanoscience and Materials of Aragon (INMA)

Pilar Martín‐Duque, ARAID

Manuel Arruebo, Institute of Nanoscience and Materials of Aragon (INMA)

Asier Unciti‐Broceta, Cancer Research UK Edinburgh Centre

Jesús Santamaría, Institute of Nanoscience and Materials of Aragón (INMA)

Introduction: The transformational impact of bioorthogonal chemistry has inspired the development of new and exciting strategies for the in vivo synthesis of bioactive agents through non‐natural means. Among these, Palladium (Pd) catalysts have played a key role in the growing subfield of bioorthogonal catalysis by producing uncaging biomolecules in living systems and providing them with new functional properties. Exosomes are proposed to be ideal vehicles for targeting novel therapies. However very little is known about the selectiveness and specificity of the transference processes involving exosomes released from different cells.

Methods: Exosomes derived from A549 and U87‐MG cells were isolated and loaded with Pd‐ nanosheets using a CO mediated reduction procedure. Pd‐loaded exosomes (Pd‐Exos) were thoroughly characterized by CryoTEM, UV‐VIS, XPS, Western Blot, Zeta potencial, etc. Their catalytic activity was evaluated by fluorogenic studies and time‐lapse microscopy. Furthermore, we assessed the biocompatibility and the internalization of the Pd‐Exos by metabolic assays and confocal microscopy. Finally, the intracellular catalytic activation of a non‐active prodrug into Panobinostat was evaluated in A549 and U87 cells.

Results: A bio‐artificial device consisting of cancer‐derived exosomes loaded with Pd catalysts (Pd‐Exos) has been created by a novel method based on a CO mediated reduction of Pd within exosomes to obtain catalytically active nanostructures inside the extracellular vesicles. Pd‐Exos do not exhibit cytotoxicity at the studied doses and they are co‐localized in the cell cytoplasm and particularly, in the endosomal‐exosomal pathway. This new hybrid system mediates Pd‐triggered dealkylation reactions in vitro (inside glioblastoma cells (U81‐MG) and lung cancer cells (A549)) and serves as Trojan Horse, having a preferential tropism and fingerprint for its progenitor cells. This bioorthogonal reaction leads to cancer cell death in an effective and selective way.

Summary/Conclusion: This study illustrates the therapeutic potential of combining the exosome‐mediated catalysts and bioorthogonal uncaging chemistries to activate bioactive substances (such as the recently approved anticancer drug Panobinostat) in a spatio‐temporal selective way.

OD21.01. Reduced, Reuse, Recycle: Replenishing extracellular vesicles lost through degeneration‐induced depletion as a novel therapy for the treatment of Age‐related macular degeneration

Yvette S M Wooff, The Australian National University

Yvette S M Wooff, The Australian National University

Adrian Cioanca, The Australian National University

Joshua A. Chu‐Tan, The Australian National University

Riccardo Natoli, The Australian National University

Introduction: We have previously demonstrated that retinal degeneration is associated with the depletion of extracellular vesicles (EV) and impaired miRNA shuttling via EV within the retina. We therefore hypothesized that supplementation of healthy retinal EV or their miRNA cargo could ameliorate retinal degeneration.

Methods: To characterize the miRNA cargo of retinal EV, RNA‐seq was performed on EV from healthy and degenerating mouse retinas. EV from healthy retinas were supplemented into the degenerating retina via intravitreal injection (2.0 × 1010 EV/eye) at day 2 of a 5‐day photo‐oxidative damage paradigm. To investigate the role of EV‐miRNA in regulating tissue homeostasis, EV abundant miRNA miR‐124‐3p was administered into the degenerating retina by intravitreal injection. Electroretinography and optical coherence tomography were used to assess retinal function and morphology while TUNEL and IBA‐1+ staining was conducted to measure cell death and inflammation.

Results: The top 10 most abundant EV‐miRNA made up ∼67% of the EV miRNA content, with miR‐124‐3p alone accounting for 18%. Bioinformatic pathway analysis revealed that these miRNA were associated with the regulation of inflammatory and cell survival pathways known to be heavily involved in retinal degenerations. Compared to controls, mice injected intraocularly with either retinal EV or encapsulated miR‐124‐3p had significantly higher retinal function, reduced inflammation and decreased photoreceptor cell death.

Summary/Conclusion: Taken together, this data supports a central hypothesis in which a loss of EV‐miRNA bioavailability is correlated to progressive retinal degeneration. Further, that replenishing levels of retinal EV and their highly abundant EV‐miRNA such as miR‐124‐3p can reduce the pathological features of degeneration. Results from this work therefore support the use of EV‐based therapies to restore homeostatic communication pathways and slow the progression of retinal degenerations.

OD21.02. Extracellular vesicles as biomarkers in precision medicine: profiling of stroke patients by Surface Plasmon Resonance imaging

Alice Gualerzi, PhD, IRCCS Fondazione Don Carlo Gnocchi ONLUS

Silvia Picciolini, IRCCS Fondazione Don Carlo Gnocchi

Arianna Iannone, IRCCS Fondazione Don Carlo Gnocchi

Cristiano Carlomagno, IRCCS Fondazione Don Carlo Gnocchi

Francesca Rodà, IRCCS Fondazione Don Carlo Gnocchi

Angelo Montesano, IRCCS Fondazione Don Carlo Gnocchi

Marzia Bedoni, IRCCS Fondazione Don Carlo Gnocchi

Introduction: Stroke is the second leading cause of death worldwide. The local hypoxia induced by stroke damages the brain tissue and patients that survive after the event may present disabilities that can persist for a long time or permanently after it. The clinical approach to regenerative rehabilitation of stroke currently lacks of easily accessible and sensitive biomarkers to evaluate the optimal rehabilitation and therapy. In this study we exploited the potentiality of Extracellular Vesicles (EVs) as carriers of stroke markers and the innovative technique of Surface Plasmon Resonance imaging (SPRi) that can guarantee a multiplexed and sensitive analysis of EVs isolated from liquid biopsies for the discovery of disease‐related markers.

Methods: The isolation of circulating EVs from serum of ischemic stroke patients and healthy controls (age and sex matched) was obtained by size exclusion chromatography and ultracentrifugation. A SPRi biosensor was developed for the detection of EVs with different cellular origin (brain and non‐brain cells), the relative quantification of specific surface molecules related to pathological or regeneration processes was accomplished. SPRi results obtained were then correlated with clinical parameters. In parallel, EV physico‐chemical characterization following MISEV2018 requirements and quantification of serum inflammatory cytokines by ELISA assay were performed.

Results: Effective isolation of EVs was obtained. Stroke patients presented higher concentrations of EVs in serum, in particular EVs released by astrocytes and endothelium. Our results demonstrated that specific antigens, like Klotho and Translocator Protein (TSPO) expressed on EVs, can be potential markers for the prediction of a better functional recovery of stroke patients, as evaluated by clinical scales. Besides, EV and non‐EV serum markers related to inflammation were demonstrated to correlate with a stronger response to damage and consequent better physical conditions of patients during the acute phase.

Summary/Conclusion: EV associated proteins were proved as potential markers of different stroke outcomes and laid the foundation for further investigations on a wider cohort. Our results demonstrated the ability of the SPRi biosensor to reveal differences in the relative amount of specific cell‐derived EV subpopulations and in their cargo during disease progression and rehabilitation induced recovery, providing support for using the proposed SPRi‐based biosensor to foresee patients’ outcome after rehabilitation protocols and regenerative therapies.

OD21.03. Embryonic Stem Cells Derived‐Small Extracellular Vesicles Regulate Tregs to Protect against Ischemic Stroke

Yuguo Xia, Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Guowen Hu, Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Qing Li, Institute of Microsurgery on Extremities, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Yang Wang, Institute of Microsurgery on Extremities, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Zhifeng Deng, Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital

Introduction: Stem cells derived‐small extracellular vesicles (sEVs) are proven to promote neurological recovery after stroke. Recent studies demonstrate a phenomenal tissue repair ability in embryonic stem cells‐derived sEVs (ESC‐sEVs). However, whether ESC‐sEVs could protect against ischemic stroke remains unknown. Immune responses play an essential role in the pathogenesis of ischemic stroke and modulating post‐stroke immune responses ameliorate ischemia‐induced brain damage. In this study, we aim to determine the therapeutic function of ESC‐sEVs, specifically, focusing on their role in immunomodulation after ischemic stroke.

Methods: ESC‐sEVs are intravenously administered after transient middle cerebral artery occlusion (MCAO) in C57/BL6 mice. Infarct volume, immune cells infiltration, neural death, and Tregs population were assessed 3 days after MCAO, neurological recovery was evaluated up to 28 days after stroke. Proteomics analysis was utilized to determine the key factors in ESC‐sEVs‐afforded increase of Treg and the following molecular pathway.

Results: ESC‐sEVs significantly decrease leukocytes infiltration, inflammatory cytokines expression, neuronal death, and infarct volume as well as alleviating long‐term neurological deficits and tissue loss after ischemic stroke. Interestingly, ESC‐sEVs induce a marked increase in Tregs after stroke. Further, ESC‐sEVs‐afforded immunomodulatory function and neuroprotection against stroke are dependent on Tregs as the depletion of Tregs almost completely abrogates the protective effects. Mechanistically, proteomic analysis reveals the enrichment of TGF‐β, Smad2, and Smad4 proteins in ESC‐sEVs which could be delivered to activate TGF‐β/Smad pathway in CD4+ T cells and therefore induce Tregs expansion. ESC‐sEVs modulate neuroinflammation and protect against ischemic stroke through the expansion of Tregs, a process which is partially dependent on the activation of TGF‐β/Smad signaling pathway by the transfer of TGF‐β, Smad2, and Smad4.

Summary/Conclusion: This study highlights ESC‐sEVs as a novel and promising cell‐free therapeutic for ischemic stroke, potentially other CNS injuries, and autoimmune diseases.

OD21.04. A novel biosensor based on plastic antibodies for sensing an extracellular vesicle from neuronal cells

Ana P.M. Tavares, Biomark@UC

M.Goreti.F. G. Sales, Biomark@UC

Introduction: Extracellular vesicles are nanostructures containing a lipid bilayer with transmembrane proteins. They play a critical role in intercellular communication and depending on the cell origin their transmembrane proteins and their cargos may vary. So, the composition of each EV allows distinguishing its origin through its proteins or their cargos. EVs circulating in plasm may also come from the brain and provide valuable information about the impact of brain on the overall health status of the body. Thus, monitoring brain‐derived EVs may provide additional insights into neuroscience.

Methods: This work proposes a biomimetic polymer for EV capture from neuronal cells, acting as a plastic antibody. This material was obtained through molecular imprinting by electropolymerized Pyrrole around a glutamate Ionotropic Receptor AMPA Type Subunit 3 (GRIA3) that is a surface protein expected to be present in EVs from neuronal origin. Then GRIA3 is removed from the polymeric network, creating complementary cavities to this protein. The resulting is a molecular imprinting polymer (MIP). In parallel, a non‐imprinted polymer (NIP) was also produced, but without the protein.

Results: The analytical features of the MIP assembly process and its behaviour as sensing material were evaluated by electrochemical impedance spectroscopy (EIS). The MIP material was tested by using different concentrations of GRIA3 from 10ng/mL to 10mg/mL during the recognition process. The results showed a good linear response between 100 ng/mL and 10 mg/mL with ‐0.19 W/decade of slope and square correlation coefficient >0.99. The detection limit was 38.4ng/mL and the quantification limit was 100ng/mL.

Summary/Conclusion: Overall, the MIP obtained was sensitive and selective to the external protein GRIA3 from EVs, which can be promising to intact EVs monitoring.

OD21.05. Highly efficient intercellular spreading of protein misfolding by viral ligand‐decorated extracellular vesicles

Ina M. Vorberg, University Bonn

Shu Liu, DZNE

Andre Hossinger, DZNE

Stefanie Heumüller, DZNE

Annika Hornberger, DZNE

Oleksandra Buravlova, DZNE

Katerina Konstantoulea, KU Leuven, Switch Lab

Stephan Müller, DZNE Munich

Lydia Paulsen, DZNE

Stefan Lichtenthaler,DZNE Munich

Frederic Rousseau, KU Leuven, Switch Lab

Joost SchymkowitzKU Leuven, Switch Lab

Manuela Neumann, DZNE Tübingen

Philip Denner,DZNE

Introduction: Aberrant folding and aggregation of host‐encoded proteins into ordered protein assemblies is a pathological hallmark of neurodegenerative diseases such as prion diseases and Alzheimer's disease. Pathologic protein aggregates have the ability to transmit to unaffected cells, thereby templating and propagating their own aberrant conformation onto soluble homotypic proteins. Proteopathic seeds can be released into the extracellular space, secreted in association with extracellular vesicles (EV) or exchanged by direct cell‐to‐cell contact. The extent to which each of these pathways contributes to the prion‐like spreading of protein misfolding is unclear.

Methods: Exchange of cellular cargo by both direct cell contact or via EV depends on receptor‐ligand interactions. We hypothesized that enabling these interactions through viral ligands enhances intercellular proteopathic seed transmission. We used different cellular models to study the effect of viral glycoproteins on intercellular exchange and propagation of protein aggregates. Induction of protein aggregates in recipient cells, either cocultured with donor cells or exposed to EV from donor cells, was analyzed using automated confocal microscopy and image analysis. Protein aggregates included self‐replicating model prion aggregates, transmissible spongiform encephalopathy agents and pathologic Tau.

Results: We here expressed vesicular stomatitis virus VSV‐G in three different cellular models that propagate different protein aggregates. Coculture of VSV‐G‐expressing donor cells with recipient cells strongly increased protein aggregate induction in the latter. Further, expression of VSV‐G also promoted the secretion of VSV‐G‐coated EV with drastically enhanced aggregate‐inducing capacity in recipient cells. Intriguingly, interactions between SARS‐CoV‐2 spike S protein and its receptor ACE2 similarly contributed to the spreading of prions and Tau aggregates.

Summary/Conclusion: We conclude that efficient intercellular proteopathic seed transfer is strongly controlled by receptor‐ligand interactions. Our data raise the intriguing possibility that viral glycoproteins, expressed during acute or chronic infection, could facilitate the spreading of protein misfolding in vivo.

OD21.06. Extracellular vesicles cargo as biomarker of mitochondrial dysfunction in Huntington's Disease

Margarida Beatriz, CNC‐Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Institute for Interdisciplinary Research of the University of Coimbra (IIIUC), Coimbra, Portugal

Rita Vilaça, CNC‐Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Institute for Interdisciplinary Research of the University of Coimbra (IIIUC), Coimbra, Portugal

George Daley, Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA; Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Medical School, Harvard Stem Cell Institute, Boston, MA, USA

Cristina Januário, Neurology Service, Coimbra Hospital and Universitary Centre, Coimbra, Portugal; FMUC‐Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Thorsten Schlaeger, Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA; Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA

A. Cristina Rego, CNC‐Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; FMUC‐Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Carla Lopes, CNC‐Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Institute for Interdisciplinary Research of the University of Coimbra (IIIUC), Coimbra, Portugal

Introduction: Huntington's disease (HD) is a neurodegenerative disorder caused by an expansion of a CAG repeat in the HTT gene that encodes for a mutant form of the huntingtin protein. The role of extracellular vesicles (EV) in HD progression is currently being investigated and our results showed the presence of mitochondrial components (DNA, proteins) in EV released from HD patient's cells.

Methods: EV were isolated from media of human fibroblasts (Fb) (3 CTR, 3 presymptomatic (pHD) and 2 late‐stage HD (HD)) through differential ultracentrifugation and analyzed by nanosight tracking analysis, transmission‐electron microscopy (TEM) and immunoblotting for characterization of exossomal markers. Live‐imaging was used to evaluate intercellular trafficking of mitochondrial proteins via EV and RT‐qPCR to quantify EV mitochondrial DNA (mtDNA) copy number.

Results: We detected the presence of TFAM (mtDNA transcription factor) by TEM and the incorporation of mtDNA in EV released from HD cells. Live‐cell imaging of GFP tagged EV and DsRed labelled mitochondria showed a colocalization in Fb suggesting that EV can transfer mitochondrial proteins intercellularly. An increased secretion of EV was observed in Fb from patients with higher CAGs number. The number of mtDNA copies were higher in pHD vs HD Fb and released EV, displaying a positive correlation between them.

Summary/Conclusion: Human Fb exchange EV loaded with mitochondrial components. Additionally, the increased number of mtDNA copies in cells and EV from presymptomatic HD suggests a pathogenic role for mitochondrial cargo within EV in HD progression.

PS01.01. KIM‐1‐mediated exosomes uptake is crucial for renal tubulointerstitial inflammation induced by hypoxia

Jun Chen, Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China

Zuo‐Lin Li, Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China

Tao‐Tao Tang, Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China

Jing‐Yuan Cao, Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China

Cui Wang, Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China

An‐Ran Shen, Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China

Xin Zhong, Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China

Bi‐Cheng Liu, Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China

Lin‐Li Lv, Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China

Introduction: Renal tubular epithelial cells (RTECs) exposed to hypoxia during kidney injury communicate with interstitial inflammatory cells and contribute to tubulointerstitial inflammation. However, the role and underlying mechanism of the communication among RTECs in renal inflammation induced by hypoxia are not fully understood. The purpose of our study is to explore the role of exosomes secreted by hypoxic RTECs in the development of renal tubulointerstitial inflammation and the related mechanism.

Methods: Ischemia reperfusion(I/R) induced acute kidney injury (AKI) and unilateral ureter obstruction (UUO) model were established. HIF‐1α (a marker of hypoxia), inflammatory factors, KIM‐1, CD63 and ALIX (markers of exosomes) were detected. Immunofluorescence staining was used to observe the localization between KIM‐1 and exosomes. In Vitro, HIF‐1α, inflammatory factors, KIM‐1, Rab27a and markers of exosomes were detected in hypoxic RTECs. Furthermore, we detected inflammatory factors after silence of KIM‐1 and Rab27a of hypoxic RTECs. Besides hypoxic RTECs derived exosomes with or without fluorescent probe labelled were cocultured with RTECs with or without inhibition of KIM‐1. Then Q‐PCR and flow cytometry were separately performed to test inflammatory factors and uptake efficiency of exosomes of recipient RTECs. In vivo, hypoxic RTECs derived exosomes with or without fluorescent probe labelled were injected into kidney with or without I/R injury. Finally, phosphatidylserine on the surface of hypoxic exosomes was detected by nanoflow cytometry (NanoFCM).

Results: Tubulointerstitial inflammation, increased exosome production and KIM‐1 expression in tubules were observed in AKI and UUO injured kidneys. Interestingly, more exosomes were enriched in KIM‐1 positive renal tubules. In vitro study showed that hypoxia could increase exosomes production and KIM‐1 expression in RTECs. Interestingly, hypoxia induced inflammation could be attenuated when KIM‐1 and Rab27a were inhibited. Phosphatidylserine (PS) was found in parts of hypoxic exosomes as detected by NanoFCM. What is more, hypoxic exosomes could induce inflammatory reaction in normal RTECs and aggravate inflammatory reaction in hypoxic RTECs, which could be relieved when KIM‐1 was knock down via siRNA transfection. Correspondingly, in vivo, exogenous hypoxic exosomes could induce inflammatory reaction in normal kidneys and aggravate inflammatory reaction in I/R injured kidneys. Most importantly, there was a co localization relationship between KIM‐1 expressing tubules and injected exosomes.

Summary/Conclusion: Our studies demonstrate that KIM‐1 expressed by injured tubules mediates exosomes uptake via recognizing PS, which participates in mutual dialogue among RTECs and augments the inflammation response in tubulointerstitial inflammation induced by hypoxia.

PS01.02. Topical application of mesenchymal stem cell exosomes alleviates the imiquimod induced psoriasis‐like inflammation

Poster Presenter

Bin Zhang, Institute of Molecular and Cell Biology

Ruenn Chai Lai, Institute of Molecular and Cell Biology

Wei Kian Sim, Institute of Molecular and Cell Biology

Andre Boon Hwa Choo, Bioprocessing Technology Institute

E Birgitte Lane, Skin Research Institute of Singapore

Sai Kiang Lim, MDPhD, Institute of Medical Biology, Agency for Science, Technology and Research, Singapore. Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Introduction: Severe psoriasis, a chronic inflammatory skin disease is increasingly being effectively managed by targeted immunotherapy but long‐term immunotherapy poses health risk and loss of response. Therefore, there is a need for alternative therapy strategies.

Methods: Mesenchymal stem/stromal cell (MSC) exosomes are widely known for their potent immunomodulatory properties. Here we investigated if topically applied MSC exosomes could alleviate psoriasis‐associated inflammation.

Results: Topically applied fluorescent exosomes on human skin explants were confined primarily to the stratum corneum with < 1% input fluorescence exiting the explant over a 24‐hour period. Nevertheless, topically applied MSC exosomes in a mouse model of imiquimod (IMQ) psoriasis significantly reduced IL‐17 and terminal complement activation complex C5b‐9 in the mouse skin. MSC exosomes were previously shown to inhibit complement activation, specifically C5b‐9 complex formation through CD59. Infiltration of neutrophils into the stratum corneum is characteristic of psoriasis and neutrophils are a major cellular source of IL‐17 in psoriasis through the release of neutrophil extracellular traps (NETs).

Summary/Conclusion: We propose that topically applied MSC exosomes inhibit complement activation in the stratum corneum and this alleviates IL‐17 release by NETS from neutrophils that accumulate in and beneath the stratum corneum.

PS01.03. Exosomes isolated from mycobacteria‐infected T lymphocytes activated immune responses in macrophages

Qian Qiu, Chongqing Public Health Medical Center

Yanlin Zhao, National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention

Ping He, National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention

Yaokai Chen, Division of Infectious Diseases, Chongqing Public Health Medical Center, Southwest University

Introduction: Approximately 2 billion people are infected with Mycobacterium tuberculosis worldwide, the etiological agent of tuberculosis, and 1.5 million of whom die annually. Macrophages are the primary host cells for Mycobacterium tuberculosis in humans, which has a remarkable capacity to survive within the hostile environment of macrophages. Macrophages infected with mycobacterium release exosomes that promote recruitment and activation of immune cells during granuloma formation was well known, however, the effects of exosomes released by infected lymphocytes on the capacity of macrophages were not well understood.

Methods: We identified exosomes from cytotoxic T lymphocytes infected with M. tuberculosis early secretory antigenic target‐6 (ESAT‐6) and culture filtrate proteins (CFP) or left uninfected as control. Relative immune protein expression changes in macrophages (i.e. RAW 264.7 cells) treated by infected and uninfected exosomes were investigated via in‐depth proteomics approach. Pathway enrichment analysis was carried out through the Gene Ontology function, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis to identify pathways and functional annotation of the differential expression of immune proteins. Furthermore, assay of colony‐forming units was also used to measure the effects against mycobacteria (i.e. M. tuberculosis H37Rv) in macrophages.

Results: Exosomes purified from uninfected T lymphocytes were found to induce antigen specific IL‐2 and IL‐2Rα expression in macrophages, while IL‐2 and IL‐33 were induced by exosomes derived from M. tuberculosis ESAT‐6 and CFP infected lymphocytes IFN‐γ was not found in either group. The production of IL‐2 however was greatly increased in induction by infected lymphocyte exosomes. The mostly enriched proteins in macrophages induced by infected exosomes were found to be related to positive regulation of T cell activation, positive regulation of immunoglobulin production, negative regulation of cell migration in biological processes. They were also especially involved in cytokine‐cytokine receptor interaction, Th1 and Th2 immune responses. In comparison, uninfected exosome induced a more limited Th1 response. Infected exosomes were found to be superior to control exosomes in inhibition of mycobacteria within macrophages.

Summary/Conclusion: Our study revealed that exosomes derived from mycobacteria‐infected T lymphocytes may play an important role in regulating Th1 and Th2 responses in macrophages during M. tuberculosis infection, which suggested exosomes might serve as a novel cell‐free vaccine against an M. tuberculosis infection.

PS01.05. LL‐37 ameliorates mouse sepsis by inducing the section of antimicrobial microvesicles from neutrophils

Yumi Kumagai, Juntendo University

Soichiro Kakuta, Juntendo University

Kyoko Kuwahara, Juntendo University

Etsuo Susaki, Juntendo University

Isao Nagaoka, Juntendo University

Introduction: Extracellular vesicles (EV), such as exosomes and microvesicles (MV), secreted upon microbial infection modulate immune and infectious responses. Sepsis is a life‐threatening multiple organ dysfunction caused by a systemic dysregulated inflammatory response to infection. Nevertheless, numerous therapeutic trials concerning immune dysfunction have still been disappointing. We previously revealed that LL‐37, a human cathelicidin host‐defense peptide, improves the survival of cecal ligation and puncture (CLP) septic mice. We herein investigated the potential of LL‐37 to secrete EV and the functions of EV in CLP.

Methods: EV isolated from the peritoneal exudates of CLP mice and the supernatant of LL‐37‐stimulated mouse bone marrow neutrophils by differential centrifugation or size exclusion chromatography were analyzed by transmission electron microscopy (TEM), flow cytometry, western blotting, and resistive pulse sensing. The antibacterial activity of EV was evaluated by incubating with Escherichia coli.

Results: TEM revealed particles with sizes of 50–1000 nm of EV fractions isolated from both PBS‐ and LL‐37‐injected CLP mice. The level of EV, especially neutrophil‐derived MV, was enhanced by LL‐37 administration. Interestingly, EV isolated from LL‐37‐injected CLP mice contained higher amounts of neutrophil‐derived antibacterial molecules and exhibited higher antibacterial activity compared to EV from PBS‐injected CLP mice. When exosomes and MV were partially separated by differential centrifugation, the MV fractions mainly possessed the antibacterial molecules and the antibacterial activity. Furthermore, LL‐37 stimulated neutrophils to secret EV with antibacterial potential, and the administration of EV isolated from LL‐37‐stimulated neutrophils reduced the bacterial load and improved the survival of CLP mice.

Summary/Conclusion: LL‐37 induces the secretion of antimicrobial EV, predominantly MV from neutrophils, in septic mice, thereby reducing the bacterial load and protecting mice from lethal septic conditions.

PS01.06. Extracellular vesicles are associated with C‐reactive protein during sepsis

René Weiss, Danube University Krems

Birgit Fendl, Center for Biomedical Technology, Danube University Krems

Tanja Eichhorn, Center for Biomedical Technology, Danube University Krems

Silke Huber, Division of Hygiene and Medical Microbiology, Medical University of Innsbruck

Viktoria Weber, Center for Biomedical Technology, Danube University Krems

Introduction: We characterized the association of C‐reactive protein (CRP) with extracellular vesicles (EVs) in plasma from sepsis patients and assessed the ability of a commercial CRP adsorbent (Pentrasorb, Pentracor, Hennigsdorf, Germany) to deplete free and EV‐associated CRP. In addition, we characterized the potential pro‐inflammatory effects of EV‐bound CRP on monocytes.

Methods: The association of EVs with CRP was characterized by flow cytometry and Western Blotting. Plasma CRP levels were quantified using ELISA. To deplete CRP, plasma from sepsis patients was incubated with Pentrasorb (10 vol%) for 60 min in vitro. Primary human monocytes were stimulated with isolated EVs (20,000 g, 30 min) and monocyte IL‐8 secretion was quantified by ELISA to assess the biological effect of CRP depletion.

Results: Septic plasma (n = 30) contained 227.0±88.6 mg/L CRP vs. 0.7±0.4 mg/L for healthy controls (n = 5). Both, total EVs and CRP+ EVs were significantly elevated in septic plasma as compared to healthy controls (14,732±14,657 EVs/μL with 45.9±17.2% CRP+ EVs vs. 3,741±2,328 EVs/μL with 0.2±0.2% CRP+ EVs). Incubation of septic plasma with Pentrasorb resulted in depletion of free CRP (247.2±72.6 mg/L before vs. 1.8±0.7 mg/L after adsorption) as well as in a significant reduction in CRP+ EVs (15,053±3,992 EVs/μL with 61.0±5.0% CRP+ EVs before vs. 6,097±1,973 EVs/μL with 1.8±1.3% CRP+ EVs after adsorption; n = 3). Septic EVs induced a significant release of IL‐8 in monocytes as compared to EVs from healthy donors (3,409.0±3,545 pg/mL, n = 7 vs. 1,333.0±202.9 pg/mL, n = 4). EVs from CRP‐ depleted septic plasma induced significantly lower IL‐8 levels.

Summary/Conclusion: Treatment of septic plasma with Pentrasorb efficiently removes free CRP and detaches CRP from the EV surface, resulting in reduced proinflammatory effects.

PS01.07. Extracellular vesicles from activated platelets induce a shift towards proinflammatory monocyte subsets

Tanja Eichhorn, Center for Biomedical Technology, Danube University Krems

Birgit Fendl, Center for Biomedical Technology, Danube University Krems

René Weiss, Danube University Krems

Andreas Spittler, Core Facility Flow Cytometry & Surgical Research Laboratories, Medical University of Vienna

Viktoria Weber, Center for Biomedical Technology, Danube University Krems

Introduction: Circulating monocytes comprise classical (CM, CD14++CD16‐), intermediate (IM, CD14++CD16+), and non‐classical (NCM, CD14+CD16++) subsets. Changes in subset distribution, have been described in various pathologies including sepsis. We analyzed the distribution of monocyte subsets following monocyte isolation from whole blood and the potential influence of platelets and platelet‐derived extracellular vesicles (EVs) on monocyte subset distribution. Additionally, we assessed the immunomodulatory properties of mesenchymal stem cells (MSCs) on monocyte subsets.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from freshly drawn human whole blood using Ficoll gradient centrifugation. Primary human monocytes were isolated from PBMCs by negative depletion of non‐monocytes using magnetic beads labeled with anti‐CD3 and anti‐CD7 to label T cells, anti‐CD16 and anti‐CD123 (granulocytes), anti‐CD19 (B cells), anti‐CD56 (NK cells, T cells), as well as anti‐CD235a to label red blood cells. The association of monocytes with platelets (CD41+) and platelet‐derived EVs (CD41+lactadherin+) was assessed by flow cytometry.

Results: Monocyte subset distribution post isolation (83.7±3.3% CM, 5.4±2.8% IM, 10.9±5.0% NCM; n = 4) did not differ from the distribution in whole blood. Isolated monocytes contained residual platelets (monocyte‐to‐platelet ratio of 1:2) and platelet‐derived EVs. Overnight storage of isolated monocytes, but not of whole blood, led to a significant increase in IM (86.4±6.2% vs. 50.5±11.8% CM, 5.4±2.6% vs. 47.1±13.4% IM, and 8.2±4.2% vs. 2.4±2.0% NCM at 0h vs. 15h).

Summary/Conclusion: Storage of isolated monocytes induces a shift towards CD16 expressing proinflammatory monocytes, which seems to be mediated by residual platelets and platelet‐derived EVs. The mechanisms by which platelet EVs can trigger this shift remain to be clarified, and we are currently also assessing whether MSCs and MSC‐derived EVs can revert this shift in an inflammatory setting.

PS01.08. Human and murine macrophages show differential activation responses to extracellular vesicles released by Leishmania (Leishmania) amazonensis promastigotes with distinct virulence profile

Isabelle Carlos de Souza Perez, Universidade Federal de São Paulo campus Diadema

Natasha FC. Ferraz de Campos Reis, Universidade Federal de São Paulo campus Diadema

Talita V. Vieira Dupin, Universidade Federal de São Paulo campus Diadema

Rogeria Cristina Zauli, Universidade Federal de São Paulo campus Diadema

Ana Claudia Claudia. Torrecilhas, Universidade Federal de São Paulo campus Diadema

Patricia X. Xander, Universidade Federal de São Paulo campus Diadema

Introduction: Leishmaniasis is a heterogeneous group of diseases caused by Leishmania protozoan that affects about 700,000 to 1.2 million people annually. The infection initiates when during blood meal infected female sandfly injects promastigote form into vertebrate host. The parasites are then phagocytized by specialized cells, such as macrophages, and within these cells the parasite differentiate into amastigote form. Leishmania subverts the activation of the immune system by several mechanisms including the release of virulence factors in extracellular vesicles (EVs). In this work we evaluated the EVs released by Leishmania (Leishmania) amazonensis with different virulence profile on human (THP‐1 lineage) and murine (RAW 264.7 lineage) macrophages activation.

Methods: Virulent L. amazonensis promastigotes were recovered by consecutive and successive infection in animals and attenuated parasites were obtained after long live period in vitro culture (100 passages in culture). After 24 hours of stimulation with EVs released from virulent and attenuated parasites, human and murine macrophages were infected with the parasites for 24 hours. The percentage of infection, mean of internalized parasites, phagocytic index, and the cytokine expression were the parameters evaluated.

Results: Our results showed that both human and murine macrophages showed a significant increase in the percentage of infection, mean of internalized parasites and phagocytic index, as compared to no‐stimulated macrophages infected with the parasites. The cytokine expression showed that RAW 264.7 cells treated with virulent and attenuated EVs had a significant increase in IL‐10 cytokine expression and a significant decrease in TNF‐α expression. However, murine cells stimulated with EVs from virulent parasites showed higher expression of IL‐10. On the other hand, THP‐1 cells stimulated with EVs released by virulent or attenuated L. amazonensis showed a significant increase in TNF‐α expression but a significant decrease in IL‐10 expression.

Summary/Conclusion: These results suggest that human and murine macrophages showed different cytokine expression profile after stimulation with L. amazonensis EVs. In addition, the EVs released by L. amazonensis modulated macrophages favoring parasite infection. A better understanding of the role of these EVs in immune system modulation and phagocytic cell activation may contribute to uncover the mechanisms of EVs involved in the parasite‐host relationship in leishmaniasis.

PS01.09. Extracellular vesicles released by tumor cells exposed to cigarette smoke promotes the shift from Th9 differentiation to Foxp3 regulatory T cells

Paula Barbim Donate, University of São Paulo

Carlos Wanderley, University of São Paulo

Fernanda Turaça, University of São Paulo

Fausto Almeida, University of São Paulo

José Alves‐Filho, University of São Paulo

Thiago Cunha, University of São Paulo

Fernando Cunha, University of São Paulo

Introduction: Smoking is a major risk factor contributing to diseases development, and poor response to therapy. In cancer, most of smoking effects are related to its mutagenic potential. Extracellular vesicles (EVs) as emerged as important carriers of bioactive molecules related to cancer progression. However, the direct effects of exposure to cigarette smoke on EVs content, release and function are limited. Considering that cigarette smoke is a potent immune response modifier, we hypothesized that exposure of tumor cells to cigarette components would impact the immunomodulatory EVs functions and contributes to tumor immunological escape.

Methods: Tumor cell line B16 were cultured in the presence or absence of a Cigarette Smoke Enriched Medium (CSEM). After 48 h the cells were evaluated for the expression of Ki67 and PDL‐1 by flow cytometry. Extracellular vesicles (EVs) from supernatant were isolated by ultracentrifugation and submitted to western blot and nanosight analysis. MicroRNA expression was also evaluated by real‐time PCR. Naïve T cells was polarized in vitro for Th9 in the presence of B16‐derived EVs. C57/BL6 mice were submitted to melanoma experimental model and exposed or not to cigarette smoke.

Results: The exposure of mice to cigarrete smoke in melanoma experimental model increases tumor size. In vitro, the presence of CSEM in cultured B16 tumor cells increased their expression of Ki67 and PDL‐1, but EVs size and quantity were not affected. Besides the presence of the CD63 marker, EVs also expressed Ago2 protein. EVs derived from B16 cells exposed to CSEM modulates some microRNAs and increase the numbers of T CD4+ Foxp3+ cells during the differenciation for Th9.

Summary/Conclusion: The increased tumor size in melanoma experimental model by cigarette smoke exposure indicate a role in disease progression. The tumor microenvironment is a dinamic and complex place and its modulation can promote tumor immunological scape. In this context, EVs are important molecules involved in cell‐cell communication and the induction of regulatory T cell in place of the Th9 cells, recognized for their anti‐tumor effects, can contribute to tumor development. Our data suggest that smoking can exert its function through EVs immunomodulatory functions mediated, in parts, for their microRNAs content.

PS01.10. Effect of menthol and audiovisual cue on nicotine metabolism, smoking‐associated oxidative stress, and inflammation

Asit Kumar, Ph.D., University of Tennessee Health Science Center

Namita Sinha, UTHSC

Sanjana Haque, University of North Carolina at Chapel Hill

sunitha kodidela, UTHSC

Tengfei Wang, UTHSC

Angel Garcia G. Martinez, UTHSC

Hao Chen, UTHSC

Santosh Kumar, UTHSC

Introduction: Tobacco products such as e‐cigarettes pose potential adverse health effects caused by direct exposure to aerosolized nicotine and flavorant such as menthol. The interaction between nicotine and an audiovisual (AV) cue was also studied. In this study, we aimed to investigate whether nicotine and menthol as flavor cue modulate nicotine‐metabolizing enzyme CYP2A6, α7 nAChR, and antioxidant enzymes such as SOD1 and catalase in plasma extracellular vesicles (EVs). Modulation of these enzymes would eventually lead to nicotine‐induced toxicity and HIV‐1 pathogenesis via EVs.

Methods: In this study, rats were assigned into 3 different groups: (a) rats with self‐administered nicotine in response to flavor cue; (b) rats with self‐administered nicotine in response to AV cue; (c) rats with self‐administered nicotine in response to AV and flavor cues. Also, blood samples were collected before self‐administered nicotine from each group. We isolated and characterized EVs as per ISEV guidelines from rat plasma before and after self‐administered nicotine with either flavor cue, AV, or both. Protein associated with CYP2A6, SOD1, and catalase were quantified by western blot. Cytokine and chemokine profiling in plasma and EV before and after self‐administered nicotine was performed using multiplex ELISA.

Results: We measured the size, total protein, and AChE activity of EVs and found no significant difference in these characteristics before and after self‐administered nicotine. We evaluated the expression of EV markers CD9 and CD63. The results showed that self‐administered nicotine increased the levels of CD9 (p ≤ 0.05), the marker of small vesicles in response to AV and flavor cues. Expression of CYP2A6 was significantly increased (P ≤ 0.001) after self‐administered nicotine in response to AV and flavor cues. The expression of nicotine receptor α7 nAChR did not change under any conditions used. Despite the noticeable effect on SOD1 and catalase, statistical significance was not observed following self‐administered nicotine. Among cytokine and chemokine profiling, we found a significant increase in the levels of MCP‐1 in EV after self‐administered nicotine in response to response to AV and flavor cues. Further investigation underlying the effect of self‐administered nicotine in response to AV and flavor cues on HIV pathogenesis is underway.

Summary/Conclusion: Nicotine self‐administration increased, though not statistically significant, the levels of circulatory EVs. Moreover, the study provided evidence that nicotine in response to AV and flavor cues increased nicotine metabolizing CYP2A6 in all the groups and AOEs and cytokines in specific groups.

PS01.11. Development of in vitro functional assays to assess the immunomodulatory effects of mesenchymal stromal cells derived‐extracellular vesicles

Giada De Lazzari, Department of Women's and Children's Health, University of Padova, Padua, Italy

Ricardo Malvicini, Instituto de Medicina Traslacional, Trasplante y Bioingenieria (IMeTTyB‐CONICET)

Anna Maria Tolomeo, L.i.f.e.L.a.b. Program, Consorzio per la Ricerca Sanitaria (CORIS), Veneto Region, Padua, Italy

Marcin Jurga, Exo Biologics, Niel, Belgium

Michela Pozzobon, Department of Women's and Children's Health, University of Padova, Padua, Italy

Maurizio Muraca, Department of Women's and Children's Health, University of Padova, Padua, Italy

Gustavo Yannarelli, Instituto de Medicina Traslacional, Trasplante y Bioingenieria (IMeTTyB‐CONICET), Buenos Aires, Argentina.

Introduction: There is increasing interest in using extracellular vesicles derived from mesenchymal stromal cells (MSC‐EVs) as therapeutic tools, mainly due to their immunomodulatory properties. However, it is well established that the functional capabilities of these EVs are affected by a large variability, similarly to their cells of origin. Therefore, a potency assay is required to verify that the cellular product exerts the intended effect in a dose‐dependent fashion. Such assays are difficult to standardize due to the inherent inconsistency of biological systems, especially with primary cells in vitro. In the present work, we evaluated the feasibility of a macrophage and a lymphocyte cell line as reproducible tools to measure some modulatory effects of MSC‐EVs, respectively, on the innate and acquired immune system.

Methods: Clinical‐grade, Wharton Jelly‐derived MSC‐EVs were provided by Exo Biologics (Niel, Belgium).

For the macrophage assay, the Raw 264.7 cell line was challenged with increasing LPS doses at different time points in a 96‐well plate, measuring NO2‐ production by Griess assay as marker of M1 polarization. M1 polarization was confirmed by FACS analysis of CD80 and CD86.

To set the optimal condition for the T cell assay, Jurkat cells (clone E6‐1) were seeded in a 96‐well plate and stimulated with different anti‐CD3/CD28 beads with different beads/cell ratio on different time points. As read‐outs, IL‐2 production was measured in culture supernatant by ELISA, and cells were characterized by FACS to evaluate the activation marker CD69 and the ratio Treg (CD25+CD127‐ or FOXP3+)/Teff (CD25‐CD127+).

Both assays were tested with increasing doses of dexamethasone (Dex) (0.5, 1, 2 ug/mL) or MSC‐ EVs (5E7, 5E8, and 5E9/mL, determined by RPS.

Results: Macrophage stimulation with 10ng/mL LPS for 16h resulted in strong induction of NO production that was inhibited up to 60% by Dex in a dose‐dependent fashion. A similar dose‐dependent inhibition was observed with increasing amounts of MSC‐EVs. Inhibition of NO production was associated with a reduced expression of M1 markers.

Jurkat cell stimulation resulted in increased IL‐2 secretion and CD69 expression. Dex inhibited IL‐2 secretion by 70% and CD69 expression by 40%, in association with an increase of Treg/Teff ratio. Again, the addition of MSC‐EVs resulted in similar, dose‐dependent effects.

Summary/Conclusion: We set up a combination of two simple in vitro functional assays, representative of both innate and acquired immunity, to assess the immunomodulatory effects of MSC‐EVs. Although these tests need to be further evaluated on a large scale, we propose that the use of cell lines with a positive internal control (Dex) should ensure both adequate precision and robustness.

PS01.12. Immunomodulation by extracellular vesicles from Trichinella spiralis muscle larvae: increasing tolerogenic properties of human dendritic cells

Sofija Glamočlija, Institute for Application of Nuclear Energy, INEP, University of Belgrade

Natasa Ilić, Institute for the Application of Nuclear Energy, INEP, University of Belgrade

Alisa Gruden‐Movsesijan, Institute for the Application of Nuclear Energy, INEP, University of Belgrade

Ljiljana Sabljić, Institute for the Application of Nuclear Energy, INEP, University of Belgrade

Sergej Tomić, Institute for Application of Nuclear Energy, INEP, University of Belgrade

Ljiljana Sofronić‐Milosavljević, Institute for the Application of Nuclear Energy, INEP, University of Belgrade

Maja Kosanović, Institute for the Application of Nuclear Energy, INEP, University of Belgrade

Introduction: Excretory‐secretory products (ES) of parasitic worms (helminths) shift hosts’ immunological balance toward Th2 and regulatory responses thus acting beneficial in chronic inflammations, i.e. autoimmune diseases. As shown in several helminths, extracellular vesicles (EVs) are active immunomodulatory component of ES. We found that Trichinella spiralis produces EVs (TsEVs) which influence cytokine production by PBMC. Now we aim to show how TsEVs influence human dendritic cells (DC), as key players in initiation, progression and regulation of immune response.

Methods: EVs were enriched from conditioned medium of T. spiralis muscle larvae (ES L1) by differential centrifugation. Human monocyte derived dendritic cells (DCs) were treated with TsEVs and subsequently co‐cultivated with allogenic T cells. Phenotypes and cytokine production of DC and T cells were determined by flow cytometry.

Results: TsEVs induce stable tolerogenic phenotype of DCs, reflected in the expression of surface markers (HLA‐DR, CD‐40, CD‐86) almost at the level of the control, except for slight elevation in the surface CD‐83, and significantly increased ILT‐3 and CCR‐7. Stimulated DCs produce significant amounts of IL‐10 and TGF‐β, and polarize immune response of T cells towards Th2 and regulatory type. T cells co‐cultured with TsEVs stimulated DCs show significant increase in the production of IL‐4 and IL‐10 with the production of IFN‐g at the level of control. Moreover, TsEVs stimulated DCs induce expansion of CD4+CD25+Foxp3+ regulatory T cells.

Summary/Conclusion: TsEVs influence viability, differentiation, maturation potential of DCs and their capacity to regulate T cell‐mediated immune response, similar as ES L1 of T. spiralis do. They induce tolerogenic phenotype of DCs and regulatory response of T cells. Starting from this capacity of TsEVs to convey immunomodulatory properties of ES L1, new therapeutics, based on TsEVs could be designed as novel therapy for autoimmune diseases.

PS01.13. Air pollution Particulate Matter and EVs: involvement of PM‐fraction and PM‐activated toxic signaling pathways in EVs released by pulmonary epithelial cells

Stéphanie Alkoussa, Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV, EA 4492)

Sylvain billet, Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV, EA 4492)

nour jaber, 1Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV, EA 4492)

Perrine J. J. Martin, 1Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV, EA 4492)

Introduction: Poor air quality associated with high levels of particulate matter (PM) is one of the five greatest environmental risks for health, causing millions of premature deaths each year (cardiovascular diseases, cancer, COPD,…). PM is composed of hundreds of different chemicals, such as organic (Polycyclic Aromatic Hydrocarbons, PAHs) and inorganic or hydrosoluble compounds (ions and metals), as well as biological species (bacteria). Due to their diameter < 2.5 μm, PM2.5, or fine particles, can penetrate deep into the lung alveoli. Exposure of lung epithelial cells to PM2.5 triggers the activation of toxic pathways such as: (1) the AhR signaling pathway involved in the metabolic activation of PAHs, (2) the TLR4 signaling pathway involved in the inflammatory response, and (3) the production of Reactive Oxygen Species (ROS), which cause severe damage to cellular macromolecules. Numerous studies also show that PM2.5 induces the secretion of EV by exposed cells, but neither the fraction of PM nor the signaling pathways involved are known. Answering these questions is the objective of this study.

Methods: First, BEAS‐2B lung epithelial cells were exposed to PM2.5 and their organic and hydrosoluble extracts for 24 and 48 hours. Second, in order to determine the signaling pathways involved, BEAS‐2B cells were pre‐treated prior to exposure to PM2.5 with optimized concentrations of the following three specific inhibitors: CH223191 (AhR antagonist), TAK‐242 (TLR4 antagonist) and NAC (antioxidant). At the end, the EVs were isolated by SEC method, quantified by Nanosight and validated by western‐blot.

Results: The organic and biological fractions of the PM2.5 induce the release of EVs from exposed cells and the pre‐incubation with the three inhibitors prior to exposure has an impact on this release.

Summary/Conclusion: Our study highlights, for the first time, the involvement of organic and biological fractions of PM2.5 and of the induced toxic signaling pathways in EV release.

PS01.14. T cell‐derived extracellular vesicles in the allergic airway

Kaitlyn E. Bunn, Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center

Heather H. Pua, Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center

Introduction: T cells are immune cells known to secrete extracellular vesicles (EVs) following stimulation that carry cell‐derived cargoes. In vitro, it has been shown that T cell‐derived EVs can induce pro‐inflammatory behaviors in target cells. However, the presence and relevance of T cell‐derived EVs in allergic airway pathology, in vivo, is not known. We previously found that immune cell‐derived EVs are increased in the airways of mice with induced allergic airway inflammation compared to control mice. Given that T cells are major drivers of inflammation in the allergic airway, the goal of this study was to determine if T cells contribute to immune cell‐derived EVs present in the allergic airway.

Methods: Allergic airway inflammation was induced in mice by ovalbumin sensitization and challenge. Bronchoalveolar lavage fluid (BALF) was collected from control mice and mice challenged with allergen in the airways. BALF was serially centrifuged to remove cells and debris. T cell‐specific labeling of membranes in vivo coupled with high sensitivity vesicle flow cytometry was used to detect the presence of T cell‐derived EVs in the BALF.

Results: T cell‐derived EVs were present in the BALF of mice challenged with allergen in the airways but absent from BALF of control mice. Using T cell‐specific membrane labeling and high sensitivity vesicle flow cytometry, we identified that 3% of fluorescence positive vesicles in the BALF of mice with induced allergic airway inflammation were of T cell origin and recovered over 6 million T cell‐derived vesicles per airway.

Summary/Conclusion: These results provide evidence that T cells recruited to the airways during allergic airway inflammation contribute to the population of immune cell‐derived EVs in the allergic airway. Now that the presence of T cell‐derived EVs in the allergic airway has been established, we can start to understand how they may mediate intercellular communication in the immune system through the cargoes they carry and contribute to allergic and asthma pathology.

PS01.15. CD24 and IgM stimulation of B cells triggers transfer of functional CD24 and B cell receptor to B cell recipients via extracellular vesicles

Hong‐Dien Phan, Department of Biochemistry, Memorial University of Newfoundland

Delania J.B. Gormley, Memorial University

Reilly H. Smith, Memorial University of Newfoundland

Modeline N. Longjohn, Department of Biochemistry, Memorial University of Newfoundland

May Dang‐Lawson, University of British Columbia

Linda Matsuuchi, The University of British Columbia Vancouver

Michael R. Gold, PhD, University of British Columbia

Sherri L. Christian, Memorial University of Newfoundland

Introduction: Extracellular vesicles (EVs) are membrane‐encapsulated nanoparticles that carry bioactive cargo, including proteins, lipids and nucleic acids. Once taken up by target cells, EVs can modify the physiology of the recipient cells. In past studies, we reported that engagement of the glycophosphatidylinositol‐anchored receptor CD24 on B lymphocytes (B cells) causes the release of EVs. However, a potential function for these EVs was not clear. Thus, we investigated whether EVs derived from CD24 or IgM‐stimulated donor WEHI‐231 murine B cells can transfer functional cargo to recipient cells.

Methods: We employed a model system where donor cells expressing palmitoylated GFP (WEHI‐231‐GFP) were co‐cultured, after stimulation, with recipient cells lacking either IgM (WEHI‐303 murine B cells) or CD24 (CD24 knock‐out (CD24KO) mouse bone marrow B cells). Uptake of lipid‐associated GFP, IgM, or CD24 by labeled recipient cells was analyzed by flow cytometry.

Results: We found that EVs released in response to stimulation of either CD24 or IgM on the donor cells could mediate the transfer of lipids, as well as both CD24 and IgM, to recipient cells. Importantly, we found that the transferred receptors are functional in recipient cells, thus endowing recipient cells with a second BCR or sensitivity to anti‐CD24‐induced apoptosis.

Summary/Conclusion: Overall, these data show that extracellular signals received by one cell can change the sensitivity of neighboring cells to the same or different stimuli, which may impact B cell development or activation.

PS02.01. Exosomes from 2D and 3D‐Organized cardiac explant cells show differences in miRNAs content and cytoprotective effects

Federico Buccino, Cardiocentro Ticino Institute

Edoardo Lazzarini, Cardiocentro Ticino Institute

Vanessa Biemmi, Cardiocentro Ticino Institute

Sara Bolis, Cardiocentro Ticino Institute

Giuseppe Vassalli, Cardiocentro Ticino Institute

Lucio Barile, Cardiocentro Ticino Institute

Carolina Balbi, Cardiocentro Ticino

Introduction: Exosome (Exo) from Cardiac progenitor cells (CPCs) are well known cardioprotective agents. The role and mechanism of action of such bioactive vesicles was associated to their miRNA content. Different culturing condition, such us hypoxia, could modulate the miRNA content, and consequentially the effect, of exosome. Here we report, for the first time, a head‐to‐head comparison of exosomes between CPC cultured in standard condition (2D‐Exo) and produced from 3D culture (3D‐Exo).

Methods: CPC were derived from atrial appendage explants from patients who underwent heart valve surgery. The same number of cells was spitted and growth on classical monolayer (2D culture) or in cardiosphere conformation (3D culture). Exo were obtained from serum free conditioned media and isolated by density gradient ultracentrifugation. Exo were characterized by western blot, NTA and facs analysis. Functional experiments of cardioprotection was performed on HL‐1 cardiomyocyte cell line. The miRNA content of Exo was evaluated by RT‐realtimePCR.

Results: Resultes: Western blot analysis, NTA and FACS confirmed the enrichment of Exo in fractions 4 and 5 among the 8 obtained by density gradient ultracentrifugation. Exo were positive for TSG101, ALIX and SYNTENIN‐1, while negative for GRP‐94 used as contaminant control, by western blot. Positivity for CD9, CD63 and CD81 was confirmed by FACS. 2D‐Exo, but not 3D‐Exo, increased cell viability and reduced ROS formation of HL‐1 cells. ERK phosporilation after 30 minutes of treatment as also observed only with 2D‐Exo. RT‐realtimePCR showed an increase of miRNA132‐3p; 146a‐5p and 181 in 2D‐Exo compared to the 3D.

Summary/Conclusion: These preliminary results showed how a different culture condition can change the content and role of produced exosome in the same cells.

PS02.02. EVs bearing tissue factor improve outcome after collagenase‐induced intracranial hemorrhage

Fanny Potzeha, PhD Student in INSERM U1237 ‐ PhIND ‐ Cyceron ‐ Caen

Merve YETIM, INSERM U 1237 ‐ PhIND ‐ Cyceron ‐ Caen

Thomas Gaberel, INSERM U 1237 ‐ PhIND ‐ Cyceron ‐ Caen

Denis Vivien, INSERM U 1237 ‐ PhIND ‐ Cyceron ‐ Caen

Maxime Gauberti, INSERM U 1237 ‐ PhIND ‐ Cyceron ‐ Caen

Sara Martinez de Lizarrondo, INSERM U 1237 ‐ PhIND ‐ Cyceron ‐ Caen

Introduction: Hemorrhagic stroke, defined as a bleeding within the brain parenchyma remains a major cause of mortality and permanent disability. There are unmet needs for effective therapies to improve outcomes after hemorrhagic stroke, in particular, intracerebral hemorrhage (ICH), the most severe form of stroke. Early hematoma growth in ICH occurs in one third of patients within 3 hours of stroke onset and is predictor of poor outcome. Clinical trials have shown that untargeted hemostatic therapy using recombinant activated coagulation Factor VII (rFVIIa) reduced hematoma growth but conveyed an unacceptable rate of side effects. Targeted treatments able to selectively promote hemostasis at the site of bleeding are therefore necessary. Previous studies reported that monocyte‐derived extracellular vesicles (mEVs) bearing tissue factor (TF) accumulate during thrombus formation and promote thrombin generation.

The aim was to generate large amount of pro‐coagulant mEVs to be used as hemostatic patches in preclinical models of ICH.

Methods: Human monocytic cell‐line THP‐1 were grown in bioreactors and treated with Tumor Necrosis Factor (TNF) to generate TF+ mEVs. Isolated mEVs were finely characterized by number, size and surface antigens (TF, CD14 and P‐Selecting Ligand1) by laser‐scanning confocal microscopy and flow cytometry. Functional studies were performed using human plasma clotting, ROTEM and thrombin generation assays and TF‐activity.

In vivo, mice received an intrastriatal injection of collagenase VII to promote intraparenchymal hemorrhage. 30 minutes thereafter, exogenous mEVs were injected intravenously. Hematoma volume was quantified by Magnetic Resonance Imaging at 24h and neurological deficits were measured at 4h and 24h post‐stroke.

Results: Large amounts of mEVs were generated in supernatants from TNF‐stimulated monocytes in bioreactors (Celline). Those mEVs presented monocyte‐specific characteristics: a mean size of 430 nm and a high pro‐coagulant activity reducing the clotting time in a dose‐ and TF‐dependent manner. In preclinical studies of ICH, intravenous injection of mEVs improved stroke outcome in a dose‐dependent manner. mEVs at 1 mg/kg prevented hematoma growth by 43% and improved neurological score at 4h and 24h compared to control mice (p < 0.01, n = 15/group). This beneficial effect was also present in a more severe model of ICH in enoxaparin‐treated mice and was showed in a TF‐inhibition in vivo experiment. Immunohistological studies and 2‐photon imaging revealed accumulation of fluorescently‐labeled mEVs specifically at the bleeding site.

Summary/Conclusion: EVs bearing TF improve outcome after collagenase‐induced ICH by acting as intravascular patches.

PS02.03. The Efficacy of EV Encapsulated Drugs is Associated with Loading Methods, EV sources and EV Uptake Efficiency in Pancreatic Cancer Cells

Wei‐Qun Ding, University of Oklahoma Health Sciences Center

Haoyao Sun, The Affiliated Suzhou Hospital of Nanjing Medical University

Kritisha Bhandari, University of Oklahoma Health Sciences Center

Stephanie Burrola, University of Oklahoma Health Sciences Center

Jingchang Wu, The Second Affiliated Hospital of Xuzhou Medical University, Xuzhou

Introduction: Pancreatic cancer is the third leading cause of cancer related death in the United States and the overall 5‐year survival rate of patients with pancreatic cancer is around 9%. Therapeutic options against pancreatic cancer are rather limited compared with other solid tumors. New strategies in therapeutic development is desperately needed. Recent advancement in extracellular vesicle (EV) biology has indicated that certain type of EVs, such as small EVs, possess tumor homing propensity with lower immunogenicity, and are potential drug carriers to effectively deliver cancer therapeutics.The aim of this study is to determine the efficiency of EV drug loading and the efficacy of EV encapsulated drugs in pancreatic cancer cells.

Methods: Small EVs were isolated from culture medium of various human cell lines using an established protocol. Paclitaxel and Gemcitabine, two commonly used chemotherapeutics, were incorporated into small EVs via Incubation, Sonication, and Electroporation. Loading efficiency was evaluated by spectrometric measurements. EV drug efficacy was analyzed by MTS assay. EV uptake was assayed by florescent microscopy with PKH‐67 stained small EVs.

Results: Small EVs were successfully isolated and verified. Compared with Electroporation and Incubation, Sonication is the most efficient method to incorporate chemotherapeutics into small EVs. However, Incubation led to more cellular uptake of the EV drugs, especially when the drugs encapsulated by the small EVs derived from HPNE cells. Furthermore, compared with Sonication and Electroporation, the EV drugs derived from Incubation is more efficacious in killing pancreatic cancer cells when applied at equivalent drug concentrations.

Summary/Conclusion: The efficiency of small EV encapsulation of chemotherapeutics varies among different loading methods, and the efficacy of EV encapsulated chemotherapeutics is associated with the loading methods, EV sources, and EV uptake efficiency.

PS02.04. Development of Engineered extracellular vesicles with targeting peptide for specific delivery to pancreas tissue

Hiroaki Komuro, Department of Biomedical Engineering, Michigan State University

Yuki Harada, Institute for Quantitative Health Science and Engineering (IQ), Michigan State University

Shakhlo Aminova, Institute for Quantitative Health Science and Engineering (IQ), Michigan State University

Nathaniel Pascual, Institute for Quantitative Health Science and Engineering (IQ), Michigan State University

Christopher Contag, Department of Biomedical Engineering, Michigan State University

Masako Harada, Department of Biomedical Engineering, Michigan State University

Introduction: One of the main hurdles of gene therapy is to develop nonviral carriers for efficient and safe delivery. Extracellular vesicles (EVs) play an essential role in several physiological and pathological functions through intercellular communication. One advantage of using EVs as a carrier is the ability to engineer their surface to display targeting moieties. Here we introduce enriched delivery of engineered EVs displaying an organ targeting peptide specific to the pancreas.

Methods: Gene fusion of β‐cell‐specific recombinant peptide p88 to the EV‐binding domain of lactadherin (C1C2) allows the generation of EVs harboring the peptide on their surface upon transfection and isolation from HEK‐293T cells. EVs were isolated by differential ultracentrifugation and characterized using nanoparticle tracking analysis, immuno‐transmission electron microscopy (TEM), and western blot. To evaluate the targeting capacity of the EVs, Gaussia luciferase (gLuc) activity was measured in in vitro and in vivo using an in vivo imaging system (IVIS). The pDNA copy numbers recovered from each organ were calculated by qPCR.

Results: We successfully displayed the peptide on the EV surface and characterized the properties of the EVs. The presence of peptides did not affect the EV size and morphology. The immune‐TEM and western blot assay showed these EVs contained exosome marker (CD63) and C1C2 fusion protein (HA). We demonstrated higher binding of EVs to the pancreatic β‐cell line NIT1 in the presence of the peptide compared to the non‐β 4T1 cells using an in vitro bioluminescent assay. Furthermore, in vivo biodistribution assay following intravenous injection of EVs to mice showed enriched localization of the engineered EV in the pancreas. The pancreas of mice that received pancreas targeting p88‐EVs featured an accumulation of p88‐pDNA. These results indicate the importance of targeting peptide for the binding of EVs in both in vitro and in vivo models.

Summary/Conclusion: Our EV engineering technique is simple, robust, and efficient. This study demonstrates that small peptide‐based ligands can impart affinity to specific organs when displayed on the surface of EVs. We believe that the EV‐mediated targeted delivery will improve the development of therapeutics for human pancreatic diseases.

PS02.05. Extracellular Vesicle‐Mediated siRNA Delivery, Protein Delivery, and CFTR Complementation in Well‐Differentiated Human Airway Epithelial Cells

Ashley Cooney, Department of Pediatrics, The University of Iowa

Sateesh Krishnamurthy, Department of Pediatrics, The University of Iowa

Patrick Sinn, Department of Pediatrics, The University of Iowa

Brajesh K. Singh, Department of Pediatrics, University of Iowa

Introduction: Primary cultures of well‐differentiated human airway epithelial cells (HAE) are a robust model for studying epithelial cell biology. However, well‐differentiated HAE cells are refractory to transfection techniques for delivering expression plasmids, small interfering RNA molecules (siRNA), and single‐stranded oligonucleotides. Viral‐based vectors (such as adenovirus, lentivirus, or adeno‐associated virus) are typically employed to deliver genetic material to HAE which can be expensive and time‐consuming to generate.

Methods: Exosomes (Exos) and microvesicles (MVs) were isolated from culture media of HEK‐293T or A549 cells using differential centrifugation. The exosomes were electroporated with siRNAs and their ability to deliver encapsulated siRNAs into well‐differentiated HAE was examined either by confocal microscopy or by QRT‐PCR. To deliver proteins, EVs were isolated from the conditioned media of A549 cells transfected with an expression plasmid or transduced with an adenovirus expressing mCherry (Ad5‐mCherry) or cystic fibrosis transmembrane conductance regulator (Ad5‐CFTR). Delivery of functional CFTR to HAEs from CF human donors was examined by measuring the change in anion channel activity in Ussing chambers.

Results: Our results suggest that exosomes are readily taken up by the cells in well‐differentiated HAE and they can efficiently deliver siRNA. We used both MVs and exosomes to deliver proteins in HAE. Although only MVs were capable of delivering large proteins like CFTR. We showed MV‐mediated delivery of functional CFTR protein to correct the transepithelial Cl' current in well‐differentiated HAE from CF donors.

Summary/Conclusion: Our study showed the potential use of EVs in manipulating the gene expression in an important in vitro airway model system. Exosomes effectively deliver siRNAs to modulate endogenous gene expression, whereas MVs can be used to deliver large protein cargos. Our data show that EVs can provide a rapid, inexpensive, and robust tool to deliver small RNAs and proteins into an important model system.

PS02.06. Platelet derived‐extracellular vesicles as drug delivery system of anti‐cancer agents targeting glioblastoma

Deng‐Yao Lee, Graduate Institute of Biomedical materials & Tissue Engineering, Taipei medical University

Yu‐Wen Wu, Graduate Institute of Biomedical materials & Tissue Engineering, Taipei medical University

Ariane Sharif, University of Lille, Lille, France · Lille Neuroscience and Cognition Research Center

Thierry Burnouf, Professor, PhD, Graduate Institute of Biomedical materials & Tissue Engineering, Taipei medical University

Introduction: Extracellular vesicles (EVs) are physiologically instrumental for intercellular communications. They may also be of great interest for clinical applications as targeted drug delivery system (TDDS). EVs derived from platelets (PEVs), which express various membrane glycoprotein markers interacting with cancer cells, may be of particular value as a DDS able to target and be retained within the tumor microenvironment. However, methods to generate PEVs from platelets and to prepare drug‐loaded PEVs still need to be developed and optimized. In this study we evaluated various methodologies for generating PEVs. We also studied the capacity of the PEVs to be internalized by glioblastoma cells.

Methods: Therapeutic‐grade human platelet concentrates were centrifuged to pellet the platelets (PLTs). Platelets were resuspended in platelet additive solution (PAS) with 6% Dimethyl sulfoxide (DMSO) for frozen storage. PEVs were generated using 5 different methods: thrombin activation (0.1 U/ml; 37°C; 1 hr); sonication (40 kHz; 30 min); glass bead activation (37°C; 1 hr); freeze/thaw treatment activation (3 cycles of ‐80°C/1hr, 37°C/3 min); and room temperature incubation (20‐22°C; 1 hr). Particle sizes of PEVs were characterized by Dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) and Tunable resistive pulse sensing (TRPS; Q‐nano), and concentration by NTA and TRPS. PEVs membrane markers expression was determined by western blot. 5‐(and‐6)‐Carboxyfluorescein Diacetate, Succinimidyl Ester (5(6)‐CFDA, SE) was used to stain PEVs and observe their internalization by U87MG glioblastoma cells using fluorescent microscopy.

Results: The particle sizes of PEVs generated by all five methods were in the range 180–200 nm. There were approximately 510 PEVs produced from each PLT when using the freeze‐thaw procedure, compared to less than 200 for the four other methods. All types of PEVs expressed CD41, CD62p, CXCR4, CD9 and CD63 glycoprotein markers. All 5 types of PEVs stained by 5(6)‐CFDA fluorescent dye could be internalized by U87MG cells within 24hrs of incubation and were found to accumulate near the nucleus.

Summary/Conclusion: All procedures evaluated (thrombin activation; sonication; glass beads activation, freeze and thaw; and room temperature incubation) can generate PEVs with similar size and membrane marker expression. The highest number of PEVs generated per naïve PLT was achieved by the freeze‐thaw procedure. All types of PEVs were able to be internalized by U87MG cells. Further studies will be carried out to select the optimal procedure for anti‐cancer agent loading within PEVs, and to determine the cytotoxicity against various glioblastoma cell lines.

PS02.07. Bovine milk‐derived extracellular vesicles as potential nanocarriers of bioactive miRNAs

Lorena del Pozo‐Acebo, IMDEA Food

María del Carmen López de las Hazas, IMDEA Food

Almudena García‐Ruiz, IMDEA Food

Alberto Dávalos, IMDEA Food

Introduction: MicroRNAs (miRNAs) are small non‐coding RNAs with a known role as mediators in crucial biological processes, which converts them into high potential powerful candidates for therapeutic intervention. However, circulating miRNAs are unstable and rapidly degraded by endogenous enzymes, diminishing the possibility of successfully exerting a biological function in distant target cells. To achieve the therapeutic potential of miRNAs, efficient, tissue‐specific and nonimmunogenic delivery technologies must be developed. Since the discovery that miRNAs are naturally transported within exosomes, a type of extracellular vesicles that confer protection against RNase digestion and increase miRNA stability, exosomes have been proposed as delivery vehicle for miRNA‐based therapy.

Methods: In this study, we aimed to evaluate the use of milk‐derived extracellular vesicles as potential vehicle for extracellular RNA drug delivery. With this purpose, exosomes were isolated from raw bovine milk, combining ultracentrifugation and size exclusion chromatography (SEC) methodology. Isolated exosomes were then loaded with exogenous hsa‐miR148a‐3p, a highly expressed miRNA in milk exosomes. The suitability of exosomes as delivery vehicles for extracellular RNAs was tested by evaluating the absorption of miR‐148a‐3p in hepatic (HepG2) and intestinal (Caco‐2) cell lines. The potential exertion of a biological effect by miR‐148a‐3p was assessed by gene expression analysis, using microarrays.

Results: In‐vitro uptake analysis of exogenous hsa‐miR‐148a‐3p (loaded into bovine milk exosomes) revealed statistically significant concentration increases in both HepG2 and Caco‐2 cell lines. The increment found between 2h and 24h of exosome exposure suggests that the absorption of bovine milk‐derived exosomes is time dependent in these cells. Furthermore, the miRNA transported within exosomes can exert a biological effect through the modulation of gene expression.

Summary/Conclusion: Results support that bovine milk is a cost‐effective source of exosomes which can be used as nanocarriers of bioactive miRNAs with a potential use in RNA‐based therapy.

PS02.08. Characterization, Biodistribution and Functional Studies of Immune‐Therapeutic Exosomes: Implications for Acute Respiratory Distress syndrome and COVID19

Mahmoud Elashiry, DDS, MDS, MD, PhD, Augusta University

Ranya Elsayed, DDS, MBA, PhD, Augusta University

Christopher cutler, DDS, PhD, Augusta university

Introduction: Dendritic cell (DC)‐derived exosomes (DC EXO), natural nanoparticles of endosomal origin, are under intense scrutiny in clinical trials for various inflammatory diseases. DC EXO are eobiotic, meaning they are well‐tolerated by the host; moreover, they can be custom‐tailored for immune‐regulatory or ‐stimulatory functions, thus presenting attractive opportunities for immune therapy. Previously we documented the efficacy of immunoregulatory DCs EXO (regDCs EXO) as immunotherapy for inflammatory bone disease, in an in‐vivo mice model. We showed a key role for encapsulated TGFB1 in promoting a bone sparing immune response. However, the on‐ and off‐target effects of these therapeutic regDC EXO and how target signaling in acceptor cells is activated is unclear.

Methods: In the present report, therapeutic murine regDC EXO were analyzed by high throughput proteomics, with non‐therapeutic EXO from immature DCs and mature DCs as controls, to identify shared and distinct proteins and potential off‐target proteins, as corroborated by immunoblot. Live animal imaging using SPECT/CT was done to track the biodistribution of regDCs EXO injected via tail vein of Black 6 mice. Mechanistic and functional studies were done to explore the mode of action of regDCs EXO on acceptor cells Invitro.

Results: The predominant expression in regDC EXO of immunoregulatory proteins as well as proteins involved in trafficking from the circulation to peripheral tissues, cell surface binding, and transmigration, prompted us to investigate how these DC EXO are biodistributed to major organs after intravenous injection. Invivo imaging showed preferential accumulation of regDCs EXO in the lungs, followed by spleen and liver tissue. In addition, TGFB1 in regDCs EXO sustained downstream signaling in acceptor DCs. Blocking experiments suggested that sustaining TGFB1 signaling require initial interaction of regDCs EXO with TGFB1R followed by internalization of regDCs EXO with TGFB1‐TGFB1R complex. Finally, these regDCs EXO that contain immunoregulatory cargo and showed biodistribution to lungs could downregulate the main severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) target receptor, ACE2 on recipient lung parenchymal cells via TGFB1 in‐vitro.

Summary/Conclusion: These results in mice may have important immunotherapeutic implications for lung inflammatory disorders and COVID19 induced acute respiratory distress syndrome.

PS02.09. GE11‐expressing bovine milk‐derived extracellular vesicles for EGFR targeted delivery of oxaliplatin to colorectal cancer

Gyeongyun Go, Department of Biochemistry, Soonchunhyang University College of Medicine, Cheonan 31151, Korea

Sang Hun Lee, Department of Biochemistry, Soonchunhyang University College of Medicine, Cheonan, 31151, Republic of Korea

Introduction: Anticancer drugs, such as fluorouracil, oxaliplatin are commonly used to treat colorectal cancer. However, owing to their low response rate and adverse effects, the development of efficient drug delivery systems is required. Bovine milk derived extracellular vesicles (milk EV) can be a potential drug delivery systems since they can be obtained with a large amount and are known to be safe after system administration. However, studies for surface modification of milk EV are limited.

Methods: Milk EV were isolated by using differential centrifugation and ultracentrifugation. To display GE11 peptide onto the surface of milk EV, the cholesterol‐PEG‐DBCO was incorporated into the membrane of milk EV. Then, azide‐modified GE11 peptides were treated to the DBCO‐modified milk EV. The GE11‐expressing milk EV (GE11‐milk EV) were loaded with oxaliplatin using simple incubation and the amount of loaded oxaliplatin was quantified using ICP‐MS.

Results: GE11‐Milk EV were spherical nanoparticles with an average diameter of 200 nm. GE11‐milk EV showed superior drug delivery efficiency to EGFR overexpressing colorectal cancer cells and breast cancer cells compared to bare milk EV. In the colorectal cancer xenograft mouse model, intravenous administration of oxaliplatin‐loaded GE11‐milk EV resulted in greater inhibition of tumor growth as compared to the treatment of equivalent amount of free oxaliplatin.

Summary/Conclusion: In this study, we demonstrated the surface modification of milk EV with GE11 peptide using DBCO‐azide click chemistry reaction. The oxaliplatin‐loaded GE11‐milk EVs showed excellent antitumor effects and have the potential to replace existing anticancer drugs, such as 5‐FU, oxaliplatin.

PS02.10. BioDrone, a novel drug delivery platform: From the basic science to potential therapeutic promises

Dong Woo Han, MDimune Inc

Jinhee Park, MDimune Inc

Jinju Lee, MDimune Inc

Jun‐Sik Yoon, MDimune Inc

Dayeon Kim, MDimune Inc

Hui‐Chong Lau, MDimune.Inc

Jeong Seon Yoon, MDimune Inc

Seung Wook Oh, PhD, MDimune Inc

Introduction: Previously, we have successfully demonstrated the establishment of a manufacturing‐scale extrusion process to allow the production of a large number of nanovesicles, cell‐derived vesicles (CDVs). Here, we present the compatibility of extrusion technology to produce CDVs from multi‐cell sources, including surface engineered cells. Productivity, one of the unique advantages of BioDrone technology, was evaluated at a single particle level. The therapeutic potential of CDVs was assessed by examining key characteristics as a drug carrier in vitro and in vivo.

Methods: Different cell sources such as immune cells, mesenchymal stem cells, red blood cells, and others were used to produce CDVs by a serial extrusion. CDVs produced from these cells were subject to a single particle analysis using flow cytometry. Additionally, intracellular uptake and trafficking of CDVs and their RNA cargos were visualized using the confocal microscope. In vivo biodistribution and tissue penetration of CDVs were assessed in mice as well. All the experiments were conducted in comparison with exosome or other drug carriers

Results: The extrusion efficiently produced CDVs from various cell sources with consistent quality. Comprehensive single particle analysis revealed genuine productivity of CDVs and conservation of key surface markers of cells, including engineered components. Robust encapsulation of small RNAs into CDVs produced from different cell sources showed promising therapeutic potential of BioDrone as a drug carrier. Additionally, efficient cellular uptake and tissue penetration of CDVs are presented here.

Summary/Conclusion: We have demonstrated that the extrusion technology enables the mass production of CDVs from various cell sources. This study highlights the expandability and versatility of BioDrone platform technology and its therapeutic potential as an innovative drug carrier in broad areas.

PS02.11. Cellular response to a Listeriolysin O mutant Y406A

Apolonija Bedina Zavec, National institute of Chemistry

Rebeka Podgrajšek, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology

Ana Špilak, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology

Matic Kisovec, Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry

Maja Jamnik, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology

Veronika Kralj‐Iglič, Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana

Gregor Anderluh, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology

Marjetka Podobnik, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology

Introduction: Listeriolysin O (LLO) is a toxin from the intracellular pathogen Listeria monocytogenes, which forms pores in cholesterol‐rich lipid membranes of host cells. Large β‐barrel pores formed by LLO indicate significant plasticity, from arc‐ or slit‐shaped pores to supramolecular assemblies generating large defects in membranes. LLO has pH optimum at pH 5.5, a condition found in late endosomes, while also at neutral pH it can bind to the membrane and form pores, and damage cells. LLO mutant protein Y406A with specific activity at acidic pH that could be interesting for the applications in medicine and biotechnology was generated by our group. Mutant Y406A with substitution (Try to Ala) at the site 406 is able to bound to membranes and oligomerized similarly to the wtLLO, but the final membrane insertion step requires acidic pH. Mutant Y406A has pH optimum at pH 5–6. The cytotoxicity and the release of extracellular vesicles (EVs) was used to examine the response of the cells to LLO and its mutant Y406A.

Methods: The cell line K562 was used. Cells were incubated with LLO or its mutant Y406A for 30 min at 37°C. The cytotoxicity was measured by quantifying cellular membrane integrity (propidium iodide and trypan blue staining), cell viability test (Presto Blue), and cell apoptosis. ROS (reactive oxygen species)‐generation activity was also measured. EVs were isolated by differential centrifugation. The concentration of larger EVs in the samples was measured by flow cytometry using Annexin‐FITC. The concentration of smaller EVs in the samples were measured by DLS.

Results: The effects of wtLLO and its mutant Y406A were tested on myelogenous leukemia cell line K562, which is highly sensitive in vitro target for the natural killer cells. The cytolethal concentration of wtLLO was between 1 nM and 10 nM. Vesiculation level was increased at cytolethal concentration and at 10‐fold lower concentration than cytolethal. At 100‐fold lower concentrations than cytolethal, the effect was reversed and cells shedding less EVs than control cells. On the other hand, the viability of cells was not affected after treating with Y406A at neutral pH, even at high concentrations of protein, while at pH 6.0 Y406A showed almost the same citotoxicity as wtLLO at pH 7.4. However, a low pore‐forming activity was detected at higher concentrations of Y406A (100 nM) at neutral pH. Besides, the level of EV secretion was slightly increased at higher concentrations of protein (100 nM) at neutral pH.

Summary/Conclusion: Mutant Y406A is significantly less toxic than wtLLO under physiological conditions and becomes toxic under acidic conditions; this makes it a potential candidate for stimuli responsive applications and cancer treatment.

PS02.12. Good things come in small packages: a new hope for preterm babies brains

Mhoyra Fraser, PhD, The University of Auckland

Teena K. Gamage, The University of Auckland

Sam Mathai, PhD, The University of Auckland

Introduction: Oxygen deprivation occurring in the womb or during delivery leads to permanent functional impairment of the brain and is one of the most common challenges faced by infants born preterm. Because of major advances in neonatal intensive care, survival of these preterm infants with brain injury has increased significantly. Extracellular vesicles (EVs), are naturally capable of penetrating the blood brain barrier, and can communicate with the microenvironment through transfer of proteins, miRNA and other nucleic acids. Importantly, they have an intrinsic neuroprotective therapeutic activity and are ideal candidates to deliver targeted therapeutic molecules of choice through modifications to enhance delivery. Using our well‐established fetal sheep model of preterm brain injury, we sought to examine the intrinsic therapeutic potential of unmodified human fetal neural stem cell‐derived extracellular vesicles (hFNSC‐EVs) to ameliorate injury.

Methods: Fetal sheep at 0.7 gestation (day 103–104; term ∼145 days) received intranasal infusions of sterile hFNSC‐EVs (hFNSC‐EVs‐occlusion) or vehicle (vehicle‐occlusion and vehicle sham‐occlusion) commencing 60 minutes following a 25 minute umbilical cord occlusion (UCO). After 3 days recovery in utero, ewes were killed and fetal brains collected for histopathology.

Results: Umbilical cord occlusion was associated with significant brain injury to areas commonly affected by asphyxia in preterm infants. We are currently comparing treatment outcomes to determine whether intranasal delivery of hFNSC‐EVs can improve survival of immature and mature oligodendrocytes (Oligo2, CNPAse) and if so does this occur in association with reduced apoptosis and astrogliosis within white matter regions of the injured preterm brain.

Summary/Conclusion: To the best of our knowledge, this study is the first to determine the usefulness of intranasal delivery of unmodified hFNSC‐EVs in a fetal sheep model of preterm brain injury. Our study has the potential to revolutionise treatment strategies and offer the possibility for amelioration and recovery in the injured preterm brain.

PS03.01. Human umbilical cord mesenchymal stem cells derived exosomes alleviate diabetic retinopathy by delivering miR‐5068 and miR‐10228 to regulate HIF‐1α/PGC‐1α pathway

Fengtian Sun, Jiangsu University

Hui Qian, jiangsu university

Wenrong Xu, Jiangsu University

Introduction: Diabetic retinopathy (DR) that is a leading cause of vision decline and blindness in adults still lacks of satisfactory treatments. Human umbilical cord mesenchymal stem cells derived exosomes (hucMSC‐Ex) are considered as novel therapeutic approaches and promising nanomaterials in regenerative medicine. Engineered exosomes have become an important approach to improve the curative effect.

Methods: In this study, we established a streptozocin‐induced DR rat model to evaluate the effect of hucMSC‐Ex on the repair of retinal damage. Meanwhile, human retinal microvascular endothelial cells (hRMECs) and adult retinal pigment epithelial cell line‐19 (ARPE‐19) were stimulated by 30 mM glucose medium to induce the high glucose environment in vitro followed by the treatment of hucMSC‐Ex to study the therapeutic mechanism. Therapeutic miRNAs were loaded into exosomes by electroporation.

Results: HucMSC‐Ex ameliorated hyperglycemia‐induce retinal injury by relieving apoptosis, inflammation, oxidative stress and pathological angiogenesis. Mechanistically, high glucose induced the increased expression and nuclear localization of hypoxia inducible factor‐1α (HIF‐1α) that inhibited peroxisome proliferator‐activated receptorγcoactivator‐1α (PGC‐1α) activation by binding with enhancer of zeste homolog 2 (EZH2) to accelerate DR progression. HucMSC‐Ex delivered miR‐5068 and miR‐10228 to reverse the upregulation of HIF‐1α and promote the expression of PGC‐1α, thereby exerting therapeutic functions in DR. Importantly, engineered hucMSC‐Ex loaded with mimics of miR‐5068 and miR‐10228 by electroporation further enhanced retinal repairing effects.

Summary/Conclusion: HucMSC‐Ex alleviate DR by transporting miR‐5068 and miR‐10228 to regulate HIF‐1α/PGC‐1α pathway, and engineered hucMSC‐Ex with miR‐5068 and miR‐10228 further strengthen the therapeutic outcome.

PS03.02. Effect of adverse pregnancy events on the protein cargo and surface marker distribution of human amniotic epithelial cell derived Extracellular Vesicles

Mehri Barabadi, Ritchie Centre

Naveen Kumar, Ritchie Centre

Gina D. Kusuma, PhD, Ritchie Centre

Dandan Zhu, Ritchie Centre

David W Greening, Baker Institute

Rebecca Lim, Ritchie Centre

Introduction: Human amniotic epithelial cell (hAEC) derived extracellular vesicles (EVs) have shown to exhibit therapeutic potentials such as immunomodulatory and repair properties. Adverse pregnancy events reflecting placental dysfunctions can induce changes in placental EVs. This study aimed to investigate the effect of pregnancy complications such as hypertension, diabetes, intrauterine growth retardation (IUGR), and preeclampsia on the protein cargo and surface markers of hAEC‐derived EVs.

Methods: hAECs were isolated from healthy term placenta (n = 6) as well as pregnancy complications such as Gestational Diabetes Mellitus on diet (GDM+D, n = 4), GDM on insulin (GDM+I, n = 5), hypertension (n = 5), preeclampsia (n = 4), and IUGR (n = 2). The cells were cultured and EVs were isolated using tangential flow filtration (TFF) coupled with size exclusion chromatography (SEC) techniques. The protein composition of EVs was characterised using mass spectrometry analysis. Furthermore, single interferometric reflectance imaging sensing (IRIS) technology was used to characterise EVs positive for tetraspanins CD9, CD81, and CD63 to determine size distribution and particle counts.

Results: IRIS technology indicated that size distribution and quantity of EVs present with CD63, CD81, CD9 tetraspanins were not significantly affected by the pregnancy complication events. Differentially expressed proteins (DEPs) were identified using mass spectrometry analysis by conducting Welch's t‐test. The DEPs from each group were subjected to functional enrichment analysis and gene sets corresponding to identified proteins were annotated using Reactome, KEGG, and Gene Ontology (GO) databases. We observed an over‐representation of DEPs involved in dysregulated neutrophil‐mediated immunity and leukocyte activation in the Preeclampsia group. We also observed dysregulating ECM organisation in EVs from GDM+I pregnancies and those affected by pregnancy‐associated hypertension. Furthermore, we observed an over‐representation of DEP associated with the dysregulation of cellular metabolic process in EVs from pregnancies in the GDM+D group.

Summary/Conclusion: In summary, these preliminary data suggest that pregnancy complications do not have a significant effect on the distribution and particle numbers of hAEC‐EVs, however, proteomic profiling in pregnancy complication groups changes compared to the healthy control.

PS03.03. Isolation and proteome profiling of plasma‐derived extracellular vesicles from the non‐obese diabetic (NOD) mouse

Isabel M. Diaz lozano, Karolinska Institutet

Helena Sork, PhD, Institute of Technology, University of Tartue

Virginia Stone, PhD, Karolinska Institutet

Maria Eldh, Karolinksa Institutet

Susanne Gabrielsson, Karolinska Institutet

Malin Flodström ‐Tullberg, Professor, Karolinska Institutet

Introduction: The mechanism(s) through which pancreatic beta‐cells are destroyed in type 1 diabetes (T1D) remain to be fully understood but may comprise of more than one distinct pathophysiological mechanism (known as T1D endotypes). Discovery of one or several endotype‐specific biomarkers during the pre‐diabetic period may open up the potential of personalized disease interventions. Extracellular vesicles (EVs) may harbor biological markers reflecting the pre‐diabetic stages of T1D. Here, we describe the enrichment and characterization of EVs from plasma collected from non‐obese diabetic (NOD) mice, a model for T1D.

Methods: EVs were enriched from prediabetic NOD mouse plasma by size exclusion‐chromatography (SEC) and membrane affinity purification (MA) based methods. EVs were characterized by nanoparticle trafficking analysis (NTA), protein concentration measurements, transmission electron microscopy (TEM) and LC‐MS/MS analysis, before or after affinity‐based depletion of highly abundant plasma proteins.

Results: SEC enriched a larger number of particles exhibiting a “characteristic” cup‐shaped EV morphology than MA. MA‐purified EV samples had a higher protein content and relatively higher levels of proteins that are abundant in plasma as compared to SEC‐purified EVs. The column‐based depletion successfully removed abundant plasma proteins, but the protein yield obtained by MS increased only in whole plasma samples and not in EV samples. An optimized LC‐MS/MS based analysis of SEC‐enriched EVs yielded a total of 680 proteins including canonical EV markers.

Summary/Conclusion: SEC can be employed to enrich EVs from NOD mouse plasma. The established method may be used to identify new biomarkers specific for the prediabetic stage, thereby serving as a tool to differentiate disease endotypes and patient stratification in intervention studies.

PS03.04. Single‐cell sequencing analysis the role of hucMSC exosomes in inhibiting renal interstitial fibrosis and preventing diabetic kidney diseases

cheng ji, jiangsu university

jiahui zhang, jiangsu university

Hui Qian, jiangsu university

Introduction: Diabetic kidney diseases (DKD) are characterized by progressive fibrosis and lead to the end‐stage renal disease (ESRD). mesenchymal stem cells (MSC) exosomes have obvious repair effects in acute and chronic kidney injury. Based on 10x genomics single‐cell sequencing technology, this article aims to explore the molecular mechanism of human umbilical cord mesenchymal stem cells derived exosomes (hucMSC‐Ex) to inhibit renal fibrosis and delay DKD progression.

Methods: A T2DM rat model was constructed with 45% high‐fat diet combined with streptozotocin (STZ, 35mg/kg) tail vein injection. Then injected with PBS as control, the experimental group was injected with hucMSC‐Ex (10mg/kg, every 3 days/time) through the tail vein to observe the therapeutic effect in DKD. Collect and separate kidney tissue single‐cell suspensions for 10x genomics single‐cell sequencing to analyze the changes in renal cell communities, numbers and gene levels in the state of diabetic nephropathy. It was found that hucMSC‐Ex could promote the increase of ubiquitin molecules bound by YAP, and the CK1δ/β‐TRCP kinase ubiquitin system with significantly high expression in hucMSC‐Ex was screened by protein profiling analysis. and promote its ubiquitination degradation.

Results: Through 10x genomics single‐cell sequencing analysis, it was found that the number of macrophage colony cells in the kidney tissue of DKD rats increased significantly. By activating the TGF‐β1/Smad2/3/YAP signal axis, it promotes the change of mesangial cells to myofibroblast‐like cells, and tissue immunity Fluorescence and histochemical experiments found that YAP protein in kidney tissue was significantly activated, and the expression of α‐SMA increased and the progression of fibrosis was aggravated. The hucMSC‐Ex treatment inhibited the decrease of YAP level and α‐SMA expression in mesangial cells. HucMSC‐Ex treatment resulted in significant up‐regulation of Ser381‐YAP and Ser127‐YAP in the cytoplasm. YAP decreases. HucMSC‐Ex is rich in CK1δ/β‐TRCP kinase ubiquitin system, which promotes YAP ubiquitination degradation and inhibits YAP signaling pathway, improves renal fibrosis and delays the process of diabetic nephropathy.

Summary/Conclusion: The DKD associated macrophages promoted the transformation of mesangial cells into myofibroblast‐like cells by activating the TGF‐β1/Smad2/3/YAP signal axis to accelerate the progression of renal interstitial fibrosis. And hucMSC‐Ex promotes the YAP ubiquitination and degradation by delivering the CK1δ/β‐TRCP kinase ubiquitin system to inhibit renal interstitial fibrosis, improve renal function and delay the progression of DKD. This experiment provides a new treatment strategy and experimental basis for the treatment of DKD with mesenchymal stem cell exosomes.

PS03.05. Lipopolysaccharide‐enriched small extracellular vesicles from metabolic syndrome patients trigger endothelial dysfunction by activation of Toll Like Receptor 4

SakinaAli – INSERM U1063

Marine Malloci – INSERM U1063

Zainab Safiedeen – INSERM U1063

Raffaella Soleti – Engineer, INSERM U1063

Luisa Vergori – Engineer, INSERM U1063

Xavier Vidal Gómez – INSERM U1063

Charlène Besnard – INSERM U1063

Séverine Dubois – INSERM U1063, Centre Hospitalo‐Universitaire d'Angers

Frederic Gagnadoux – INSER U1063, Centre Hospitalo‐Universitaire d'Angers

Soazig Le Lay – INSERM U1063

Jérôme Boursier – Centre Hospitalo‐Universitaire d'Angers

Arnaud Chevrollier – CNRS 6015, INSERM U1083, Centre Hospitalo‐Universitaire d'Angers

Gilles Simard – INSER U1063, Centre Hospitalo‐Universitaire d'Angers

Ramaroson Andriantsitohaina – Director of Research INSERM, INSERM U1063 SOPAM

Paul Calès – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Frédéric Oberti – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Isabelle Fouchard‐Hubert – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Adrien Lannes – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Ingrid Allix – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Pierre‐Henri Ducluzeau – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Wojciech Trzepizur – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Nicole meslier – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Pascaline Priou – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Samir Henni – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Georges Leftheriotis – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Pierre Abraham – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Christophe Aubé – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Gilles Hunault – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Odile Blanchet – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Belaid sekour – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Jean‐Marie Chrétien – Metabol study Group, Centre Hospitalo‐Universitaire d'Angers

Carmen Martínez – research scientist, INSER U1063, Centre Hospitalo‐Universitaire d'Angers

Introduction: Metabolic syndrome (MetS) is characterized by a cluster of interconnected risk factors leading to an increased risk of cardiovascular events. Small extracellular vesicles (sEVs) can be considered as new biomarkers of different pathologies, and they are involved in intercellular communication. Here, we hypothesize that sEVs is implicated in MetS‐associated endothelial dysfunction.

Methods: Circulating sEVs of non‐MetS subjects and MetS patients were isolated from plasma and characterized. Thereafter, sEVs effects on endothelial function were analyzed by measuring nitric oxide (NO) and reactive oxygen species (ROS) production and mitochondrial dynamic proteins, on human endothelial aortic cells (HAoECs).

Results: Circulating levels of sEVs positively correlated with anthropometric and biochemical parameters including visceral obesity, glycaemia, insulinemia, and dyslipidemia. Treatment of HAoECs with sEVs from MetS patients decreased NO production through the inhibition of the endothelial NO‐synthase activity. Injection of MetS‐sEVs into mice impaired endothelium‐dependent relaxation induced by acetylcholine. Furthermore, MetS‐sEVs increased DHE and MitoSox‐associated fluorescence in HAoECs, reflecting enhanced cytosolic and mitochondrial ROS production which was not associated with mitochondrial biogenesis or dynamic changes. MetS patients displayed elevated circulating levels of LPS in plasma, and, at least in part, it was associated to circulating sEVs. Pharmacological inhibition and down‐regulation of TLR4, as well as sEV‐carried LPS neutralization, results in a substantial decrease of ROS production induced by MetS‐sEVs.

Summary/Conclusion: These results provide evidence that sEVs from MetS patients as potential new biomarkers for this syndrome and that activation of the TLR4 pathway by sEVs provides a link between the endothelial dysfunction and metabolic disturbances in MetS.

PS03.06. Adipocyte‐Derived Exosome Induced Obesity‐Mediated Insulin Resistance and Lipotoxicity

Yujeong Kim, Division of Food and Nutrition, Chonnam National University

Ok‐Kyung Kim, Division of Food and Nutrition, Chonnam National University

Introduction: Obesity is directly or indirectly associated with the development of insulin resistance that causes type 2 diabetes. Recently, extracellular vesicle from adipose tissue has been shown to be involved in the development of insulin resistance. Here, we performed the isolation of exosomes from adipose tissue in obese conditions and investigated the effect of adipocytes‐derived exosome in the mechanism of insulin resistance development during obese conditions.

Methods: We isolated exosome from adipose tissue in normal diet‐treated mice or high fat diet‐induced obese mice, and confirmed the vesicle size, exosome markers, and uptake into differentiated C2C12 cells. We investigated whether exosome from adipose tissue in high fat diet‐induced obese mice induces insulin resistance and lipotoxicity in differentiated C2C12 cells.

Results: We found that the culture medium of differentiated 3T3‐L1 cells induced insulin resistance in differentiated C2C12 cells, whereas the culture medium from exosome inhibitor treated‐differentiated 3T3‐L1 cells improved insulin resistance. Exosome secretion was increased in adipose tissue from high fat diet‐induced obese mice, compared to normal diet. In addition, exosome from adipose tissue in high fat diet‐induced obese mice caused directly insulin resistance and lipotoxicity in differentiated C2C12 cells, compared to normal diet.

Summary/Conclusion: In conclusion, we suggest that adipocytes‐derived exosome in obese condition lead to insulin resistance and lipotoxicity in muscle tissue. This finding assessed the role played by exosome derived from adipocytes in the development of insulin resistance and potential strategies for the development of therapeutics aimed at obesity and metabolic disorders such as type 2 diabetes.

PS03.07. Identification and quantification of key metabolites of the steroid hormone biosynthesis pathway in extracellular vesicles

Guillermo Bordanaba, CIC bioGUNE

Sebastiaan van Liempd, CIC bioGUNE

Diana Cabrera, CIC bioGUNE

Félix Royo, CIC bioGUNE

Juan Manuel Falcón‐Pérez, CIC bioGUNE

Introduction: Prostate cancer (PCa) is among the most frequently diagnosed type of cancer worldwide. The lack of sensitive diagnostic tools and knowledge in its mechanisms of emergence and progression are a major challenge. In this regard, the metabolomic analysis of PCa provides unprecedent pathophysiological information about metabolic changes and responses to the microenvironment in healthy and tumorigenic cells. Such data often remains hidden in genomics, transcriptomics and proteomics approaches. Moreover, extracellular vesicles (EVs) are heterogeneous lipid containers with a complex cargo of molecules that are a valuable asset for cell‐to‐cell communication and signalling. EVs play a key role in pathophysiological processes by actively triggering various genetic or metabolic responses. Remarkably, PCa cells secrete EVs that participate in driving tumour growth and metastasis towards healthy recipient cells by building up a local environment, which increases tumour chemotaxis. There is literature reporting the transport of metabolic components using EVs as carriers; however, the manner their cargo (metabolites and enzymes) interacts with PCa metabolism is yet to be properly described. In previous research from our group, an increased level of dehydroepiandrosterone sulphate (DHEAS) in urinary EVs derived from PCa patients samples was detected. Moreover, steroid‐related metabolites and enzymes have been reported as important modulators of PCa progression.

Methods: In this work, we have developed an assay for the extraction and simultaneous identification of eleven metabolites of the steroid hormone biosynthesis pathway from cellular matrices and EVs. The metabolites included as chemical standards are pregnenolone, pregnenolone sulphate, DHEA, DHEAS, androsterone, androsterone sulphate, aldosterone, cortisol, estrone, testosterone and dihydrotestosterone. The metabolites were extracted using a liquid biphasic method and this process was optimized by testing the effect of different solvent combinations.

Results: The identification method was performed using hydrophobic integration chromatography coupled with time‐of‐flight mass spectrometry (UPLC‐MS) and has a run time of 5 min. In addition, this chromatographic method was optimized by testing various phases and gradients over the run. So far, this UPLC‐MS method has successfully identified steroid‐related metabolites in a panel prostate cell lines (PC‐3, DU145, 22Rv1, LnCaP and BPH‐1). Furthermore, EVs derived from this panel of cells and EVs isolated from urine fluids have been assayed using the method.

Summary/Conclusion: In summary, we present an optimized and rapid assay for the extraction and identification of steroid‐related metabolites from cells and extracellular vesicles.

PS03.08. Exercise modulation of Extracellular Vesicles’ content: focus on Nrf2 and antioxidant enzymes

Veronica Lisi, University of Rome Foro Italico

Chantalle Moulton, University of Rome Foro Italico

Ambra Antonioni, University of Rome Foro Italico

Elisa Grazioli, University of Rome Foro Italico

Cristina Fantini, University of Rome Foro Italico

Flavia Guidotti, University of Rome Foro Italico

Laura Capranica, University of Rome Foro Italico

Luigi Di Luigi, University of Rome Foro Italico

Paolo Sgro, University of Rome Foro Italico

Ivan Dimauro,University of Rome Foro Italico

Daniela Caporossi, University of Rome Foro Italico

Introduction: Extracellular vesicles (EVs) are lipid‐bound vesicles secreted by cells that mediate intercellular communication by shuttling functional molecules, such as different RNA species, lipids, DNA, and proteins. In literature it's well described that physical activity (PA) stimulates the release of molecules into circulation as EV cargo. The transcription NF‐E2‐related factor 2 (Nrf2) plays an important role in maintaining Redox homeostasis (RH) by regulating downstream antioxidants. The function of PA to trigger Nrf2, in response to the increase in ROS, is already known. Considering the role of RH in exercise‐induced signaling and adaptation, we focused on the exercise‐related intercellular communication of redox components mediated by EVs, including upstream and downstream factors.

Methods: Plasma EVs were isolated from trained and untrained healthy males (n = 14, 20–35 yrs) before and after (3 and 24 hours) an acute bout of endurance exercise (70% HRmax for 30’), or a short‐term endurance training (70% HRmax for 30’/day for 5 consecutive days), and analyzed for Nrf2, Catalase (CAT), Glutathione Peroxidase 1 (GPX1), Thioredoxin reductase 1 (TrxR1), Thioredoxin reductase 2 (TrxR2), Thioredoxin 2 (Trx2), SOD1, MnSOD (SOD2) and oxidative stress markers, as protein carbonilation and lipid peroxidation (4‐HNE), content.

Results: Our results showed that plasma EVs contain Nrf2 and antioxidant enzymes differently modulated by the fitness level and the exercise intervention. While no specific modulation was detected for the Nrf2 content in EVs, our data highlighted that SOD2 (p = 0.05) and CATALASE (p = 0.005) content in EVs’ cargo is decreased in trained with respect to the untrained subjects, with no significant effects exerted by a single acute exercise. When untrained subjects were submitted to 5 days of endurance training, CATALASE (p = 0.05) and SOD2 (p = 0.05) content were decreased, reaching levels similar to those found in trained subjects.

Summary/Conclusion: This study shows the presence of Nrf2 and antioxidant enzymes in plasma EVs, indicating a cross‐tissue molecular system to maintain and restore RH, and possibly to counteract, at systemic level, the oxidative stress derived by poor fitness levels or during physical exercise.

PS03.09. Extracellular Vesicle‐derived Epigenetic Biomarkers for Early Type 2 Diabetes Diagnosis from Saliva

Poster Presenter

Ulrike Kegler, AIT Austrian Institute of Technology

Manuela Hofner, AIT Austrian Institute of Technology

Nathalie Ropek, AIT Austrian Institute of Technology

Anja Buhmann, AIT Austrian Institute of Technology

Michael Leutner, Medical University of Vienna

Alexandra Kautzky‐Willer, Medical University of Vienna

Julie Krainer, AIT Austrian Institute of Technology

Klemens Vierlinger,AIT Austrian Institute of Technology

Christa Noehammer, AIT Austrian Institute of Technology

Introduction: Saliva is a readily and even within short time intervals repeatedly available body fluid, which can be obtained via non‐invasive, painless collection. The aim of this study was to define salivary extracellular vesicle (EV)‐derived epigenetic biomarkers suitable for early Type 2 diabetes (T2D) diagnosis.

Methods: A diabetic patient cohort (T2D, pre‐diabetes, gestational diabetes, healthy) was recruited. Plasma and cell‐free saliva was collected and EVs thereof prepared. Genome‐wide DNA methylation profiling was performed using EV‐derived DNA on Illumina EPIC arrays, whereas EV‐RNA was used for small RNA sequencing after several library preparation as well as EV isolation kits had been evaluated.

Results: After having identified the best suited EV isolation kit as well as the in the body fluids cell‐free saliva and plasma best performing small RNA library preparation kit, a variety of smallRNA /miRNA candidate biomarkers could be identified. In addition, we were able to successfully run a genome‐wide DNA methylation study from only 100 ng of salivary EV DNA and identified corresponding DNA‐methylation based candidate biomarkers. Verification of the identified potential epigenetic biomarkers for T2D is currently ongoing and performed in independent patient samples.

Summary/Conclusion: This study once more demonstrated saliva to be a most promising sample matrix for disease diagnostics which by far is not limited to oral diseases. Further genome‐wide high‐end profiling technologies such as methylation bead arrays and small RNA Seq could be successfully applied to cell‐free body fluids despite low amount of circulating DNA and RNA present there.

PS03.10. Extracellular Vesicles in Hypermobile Ehlers‐Danlos syndrome: deconstructing the fibroblast secretome to define bioactive molecules and disease mechanisms

Miriam Romano, Department of Molecular and translational Medicine‐University of Brescia, Italy

Nicoletta Zoppi, University of Brescia, Department of Molecular and Translational Medicine

Marco Giuseppe Ritelli, University of Brescia, Department of Molecular and Translational Medicine

Nicola Chiarelli, University of Brescia, Department of Molecular and Translational Medicine

Paolo Bergese, Dipartimento di Medicina Molecolare e Traslazionale, Università degli Studi di Brescia, 25123 Brescia, Italy

Annalisa Radeghieri, Department of Molecular and Tranlational Medicine‐Università degli Studi di Brescia

Marina Colombi, University of Brescia, Department of Molecular and Translational Medicine

Introduction: Hypermobile Ehlers‐Danlos syndrome (hEDS) is a multisystemic connective tissue disorder without known molecular bases. hEDS dermal fibroblasts show widespread extracellular matrix (ECM) disarray, including that of fibronectin (FN), increased levels of matrix metalloproteinase 9 (MMP9), and a myofibroblast‐like phenotype with α‐smooth muscle actin (α‐SMA) cytoskeleton organization. Control fibroblasts treated with hEDS conditioned media (CM) acquire this phenotype, indicating that patient cells’ CM contains key factors controlling this phenotypic switch. We hence dissected hEDS cells’ secretome into its nanoscale components, i.e., extracellular vesicles (EVs) and nanosized macromolecular complexes (nMC), to explore their possible role in the hEDS pathomechanisms.

Methods: Culture media of control and hEDS fibroblasts were treated by differential ultracentrifugation (0.8k g x 30’,16k g x 45’ and 100k g x 4h). Pellets and relative supernatants (SNs) were characterized according to the MISEV 2018 guidelines by AFM imaging, colorimetric nanoplasmonic assay (CONAN), and Western blotting (WB). Functional assays were performed by treating control fibroblasts with the different CM fractions from control and patient cells.

Results: We observed that both 16k and 100k control and hEDS pellets were composed of round‐shaped objects with average diameters < 150 nm. AFM imaging and CONAN assay confirmed the absence of exogenous protein contaminants. WB proved the presence of EVs and showed the presence of MMP9 in the 100k hEDS pellet. Functional assays revealed that the treatment of control cells with either 16k and 100k pellets or nMCs derived from hEDS cells partly induces the phenotypic switch (i.e., partial FN‐ECM degradation with few α‐SMA‐positive cells), whereas the different control CM fractions did not display any effect.

Summary/Conclusion: Overall, these data suggest that both EVs and nMCs fractions synergistically contribute to the hEDS fibroblast‐to‐myofibroblast transition.

PS03.11. Purine metabolism: new regulator of senescence associated with the secretory phenotype mediated by exosome‐like particles

Juan Fafián‐Labora, Universidade da Coruña

Rocio Mato Basalo, Universidade da Coruña

Miriam López‐Morente, Universidade da Coruña

Onno Arntz, Radboudumc

Fons van de Loo, Radboud University Medical Centre

María C. Arufe, University of La Coruña

Introduction: Cellular senescence is a hallmark of ageing and characterized by cell cycle arrest and production of cytokines, interleukins, lipid mediators and extracellular vesicles. Exosome‐like particles are a group involved in the transmission of paracrine senescence and rejuvenation and they are an attractive target to therapeutic application. In the last years, the metabolic changes had revealed crucial in the senescence signature in ageing and premature ageing and our group has focused to determined what metabolism pathways were altered in Hutchinson‐Gilford progeria syndrome (HGPS). This disease is a very rare fatal disease characterized for accelerated aging. Because of that, we focused our study on how the alteration of purine‐metabolism could affect the SASP mediated by exosome‐like particles.

Methods: In this study, we work with several models of cellular senescence: oncogenic‐induced senescence and DNA‐damage induced senescence (DDis) in mesenchymal stem cells, chondrocytes and fibroblasts from human origin. During the senescence induction the medium was supplemented with S‐adenosyl‐methionine (SAMe), a metabolite that is an alternative source of purine and besides the exosome‐like particles from that senescent cell models were used to treat proliferative cells.

Results: It was observed that the production of exosome‐like particles characterized by NTA from senescence signature models were decreased in a significantly way. It was observed that the production of exosome‐like particles characterized by NTA from senescence signature models were decreased in a significantly way. Besides, the transmission of paracrine senescence, which occurs through exosome‐like particles, it is no happens when cells are treated with SAMe as indicated in our results on proliferation using crystal violet and b‐galactosidase activity.

Summary/Conclusion: Altogether, our data suggests that purine metabolism is altered in premature aging through SASP mediated by exosome‐like particles. This opens a new window for the therapeutic treatment of the age‐related disease and HGPS

PS03.12. Endothelial shear stress affects extracellular vesicles release and biodistribution

Cecile Devue, InsermU970‐ Paris Cardiovascular Research Center

Michael Robillard, Inserm U970‐ Paris Cardiovascular Research Center

Pierre‐Michaël Coly, Université de Paris, INSERM U970, Paris Cardiovascular Research Centre, Paris, France

Chantal Boulanger, Inserm U970‐ Paris Cardiovascular Research Center

Xavier LOYER, INSERM U970‐PARCC

Introduction: Atherosclerosis initiates at endothelial level at specific areas where blood flow is low and disturbed contributing to the recruitment of inflammatory cells. Extracellular vesicles (EVs) emerged as new mediators of intercellular crosstalk. Additionally endothelial atheroprone conditions (shear stress‐ SS) have been showed to influence large EVs release. During their formation, EVs package and carry some material, especially miRNAs, from originating cells to recipient cells, in which they contribute to specific phenotypical changes through regulation of gene expression. To date, mechanisms linking EVs‐mediated transfer of information onto the development of atherosclerosis have not been characterized so far. We hypothesize that endothelial‐derived EVs can selectively be released as function of endothelial shear stress and transferred to the spleen given its recently recognized importance in regulating the immune response in atherosclerosis.

Methods: Confluent mouse endothelial cells (SVEC) were exposed to either Low SS or High SS for 24 hours in a serum free medium. Large (>200 nm) and small EVs were isolated from conditioned medium by sequential centrifugation; they were then characterized by Western blot and tunable resistive pulse sensing. In vitro EV biodistribution to splenocytes was assessed after co‐culture for 12hours by flow cytometry using labelled EVs with Vybrant DID (VD) or Membright. miRNAs content have been analyzed by microarray and qPCR.

Results: Levels of large and small EVs were not modulated by low SS conditions. In response to High SS conditions EVs " both large and small " levels were further increased to LSS conditions. No changes in EVs size were observed as a function of SS levels. Western blot analysis revealed that EV markers were not affected by SS conditions. Labelling EVs with VD or Membright do not alter EVs size, concentration and EVs markers expression. Analysis of EVs biodistribution co‐cultured with splenocytes revealed that large EVs derived from low SS conditions are preferentially transferred to myeloid cells including macrophages, monocytes and neutrophils as compared to EVs derived from high SS. Small EVs do not exhibit any specific tropism and are poorly transferred to cells. EVs miRNAs content analysis revealed that EVs selectively contain miRNAs into large and small EVs. Among such miRNAs, we showed that miR‐24 is selectively packaged into large EVs upon LSS. In vitro studies showed that specific miR‐24 transfer could modulate defined targets into splenocytes.

Summary/Conclusion: Altogether, these findings revealed that shear stress affect selectively EVs release, miRNAs content and biodistribution to splenocytes. Future perspectives will aim to decipher the in vivo role of such EVs in the context of atherosclerosis.

PS04.01. Exosomes derived from covid‐19 infected mesenchymal stem cells can activate antigen‐specific immune response against covid‐19 virus

zahra naseri, Department of molecular biology, Faculty of Curative Medicine, Khatam‐al‐nabieen University, Kabul, Afghanistan.

hossein Rahimi, Department of Biology and Microbiology, Medical laboratory Science Institute, Khatam‐al‐nabieen University, Kabul, Afghanistan.

Introduction: Due to the rapid spread of covid‐19 infection around the world, there is a fundamental need to design an effective vaccine. Mesenchymal stem cells (MSCs) are unique multipotent progenitor cells that are presently being exploited as gene therapy vectors for a variety of conditions. MSCs have also demonstrated some success in anti‐microbial prophylactic vaccines. MSCs exert their therapeutic effects via secretion of soluble paracrine factors and exosomes. Exosomes are known as naive nanovesicles that play an important role in intercellular communications. Some studies have shown the presence of some viral components in exosomes secreted by virus‐infected cells. So, we hypothesized that covid‐19 infected mesenchymal stem cells produce exosomes carrying viral components that can be used as an effective vaccine against covid‐19 infection.

Methods: The mouse bone marrow‐extracted MSCs became infected with the covid‐19 virus obtained from positive patients. Then, the exosomes were extracted from the infected cells by ultracentrifugation and characterized. The secreted exosomes were then injected to balb/c mice intranasally to evaluate the antisera.

Results: The extracted MSCs had a defined‐shaped fibroblastic morphology with moltipotent nature that were positive for for CD29, CD44, CD90, CD105 and negative for CD34 and CD45. TEM analysis verified the disc‐shaped of MSCs‐Exo and showed that the exosomes had average size between 30–150 nm. Expression of some important exosomal markers such as CD81 and CD63, and not expression of Calnexin, endoplasmic reticulum marker, were characterized using western blot. In addition, the expression of a specific SARS‐CoV‐2 spike antigen, S protein, was confirmed using specific antibody in western blotting. Interestingly, intranasal administration of mice with the exosomes isolated from covid‐19 virus infected MSCs induced neutralizing antibody titers that confirmed by ELISA and plaque reduction neutralization (PRNT) assays.

Summary/Conclusion: In conclusion, our results suggest that the exosomes may be a promising vaccine candidate against covid‐19 infection.

PS04.02. Extracellular Vesicles derived from Antigen Presenting Cells pulsed with inactivated Foot and Mouth Disease VIrus express a high level of viral proteins and induce a specific antiviral response

Florencia Menay, National Research Council of Argentina (CONICET)

Federico Cocozza, Institut Curie / INSERM U932

Maria Jose Gravisaco, INTA

Analia Elisei, INTA

Javier Re, INTA

Claudia Waldner, National Research Council of Argentina (CONICET)

Pura Sampedro, Universidad de Moron

Alejandra Ferella, National Research Council of Argentina (CONICET)

Claudia Mongini, National Research Council of Argentina (CONICET)

Introduction: Foot and mouth disease (FMD) is a worldwide economically important infection caused by FMD‐virus (FMDV).The main strategy for the control is vaccination with FMDV chemically inactivated with binary ethylenimide (FMDVi). In FMDV infection and in vaccination, the B cell response plays a major role by providing neutralizing/protective antibody in both animal models and natural hosts. EVs secreted by antigen presenting cells (APC) participate in the activation of B and T cells through the presentation of native antigen membrane associated (to B cells) or by transferring MHC‐peptide complexes (to T cells) and even complete antigen from DCs. We aimed to evaluate the immune properties of EVs derived from APC pulsed with FMDVi in a murine model.

Methods: Bone marrow‐derived DCs differentiation. In Vitro APC pulse load with BEI‐FMDVI. EVs isolation by differential centrifugation ultrafiltration, and ultracentrifugation. EVs immunofluorescence staining and Flow cytometry. Size distribution by Nanoparticle Tracking Analysis. Expression of viral protein on EVs at different secretion and pulse time. Carboxyfluorescein Succinimidyl Ester (CFSE) Proliferation Assay and Flow cytometry.

Results: APC differentiated from bone marrow cells internalized FMDVi labelled with FITC after incubation for 60 min. EVs were isolated after 24h from APC pulse with FMDVi. By flow cytometry EVs expressed the EVs (CD9, CD81, CD63) and APC markers (MHC‐II and CD86). Remarkably, FMDV antigens were expressed on >89% EVs. We demonstrated that EVs‐FMDVi induced specific proliferation in vitro in splenocytes sensitized with FMDVi, EVs‐FMDVi induced specific B cell (16.05% ± 0.61 p < 0.001) and T cell proliferation (8.5% ± 0.81 p < 0.01) when compared to the control (9.66% ± 0.17 and 5.70% ± 0.15, respectively) detected by CFSE dilution.

Summary/Conclusion: We demonstrated that APC cells can internalize FMDVi and release EVs expressing APC markers and high level of viral proteins. Our results revealed that EVs‐FMDVi could present FMDV proteins in native conformation or partially processed. These peptides can be recognized by the BCR and stimulate specific B cell response against viral infection. In addition, EVs‐FMDVi activate direct or indirectly a T cell response that could collaborate in B cell activation. The knowledge derived from this work will serve to deepen the knowledge of the interrelation between the FMDV and the immune system for the rational design of vaccines.

PS04.03. Let‐7b in extracellular vesicles secreted by human airway epithelial cells increases the ability of beta‐lactam antibiotics to reduce P. aeruginosa biofilm formation

Katja Koeppen, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth

Amanda Nymon, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth

Roxanna Barnaby, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth

Laura Bashor, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth

Zhongyou Li, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth

Thomas H. Hampton, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth

Amanda E. Liefeld, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth

Fred W. Kolling, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth

Ian S. LaCroix, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth

Scott A. Gerber,Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth

Deborah A. Hogan, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth

Swetha KasettyDepartment of Biological Sciences, Dartmouth College

Carey D. Nadell, Department of Biological Sciences, Dartmouth College

Bruce A. Stanton,Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth

Introduction: P. aeruginosa is an opportunistic pathogen that forms antibiotic‐resistant biofilms which facilitate chronic infections in immunocompromised hosts. We have previously shown that P. aeruginosa secretes outer membrane vesicles that deliver a small RNA to human airway epithelial cells where it suppresses the innate immune response. Here, we demonstrate that inter‐domain communication through small RNA‐containing membrane vesicles is bi‐directional and that miRNAs in extracellular vesicles (EV) secreted by human airway epithelial cells regulate protein expression, antibiotic sensitivity and biofilm formation by P. aeruginosa.

Methods: We used RNA‐seq to characterize the RNA content of EVs secreted by human airway epithelial cells and to show delivery of human miRNAs from EVs to P. aeruginosa. We evaluated the response of P. aeruginosa to human EVs and to let‐7b‐5p using biotic and abiotic biofilm formation assays. Sensitivity to the beta‐lactam antibiotic aztreonam in the presence and absence of EVs was determined using growth curve assays. Targets of let‐7b‐5p in P. aeruginosa were predicted using IntaRNA and validated in proteomics experiments.

Results: We found that human EVs deliver let‐7b‐5p, a 22‐nt miRNA, to P. aeruginosa, which systematically decreases the abundance of proteins essential for biofilm formation, including PpkA and ClpV1, and increases the ability of beta‐lactam antibiotics to reduce biofilm formation by targeting the beta‐lactamase AmpC. Let‐7b is bioinformatically predicted to target PpkA, ClpV1 and AmpC not only in P. aeruginosa, but also the corresponding orthologs in Burkholderia cenocepacia, another notorious opportunistic lung pathogen, suggesting that the ability of let‐7b‐5p to reduce biofilm formation and increase beta‐lactam sensitivity is not limited to P. aeruginosa.

Summary/Conclusion: To our knowledge, this is the first direct evidence for transfer of miRNAs in EVs secreted by eukaryotic cells to a prokaryotic organism, resulting in subsequent phenotypic alterations in the prokaryote as a result of this transfer. Since let‐7 family miRNAs are in clinical trials to reduce inflammation and, given that chronic P. aeruginosa lung infections are associated with a hyper‐inflammatory state, treatment with a combination of let‐7b‐5p and a beta‐lactam antibiotic in nanoparticles or EVs are predicted to benefit patients with antibiotic‐resistant P. aeruginosa infections.

PS04.04. Urinary SARS‐CoV‐2 RNA is an indicator for the progression and prognosis of COVID‐19 disease

Huiming Wang, Renmin Hospital of Wuhan University

Lu Zhang, Renmin Hospital of Wuhan University

Maoqing Tian, Renmin Hospital of Wuhan University

Introduction: SARS‐CoV‐2 RNA can be detected in the urine of Coronavirus disease 2019 (COVID‐19) patients. However, it is unclear whether urinary SARS‐CoV‐2 RNA (URNA+) is associated the severity and clinical manifestations of hospitalized COVID‐19 patients.

Methods: In this study, we analyzed the demographic and clinical data of COVID‐19 patients and detected SARS‐CoV‐2 RNA in urine sediments collected from 53 COVID‐19 patients enrolled in Renmin Hospital of Wuhan University from January 31, 2020 to February 18, 2020 with qRT‐PCR analysis, and then classified those patients based on clinical conditions (severe or non‐severe syndrome) and urinary SARS‐CoV‐2 RNA (URNA‐ or URNA+).

Results: We found that COVID‐19 patients with severe syndrome (severe patients) showed significantly higher positive rate (11 of 23, 47.8%) of urinary SARS‐CoV‐2 RNA than non‐severe patients (4 of 30, 13.3%, p = 0.006). URNA+ patients or severe URNA+ subgroup exhibited higher prevalence of inflammation and immune discord, cardiovascular diseases, liver damage and renal disfunction, and higher risk of death than URNA‐ patients. To understand the potential mechanisms underlying the viral urine shedding, we performed renal histopathological analysis on postmortems of patients with COVID‐19 and found that severe renal vascular endothelium lesion characterized by increase of the expression of thrombomodulin and von Willebrand factor, markers to assess the endothelium dysfunction. We proposed a theoretical and mathematic model to depict the potential factors determining the urine shedding of SARS‐CoV‐2.

Summary/Conclusion: This study indicated that urinary SARS‐CoV‐2 RNA detected in urine specimens may be used to predict the progression and prognosis of COVID‐19 severity.

PS04.05. Bacterial lipid content of extracellular vesicles released by macrophages infected by Mycobacterium tuberculosis

Pierre Boyer, Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, Université Paul Sabatier

Jérôme Nigou, CNRS

Emilie Layre, Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, Université Paul Sabatier

Introduction: Tuberculosis is one of the top ten causes of death worldwide. Mycobacterium tuberculosis (Mtb) is an intracellular pathogen of alveolar macrophages that has evolved strategies to adapt to and subvert host defenses. In most cases of infection, the host immune responses only controls the bacillus dissemination, leading to latent tuberculosis. Understanding the molecular bases of the complex cross‐talk between the host and the bacillus is key to design new anti‐TB strategies. The lipids, glycolipids, lipoproteins and lipoglycans that compose Mtb envelope play a major role in host‐pathogen interactions, acting as Pathogen Associated Molecular Patterns, antigens or virulence factors. If the functions of these molecules is conceived at the bacillus surface, a yet incompletely characterized repertoire of bacterial lipid traffic within infected cells and out of the infected cells within extracellular vesicles (EV) that have the potential to modulate the response of bystanders cells. A better characterization of the lipid content of EV released by infected cells will provide better insight into the role of mycobacterial lipids and EV themselves in host‐pathogen interactions.

Methods: The populations of EV released by infected macrophages were purified by combining centrifugal centrifugation and density gradient. Purified vesicles were characterized by nanosight, microscopy, FACS and western blot. Their content in mycobacterial lipids and lipoglycans was characterized by western blot and using a high sensitivity and last generation mass spectrometry‐based approach.

Results: EV populations were purified from cultures of infected macrophages. Using western blot analyses and a targeted mass spectrometry approach, we performed a comprehensive analysis of EV content in mycobacterial lipidic molecules. We highlighted the presence of additional mycobacterial lipid families. Using different bacterial species, we have also assessed whether the lipid content of EV released by infected cells differs with the bacillus virulence degree, which would subsequently result in different immunomodulatory properties.

Summary/Conclusion: Several families of immunomodulatory mycobacterial lipids, lipoproteins and lipoglycans, traffic within vesicles released by macrophages, by which they likely modulate immune responses beyond the infected cells. Further investigations are required to assess the diversity of EV populations, their fine composition and immunomodulatory properties.

PS04.06. Plasma‐derived EV analysis indicates the persistency of fibrogenic signals in HCV DAA‐treated patients that reached sustained virological response

Cecilia Battistelli, SapienzaUniversity of Rome

Claudia Montaldo, Spallanzani National Institute for Infectious Diseases

Michela Terri, Sapienza University of Rome

Veronica Riccioni, Sapienza University of Rome

Veronica Bordoni, Spallanzani National Institute for Infectious Diseases

Gianpiero D'Offizi, Spallanzani National Institute for Infectious Diseases

Maria Giulia Prado, Sapienza University of Rome

Tiziana Vescovo, Spallanzani National Institute for Infectious Diseases

Eleonora Tartaglia, Spallanzani National Institute for Infectious Diseases

Raffaele Strippoli,Sapienza University of Rome

Chiara Agrati, Spallanzani National Institute for Infectious Diseases

Marco TripodiSapienza University of Rome

Introduction: HCV SVR achievable now by means of DAA therapy identifies a new class of patients requiring medical surveillance to be designed in relation to the liver disease stage advancement. To this end, identification of both disease biomarkers and therapeutic target appears necessary.

Methods: Extracellular Vesicles (EVs) purified from plasma of 5 healthy donors (HD), and 5 HCV infected patients before (T0) and after (T6) DAA treatment have been utilized for functional and miRNA cargo analysis. EVs purified from plasma of 13 HD and 13 T0 and T6 patients have been employed for proteomic and western blot analysis. Functional analysis in LX2 cells measured fibrotic markers (mRNAs and proteins) in response to EVs. Structural analysis was performed by qPCR, mass spectrometry (orbitrap) and Western blot.

Results: On the basis of observations indicating functional differences of plasma‐derived EVs from HD, T0 and T6, we endeavour EV structural analysis. We found consistent differences in terms of both miRNAs and proteins cargos; (i) antifibrogenic miR204‐5p, miR93‐5p and miR181a‐5p were found statistically underrepresented in T0 EVs with respect to HD and miR204‐5p and miR143‐3p were found statistically different between HD and T6 (p‐value < 0.05) (ii) proteomic analysis highlighted, in both T0 and T6, the modulation of several proteins with respect to HD; among them the fibrogenic DIAPH1 was found upregulated by western blot (4.4 Log2 fold change).

Summary/Conclusion: Taken together, these results highlight structural EVs modifications, conceivably causal for long‐term liver disease progression in HCV patients that persist despite the DAA‐mediated HCV SVR.

PS04.07. Syntenin facilitates dissemination of ZIKV through regulating exosomes release and uptake

RuiZhang, Nan Jing university, China

Min Cheng, NanJing University

Zhiwei Wu, NanJing University

Introduction: Exosomes was considered as small membrane‐encapsulated vesicles that was loaded with proteins, lipids and RNAs that compose ‘a signature’ of the cell of origin and potentially can reprogram‐alter recipient cells. One of the main exosomal functions is to mediate intercellular communication during viral pathogenesis and immune responses. Zika virus (ZIKV), a re‐emerging mosquito‐borne flavivirus, is an enveloped, positive single‐stranded RNA virus, had reported continuing vector‐borne transmission of ZIKV. ZIKV infection is the association of some viral strains with neurological diseases. During viral infection, some exosomes regulatory proteins regulate the release and uptake of viral exosomes, In particular, syntenin plays a role in exosome biogenesis. Given the pivotal role played by syntenin in exosome biogenesis, here we studied the role of syntenin in the spread of exosome‐zika.

Methods: A549 cells were infected with Zika, and the secretion of exosomes was significantly increased. The role of Syntenin in the process of viral exosome generation was explored by CRISPA‐CAS9 technique.

Results: We found that Zika virus (ZIKV) infection increased the release of exosomes in A549 cells, and exosomes derived from zika virus infected cells have the ability to re‐establish infection in vivo and in vitro (in a manner independent of traditional viral receptor‐dependent pathways). In addition, we also found that ZIKV infection up‐regulates syntenin (which was supported the formation of intraluminal vesicles that compose the source of a major class of exosomes, supported the recycling of these same components to the cell surface.), and syntenin controlled re‐infection of exosome‐zika by affecting exosome release and uptake.

Summary/Conclusion: Our study significantly extended RNA virus (Zika) infection and regulated exosome secretion, and the related molecular protein (syntenin) regulated the biological significance of exosome‐zika.

PS04.08. Outer membrane vesicles from Hypervirulent Klebsiella pneumoniae mediate virulence factor transfer

Yuneng Hua, Southern Medical University

Introduction: Outer membrane vesicles (OMVs) are potent virulence factors, naturally secreted by gram‐negative bacteria, acting as mediators of inter‐ and intra‐species communication. Since Hypervirulent Klebsiella pneumoniae(HvKp) has emerged as an important nosocomial community‐acquired pathogen, because of more virulent than classical K. pneumoniae (CKp), it is crucial to investigate its pathogenetic mechanism as it relates to OMVs.

Methods: NTA;WB;TEM;BCA;PCR;flow cytometry;proteomics;CLSM

Results: In this work, we indicate that HvKp OMVs may contain several virulence factors that contribute to virulence and disease pathology. HvKp OMVs are mediating virulence factors transfer and allowing increase in the virulence level of CKP or CRKP.

Summary/Conclusion: In this work, we indicate that HvKp OMVs may contain several virulence factors that contribute to virulence and disease pathology. HvKp OMVs are mediating virulence factors transfer and allowing increase in the virulence level of CKP or CRKP.

PS04.09. Characterisation of membrane vesicles in bacterial sepsis pathogens

Christian Grätz, Technische Universität München

A. Ronja D. Binder, Technische Universität München

Anja Lindemann, University Hospital, Ludwig‐Maximilians‐Universität München

Genia Lücking, Technische Universität München

Michael W. Pfaffl, PhD, Chair of Animal Physiology & Immunology

Gustav Schelling, University Hospital, Ludwig‐Maximilians‐Universität München

Marlene Reithmair, University Hospital, Ludwig‐Maximilians‐Universität München

Introduction: Sepsis is defined as a life‐threatening organ dysfunction due to a dysregulated host response to infection with bacteria as the most common infectious agent. Gram‐positive (G+) and Gram‐negative (G‐) bacteria shed membrane vesicles (MVs) which carry various types of RNA. By merging with eukaryotic cells, these may induce a systemic inflammatory response. After antibiotic treatment, it is often impossible to identify life bacteria by cultivation. MV‐associated RNA shed by these bacteria may be used as a more reliable indicator of bacterial infection.

Methods: Here, we characterised MVs and their RNA cargo from six bacteria, five of them commonly involved in sepsis pathogenesis: S. saprophyticus (G+), S. aureus (G+), C. jeikeium (G+), H. influenzae (G‐) and H. parainfluenzae (G‐). The pathogenic E. coli strain O1:K1:H7 served as a control although it is not necessarily associated with sepsis. MVs were isolated from the sterile‐filtered bacterial culture supernatant using ultracentrifugation and for further purification a density gradient step was added. Nanoparticle tracking analysis (NTA) was used to measure particle number and size distribution of the isolated MVs, which were then visualised using transmission electron microscopy (TEM). Western blotting identified various MV markers. RNA from selected samples was analysed by next‐generation sequencing (NGS).

Results: All six bacteria shed membrane‐enclosed vesicles, with a tendency to a higher MV number in G‐ bacteria. In the supernatant of E. coli and C. jeikeium, a high number of flagella was found. MV density varied between the six bacteria, ranging from 1.12 to 1.21 g/ml. The MV marker groEL was detected in the purified MVs of all G‐ bacteria. Preliminary NGS results for E. coli and H. influenzae showed bacteria‐specific rRNA transcripts at the top 15 genes with the most generated reads, followed by protein coding genes.

Summary/Conclusion: All pathogens produced high numbers of RNA‐containing MVs. The uptake of those MVs and their RNA cargo into human blood cells will be assessed in a follow‐up study. It remains to be shown that bacterial MVs or their RNA can be detected in blood samples from sepsis patients, but MV‐associated RNA remains a promising sepsis biomarker.

PS04.10. Differential shedding of EVs by distinct Trypanosoma cruzi strains during metacyclogenesis

Ana Claudia Claudia. Torrecilhas, Universidade Federal de São Paulo campus Diadema

Paula Meneghetti Meneghetti, UNIFESP

Rafael Pedro Madeira, UNIFESP

João Paulo Ferreira, UNIFESP

Nobuko Yoshida, UNIFESP

Introduction: The protozoan parasite Trypanosoma cruzi spontaneously releases extracellular vesicles (EVs). In a previous research, we have shown that T. cruzi EVs are shed from epimastigote forms during interaction with triatomine bugs Rhodnius prolixus and Triatoma infestans. The EVs delayed parasite migration to rectum only in the gut of R. prolixus. The aim of our research is the characterization of EVs released during metacyclogenesis of different T. cruzi strains (Y, CL and G)

Methods: Methods The epimastigotes forms from T. cruzi strains (Y, CL and G) were cultivated during 30 days to follow the parasite growth, metacyclogenesis, and release of EVs. Nanoparticle tracking analysis (NTA) was performed to check the size and concentration of the EVs isolated from different strains during metacyclogenesis.

Results: Results The size of EVs isolated from CL strains was large than Y and G strains. Y strain parasites released more EVs (1 × 109 particles/ mL) than strains G (6 × 108 particles / mL) and CL (5 × 108 particles / mL).

Summary/Conclusion: Conclusion During metacyclogenesis, T. cruzi strains Y, CL and G released EVs of different sizes and their numbers varied between strains. This may be associated with the parasite development and differentiation.

PS04.11. Circulatory EVs as potential biomarkers of HIV‐drug abuse interactions and neurological dysfunction in HIV‐Infected subjects and alcohol/tobacco Users

Sunitha Kodidela, UTHSC

Namita Sinha, UTHSC

Asit Kumar, Ph.D., University of Tennessee Health Science Center

Prashant Kumar, UTHSC

Santosh Kumar, UTHSC

Introduction: Abuse of alcohol and tobacco can exacerbate HIV pathogenesis by transferring materials through extracellular vesicles (EVs). EVs present a stable and accessible source of biological information from one cell to various types of cells, including brain cells. Therefore, we aimed to study the plasma EVs proteins, which are altered in both HIV and drug abusers to identify a physiological marker to indicate the immune status and neuronal damage of HIV‐positive drug abusers

Methods: EVs were isolated from plasma samples of the following subjects by double isolation method to improve their purity: a) Healthy b) HIV c) Alcohol drinkers d) Smokers e) HIV+drinkers f) HIV+smokers. Quantitative proteomic profiling of EVs was performed by mass spectrometry and potential EV proteins associated (G‐FAP and L1‐CAM) with glial and neuronal dysfunction were quantified by western blot.

Results: The EVs were characterized according to the MISEV guidelines. Comparison of proteins among all the study groups revealed that hemopexin was significantly altered in HIV+drinkers compared to drinkers and HIV subjects. Further, our study is the first to show properdin expression in plasma EVs, which was decreased in HIV+smokers and HIV+drinkers compared to HIV patients. The levels of astroglial marker (GFAP) were elevated in plasma EVs from HIV subjects compared to healthy subjects. Further, the levels of GFAP and L1‐CAM were increased in drinkers and smokers without HIV infection compared to healthy subjects

Summary/Conclusion: The present findings suggest that plasma EVs serve as potential markers for complications associated with substance abuse in HIV‐subjects. In particular, hemopexin, and properdin show potential as markers for HIV‐drug abuse interactions. The present study has also established that the astrocytic and neuronal‐specific markers (GFAP and L1CAM), which have the potential to play a role in neurological dysfunctions, can be packaged in EVs and circulated in plasma.

PS04.12. Extracellular vesicles from ethanol‐treated and HIV‐infected macrophages induce inflammasome activation in hepatocytes

Edward Makarov, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA 68105

Moses New‐Aaron, Research Department, Veteran Affairs Medical Center, Department of Environmental, Agriculture and Occupational Health, University of Nebraska Medical Center, Omaha, NE, USA 68105

Murali Ganesan, Research Department, Veteran Affairs Medical Center, Internal Medicine Department, University of Nebraska Medical Center, Omaha, NE, USA 68105

Larisa Y. Poluektova, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA 68105

Natalia A. Osna, Research Department, Veteran Affairs Medical Center, Internal Medicine Department, University of Nebraska Medical Center, Omaha, NE, USA 68105

Raghubendra S. Dagur, PhD, University of Nebraska Medical Center

Introduction: Hepatic inflammation is a common trigger of hepatic steatosis and progresses rapidly to cirrhosis, and finally, hepatocellular carcinoma in HIV‐patients who have a history of alcohol abuse. During the disease conditions, macrophages are quickly recruited to the liver and produce many extracellular vesicles (EVs). How these EVs from alcohol and HIV‐infected macrophages regulate hepatocyte survival is not clear and is our study's focus.

Methods: Monocyte‐derived macrophages (MDM) were exposed to 50 mM ethanol (EtOH) before infecting the cells with the HIV‐1ADA strain at a multiplicity of infection of 0.1. HIV‐infected cells were further exposed to 25mM EtOH, and the conditioned medium was collected from 4 groups of cells: untreated, HIV‐, EtOH‐ and EtOH+HIV. Quantification of EVs number and size was evaluated with Nanosight and characterized for EVs markers following the Minimal Information for Studies of Extracellular Vesicles guidelines, 2018. To assess EV‐mediated liver inflammation, we exposed human hepatocyte Huh7.5CYP2E1 [hepatoma cells stably transfected with CYP2E1 designated as RLW cells] to MDM‐EVs and then measured inflammasome activation based on NLRP3, caspase 1, and IL‐1β mRNAs with qPCR.

Results: Alcohol treatment stimulated the EVs secretion from HIV‐infected macrophages. Size distribution assessed by Nanosight revealed more than 90% of particles distributed in the range of 50 to 200 nm. Western blotting of MDM‐EVs demonstrated positivity for small EVs enriched proteins Alix, TSG 101, and CD9 and negative for endoplasmic reticulum protein calnexin and HIV protein. The uptake of MDM‐EVs by hepatocytes was apparent, as demonstrated by immunofluorescence. Transfer of MDM‐EVs from the EtOH+HIV group, but not from other groups, significantly increases the activation of NLRP3 inflammasome in hepatocytes. The activation of NLRP3 was accompanied by an increase in the expression of inflammatory caspase 1 and IL‐1β.

Summary/Conclusion: We conclude that alcohol treatment stimulates EVs secretion from HIV‐infected macrophages. The uptake of EtOH‐and HIV‐ modified MDM‐EVs leads to activation of canonical NLRP3 inflammasome signaling in hepatocytes to promote liver inflammation and may trigger fibrosis development.

PS04.13. Pseudomonas aeruginosa extracellular vesicles from cystic fibrosis sputum induce inflammation in human bronchial epithelial cells

Andrea L. Hahn, MD, Children's National Hospital

Aszia Burrell, BS, Center of Genetic Medicine, Children's National Research Institute

Kylie I. Krohmaly, BS, Institute of Biomedical Sciences, George Washington University

Kayla Authelet, BS, Center of Genetic Medicine, Children's National Research Institute

Claire Hoptay, PhD, Center of Genetic Medicine, Children's National Research Institute

Robert J. Freishtat, MD, MPH, Center of Genetic Medicine, Children's National Research Institute

Introduction: Pseudomonas aeruginosa (Pa) is an important chronic pathogen associated with intermittent pulmonary exacerbations (PEx) and lung inflammation in persons with cystic fibrosis (CF), likely mediated in part by extracellular vesicles (EVs). We hypothesized that Pa EVs obtained from the sputum of persons with CF would induce inflammation in CF human bronchial epithelial (HBE) cells.

Methods: Following IRB approval and informed consent, sputum samples were collected from three persons with CF, homogenized, and centrifuged at 12000g x 10 minutes. EVs were isolated from the supernatants using precipitation and size exclusion chromatography. Pa antibodies were attached to magnetic beads to isolate Pa‐specific EVs. Life extended CF HBE cells (F508del/F508del) were grown to 80% confluency, and equivalent volumes of Pa EVs or PBS (250 μL) were added and incubated for 22 hours before cell harvesting. Cells were immediately lysed with TRIzol and RNA extracted with a DirectZol MiniPrep kit. RNAseq was performed using a Mid‐Output, 75 cycle kit on a NextSeq 500. A Galaxy workflow incorporating HISAT2, Stringtie, Gffcompare, featureCounts, and DESeq2 was used to determine differential gene expression. Ingenuity pathway analysis (IPA) was used to identify differences in canonical pathways.

Results: RNA quality assessment showed RIN values near 10, with an average of 19 million reads per sample (range 14–25 million). Differential expression of 394 transcripts were identified using an unadjusted p value of < 0.05. When filtering for lung tissue, 8 canonical pathways with a Z‐score of ‐2 were upregulated in HBE cells exposed to Pa EVs compared to PBS exposure. This included several signaling pathways associated with inflammation and immunity: G Beta Gamma (Z‐score +2, genes GNB1L, GNG11, PRKAG2, PRKCD, RRAS), P2Y Purigenic Receptor (Z‐score +2, genes GNB1L, GNG11, PRKAG2, PRKCD, RRAS), IL‐8 (Z‐score +2, genes GNB1L, GNG11, NCF2, PRKCD, RRAS), and HMGB1 (Z‐score +2, genes IL1A, PLAT, RRAS, TGFB2).

Summary/Conclusion: CF HBE cells exposed to Pa EVs isolated from the sputum of persons with CF demonstrated upregulation of inflammatory and immunity pathways compared to PBS exposure. Future studies comparing the effect of Pa EVs isolated during times of wellness and PEx may provide insight into how Pa EVs impact variations in inflammation with chronic infection.

PS04.14. miRNAs in extracellular vesicles, novel therapeutic agents for COVID‐19

JisookMoon, College of Life Science, Department of Biotechnology, CHA University

Jae Hyun Park, CHA University

Yuri Choi, CHA University

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) causes causes severe respiratory failure and there is no treatment yet. Micro‐RNAs (miRNAs) are potential novel anti‐viral agents because of their ability to degrade viral RNAs. Extracellular vesicles (EVs) is a small vesicle that secreted from cell and can transfer miRNAs to recipient cells and regulate conditions within them.

Methods: Mesenchymal stem cell derived EVs (MSC‐EVs) contain anti‐viral miRNAs that play important roles in the virus‐infected host cells. Here, we examined their potential impact on viral and immune responses. Here, we identified candidate therapeutic miRNAs with important roles in the biological functions of virusinfected host cells, and characterized the antiviral effects of miRNAs derived from placenta‐derived MSC‐EVs

(pMSC‐EVs) or placenta EVs, which exhibit potent regenerative and anti‐inflammatory effects.

Results: MSC‐EVs contained 18 miRNAs predicted to interact directly with the 3’ UTR of SARS‐CoV‐2. In addition, five major miRNAs in MSC‐EVs suppressed viral 3’ UTR sequence in a luciferase reporter assay and these EVs suppressed SARS‐CoV‐2 infection in Vero E6 cells.

Moreover, MSC‐EVs showed anti‐inflammatory effect which may prevent lethal cytokine storms caused by viral infection and we confirmed that MSC‐EVs suppressed inflammatory responses by several cell types.

Summary/Conclusion: miRNAs in MSC‐EVs have several advantages as therapeutic agents against SARS‐CoV‐2: 1) they can inhibit viral 3’ UTR and suppress viral replication; 2) because the 3’ UTR is highly conserved and rarely mutates, MSC‐EV miRNAs could be used against new variants of SARS‐CoV‐2; and 3) unique cargoes carried by MSC‐EVs can have immunomodulatory effect.

PS04.15. Rapid Isolation Protocol for Circulating Extracellular Vesicles in Chronic Chagas Disease Patients

Ana Claudia Claudia. Torrecilhas, Universidade Federal de São Paulo campus Diadema

Rafael Pedro Madeira, UNIFESP

Lucas Alexandre Barros, UNIFESP

Introduction: Extracellular vesicles are lipid bilayer envelopes that encase several types of molecules in their interior in addition to those in their membranes. Their contents mostly reflect their cell of origin and possible targets at other places in the organism and can also be modified in pathological conditions to interfere with intercellular communication, promoting disease establishment and development. These characteristics, in addition to their presence in virtually all body fluids, make them ideal for biomarker research

Methods: In Chagas disease where no biomarker exists to date to infer prognosis in indeterminate stage chronic patients, our work proposed establishing a protocol for circulating extracellular vesicle isolation in this population. For this, we isolated extracellular vesicles from blood collected with different anticoagulants by centrifugation, quantified their protein content and, through nanoparticle tracking analysis, characterized their size and concentration

Results: We observed that while anticoagulants did not interfere with the parameters analyzed, the occurrence of Chagas disease had an impact on the mean size and size dispersion. Altogether, our protocol is adequate for the isolation of circulating extracellular vesicles and an important basis for further studies on biomarker research.

Summary/Conclusion: Considering that monitoring of chronic Chagas disease cardiac burden and prognosis can only be done after the onset of symptoms and that just one study has been performed to date to assess circulating EVs in patients with chronic Chagas disease, but in this case concerning one patient with reactivation after a heart transplant, our study proposed to develop a rapid and reliable isolation and characterization method for chronic Chagas patients’ circulating EVs

PS05.01. Structural and mechanical characterization of the extracellular vesicles from two protozoan microorganisms using atomic force microscopy and force spectroscopy

Lissette Retana Moreira, Departamento de Parasitología, Facultad de Microbiología, Universidad de Costa Rica

Fátima Linares, Centro de Instrumentación Científica, Universidad de Granada, Spain

Antonio Osuna, 1. Instituto de Biotecnología. Universidad de Granada, Spain

Elizabeth Abrahams Sandí, Departamento de Parasitología, Facultad de Microbiología, Universidad de Costa Rica

Introduction: The use of atomic force microscopy (AFM) in the analysis of biological samples has been growing. AFM is a versatile technology employed to study samples at nanoscale. In AFM, a cantilever with a very sharp tip is employed to scan over the sample. Both attractive and repulsive forces between the surface and the tip make the cantilever deflect and these deflections are detected using a laser beam. In this way, changes in the direction of the reflected beam are tracked with a position‐sensitive photodiode. In biology, most of the studies using AFM have focused in imaging and characterizing the structure of DNA and proteins, the ultrastructure of organelles and the dynamics of the cell membranes, among others (Schafer etal., 2002; Heinisch etal., 2012). Regarding the extracellular vesicles (EVs), AFM has been employed during the last decade and most of the research has focused in performing structural and mechanical characterizations of exosomes isolated from different sources such as saliva (Sharma etal., 2010) and malignant (metastatic and non‐metastatic) cells (Xiao etal., 2013; Whitehead etal., 2015), and to compare how different isolation methods affect the surface structure and size of exosomes (Woo etal., 2016). In the specific area of parasitology, only few groups have employed this technology.

Methods: In this work, we employed AFM for imaging the EVs (mostly exosomes) secreted by epimastigotes and trypomastigotes of the protozoan parasite Trypanosoma cruzi, as well as from trophozoites of the free living amoeba Acanthamoeba sp. incubated at different temperatures. Besides topographic images, we employed force spectroscopy to determine the mechanical properties (stiffness, adhesion, Young modulus) of the EVs secreted from each organism and to compare the results between the stages or the incubation conditions. The analyses were performed using a Park Systems NX‐20 instrument (CIC, Universidad de Granada).

Results: The results obtained reflect significant differences in the nanomechanical properties of the EVs of trypomastigotes and epimastigotes of T. cruzi, which could be related to the protein cargo assessed by proteomics. In the case of Acanthamoeba sp., drastic differences in adhesion were observed in EVs isolated at 28°C and 37°C. These differences could have implications in the survival and damaging potential of these protozoan microorganisms in different biological environments.

Summary/Conclusion: Atomic force microscopy is a technology that could be employed to assess possible differences between extracellular vesicles secreted by different stages or different inubation conditions of protozoan microorganisms.

PS05.03. Charge optimization in electrokinetic sensor for highly sensitive surface protein profiling of cancer cell derived extracellular vesicles

Siddharth S. Sahu, Department of Electrical Engineering, The Angstrom Laboratory, Uppsala University, Uppsala, Sweden

Petra Hååg, Department of Oncology/Pathology, Karolinska Institutet, Stockholm, Sweden

Amelie E. Karlström, Department of Protein Science, KTH Royal Institute of Technology, Stockholm, Sweden

Kristina Viktorsson, Department of Protein Science, KTH Royal Institute of Technology, Stockholm, Sweden

Rolf Lewensohn, Department of Oncology/Pathology, Karolinska Institutet, Stockholm, Sweden

Jan Linnros, Department of Applied Physics, KTH Royal Institute of Technology, Stockholm, Sweden

Apurba Dev, Department of Electrical Engineering, Uppsala University, Uppsala, Sweden

Introduction: Extracellular vesicles, including small extra cellular vesicles (sEVs)/exosomes play a vital role in inter and intracellular communication of tumors and have attracted a lot of interest as sources of biomarkers for cancer diagnostics and treatment monitoring. We have already shown that surface protein profiling of tumor cell derived sEVs is possible using streaming current method. However, a better understanding of the role of surface charge in electrokinetic biosensing through both theoretical and experimental means has opened up the scope of enhancing the signal from sEVs detection. Alternative surface functionalization strategies can be explored for this purpose.

Methods: EVs were isolated from conditioned cell culture media of the non‐small cell lung cancer (NSCLC) cells H1975 by size exclusion chromatography. They were profiled for size/amount by nanoparticle tracking analysis. Evaluation of EV markers and purity was performed by western blotting. sEVs from both untreated and Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (EGFR‐TKI) treated cells were used. The charge contrast between the negatively charged sEVs and the sensor surface was increased by coating the sensor surface with positively charged biotinylated copolymers of poly‐L‐lysine and polyethylene glycol. Further charge tuning was achieved by the choice of linkers (avidin/streptavidin), followed by immobilization of biotinylated antibodies/affinity capture probes against CD9, CD63, and EGFR. This method was then used for assessing change in the expression levels of the sEV surface markers prior and post EGFR‐TKI treatments.

Results: When streptavidin and avidin were used as the linkers, the surface zeta potential was ‐10.6 and ‐2.5 mV respectively. These are significantly less negative than the previously reported value of ‐32.5 mV, obtained by a silanisation based functionalization approach with glutaraldehyde linker. With the new method, the net signals against CD9 detection were 7.7 and 23.1 mV for streptavidin and avidin linkers respectively, marking 3‐fold and 10‐fold improvement against the previously reported value for the sEV concentration 3.5e9 particles/mL. This is clearly a result of the surface charge getting less negative before capturing negatively charged sEVs, and is consistent with theoretical predictions. The sEVs surface expression studies of CD9, CD63 and EGFR in samples from NSCLC cells prior and post EGFR‐TKI treatments ongoing.

Summary/Conclusion: Our results demonstrate the possibility to enhance the signal by tuning the surface charge, thus expanding the scope of surface protein profiling of cancer cell derived sEVs by electrokinetic sensing.

PS05.04. Phenol‐free extraction workflow for analysis of urinary exosomal RNA and detection of TMPRSS2:ERG in prostate cancer

Anne Bickel, Exosome Diagnostics GmbH

Anja Reichert, Exosome Diagnostics GmbH

Nike Bahlmann, QIAGEN GmbH

Georg Stoll, Exosome Diagnostics GmbH

Lisa Meyer, MSc, Exosome Diagnostics GmbH

Kurt Franzen, Exosome Diagnostics, Inc.

JAMES HURLEY, EXOSOME DIAGNOSTICS

Markus Sprenger‐Haussels, QIAGEN GmbH

Martin Schlumpberger, QIAGEN GmbH

Johan Skog, Ph.D.,Exosome Diagnostics, Inc.

Mikkel Noerholm, Exosome Diagnostics GmbH

Daniel EnderleExosome Diagnostics GmbH

Introduction: Exosomes and other extracellular vesicles (EVs) are a valuable source of RNAs from biofluids. EV messenger RNAs (mRNA) include known biomarkers that are used in tissue biopsies for oncological diseases, e.g. TMPRSS2:ERG fusion transcripts found in prostate cancer. To facilitate clinical research, reliable isolation methods for EV‐derived RNA (exoRNA) are needed that do not contain harmful chemicals like phenol or chloroform. This is especially challenging for urine samples due to the significant variability in the major constituents of this biofluid, many of which are potent inhibitors of RT‐qPCR.

Methods: We developed an improved membrane affinity‐based workflow for simple bind‐wash‐elute isolation that does not require phenol/chloroform to extract exoRNA from up to 20 mL Urine. We used this workflow to extract exoRNA from urine samples from healthy donors and confirmed prostate cancer subjects scheduled for radical prostatectomy. RT‐qPCR was used to quantify housekeeping mRNAs and prostate specific biomarkers TMPRSS2:ERG fusion (T2:E) and KLK3 (Prostate Specific Antigen mRNA, PSA).

Results: Urinary mRNAs are detected from 2–20 mL of input volume with RT‐qPCR signals scaling with input volume and depletion of mRNA signals in the remaining flow‐through. Repeated extractions on the same urine demonstrated robust performance of isolation and inhibitor removal from the sample. In addition to mRNAs, exosomal miRNAs can be analyzed in the isolates. TMPRSS2:ERG and KLK3 biomarkers were detected in urine from a cohort of subjects with high‐grade prostate cancer, consistent with observations in tissue previously reported in literature.

Summary/Conclusion: We present a new workflow for extraction of urinary RNAs that is suitable for application in routine laboratory use.

PS05.05. NanoBioAnalytical platform for the characterization of Canine Mesenchymal Stromal Cells extracellular vesicles

geetika Raizada, FEMTO‐ST Institute

Rodolphe Rakic, Vetbiobank

Céline Elie‐Caille, Institute FEMTO‐ST

Nathalie Saulnier, Vetbiobank

Wilfrid Boireau, Institute FEMTO‐ST

Introduction: Mesenchymal stromal cells (MSCs) are of clinical interest because of their validated safety profile and their tremendous biological properties. These cells also secrete a large set of paracrine factors that support the healing process mainly through their interaction with the host immune cells. Canine MSCs have been used successfully for veterinary application and represent a valuable preclinical model for naturally‐occuring diseases. The aim of this study is to characterize, through an innovative analytical platform, the EVs populations produced by canine neonatal MSCs, possibly their subset and evaluate their properties.

Technically challenging task of measuring the concentration, size and characterization of a diverse population of EVs has led researchers to explore several technologies as standalone or in combination. In this study, we have utilized the Tunable Resistive Pulse Sensing (TRPS) to obtain EVs concentration and size distribution and the NanoBioAnalytical (NBA) platform especially developed in our group (Obeid etal. 2017 and 2019) to characterize EVs from different samples of canine‐MSC conditioned media (CM).

Methods: NBA platform utilizes Surface Plasmon Resonance imaging (SPRi) for real‐time detection and characterization of EVs according to their phenotype; which were then further deeply characterized by Atomic Force Microscopy (AFM) to precise size distribution and distinguish captured EVs from other biological materials (like protein aggregates).

Results: Captured EVs from different MSC‐CM were found to be CD44+, CD81+, CD9+ and CD90+ while presenting different phenotype profiles. Moreover, based on AFM investigation of immunocaptured EVs, we could refine SPRi signals obtained, as example the anti‐CD81 immunospot was mainly due to a small‐EVs population whereas the presence of EVs, cell debris and filament like structures are mainly characterized on anti‐CD44 spots. Current progress aims to compare results from the multiplexed SPR/AFM study and in vitro functionality of various EV subsets to distinguish the most promising culture conditions for EVs bioproduction and their future use for therapeutic treatments.

Summary/Conclusion: For the first time, our NBA platform serves to establish, optimize and qualify the production line of EVs for clinical purposes. We would address in the near future genomic and proteomic analysis directly on the immunocaptured EVs that will help us in establishing a comprehensive correlation between the functional activity and the components of the conditioned media.

PS05.06. Comparison of MSC‐EV populations from different MSC sources using MSCSPlex, ExoView and Nanoimager instruments

Renata Skovronova, University of Turin

Cristina Grange, Department of Medical Sciences, University of Turin

Benedetta Bussolati, University of Turin, Department of Molecular Biotechnology and Health Sciences, Italy

Introduction: Mesenchymal stromal cells and their extracellular vesicles (MSCs‐EVs) have been in the centre of regenerative research. The current field of EV‐stem cell therapy focuses on the small‐EVs fraction of EVs, as they have been implicated to ameliorate tissue injury. This project aims to characterize trough different techniques and compare the different fraction of MSC‐EVs (small, large EVs and apoptotic bodies) from different MSC sources (bone marrow, adipose tissue, and umbilical cord).

Methods: MSCs are cultured until 80% confluency at 37°C in Alpha MEM medium with 10% of serum. To collect apoptotic EVs, apoptosis is induced using 500ng/mL Anti‐Fas antibody. Medium from overnight starved cells is collected then centrifuged at 1500g for 15mins to pellet apoptotic bodies. The supernatant is centrifuged further at 10,000g for 1h, to pellet large‐size EVs, another ultracentrifugation step of 100,000g for 1h is applied to collect small‐size EVs. EVs are pooled and kept at ‐80°C in medium with 0.1%DMSO. Nanosight is performed to analyse the concentration and size of EVs. dSTORM analysis using super‐resolution microscopy (NanoImager) is used to detect single vesicles and their markers. ExoView multiplex analysis is used to detect the size and marker expression. MACSPlex, a semi‐quantitative kit aimed to assess EV surface markers is used to test each fraction of the MSC‐EVs.

Results: The techniques used showed the EVs to be in their expected size ranges. The presence of tetraspanin (CD63, CD81, CD9) and mesenchymal (CD105, CD49e, CD44, CD29, CD146) markers were confirmed. In comparison with all fractions, umbilical cord‐derived small‐EVs showed a higher expression of CD63 and CD105. All large size EVs showed a higher expression of CD40 whereas the small size EVs of Annexin A1. Super resolution microscopy and multiplex analysis further allowed us to determine the EV particle count and size. Interestingly, overlapping results were obtained using different instruments.

Summary/Conclusion: Full and detailed characterization of EV sources and types are required for safe and effective application of stem cells and/or their bioproducts in clinical application. Specific, but often superimposable results, can be obtained using different EV‐dedicated instruments.

PS05.07. Power‐Law Characterization of EV Size Distributions

Michael Paulaitis, Johns Hopkin University School of Medicine

Olesia Gololobova, Johns Hopkins University School of Medicine

Kenneth W. Witwer, Johns Hopkins University School of Medicine

Introduction: A power‐law model is described for characterizing EV size distributions. The scaling exponent in this model captures the asymmetry of these size distributions, which are notably right‐skewed to larger vesicles, independent of the minimum detectable vesicle diameter.

Methods: This model is applied to analyze the power‐law behavior of EV size distributions measured by nanoparticle tracking analysis (NTA), microfluidic resistive pulse sensing (MRPS), nanoflow cytometry (nanoFCM), and single‐particle interferometric reflectance imaging sensing (SP‐IRIS). EVs from the human T lymphocyte line H9, the promonocytic line U937, and released apically and basally from polarized retinal pigmented epithelial cells were separated from culture media by differential ultracentrifugation and size exclusion chromatography.

Results: We show that the scaling exponent derived from model fits of the measured EV size distribution is sensitive to the sizing platform, the cell source, and treatment conditions, and insensitive to the minimum detectable vesicle size intrinsic to the different detection methods.

Summary/Conclusion: Our results establish the scaling exponent as a quantitative biophysical parameter for characterizing EV populations. In that the power‐law behavior of EV size distributions reflects changes in the membrane composition of the EVs, we can consider the measured scaling exponent to be a biophysical EV marker comparable and complementary to biochemical EV markers, such as the tetraspanins.

PS05.08. Novel approach for quantitative real‐time nanoparticle analysis of extracellular vesicles

Marie Berger, Myrade lab

Quentin Sabbagh, INSERM, CNRS, Université de Nantes

Mathilde Richard, CRCINA INSERM U1232

Gwennan ANDRE‐GREGOIRE, CRCINA UMR1232 and Integrated Center for Cancerology (ICO)

Laetitia Guevel, INSERM, CNRS, Université de Nantes

Julie Gavard, INSERM, CNRS, Université de Nantes, Institut de Cancérologie de l'Ouest

Introduction: EVs are abundant and stable in body fluids (plasma, urine, breast milk…), they emerged as promising biomarkers for the assessment of health status, but also responses to treatments and outcomes in pathological conditions. As nanosized objects, research on EV requires advanced technologies and specialized expertise to assess both their specificity and sensitivity as biomarkers, as well as their efficacy and safety as therapeutic tools. This context calls for new technological improvement for measuring easily and quickly EVs concentration and size. A new method for quick and reliable EV quantification, compatible with the use of multiple larger patient cohorts and with limited volume of liquid biopsies was developped by Myriade. Based on Interferometric Light Microscopy (ILM), this method lies on its simplicity.

Methods: We compare the quantification of EVs (recently called Vesiclemia), by ILM and the TRPS, a well‐established method for the characterization of EVs separated from serum and biological fluids. ILM was also used on a stability study at different temperature of EVs.

Results: EVs were separated from 10 plasma and urine samples using size exclusion chromatography (SEC) and analyzed upon their separation, without any step of storage. Vesiclemia was estimated either by ILM and TRPS. ILM and TRPS display a linear correlation regarding EV concentration (correlation coefficient R2 = 0.95). The stability study shows EVs should be preferentially stored and quantified by Videodrop after freezing at ‐80°C.

Summary/Conclusion: The correlation between Videodrop analysis and the reference method TRPS appeared to be robust, with high R² values.

Videodrop is particularly adapted fulfill the pre‐requisite for characterization of EVs as non‐invasive biomarkers. This fast, real‐time titration method consuming low volumes of product turns out to be suitable for EV quantification.

PS05.09. A semi‐quantitative assay for extracellular vesicles in vivo correlates EV overproduction with developmental phenotypes

Lauren Pitts, University of Denver

Katharina Beer, University of Würzburg

Gholamreza Fazeli, University of Würzburg

Julia Frondoni, University of Denver

Ann M. Wehman, University of Denver

Introduction: To clarify the roles of extracellular vesicles (EV) in vivo, it is important to visualize EVs. However, most EV reporters are also present in the releasing cell, making it challenging to visualize and count EVs in vivo.

Methods: To tackle these problems, we developed a technique to remove background fluorescence from the source cell. We use degradation motifs called degrons to target proteins for ubiquitination and degradation in the cytosol, while leaving EVs labelled. We use different degrons in the nematode model organism Caenorhabditis elegans and specifically label MVs by degron‐tagging the PI4,5P2‐binding PH domain of the cytosolic phospholipase PLC1∂1, which is primarily found at the plasma membrane.

Results: Using degron‐tagged reporters, we can visualize EVs released after fertilization as they are often trapped between different layers of the eggshell. By counting these puncta, we have developed a semi‐quantitative assay for EV release and have begun to characterize different perturbations to see their effect on EV number. For example, we have been studying the role of a lipid flippase TAT‐5 and its activating protein PAD‐1 in inhibiting EV release. Different alleles of tat‐5 and pad‐1, including FP‐knock‐ins, result in different numbers of EVs released. Intriguingly, the EV numbers correlate with cellular and developmental phenotypes such as phagocytic capacity and viability. The higher the EV numbers, the fewer endogenous cargos are successfully phagocytosed and the more likely the embryos are lethal.

Summary/Conclusion: Our results indicate the importance of tightly regulating the EV biogenesis machinery to avoid the deleterious effects of EV overproduction. The semi‐quantitative assay also reveals intermediate alleles, which will be useful genetic tools for enhancer/suppressor screens to identify both positive and negative regulators of EV biogenesis.

PS05.10. Simple density‐based method for enrichment of plasma‐EVs and its use in biomarker discovery for thrombotic antiphospholipid syndrome

Marija Holcar, Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, EU

Ula Štok, Department of Rheumatology, University Medical Centre Ljubljana, Ljubljana, Slovenia, EU

Jana Ferdin, Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, EU

Simona Sitar, Department of Polymer Chemistry and Technology, National Institute of Chemistry, Ljubljana, Slovenia, EU

Magda Tušek‐Žnidarič, Department of Biotechnology and System Biology, National Institute of Biology, Ljubljana, Slovenia, EU

Ana Plemenitaš, Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, EU

Ema Žagar, Department of Polymer Chemistry and Technology, National Institute of Chemistry, Ljubljana, Slovenia, EU

Vita Dolžan, Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, EU

Snežna Sodin‐Šemrl, Faculty of Mathematics, Natural Sciences and Information Technologies, University of Primorska, Koper, Slovenia, EU

Saša Čučnik,Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia, EU

Polona Žigon, Department of Rheumatology, University Medical Centre Ljubljana, Ljubljana, Slovenia, EU

Metka LenassiInstitute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, EU

Introduction: Extracellular vesicles (EVs) are promising minimally invasive biomarkers of various pathologies, but their isolation from blood is hindered by other nanoparticles (lipoproteins, proteins/protein aggregates, viruses) present in the blood. Here, we established a simple but reliable method for effective enrichment of EVs from human plasma and applied it prior to characterizing size and concentration of plasma‐EVs as biomarkers of thrombotic antiphospholipid syndrome (APS).

Methods: EVs were first enriched from plasma of 10 healthy subjects, using density‐based (ultracentrifugation on 20% sucrose cushion; sUC) method. Size, concentration and purity of sUC‐enriched EVs were evaluated with NTA, AF4‐MALS, TEM, ApoA1 and ApoB100 ELISA, qPCR of EV‐miRNA, and later compared to EVs enriched with size exclusion chromatography (SEC). For APS biomarker discovery, size and concentration of sUC‐enriched plasma‐EVs from 14 thrombotic APS patients, 5 aPL negative patients with idiopathic thrombosis (aPL‐ IT) and new 7 healthy subjects (HS) were determined using NTA. All subjects provided informed consent, NMEC approved the study.

Results: NTA detected 3.11*109 particles/mL of plasma (mode size 109 nm) and AF4‐MALS 0.66*109 particles/mL of plasma (2*Rgeom 195 nm) in samples after EV‐enrichment with sUC. The method was highly repeatable (NTARSD = 5.4%, AF4 MALSRSD = 2.1%) and resulted in more pure EVs with significantly fewer lipoprotein contaminants and more miRNA cargo compared to SEC‐enriched EVs. In the APS biomarker study, NTA showed similar EV sizes (110"170 nm) between the groups, but higher concentrations indicated enhanced shedding of EVs in patients with APS and aPL‐ IT compared to HS (p = 0.021 and p = 0.007, respectively).

Summary/Conclusion: The sUC method results in satisfactory purity of enriched EVs for use in biomarker studies. Quantification of plasma‐EVs enriched by sUC showed potential for future investigation of biomarkers in thrombotic APS.

PS05.11. Rapid Separation and Detection of Exosomes based on Metal Organic Framework‐Aptamers

Bo Li, Department of Laboratory Medicine, NanfangHospital, Southern Medical University

Feng Jun Jie, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University

Zheng Lei, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University

Introduction: To construct an integrated platform for the separation and detection of specific exosomes subpopulations through a new method of metal organic framework (MOF)‐aptamers and fluorescence signal amplification and detection.

Methods: ZrOCl2·8H2O and organic ligand 2‐aminoterephthalic acid (NH2‐BDC) were used as raw materials, and kept at 120°C for 12 hours to synthesize MOF material UiO‐66‐NH2. A functionalized specific nucleic acid aptamer (PO34–Spaser‐PD‐L1‐Aptamer) PSPA was modified on the surface of UiO‐66‐NH2, and the modified MOF@PSPA platform was used to isolate and enrich the breast cancer cell line MDA‐MB‐ 231 expresses a specific exosomal subset of PD‐L1 membrane protein. Then, by lipid probe identification and rapid nucleic acid isothermal detection (RNAID), the captured specific exosomes subpopulations are amplified and detected by fluorescence signals.

Results: The established MOF@PSPA@RNAID integrated platform for exosomes separation and detection successfully separated and detected exosomes secreted by breast cancer cell lines. The detection was nonlinear, Y = 75.61*X^0.163, R2 = 0.984, The detection range reaches 4.5‐900 (105particles/μL)

Summary/Conclusion: MOF@PSPA based on synthetic modification can realize rapid separation and enrichment of specific exosomes subgroups. Combined with the rapid nucleic acid isothermal detection method, the difference in the number of separated exosomes subpopulations can be converted into the difference in fluorescence signal intensity. The new integrated method for separation and detection of exosomes based on the MOF@PSPA platform can be used for rapid separation and detection of specific exosomes subgroups of tumor cells.

PS05.12. Quantification and size distribution of extracellular vesicles: a comparative study of available tools

Romain Sausset, INRAe, UMR1319, Micalis, domaine de Vilvert, Jouy en Josas, France

Eric Guédon, INRAe, Institut Agro, STLO, Rennes, France

Zuzana Krupova, Excilone, Departement R&D, 6 rue Blaise Pascal, Parc Euclide, Bat. A, 78990 Elancourt, France

Marie‐Agnès Petit, INRA (National Institute for Agronomy), France

Marianne De Paepe, INRAe, UMR1319, Micalis, domaine de Vilvert, Jouy en Josas, France

Introduction: It is now largely accepted that the intestinal microbiota has a key role in Intestinal Bowel Diseases (IBD). An imbalance in the composition and diversity of the intestinal microbiota (i.e. dysbiosis) of patients has been repeatedly pointed out by several teams. Production of extracellular vesicles (EVs), either by the microbiota or by intestinal epithelial cells, depends on the environment. Because of the stress due to chronic inflammation during IBD, the quantity of EVs could be more numerous in stools from patients. In order to address this question in large cohorts, we needed a quick and reliable tool to quantify extracellular vesicles.

Methods: Interferometric Light Microscopy (ILM), a new way to quantify nanoparticles that relies on the creation of single beam interferences between two signals from the same light path by nanoparticles such as small vesicles, was compared to Nanoparticle Tracking Analysis (NTA). For this, extracellular vesicles were purified from the stools of conventional and axenic mice and rats; and humans.

Results: We show that the use of ILM leads to quantification and size profiling similar to those given by NTA with all samples. In addition, ILM results were obtained with a significant time gain relative to NTA, facilitating large cohorts analyses.

Summary/Conclusion: ILM is particularly adapted for EV characterization, since it is a quick and handy method, consuming low volumes of product.

PS05.13. Detection and treatment monitoring of small extracellular vesicle surface proteins in liquid biopsies of lung cancer patients by a multiplexed electrokinetic sensor

Sara Cavallaro, KTH Royal Institute of Technology

Petra Hååg, Department of Oncology/Pathology, Karolinska Institutet, Stockholm, Sweden

Kristina Viktorsson, Department of Protein Science, KTH Royal Institute of Technology, Stockholm, Sweden

Rolf Lewensohn, Department of Oncology/Pathology, Karolinska Institutet, Stockholm, Sweden

Jan Linnros, Department of Applied Physics, KTH Royal Institute of Technology, Stockholm, Sweden

Apurba Dev, Department of Electrical Engineering, Uppsala University, Uppsala, Sweden

Introduction: Small extracellular vesicles (sEVs) have attracted interest as a source of biomarker for cancer diagnostics and monitoring based on liquid biopsies, as they are secreted in different body fluids and their contents (proteins, RNAs, etc.) in part reflect their parent cells. While for diagnostics it is fundamental to study the genomic alterations driving a tumor, e.g. mutated EGFR for Non‐small cell lung cancer (NSCLC), multiple pathways may co‐exist in a tumor and hence contribute to treatment response. Therefore, it is also highly relevant to monitor the oncogenic markers that are responsible for these pathways. Recently, PD‐L1 on tumors has gained increasing attention, as it is related to the capacity of the immune system to attack the tumor and offers a way to therapy. It has also been suggested that PD‐L1 is expressed in sEVs and that Tyrosine Kinase Inhibitors (TKIs) against mutated EGFR‐ or EML4‐ALK, two clinically used treatments for NSCLC, may influence PD‐L1 expression in NSCLC.

Methods: Herein, we use our multiplexed electrokinetic platform for label‐free detection and treatment monitoring of sEV surface proteins in liquid biopsies of NSCLC patients. The technique relies on the electrokinetic phenomena of streaming current and zeta potential (z*) and measures the z* change upon sEV binding on functionalized microcapillary surfaces. The current platform can measure 3–4 channels simultaneously, but can be further extended. For the analysis, we used sEVs derived from the pleural effusions (PEs) of NSCLC patients with different genetic aberrations (EGFR, EML4‐ALK or KRAS). The vesicles were isolated by size exclusion chromatography, verified to be of sEV size by NTA and confirmed by western blot to express CD9, TSG101 but not calnexin.

Results: We already demonstrated that our electrokinetic sensor successfully detects sEVs, profiling sEV surface proteins up to a change of 3% in their expression levels. Moreover, the platform has been improved in order to measure multiple capillaries simultaneously. Here, we apply the multiplexed platform to detect and compare relevant tumor markers, e.g. EGFR, PD‐L1 in sEVs from PEs of NSCLC patients. Interpatient differences were evident for all markers. In ongoing studies, we are analyzing if we can monitor changes in the expression levels of these markers at different treatment stages, as well as studying the influence of TKI‐treatment on sEV PD‐L1 expression. This data will be presented.

Summary/Conclusion: The sensor results show successful monitoring of sEVs in liquid biopsies of NSCLC patients. With further development, the platform may be used for monitoring sEV alterations during treatments.

PS05.14. Introducing the New ExoView Flex technology: Enabling easy, fast and robust characterization of extracellular vesicles using any antibody on the ExoView platform

Aditya Dhande, NanoView Biosciences

Dennis Zimmermann, NanoView Biosciences

George Daaboul, MDPhD, NanoView Biosciences

Introduction: The ExoView chip allows the characterization of single extracellular vesicles (EVs) using a combination of interferometric sizing and fluorescence imaging. Following incubation of the sample on the chip, EVs are captured on an array of target‐specific tetraspanin antibodies that are covalently linked to the chip. Once captured, these EVs can then be immunofluorescence (IF)‐stained with up to three additional probes. For users seeking to capture EVs with probes other than tetraspanins, NanoView Biosciences can provide custom chips, however this approach comes with longer lead times and higher costs.

Methods: Here we present NanoView Biosciences’ new ExoView Flex technology enabling one to design a single EV custom assay at the bench, allowing specific capture and detection of EVs using antibodies of choice. The assay proves to be extremely time‐efficient and the ExoView Flex Chip can be ready for sample incubation in about two hours.

Results: This new technology adds flexibility and has also been proven to yield highly robust and reproducible results (CV 4%). Experiments performed demonstrate a range of different EV concentrations and show a linear range of 2.5 logs. Furthermore, the ExoView Flex technology enables users to characterize EVs without purification even when challenged with matrices like biofluids and can also be used to characterize internal cargo.

Summary/Conclusion: This new multiplexed ExoView Flex technology allows sensitive detection of rare events in biomarker discovery while at the same time enables screening of antibody and biomarker candidates. The ExoView Flex technology is aligned with any of the core capabilities of the ExoView technology, such as purification‐free single‐particle detection, counting, phenotyping, sizing and biomarker colocalization of individual EV

and their biomarkers.

PS06.01. Flow Cytometric Strategies for Reproducible Extracellular Vesicles Quantitation and Phenotyping

Gabriele De Rubis, Laboratory of Cancer Cell Biology and Therapeutics, Discipline of Pharmacy, Graduate School of Health, The University of Technology Sydney, Australia

Michael Wallach, Laboratory of Cancer Cell Biology and Therapeutics, Discipline of Pharmacy, Graduate School of Health, The University of Technology Sydney, Australia

Mary Bebawy, MD PhD, Laboratory of Cancer Cell Biology and Therapeutics, Discipline of Pharmacy, Graduate School of Health, The University of Technology Sydney, Australia

Introduction: Extracellular vesicles (EVs) are mediators of cell‐to‐cell communication in many pathological conditions including cancer. Their ubiquitous presence in biofluids makes them a promising source of systemic biomarkers in the field of liquid biopsies. Among the technologies employed in EVs analysis, flow cytometry (FCM) allows rapid, multiparametric characterization of EVs at single particle resolution. However, its clinical application is still hampered by limited cross‐platform reproducibility, mainly caused by the small size of EVs and by lack of standardization. Here, we describe a reproducible and sensitive EV detection and phenotyping protocol across two commercial flow cytometers designed for EVs analysis.

Methods: Scatter resolution, enumeration accuracy and precision of the instruments were determined by analysing submicron silica reference beads (ApogeeMix). Large EVs were enriched from the conditioned supernatant of two cancer cell lines by high‐speed centrifugation (18,900g) and characterized using electron microscopy and dynamic light scattering. EVs were stained with AnnexinV for phosphatidylserine (PS) exposure and detected by FCM, using a scatter‐based triggering strategy, using two complementary size gates: a “Latex” gate (300 to 1100 nm polystyrene beads) and a “Silica” gate (180 to 1300 nm silica beads). FCMPass software was used to estimate the size of the EVs detected by these size gates. Validation of vesicular constituents was performed, and serial dilutions analysed to assess swarm detection.

Results: We observed compatible scatter resolution, enumeration accuracy (error ≤15%) and precision (CV ≤10%) across both flow cytometers. Similar estimated EV sizes are detected by the two instruments in both size gates of interest according to FCMPass software modelling. We obtained linear and cross‐platform reproducible detection of PS+ EVs, with inter‐instrument CV≤20% for the “Latex” gate and ≤10% for the “Silica” gate, across an EV range of 0.125 ‐ 2 μg/mL. This corresponds to 10–800 PS+ EVs/μL in the “Latex” gate and 500–10,000 PS+ EVs/μL in the “Silica” gate. Larger EVs amounts resulted in loss of linearity in the “Silica” gate in one of the two instruments, indicating interference of swarm detection for EVs amounts >2 μg/mL.

Summary/Conclusion: Our results show the cross‐platform reproducible FCM analysis of large EVs using a scatter‐based triggering approach. This work provides the basis for the development of robust protocols for clinically viable EV‐based liquid biopsy tests.

PS06.02. Nanoscale imaging and analysis of cerebrospinal fluid derived single EVs

ShivaniSharma, PhD, University of California Los Angeles

Introduction: EVs circulating in cerebrospinal fluid (CSF) offers unique ‘nanoscale windows’ into brain tumors, but the low abundance and nanometer‐scale dimensions of EVs pose challenges. Uncertainties exist with due to different isolation techniques employed, as well as the rigor and reliability of EV characterization. Also, there is a need to pool individual CSF for downstream bio‐molecular analysis. Besides evaluating the downstream proteomic and genomic cargoes of CSF‐derived EVs, information on quantitative, high resolution, structural‐mechanical properties of CSF EV isolates, and the impact of isolation techniques is scarce.

Methods: Using minimal (less than 100 micro‐liter) volumes of CSF, we successfully compared EVs isolated via different isolation methods within the same patient samples, in replicates using atomic force microscopy.

Results: Our results provide new biophysical insights into the effects of isolation techniques on single EVs reveal a combination of size exclusion and precipitation as the most optimal method for label‐free isolation of unperturbed EV particles from limited CSF samples, compared to either method alone.

Summary/Conclusion: Our study on nanoscale EV structural‐mechanical analysis in glioblastoma and other CSF samples highlights the novel potential implications for AFM technology in improved isolation, quantification, and single vesicle characterization of CSF EVs for glioblastoma (and other brain associated) biomarkers.

PS06.03. Quantitative multi‐parameter analysis of individual fecal extracellular vesicle via a laboratory‐built nano‐flow cytometer

HaishengLiu, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, China

Yongyu Chen, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, China

Yuhang Qin, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, China

Xiaomei Yan, PhD, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University

Introduction: Fecal extracellular vesicles (fEVs) has been implicated in physiological processes in various diseases and host immune response. To further explore their prominent biological potential, an in‐depth study of fEVs at the single‐particle level is important. Employing a laboratory‐built nano‐flow cytometer (nFCM) that facilitates multiparameter analysis of single EVs as small as 40 nm, here we report quantitative measurement of size distribution, purity, nucleic acids and surface markers of fEVs.

Methods: fEVs were isolated from stool samples collected from healthy donors via iodixanol gradient ultracentrifugation. Six fractions of 2 mL each were collected from the top of the tube (F1 " F6). TEM was used to characterize the morphology of fEVs. Monodisperse silica nanoparticles were used as the size reference standards for the size distribution measurement of fEVs via light scattering detection. The purity of fEVs was examined by measuring the particle concentration before and after Triton X‐100 treatment. Subpopulation of fEVs expressing specific surface markers, such as CD9, CD63, CD81, CD24, lipopolysaccharide (LPS) and lipoteichoic acid (LTA) were analyzed via immunofluorescent staining. SYTO 16, a cell‐permeant stain, was used to stain the DNA of fEVs before and after DNase I treatment.

Results: The purity of isolated F1 ‐ F6 fEVs via density gradient UC was ranging from 55.1% ‐ 93.2%. We found that there was almost no expression of CD9, CD63, CD81 and CD24 for fEVs. We also found that ∼20% of fEVs expressing LPS (the marker of EVs from gram‐negative bacteria) or LTA (the marker of EVs from gram‐positive bacteria). The ratio of fEVs that can be fluorescently stained by SYTO 16 had no obvious change after DNase I treatment, suggesting that all the EV‐DNA of fEVs resides in the lumen of EVs.

Summary/Conclusion: The laboratory‐built nFCM is applicable to the multiparameter biochemical analysis of individual fEV via protein and nucleic acid staining. We expect nFCM will facilitate more in‐depth studies of fEVs.

PS06.04. Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques

Marvin Droste, Department of Pediatrics II (Pediatric Nephrology), University Hospital Essen, University of Duisburg‐Essen, Essen, Germany

Tobias Tertel, Institute for Transfusion Medicine, University Hospital Essen, Germany

Stefanie Jeruschke, Department of Pediatrics II (Pediatric Nephrology), University Hospital Essen, University of Duisburg‐Essen, Essen, Germany

Robin Dittrich, Institute for Transfusion Medicine, University Hospital Essen, Germany

Evangelia Kontopoulou, Department of Pediatrics III, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany

Bernd Walkenfort, Electron Microscopy Unit, Imaging Center, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany

Verena Börger, Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg Essen, Essen, Germany

Peter F. Hoyer, Department of Pediatrics II (Pediatric Nephrology), University Hospital Essen, University of Duisburg‐Essen, Essen, Germany

Anja K. Büscher, Department of Pediatrics II (Pediatric Nephrology), University Hospital Essen, University of Duisburg‐Essen, Essen, Germany

Basant K. Thakur,Department of Pediatrics III, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany

Bernd Giebel, Prof, Institute for Transfusion Medicine, University Hospital Essen, Germany

Introduction: Urinary small extracellular vesicles (sEVs) are studied as potential biomarkers. They can be enriched with different protocols resulting in significant variance regarding purity and yield. Assuming that the evaluation of EV preparation methods largely depends on the applied analysis tools, obtained samples were analyzed by different techniques, i.e., by imaging flow cytometry (IFCM), conventional nanoparticle tracking analysis (NTA), Western Blot (WB) and transmission electron microscopy (TEM).

Methods: Cell‐free urine was first screened for the presence of CD9+, CD63+ and CD81+ objects by IFCM. Urinary sEVs were then prepared from healthy donor void urine applying five different methods based on combinations of frequently used EV preparation techniques: polyethylene glycol (PEG)‐precipitation + ultracentrifugation (UC), PEG + size exclusion chromatography (SEC), UC + SEC, ultrafiltration (UF) + SEC, or the commercial ExoEasy Maxi kit. We determined the obtained amount of CD9‐labeled sEVs as well as the average particle numbers by conventional NTA. Furthermore, we assessed the intensity of the bands of TSG101 and the contaminant protein uromodulin (UMOD) in WBs. The morphology of the particles was documented by TEM.

Results: Urine contains a prominent population of CD9+ sEVs, but hardly any CD63+ and CD81+ sEVs. The number of recorded CD9‐positive objects detected by IFCM correlated with the TSG101 WB band intensities. In contrast, average particle numbers as determined by conventional NTA correlated with the intensity of UMOD WB bands.

Summary/Conclusion: Overall, our data question the reliability of conventional NTA analyses for identifying the optimal EV preparation method. In our method comparison, the combination of SEC and UF showed the highest CD9+ object and TSG101 protein recovery, and in relation to the number of CD9‐positive objects, the lowest amount of UMOD contamination.

PS06.05. Considerations towards flow cytometric calibration of fluorescent signals from nanoparticles and extracellular vesicles by using MESF‐bead based calibrators

EstefaníaLozano‐Andrés, Utrecht University

Tina Van den Broeck, BD Biosciences, Erembodegem, Belgium

Lili Wang, Biosystems and Biomaterials Division, National Institutes of Standards and Technology (NIST), Gaithersburg, MD 20899

Majid Mehrpouyan, BD Biosciences, 2350 Qume Drive, San Jose, CA 95131

Marca H.M. H.M. Wauben, Department of Biomolecular Health Sciences, Utrecht University, The Netherlands

Ger. J.A. Arkesteijn, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Introduction: low cytometry is a powerful technique to characterize nanoparticles (NP) and Extracellular Vesicles (EV). However, in the majority of reported experiments, arbitrary units are used to indicate fluorescence intensity. This hampers comparison of results from different laboratories and different platforms. We investigated the use of calibrated Molecules of Equivalent Soluble Fluorophores (MESF)‐beads designed for cell‐based flow cytometric analysis for assignment of absolute fluorescence to NP and EV.

Methods: FITC‐MESF and PE‐MESF bead sets of 2 μm and 6 μm were evaluated and used as calibrators on different platforms (BD Influx, CytoFLEX, SORP BD FACSCelesta). 550 nm silica NP with six different FITC fluorescent intensities were used as a synthetic NP sample. EV were isolated from conditioned media of the 4T1 mammary carcinoma cell line (dUC), stained with CFSE and CD9PE followed by density gradient floatation. Various EV‐densities were collected and further analysed. Synthetic NP and stained EV were measured on the BD Influx and their respective fluorescent signals were calibrated in standardized units of FITC‐MESF, CFSE‐ERF (Equivalent Reference Fluorophores) and PE‐MESF.

Results: Fluorescence calibration, using two different sizes, brightly fluorescent calibrators designed for cell‐based flow cytometry, makes inter‐platform comparison possible. However, MESF numbers based on extrapolation into the dim fluorescence range of NP and EV vary depending on the MESF‐bead based set used. These variations ranged from 27.3 to 76.5% when calibrating different FITC fluorescent signals from synthetic NP in FITC‐MESF units and were 78.6% and 156.9% respectively, when calibrating CFSE‐ERF and PE‐MESF signals of CFSE and CD9PE stained biological EV.

Summary/Conclusion: The differences in the slopes of the regression lines between different calibrator bead sets, caused by uncertainties in the assignment of MESF to the calibrators, are exaggerated during extrapolation through linear regression into the dimmer fluorescent area. For proper assignment of low fluorescent sub‐micron particles such as EV, calibrator beads with high accuracy of MESF assignment are required; preferably in the fluorescence range of EV. Furthermore, for robust interpretation and for benchmarking studies the use of the same calibration materials is advised when possible.

PS06.06. Comparison of extracellular vesicle isolation and storage methods using high‐sensitivity flow cytometry

Sarah Deville, Flemish Institute for Technological Research (VITO), Health Unit, Boeretang 200, 2400 Mol, Belgium

Pascale Berckmans, VITO nv

Rebekka Van Hoof, KU Leuven, UHasselt, VITO

Ivo Lambrichts, Hasselt University

Anna Salvati, University of Groningen

Inge Nelissen, VITO nv

Introduction: Extracellular vesicles (EVs) are membrane‐bound carriers with complex cargoes which are released by most biological cells and are of interest for a wide variety of applications, including the early monitoring of diseases, as primary therapeutics and as drug delivery vehicles. Flow cytometry is emerging as a very promising technology for EV characterization due to its high throughput and multiplex fluorescence possibilities. As the use of high‐sensitivity flow cytometry and the availability of fluorescent dyes for labeling of EV subsets are expanding, there is a growing need for standardization efforts to enhance the reproducibility of measurements. A critical aspect which is poorly investigated, is the influence of EV storage conditions on the EV concentration and the stability of EV‐associated fluorescent labels.

Methods: We used EVs from lipopolysaccharide‐stimulated monocytic THP‐1 cells which were obtained by two different EV isolation methods, differential centrifugation and exoEasy membrane affinity spin column purification. The EV fractions were stored at 4°C and ‐80°C for up to one month, and EV concentrations were evaluated at regular time intervals using scatter‐based nanoparticle tracking analysis (NS500) and flow cytometry (BD Influx) after fluorescent EV labeling with CFDA‐SE and PKH67.

Results: Unlabeled THP‐1 cell‐derived EVs remained relatively stable after one month of storage at both 4°C and ‐80°C. When storing CFDA‐SE‐ and PKH67‐labelled EVs, those kept at 4°C lost their fluorescence intensity within one day, while the EVs stored at ‐80°C remained stable over time.

Summary/Conclusion: Good practice in EV sample storage is a major determinant for standardization of fluorescence‐based analysis methods, such as flow cytometry.

PS06.07. Plasmon‐enhanced detection and molecular profiling of single extracellular vesicles

TaehwangSon, MassachussetsGeneral Hospital

Jouha Min, Massachusetts General Hospital

Jae‐Sang Hong, Massachusetts General Hospital

Ralph Weissleder, Massachusetts General Hospital

Hakho Lee, Massachusetts General Hospital

Hyungsoon Im, Massachusetts General Hospital

Introduction: Extracellular vesicle (EV) analyses have shown the potential for molecular cancer diagnosis from non‐invasive liquid biopsies. Considering EVs’ physical and molecular heterogeneity, a sensitive and robust platform with a single EV analysis capability is still needed to further explore EVs’ potential as biomarkers and accelerate their clinical adaption. However, multiplexed single EV analysis is technically challenging due to EVs’ small sizes and weak optical signals. Here, we report a nanoplasmonic platform based on plasmon‐enhanced fluorescence (PEF) detection for single EV analyses.

Methods: Periodic Au nanohole arrays were employed for PEF detection where excitation/emission wavelengths of fluorophore overlap with plasmon resonance wavelength. EVs are captured on the nanohole surface and labeled by fluorescently labeled antibodies. We used a conventional fluorescence microscope for single EV imaging and detected both surface and intravesicular markers. We identified EVs by signals of a tetraspanin combination (CD9, CD63, and CD81), and those from glioblastoma cancer cells by signals for EGFR. Then we detected EGFRvIII mutation proteins from the cancer‐derived EV population. We reported the fraction for target markers among colocalized spots positive to EGFR and the tetraspanin combination.

Results: The PEF signals of nanohole arrays were characterized by a streptavidin monolayer conjugated with 4 different fluorophores (AF488, Cy3, Cy5, and Cy5.5). The strongest enhancement was achieved at the Cy5 channel with a 23‐fold signal enhancement compared to a plain Au. When we captured biotinylated EVs on glass and nanohole substrates and labeled them with streptavidin‐Cy5, we showed a one‐order enhancement in the numbers and intensities of EVs on Au nanoholes compared to glass. This indicates the plasmon enhancement unveils EVs with weak fluorescence signals otherwise undetected without signal enhancement (glass substrates). The feasibility of its clinical application was tested using EVs from Gli36‐WT and Gli36‐EGFRvIII cell lines spiked in human plasma. We successfully identified cancer cell‐derived EVs by EGFR signals and detected EGFRvIII signals only from Gli36‐EGFRvIII EVs.

Summary/Conclusion: We developed PEF detection for multiplexed EV molecular profiling. Fluorescence signals from multiple channels were amplified simply using Au nanohole substrates. Especially, a marker expected to be low abundant can be strategically assigned to the strongest signal enhanced channel. This approach will provide a better understanding of the molecular heterogeneity of EVs and could improve the robustness and accuracy of EV‐based cancer detection.

PS06.08. Multiscale characterisation approach to uncover the correlation between isolation methods, physicochemical composition and biological function of extracellular vesicles

Huyen T. Phan, The University of Sydney, Sydney Nano Institute, School of Pharmacy, Faculty of Medicine and Health

Shiva Kamini Divakarla, The University of Sydney, Sydney Nano Institute, Faculty of Medicine and Health, Sydney School of Pharmacy

Jia Hao Yeo, The University of Sydney, School of Chemistry, Camperdown, NSW 2006, Australia.

Qingyu Lei, The University of Sydney, Sydney Nano Institute, Faculty of Medicine and Health, Sydney School of Pharmacy

Priyanka Tharkar, The University of Sydney, Sydney Nano Institute, Faculty of Medicine and Health, Sydney School of Pharmacy

Taisa Pansani, UNESP – Univ. Estadual Paulista, Araraquara School of Dentistry, Department of Dental Materials and Prosthodontics, Araraquara, Centro 14801–903, Brazil

Karthryn G. Leslie, The University of Sydney, School of Chemistry, Camperdown, NSW 2006, Australia.

Maggie Tong, The University of Sydney, School of Chemistry, Camperdown, NSW 2006, Australia.

Victoria Coleman, National Measurement Institute Australia, Nanometrology Section, Lindfield, NSW 2070, Australia

Åsa Jamting,National Measurement Institute Australia, Nanometrology Section, Lindfield, NSW 2070, Australia.

Mar‐Dean Du Plessis, National Measurement Institute Australia, Nanometrology Section, Lindfield, NSW 2070, Australia.

Elizabeth NewThe University of Sydney, Sydney Nano Institute, Faculty of Science, School of Chemistry, Camperdown, NSW 2006, Australia

Bill Kalionis, Department of Maternal‐Fetal Medicine Pregnancy Research Centre and University of Melbourne Department of Obstetrics and Gynaecology, Royal Women's Hospital, Parkville, VIC 3052, Australia

Philip Demokritou,Harvard T.H Chan School of Public Health, Center for Nanotechnology and Nanotoxicology, Department of Environmental Health, MA 02115, USA.

Hyun‐Kyung Woo,Center for Soft and Living Matter, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea.Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea.

Yoonkyoung Cho,Center for Soft and Living Matter, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea.Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea.

Wojciech Chrzanowski,The University of Sydney, Sydney Nano Institute, Faculty of Medicine and Health, Sydney School of Pharmacy

Introduction: Extracellular vesicles (EVs) have been lauded as next generation medicines, but very few EV‐based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass‐produce EVs, i.e. to isolate, purify and characterise them effectively. Technical limitations in comprehensive characterisation of EVs leads to unpredicted biological effects of EVs.

Methods: To measure EVs size and concentration we used Particle Tracking Analysis (PTA), Dynamic Light Scattering (DLS), Nano‐flow Cytometry (nFCM), Tunable Resistive Pulse Sensing (TRPS), and Asymmetric Flow‐Field fractionation (AF4). Nanoscale infrared spectroscopy (AFM‐IR) and nFCM were used to determine EV composition at the single EV and EV sub‐population levels. For the first time, we used Resonant Mass Measurement (RMM) for the characterisation of dry mass and buoyant mass of large EVs (>100 nm) and distorted grid (DG) for the sedimentation prediction of EVs. The actual functional effects of EV isolates on cells was determined using newly developed nitric oxide fluorescent probe to measure intracellular stress in an in vitro model of acute lung injury.

Results: Here, using a range of optical and non‐optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub‐population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were shown to correlate well with the biological and functional effects of EVs on single cell and cell cultures.

Summary/Conclusion: We anticipate that our multiscale characterisation approach will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

PS06.09. Extracellular vesicles: not only size matters

Pietro Parisse, Istituto Officina dei Materiali‐CNR

Introduction: The elucidation of biophysical and biochemical characteristics of EVs is crucial for the understanding of their interaction with recipient cells and their functional activity. In particular, for therapeutic applications understanding how the process of manufacturing affects the biological function of EVs is mandatory before going to the clinical testing. The absence of standardized methodologies and technologies to establish reliable criteria has been the main hurdle for real therapeutic applications of EVs.

Methods: We focused on the analysis of biophysical properties of standardized Umbilical Cord‐MSC‐EVs preparations, to help elucidating the role of phenotypic parameters (size,morphology, structure, protein/lipid ratio) in view of possible therapeutic applications. In particular we combined different techniques to capture morphological, structural and chemical information on EVs isolated with different protocols (Tangential Flow Filtration, Ultracentrifugation, Size Exclusion Chromatography). We exploited the nanometer resolution of Atomic Force Microscopy (AFM) to visualize the morphology of single EVs combined to structural information from Small Angle Scattering experiments and to the chemical information from vibrational spectroscopies, namely Fourier Transform Infrared and Ultraviolet Resonant Raman spectroscopies, to go beyond the mere size analysis and capture novel insights on the molecular contamination/stability of the different EVs preparation.

Results: We evidenced that size distribution analysis is not sufficient to distinguish different preparations, but that the structural, biophysical and chemical fingerprints of EVs and their co‐isolation products need to be taken into account to address stability and purity issues. The structural insights obtained by scattering techniques allow for a detailed description of the EVs bilayer structure; AFM reveals the presence of particles smaller than 50 nm, usual limit for standard optical techniques, allowing to distinguish vesicles from other particles based on their morphology and nanomechanical behavior; vibrational spectroscopies allow the extraction of protein/lipid ratio giving an easy and fast screening of sample purity.

Summary/Conclusion: Our results point towards the necessity of: a multi parametric analysis, capable of giving a wide overview of the biophysical properties of EVs; monitoring the morphology of EVs also in native environment; evaluating not only size, but also purity and stability of the EV preparations.

PS06.10. The Impact of Limits of Detection (LOD) on Studies of Extracellular Vesicles (EVs) Using Flow Cytometry

Sabrina La Salvia, SL, Icahn School of Medicine at Mount Sinai

Luca Musante, University of Virginia

Emily M. Heiston, EM, 1University of Virginia, Charlottesville, VA.

Nathan R. Stewart, NRS, Rutgers University, New Brunswick, NJ.

Steven K. Malin, SKM, University of Virginia

Uta Erdbrügger, UE, University of Virginia

Joanne Lannigan, JL, Cytek Biosciences

Introduction: Use of flow cytometry (FC) is one of the most commonly used technologies to study EVs isolated from various sources and a tool for understanding the role of EVs in clinical pathologies. There is a great deal of discordance in the literature regarding the size and frequencies of EVs from clinical samples. In this study, we sought to understand the impact of two flow cytometers with different LOD on size, phenotype, and concentration of EVs isolated from patient's samples.

Methods: Plasma samples were collected from 20 adults with metabolic syndrome after an overnight fast before and after testing for insulin sensitivity (n = 10) and a treadmill exercise test (n = 10). Plasma was centrifuged at 5000 g for 15 min and supernatant (SN) at 17,000g for 10 min. Samples were labeled with CD9,63,81, FITC, CD105 PE, CD31 AF647, CD41 PacBlue, and CD45 PE‐Dazzle594, split in two aliquots and acquired on a standard Cytek 5 laser Aurora and a 5 laser Aurora modified with a small particle enhancement option. Both instruments were calibrated for size (nm) and fluorescence (MESF) using FCMPass (nanopass.ccr.cancer.gov). Size was calibrated using NIST certified polystyrene beads (Series 3000 " ThermoFisher) and [Quantum MESFTM beads; Bangs Laboratories].

Results: Size (315‐330nm vs. 122–126nm) and concentration of total events < 1 u (2.2‐6.5E9/mL vs. 6.8E10‐1.5E11/mL) were statistically different across instruments (p < 0.05). Additionally, differences in the sizes of the major subsets (CD31, CD45, CD105) were also significant (p < 0.05), with the exception of CD41+ EVs. The Tetraspanin negative subsets were significantly smaller for the subtype CD45 and CD105. However, no significant difference was observed in the concentrations when comparing same samples across instruments or between patient groups or time points. Interestingly, the trends in the patient groups as well as the trends in changes across time points appeared to be similar, suggesting some level of reproducibility.

Summary/Conclusion: EV size and concentration detection using FC can be greatly impacted by an instrument's LOD. In order to compare results across instruments, it is imperative the instruments be calibrated to determine the LOD for size and fluorescence and results reported in the context of this information. Deeper subset analysis regarding size concentrations and MESF values is needed

PS06.11. New kid on the block: Nano‐flow cytometry measures up against first‐generation technologies for EV physical characterisation

Ben Peacock, NanoFCM

Adriele Prina, Trinity College Dublin

Alice Law, NanoFCM

Dimitri Aubert, NanoFCM

Robert Vogel, PhD, Izon

Introduction: Physical characterisation of extracellular vesicles, and other polydisperse complex biological samples, is critical to assess variation in quality deriding from their production and isolation. We recently published data analysing polystyrene nanoparticles as well as EVs using six applied techniques include multi‐angle dynamic light scattering (MADLS), asymmetric flow field flow fractionation coupled with multi‐angle light scattering (AF4‐MALS), centrifugal liquid sedimentation (CLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), and high‐sensitivity nano flow cytometry (nFCM).

Methods: Ability to detect and distinguish particles of different size and concentration was investigated using monomodal samples and complex polystyrene mixtures allowing for development of reliable post‐processing data protocol. Liposomes with known physicochemical properties closer to EVs, as well as EV containing plasma samples were analysed with all the tested techniques providing insight into the measurement of biological nanoparticles.

Results: Only nFCM and TRPS were capable of detecting the smallest populations of Polystyrene populations also distinguishing them in mxture. Liposome, concentrations and size distributions, as measured with NTA, TRPS, and AF4‐MALS were in good agreement (with a coefficient of variance of 27%), whereas nFCM measured a significantly lower. EV concentrations and size distributions of plasma EVs were generally in agreement for NTA, TRPS and nFCM measurements highlighting that single particle analysis techniques are well suited to measure particle concentration of biological samples such as EVs. For CLS the measured concentration was significantly higher compared with the single particle analysis techniques, and for AF4‐MALS the concentration was below the acceptable threshold for size and concentration measurements,

Summary/Conclusion: The data should help researchers decide which methodologies to implement in their own research, with additional information on ease of use and technical parameters included within the manuscript.

Declaration of Interest Statement

RV is a contractor at IZON Science, JM and MM are employed by IZON Science and their contributions to this paper were made as part of their contract/employment.

AL, BP, DA are employees of NanoFCM and their contributions to this paper were made as part of their employment.

PS06.12. Direct Measurement of Small Extracellular Vesicles in Unprocessed Human Plasma by Imaging Flow Cytometry

Wouter W. Woud, Nephrology and Transplantation, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, The Netherlands

Erik Mul, Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

Martin Hoogduijn, Nephrology and Transplantation, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, The Netherlands

Carla Baan, Nephrology and Transplantation, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, The Netherlands

Karin Boer, Nephrology and Transplantation, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, The Netherlands

Ana Merino, Nephrology and Transplantation, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, The Netherlands

Introduction: Characterization of EVs is hampered by their small size, low epitope copy number and the use of different isolation methods which may modify the EVs of interest. Analysis of EVs in human plasma is even more complicated due to the molecular complexity of plasma (e.g. lipoproteins and soluble factors). In recent years, Imaging Flow Cytometry (IFCM) has emerged as a potential technique that is sensitive enough to discriminate and analyse single EVs. Here we present an easy to use sample protocol without prior purification / isolation of EVs from plasma samples. Additionally, IFCM instrument settings as well as a set of controls are provided to accurately quantify, phenotype and visualize single human plasma derived small EVs (< 300 nm, sEVs) while excluding potential artefacts.

Methods: Platelet‐Poor Plasma (PPP) from 5 healthy individuals was stained directly without prior purification / isolation of EVs with CFSE and antibodies directed against proteins of interest including tetraspanins and CD31. Strict controls were used for each sample to allow for the accurate detection of sEV signatures. Acquisition was performed by running each sample for 3 minutes on an ImageStreamX (ISX) MkII IFCM.

Results: Fluorescent background levels of the IFCM were established and a gating strategy to accurately analyse sEVs was developed. sEV signatures in healthy individuals were identified as double‐positive events expressing a tetraspanin marker (CD9/CD63/CD81) in conjunction with enzyme activity (CFSE+) or presence of a cellular origin marker (CD31+); 2.7E6 ± 1.64E6 objects/mL and 3.57E6 ± 6.99E5 objects/mL respectively. Detergent lysis was performed to enable discrimination of biological signals from artefacts. Serial dilution experiments demonstrated accurate quantification of single sEVs.

Summary/Conclusion: We successfully developed a method to discriminate, identify and quantify sEVs in complex mixtures such as human plasma without prior purification or isolation of EVs. In this work, we propose a set of criteria for events to be classified as true sEVs by IFCM.

PS06.13. Imaging flow cytometry‐based detection of small extracellular vesicles in the synovial fluid of Rheumatoid Arthritis and Osteoarthritis patients

Edveena Hanser, University of Basel

Diego Kyburz, University of Basel

Introduction: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic joint inflammation and progressive destruction of cartilage and bone leading to severe pain and disability. A number of publications indicate either a pro or anti‐inflammatory role of extracellular vesicles (EVs) in RA and OA. EVs are produced by almost all cells. EVs have gained significant interest as biomarkers in health and disease. These nanobioparticles are believed to transfer cargo consisting of protein, lipids, nucleic acids, thus facilitating communications among cells. Here, we have optimized a method to detect small EVs called exosomes in the synovial fluid (SF) of RA and OA patients using imaging flow cytometry (iFCM)

Methods: Synovial fluid was collected from RA (n = 6) and OA (n = 4) patients after obtaining their written informed consent. EVs were isolated from hyaluronidase‐treated cell free SF by size exclusion chromatography (SEC) using iZON qEVoriginal size exclusion columns. The fractions were pooled in and concentrated by using Amicon ultra4 10KDa cellulose ultrafiltration filter units. The concentration and size determination of enriched EVs was determined by ZetaView(R) nanoparticle tracking analyzer (NTA). The concentration of EV samples were adjusted for staining with fluorescently labelled antibodies CD63 and CD9. The samples were acquired by imaging flow cytometry (iFCM) using Amnis(R) Imagestream(R) MK II imaging flow cytometry and analyzed by IDEAS(R) and FCS Express software. The EVs were also subjected to negative staining for transmission electron microscopy

Results: The transmission electron micrograph (TEM) showed the presence of EVs isolated from synovial fluid of RA and OA patients.

Imagestream(R) depicted small EVs labelled with fluorescently conjugated antibodies CD63 and CD9, which are the surface markers of exosomes. The various controls and other calibration parameters as stated in MIFlowCytEV‐ reporting framework was taken into account while conducting experiments to ensure the standardization and reliability of data

Summary/Conclusion: We have shown here an optimized method to detect small EVs present in the synovial fluid of RA and OA patients using imaging flow cytometry‐based technique. The protocol uses iFCM to identify small EVs, it combines high fluorescence sensitivity, image confirmation ability and powerful data analysis tools. As EVs carry markers of their parent cells and reflect their parent cells from where they originate because their membrane orientation is the same as that of the donor cell. Thus, they can be considered to be miniature versions of a cell. This property of EVs can be exploited in the search of biomarkers for diagnosis, prognosis, therapeutic potential and imaging flow cytometry can be used to identify EV origin via targeted and high‐throughput phenotyping

PS07.01. The role of Extracellular Vesicles (EVs) in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD)

Elena Casarotto, Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Department of excellence 2018–2022, University of Milan, Milan (Italy)

Daisy Sproviero, Mondino Foundation – IRCCS, Pavia (Italy)

Stella Gagliardi, Mondino Foundation – IRCCS, Pavia (Italy)

Eleonora Corridori, Mondino Foundation – IRCCS, Pavia (Italy)

Fabrizio Fabbiano, Centre of Integrative Biology (CIBIO), University of Trento, Trento (Italy)

Maria Cristina Gagliani, University of Genoa, Genoa (Italy)

Marta Cozzi, Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Department of excellence 2018–2022, University of Milan, Milan (Italy)

Barbara Tedesco, Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Department of excellence 2018–2022, University of Milan, Milan (Italy)

Riccardo Cristofani, Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Department of excellence 2018–2022, University of Milan, Milan (Italy)

Veronica Ferrari,Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Department of excellence 2018–2022, University of Milan, Milan (Italy)

Marta Chierichetti, Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Department of excellence 2018–2022, University of Milan, Milan (Italy)

Paola RusminiDipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Department of excellence 2018–2022, University of Milan, Milan (Italy)

Mariarita Galbiati, Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Department of excellence 2018–2022, University of Milan, Milan (Italy)

Vito D'agostino, Centre of Integrative Biology (CIBIO), University of Trento, Trento (Italy)

Katia Cortese,University of Genoa, Genoa (Italy)

Cristina Cereda, Genomic and post‐Genomic Unit, IRCCS Mondino Foundation, Pavia, Italy

Angelo Poletti, Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Department of excellence 2018–2022, University of Milan, Milan (Italy)

Valeria Crippa, Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Department of excellence 2018–2022, University of Milan, Milan (Italy)

Introduction: ALS and FTLD are neurodegenerative diseases characterized by pathological ubiquitinated and phosphorilated inclusions in the cytosol of affected cells. In 98% of ALS and in the majority of Tau‐negative FTLD cases the main component is the TAR DNA‐binding protein of 43 KDa (TDP‐43) together with its C‐terminal fragments of 35 (TDP‐35) and 25 KDa (TDP‐25).

TDP‐inclusions are mainly removed from cells via the protein quality control (PQC) system, but they could also be secreted within extracellular vesicles (EVs).

In our work we first analysed the TDP‐content of the EVs, by comparing large (LVs) with small vesicles (SVs); then, we evaluated the presence of some PQC‐members. Finally, we investigated the effect of PQC blockage on EVs secretion and content.

Methods: We isolated EVs produced by NSC34 cells untreated or treated with MG132 or NH4Cl (proteasome and autophagy inhibitors). To isolate EVs we used the differential ultracentrifugation method. We analysed EVs size, count and morphology through the Nanoparticle Tracking Analysis and the transmission electron microscopy, and their protein content through western blot analysis.

Results: We showed that both TDP‐43 and its C‐terminal fragments (especially TDP‐35) are secreted in EVs, mainly in LVs. Interestingly, in cells TDPs are present as soluble forms, instead the secreted TDPs are mainly insoluble. We found that many PQC‐components are secreted in EVs and PQC modulation resulted in a significant increase in EVs numbers, that is paralleled by a slight increase in TDP‐content.

Summary/Conclusion: EVs may positively contribute to the clearance of insoluble TDPs species by cooperating with PQC, having a protective role for affected cells. However, they may also contribute to the prion‐like distribution of TDP‐neurotoxic forms in neighboring and more distant cells.

PS07.02. Biochemical signatures in extracellular vesicles from sporadic Amyotrophic Lateral Sclerosis patients revealed by Raman Spectroscopy and Mass Spectrometry lipidomics

Maria Chiara Mimmi, IRCCS Mondino Foundation Neurological Institute

Daisy Sproviero, PhD, IRCCS Mondino Foundation

Carlo Morasso, Maugeri Scientific Clinical Institutes IRCCS

Fabio Corsi, Maugeri Scientific Clinical Institutes IRCCS

Orietta Pansarasa, Genomic and post‐Genomic Unit, IRCCS Mondino Foundation, Pavia, Italy

Cristina Cereda, Genomic and post‐Genomic Unit, IRCCS Mondino Foundation, Pavia, Italy

Introduction: There is no validated blood‐based biomarker for sporadic Amyotrophic Lateral Sclerosis (ALS). Extracellular vesicles (EVs) have the potential to solve this unmet clinical need, as they can be involved in the pathogenesis/progression of neurodegenerative diseases (Selmaj etal., 2017). Lipids are essential molecular components of EVs, but at the moment the knowledge about their distribution and function is limited. The aim of this work was to find biomarkers of ALS by investigating biochemical composition of plasma‐derived EVs with Raman Spectroscopy (RS) and HPLC‐MS (High Performance Liquid Chromatography‐Mass Spectrometry).

Methods: We isolated small and large extracellular vesicles (sEVs and lEVs), from blood plasma of 20 sporadic ALS patients and a matched group of healthy controls, by differential centrifugation/ultracentrifugation. We characterized sEVs, lEVs and blood plasma firstly by RS and subsequently by HPLC‐MS, targeting a panel of around 200 lipids. Statistical analysis included univariate and multivariate analysis techniques such as PCA (Principal Component Analysis) and PLS‐DA (Partial Least Squares‐ Determinant Analysis).

Results: Raman spectroscopy highlighted lEVs as a particularly promising biomarker for ALS. Raman spectra showed in fact that sporadic ALS patients have a different lipid content and less intense bands relative to the aromatic amino acid phenylalanine. HPLC‐MS revealed some lipid species discriminating between ALS and healthy subjects. They were mainly phospholipids, belonging to the subclasses of phosphatidylcholines (PC), phosphatidylethanolamines (PE) and phosphatidylinositols (PI), and sphingolipids, belonging to the subclasses of ceramides (Cer), mono/di‐hexosyl‐ceramides (M/DHC). In particular the increase of PC(34:1), MHC(24:1) and Cer(24:1) was observed in either plasma, lEVs and sEVs from ALS patients. The species PI(36:3) was up‐regulated in both large and small vesicles of ALS patients.

Summary/Conclusion: Interestingly, some species significantly altered in our analysis of plasma lipidome, overlap with the ones highlighted by Blasco etal. in their work on cerebrospinal fluid (CSF) (Blasco etal. 2017), namely PC(38:2), MHC(24:1) and the plasmalogen PCO(34:1). This supports the idea of plasma and plasma‐derived EVs as easily available source of robust biomarkers. Among the other results, the perturbed sphingolipids are particularly relevant as they are involved in key pathways for ALS patogenesis, such as autophagy, energy metabolism and neuroinflammation.

PS07.04. Plasma extracellular vesicles size and concentration are altered in Alzheimer's disease, dementia with Lewy bodies and frontotemporal dementia

Antonio Longobardi, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Luisa Benussi, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Roland Nicsanu, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Sonia Bellini, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Clarissa Ferrari, Service of Statistics, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Claudia Saraceno, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Roberta Zanardini, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Marcella Catania, Neurology 5 / Neuropathology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy

Giuseppe Di Fede, Neurology 5 / Neuropathology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy

Rosanna Squitti, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Giuliano Binetti, MAC‐Memory Clinic, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Roberta Ghidoni, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Introduction: Alzheimer's disease (AD), Lewy body dementia (LBD), frontotemporal dementia (FTD), are the major neurodegenerative dementias. Abnormal protein accumulation characterizes all these diseases. An alteration in extracellular vesicles (EVs) release and or composition might be a common pathological mechanism across dementia influencing the fate of disease‐related proteins. Loss of neurotrophic factors might be one of the determinants affecting EVs release.

Methods: EVs were isolated with commercial kit from plasma of n = 30 AD, n = 30 LBD, n = 30 FTD and n = 30 controls (CTRL). Nanoparticle Tracking Analysis (NTA) was performed to evaluate EVs concentration and size distribution. PGRN, BDNF, GDNF and Cystatin C plasma concentrations were measured by Bioplex and ELISA. A classification tree (CT) was applied to detect the best predictors for discriminating CTRL vs patients’ (PTS) group. Patients provided written informed consent. Local ethics committee approval Prot. N. 111/2017.

Results: A decrease of plasma EVs concentration and an increase of EVs size were measured in AD, LBD and FTD vs CTRL. Levels of PGRN were reduced in FTD and Cystatin C levels were increased in LBD vs CTRL samples. CT revealed that EVs concentration and size are the best predictors to classify dementia patients from CTRL (96.8%). ROC analysis revealed a good diagnostic performance (AUC = 0.86) of EVs concentration/size ratio. Cystatin C was the only neurotrophic factor associated with EVs concentration.

Summary/Conclusion: Alterations in the intercellular communication mediated by EVs might be a common molecular pathway in neurodegenerative dementias. Cystatin C might be one of the determinants affecting EVs release. The identification of shared disease mechanisms is of pivotal importance to develop treatments to delay disease progression.

PS07.05. EVs size and concentration are altered in frontotemporal dementia caused by progressive loss of progranulin and C9orf72

Sonia Bellini, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Luisa Benussi, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Claudia Saraceno, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Antonio Longobardi, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Roland Nicsanu, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Roberta Zanardini, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Sara Cimini, Neurology 5 / Neuropathology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy

Giacomina Rossi, Neurology 5 / Neuropathology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy

Giuliano Binetti, MAC‐Memory Clinic, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Roberta Ghidoni, Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

Introduction: Frontotemporal dementia (FTD) is a neurodegenerative disease among the most common forms of presenile dementia, mainly affecting individuals under 65 years of age. Mutations in GRN, C9orf72 and MAPT are currently the most common causes of inherited FTD. Cutting‐edge molecular research suggests that lysosomal/exosomal dysfunctions are pathological events driven by the loss of functional proteins in GRN/C9orf72‐associated FTD. We therefore investigated whether extracellular vesicles (EVs) in cells and plasma are altered in association with progressive loss of progranulin and C9orf72.

Methods: EVs isolation was performed with a commercial kit from i) human plasma samples: 43 controls, 31 C9orf72 pathological expansion, 9 C9orf72 intermediate expansion, 72 heterozygous GRN null mutation (45 affected and 27 pre‐symptomatics), 3 homozygous GRN null mutation, 4 GRN missense mutation carriers and 10 sporadic FTD; ii) human primary fibroblast conditioned media: 3 controls, 3 C9orf72 pathological expansion, 1 C9orf72 intermediate expansion, 7 GRN null mutation carriers. Size and concentration were measured by Nanoparticle Tracking Analysis (NTA). Patients provided written informed consent. Local ethics committee approval Prot. N.44/2018.

Results: In human plasma samples EVs concentration was significantly reduced both in C9orf72 pathologically expanded and GRN+ patients compared to controls, while EVs size significantly increased in the same mutated groups. In human primary fibroblasts we observed a trend toward a progressive decrease of EVs release associated with a progressive loss of progranulin and C9orf72.

Summary/Conclusion: EVs dosage and size characterization might be a promising biomarker in GRN/C9orf72‐associated FTD. Taken together these results suggest a correlation between progressive loss of GRN/C9orf72 and lysosomal/exosomal dysfunctions in FTD.

PS07.06. Levels of neuronal factors in circulating extracellular vesicles predict the progression of preclinical subjects to Alzheimer's disease in APOE ε4 carriers

Mohamed Raâfet Ben Khedher, INRS‐CAFSB

Mohamed Haddad, INRS‐CAFSB

Danielle Laurin, University Laval

Charles Ramassamy, INRS‐Centre Armand‐Frappier Santé‐ Biotechnologie

Introduction: Background: In brain, extracellular vesicles (EVs) play an essential role in neuron‐glia interface and ensure the crosstalk between the brain and the periphery. Some studies now link EVs pathway dysfunction to apolipoprotein E4 variant (APOE ε4) and the risk of progression to Alzheimer's disease (AD). To better understand the role of APOE ε4 in pre‐clinical AD, we determined levels of pathogenic, neurotrophic and inflammatory proteins in peripheral EVs (pEVs) and in plasma from cognitively impaired‐no dementia (CIND) participants stratified upon the absence (APOE ε4‐) or the presence (APOE ε4+) of the ε4 allele of APOE.

Methods: Method: Levels of 15 neurodegenerative, neurotrophic and neuroinflammatory proteins were quantified in pEVs and compared to their plasma levels from cognitively normal and CIND participants

Results: For the first time, several neurotrophic and inflammatory markers including LCN‐2, S100B, ANGPTL‐4, NPTX‐2 and α‐synuclein were evidenced in pEVs. Some proteins such as α‐Syn, NPTX‐2 and S100B were enriched in pEVs as compared to plasma. APOE ε4 presence was associated with differential regulation of 7 markers and compromised the release of pEVs formed by an endosomal route. The pentraxin‐2/α‐synuclein ratio measured in pEVs was able to predict AD. 5 years before the onset, among APOE ε4+ CIND individuals.

Summary/Conclusion: Discussion: Our findings suggest an alteration of the endosomal pathway in APOE ε4+ carriers and that pEVs pentraxin‐2/α‐synuclein ratio could serve as a useful early biomarker for AD susceptibility.

PS07.07. Microglial depletion reduces adeno‐associated virus mediated tau propagation from the entorhinal cortex to the dentate granular cells in human APP NL‐G‐F knock‐in mice while increasing amyloid burden

Kevin A. Clayton, Boston University School of Medicine

Jean‐Christophe Delpech, INRAE

Shawn Herron, Boston University School of Medicine

Seiko Ikezu, Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine

Tsuneya Ikezu, MD, PhD, Department of Pharmacology & Experimental Therapeutics, Center for Systems Neuroscience,Boston University School of Medicine; Department of Neuroscience, Mayo Clinic Florida

Introduction: Microglia are the innate immune cells in the brain, and known to phagocytose apoptotic neurons and dystrophic neurites containing phosphorylated tau (p‐tau), possibly enhancing the spread of pathological tau via extracellular vesicles (EVs). Evidence suggests that proteinopathic stress from amyloid plaques transforms microglia into a neurodegenerative phenotype (MGnD), possessing enhanced phagocytic and exocytotic functions, which may exacerbate propagation of p‐tau in the diseased brain.

Methods: C57BL/6 (WT) and APPNL‐G‐F mice were fed with a CSF1R inhibitor (PLX5622) or control chow for one month before and after stereotaxic injections of AAV2/6‐SYN1‐P301Ltau expressing P301L tau mutant into the medial entorhinal cortex (MEC) at 5 months of age. Propagation of tau to the dentate granular cells of the hippocampus, amyloid plaque formation, and association of microglia and p‐tau with plaques were assessed by immunohistochemistry after one month. MGnD and homeostatic microglia were separately isolated from APPNL‐G‐F mouse brains by FACS and evaluated for the expression of EV marker molecules. To investigate the propensity of DAM/MGnD to hyper‐secrete EVs in vivo, we developed a novel lentivirus (pLV‐ mEmerald ‐CD9) to specifically express mEmerald fused to CD9, an exosomal marker, in microglia and co‐injected with AAV‐P301L tau into the brains of diseased and WT mice.

Results: APPNL‐G‐F mice exhibited approximately a 10‐fold increase in tau propagation compared to WT mice. Strikingly, PLX5622 treatment, which depleted ∼ 99% of microglia, showed 74 and 87% reduction of tau propagation in WT and APPNL‐G‐F groups respectively. Contrarily, PLX5622 increased intensity of plaque associated p‐tau along with increased size and number of amyloid plaques in the APPNL‐G‐F mice, suggesting their regulation by MGnD microglia. Gene expression of EV markers, CD9 and CD63 was upregulated in Clec7a+ microglia compared to Clec7‐ microglia isolated from APPNL‐G‐F mice, which was further supported by co‐localization of Tsg101, an exosome marker, with Clec7a+ MGnD microglia. The mEmerald‐CD9 lentivirus allowed for visualization of microglia‐specific EVs, which are released by DAM/MGnD at a 3‐fold higher rate than homeostatic microglia and contain pathologic pTau.

Summary/Conclusion: Tau propagation may be influenced by engulfment of pathological tau seeds by microglia and secretion through EVs, which is exacerbated in microglia activated by plaques, and mitigated by PLX5622 treatment.

PS07.08. Extracellular Vesicles: Mediator and Biomarker for Oxidative Stress in Parkinson's Disease

Adityas Purnianto, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne

Leah Beauchamp, The Florey Institute of Neuroscience and Mental Health

Eleanor Saunders, 2.The Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia

Ashley Bush, The Florey Institute of Neuroscience and Mental Health, Parkville, VIC, Australia

David Finkelstein, The Florey Institute of Neuroscience and Mental Health

Kevin Barnham, The Florey Institute of Neuroscience and Mental Health

Laura Vella, The Florey Institute of Neuroscience and Mental Health

Introduction: Parkinson's disease (PD) develops insidiously from underlying pathogenesis such as mitochondrial dysfunction and oxidative stress. Increasing evidences in diseases such as cancer and inflammation have demonstrated that oxidative stress induces release of functional extracellular vesicles that contain a source biomarkers of disease characterized by oxidative stress. As a disease characterized by oxidative stress, this phenomenon has been understudied in PD.

Methods: We are using cell models of mitochondrial dysfunction and oxidative stress in PD as well as clinical samples (nasal secretion and plasma) from newly diagnosed PD patients to explore the potential role of EVs in oxidative stress in PD. Utilizing different techniques such as western blot and mass spectrometry, we are examining the impact of oxidative stress in the metabolite, metal profiles, and mitochondrial components of EVs content. We are investigating the pathological effects of the intercellular transfer of oxidative‐stress‐modified EVs content, for example by using Seahorse assay, to determine if they contribute to the progression of disease

Results: Our initial findings showed that the EV contents of some mitochondrial electron transport chain complexes and metals were altered in EVs from SH‐SY5Y cells treated with rotenone, a mitochondrial complex I inhibitor.

Summary/Conclusion: The results of this study will generate knowledge on the biosignature of EVs in early stage PD and the role of EVs in intercellular transfer of oxidative stress to provide insight into the development of EV based biomarkers of prodromal PD.

PS07.09. Do microbiota‐derived outer membrane vesicles promote inflammation and neurodegeneration in Parkinson's disease?

Tiana F. Koukoulis, The Florey Institute of Neuroscience and Mental Health

Leah Beauchamp, The Florey Institute of Neuroscience and Mental Health

David Finkelstein, The Florey Institute of Neuroscience and Mental Health

Victoria Lawson, Department of Microbiology and Immunology, The University of Melbourne

Neil O'Brien‐Simpson, Centre for Oral Health Research, Melbourne Dental School, University of Melbourne

Maria Kaparakis‐Liaskos, Department of Physiology, Anatomy and Microbiology, La Trobe University

Kevin Barnham, The Florey Institute of Neuroscience and Mental Health

Laura Vella, The Florey Institute of Neuroscience and Mental Health

Introduction: The microbiome‐gut‐brain axis plays an important role in Parkinson's disease pathogenesis with dysbiosis of the gut microbiota proposed to initiate a proinflammatory cascade that drives neurodegeneration. The mechanisms by which gut microbes communicate with host cells to trigger inflammation in Parkinson's disease is currently unclear. We hypothesise that microbiota‐derived outer membrane vesicles (OMVs), rich in the potent immune stimulator, lipopolysaccharide (LPS), promote gastrointestinal inflammation and leaky gut, but also the systemic and neural inflammation that characterises Parkinson's disease.

Methods: OMVs were isolated from Gram‐negative bacteria in culture or faeces from Parkinson's disease animal models and characterized by density, size and morphology and LPS content. The ability of OMVs to induce a proinflammatory response in vitro was determined and compared to OMVs from WT animals and equivalent concentrations of purified LPS. In vivo studies to determine whether orally administered OMVs predispose the enteric and central nervous system to Parkinson's disease pathology are currently underway.

Results: OMVs isolated from Escherichia coli cultures are shown to be more potent at promoting immune activation than an equivalent dosage of LPS in vitro. OMV induced immune activation results in the promotion of neurodegeneration in vitro and anticipated to exacerbate gastrointestinal dysfunction in a neurotoxin mouse model of Parkinson's disease.

Summary/Conclusion: We are investigating the functional role of OMVs in Parkinson's disease with OMVs isolated from in vitro culture and the faeces from animal models of the disease. Our preliminary data suggest that OMVs from gram negative bacteria are potent immune stimulators that have the potential trigger and/or exacerbate Parkinson's disease pathogenesis.

PS07.10. Mesenchymal stem cell‐derived extracellular vesicles ameliorate Alzheimer's disease‐like phenotypes in a 5XFAD mouse model

Allaura S. Cone, Florida State University College of Medicine

Xuegang Yuan, Florida State University College of Engineering

li sun, FSU College of Medicine

Leanne Duke, Florida State University College of Medicine

Michael P. Vreones, Florida State University College of Medicine

Allison N. Carrier, Florida State University College of Medicine

Stephanie M. Kenyon, Florida State University College of Medicine

Spencer R. Carver, Florida State University College of Medicine

Sarah D. Benthem, Florida State University College of Psychology

Alina C. Stimmell,Florida State University College of Psychology

Shawn C. Moseley, Florida State University College of Psychology

Aaron A. WilberFlorida State University College of Psychology

James M. Olcese, Florida State University College of Medicine

David G. Meckes, PhD,FSU college of medicine

Introduction: Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that affects more than 44 million people worldwide. Despite the high disease burden, there is no effective treatment for people suffering from AD. Mesenchymal stem cells (MSCs) are multipotent stromal cells that have been widely studied due to their therapeutic potential. However, administration of cells has been found to have a multitude of limitations. Recently, extracellular vesicles (EVs) derived from MSCs have been studied as a therapeutic candidate, as they exhibit similar immunoprotective and immunomodulatory abilities as the host hMSCs.

Methods: To test the potential therapeutic effects of MSC EVs, human bone‐marrow derived MSCs were grown in 3D cell culture, and small EVs were harvested using differential centrifugation and PEG precipitation. These small EVs were given to non‐transgenic (NT) or 5XFAD (5 familial Alzheimer's disease mutations) mice intranasally (IN) every 4 days for 4 months. The mice were then required to perform a variety of behavioral assays to measure changes in learning and memory. Afterwards, immunohistochemistry was performed on brain slices to measure amyloid beta (Aβ) and glial fibrillary acidic protein (GFAP) levels.

Results: The data revealed that 5XFAD mice that received EV treatment behaved significantly better in cognitive tests than saline treated 5XFAD mice, with no significant change between EV‐treated 5XFAD mice and NT mice. Additionally, we found lower Aβ plaque load in the hippocampus of the EV‐treated mice. Finally, less colocalization between GFAP and Aβ plaques was found in the brain of EV‐treated mice compared to saline.

Summary/Conclusion: Taken together, these data suggest that IN administration of MSC‐derived EVs can slow down AD pathogenesis in a preclinical mouse model.

PS07.11. A Parkinson's disease‐causing LRRK2 mutation leads to an early disruption of brain exosome biogenesis

Lital Rachmany, Department of Psychiatry, New York University School of Medicine, New York, NY 10016, USA

Brainson Liemisa, Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, New York 10962, USA

Samantha F. Newbury, Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, New York 10962, USA

Adaora Aroh, Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, New York 10962, USA

Efrat Levy, Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, New York 10962, USA

Paul M. M. Mathews, Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, New York 10962, USA

Introduction: Parkinson's disease is a neurodegenerative disorder clinically characterized by motor and cognitive deficiencies with the loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of alpha synuclein aggregates. Variants of the Leucine‐Rich Repeat Kinase 2 (LRRK2) are associated with an increased risk of Parkinson's disease, with the Gly2019Ser mutation a relatively common cause of familial Parkinson's disease. Knockin mice carrying this mutation develop motor symptoms with aging and cell death in cortical neurons, mediated in part by altered macroautophagy. At a pre‐symptomatic age in these mice, we investigated the effect of this mutation on brain exosome biology, a key component of the endosomal‐lysosomal system.

Methods: Six‐month‐old mutant LRRK2 mice and wildtype controls were compared. Motor skills were studied using RotaRod and wire hanging tests. Extracellular vesicles (EVs) were isolated from brain tissue using a density‐base column and analyzed by nanoparticle tracking and electron microscopy. Specific markers for exosomes as well as alpha‐synuclein levels were determined using Western blot analysis.

Results: The six‐month‐old mice were pre‐symptomatic for Parkinsonian motor deficiencies. Lower levels of exosomes were seen in the brains of LRRK2 mice as compared to controls. While no difference in the level of alpha‐synuclein was found in brain homogenates, alpha‐synuclein levels were significantly lower in EVs in the LRRK2 mice compared to controls.

Summary/Conclusion: Our findings argue that brain exosome biogenesis is compromised by the LRRK2 mutation prior to the development of clinical symptoms in a mouse model. Additionally, our data show that alpha‐synuclein in EVs is reduced. While additional studies are needed, an alteration in exosomal alpha‐synuclein packaging may prove to be a useful biomarker of early Parkinson changes in the endosomal‐lysosomal system and reflect pathological alterations in alpha‐synuclein within neurons.

PS08.01. Optimization of culture conditions for the production of mesenchymal stromal cell‐derived extracellular vesicles towards its translation into large‐scale manufacturing

Raquel MS Cunha, Instituto Superior Técnico, University of Lisbon

Cecília Calado, ISEL ‐ Instituto Superior de Engenharia de Lisboa

Joaquim M.S. Cabral, Department of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa

Cláudia Lobato da Silva, Department of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa

Ana Fernandes‐Platzgummer, Department of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa

Introduction: Therapies based on mesenchymal stromal cells‐derived extracellular vesicles (MSC‐EV) have emerged as a potential alternative to whole cell therapies, as MSC‐EV offer specific advantages for patient safety such as the minimal predisposition to activate innate and immune responses. However, MSC‐EV productivity and efficacy are still limited, and clinical translation entails scalable and GMP‐compliant processes for their production, isolation and characterization.

Methods: To optimize MSC‐EV production, we compared different MSC tissue sources (bone marrow, adipose tissue, umbilical cord matrix) and culture conditions (feeding regime, oxygen tension, temperature, chemical cues). MSC isolated from multiple donors were expanded using Serum‐free and Xeno‐Free culture medium under static conditions. The conditioned medium (CM) was collected at different time points and MSC‐EV were isolated by ultracentrifugation or with a commercially available isolation kit and characterized according to ISEV guidelines.

Results: MSC derived from the different sources and donors were able to grow under the different culture conditions tested, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the ISCT. The feeding regime was optimized and the best time point for pre‐conditioning and collection of CM for MSC‐EV isolation was determined. To further optimize MSC‐EV production, different physical (oxygen tension, temperature) and chemical cues were tested to determine the conditions that resulted in higher EV production using techniques as NTA, protein and lipid quantification and purity assessment. Additional MSC‐EV characterization techniques included western blot, flow cytometry, imaging, FTIR and omic tools (using the cells, CM and culture medium as controls, according to ISEV guidelines).

Summary/Conclusion: In summary, this study contributes to the establishment of optimal culture conditions for MSC‐EV production using different MSC tissue sources. The optimized culture conditions are being translated into scalable processes for MSC‐EV production using bioreactor systems.

PS08.02. Immortalization strategies for human mesenchymal stromal cells for large scale production of extracellular vesicles

Yanis Mouloud, Universitätsklinikum Essen

Introduction: Mesenchymal stromal cells (MSCs) are considered as therapeutic agent for many diseases due to their immunomodulatory properties. Apparently, secreted extracellular vesicles (EVs) that MSCs also release in vitro mediate these activities. Indeed, we have successfully confirmed the therapeutic potential of EVs prepared from conditioned media of cultured MSCs in several animal models and a treatment resistant GvHD patient. Thus, MSC‐EVs provide a promising therapeutic agent for the future.

Currently, we aim to scale the MSC‐EV production process for the clinical setting. However, the scaling process is limited by the life span of EV releasing cells.

Methods: To address that issue, we have compared different strategies to immortalize primary MSCs for the production of immunomodulatory EVs.

Results: Indeed, we were able to establish immortalized clonal MSC lines which maintained their bona fide MSC features and secrete immunomodulatory active EVs.

Summary/Conclusion: To learn whether the immortalization affects the quality of released EVs, the immune modulatory capabilities of secreted EVs were analysed in a mixed lymphocyte reaction assay. EVs isolated from immortalized MSC supernatants retained their ability to modulate immune responses in the MLR assay just like EVs harvested from supernatants of the original primary MSCs. EVs produced by these clonal cell lines will now broadly be tested in various disease models. Importantly, batch‐to‐batch variations will be addressed.

PS08.03. Regenerative capacity of blood‐derived EVs on primary osteoarthritic chondrocytes coincides with the EV‐associated miRNA functional repertoire

Alexander Otahal, Danube University Krems

Karina Kramer, Danube University Krems

Olga Kuten‐Pella, Orthosera GmbH

Christoph Stotter, Danube University Krems

Markus Neubauer, Danube University Krems

Zsombor Lacza, Orthosera GmbH

Stefan Nehrer, Danube University Krems

Andrea De Luna, Danube University Krems

Introduction: Regenerative medicine increasingly focuses on blood‐derived products for osteoarthritis therapy. Frequently, citrate‐anti‐coagulated platelet‐rich plasma (CPRP) is intra‐articularly injected into diseased joints, however, cell‐free alternatives such as hyperacute serum (hypACT) are under development. Mechanisms of action of blood products are still poorly understood. The discovery of EVs in blood and EV‐associated cargo molecules such as miRNAs opened up new levels of complexity in understanding the therapeutic potential of blood products.

Methods: To investigate the role of EVs isolated from CPRP and hypACT during osteoarthritis (OA),primary human OA chondrocytes were treated with EVs enriched via ultracentrifugation (UC) from these two blood products. Chondroprotective and anti‐inflammatory effects were evaluated based on gene expression analysis via reverse transcription quantitative PCR (RT‐qPCR) and Western Blot, as well as cytokine release via ELISA, respectively. EV‐associated miRNA profiles were analysed by screening a 372 miRNA panel via RT‐qPCR in EVs purified via UC and size exclusion chromatography (SEC) as well as in the respective blood products.

Results: EVs from either blood product increased the expression of anabolic markers type II collagen (COL2A1), SRY‐box transcription factor 9 (SOX9) and aggrecan (ACAN) compared to blood products, but also the catabolic marker and tissue remodeling factor matrix metalloproteinase 3 (MMP3). CPRP blood product increased SOX9 protein expression, in contrast, CPRP EVs decreased NFκB and COX2 expression in IL1β‐stimulated chondrocytes compared to unstimulated cells. However, hypACT EVs induced SOX9 protein expression while preventing IL6 secretion compared to hypACT blood product. Analysis of the functional repertoire encoded in EV‐associated miRNAs revealed that CPRP EV‐associated miRNAs strongly target NFκB signaling and hypACT EV‐associated miRNAs were predicted to strongly affect IL6‐ and TGFβ/SMAD signaling.

Summary/Conclusion: The results indicate that blood EVs are sufficient to induce chondrogenic gene expression changes in OA chondrocytes, while preventing pro‐inflammatory cytokine release compared to full blood products. In addition, the effects observed in the biological assays can be explained by EV‐associated miRNAs. This highlights the potential of blood‐derived EVs to be regulators of cartilage extracellular matrix metabolism and inflammation as well as candidates for new cell‐free therapeutic approaches for OA.

PS08.04. Cardiac progenitor cell‐derived EVs affect human macrophage polarization

Margarida Viola, Laboratory of Experimental Cardiology, Circulatory Health Laboratory, UMC Utrecht Regenerative Medicine Center, University Medical Center Utrecht, Utrecht University, The Netherlands

Pieter Vader, CDL Research, University Medical Center Utrecht, The Netherlands

Saskia C.A. de Jager, Laboratory of Experimental Cardiology, Circulatory Health Laboratory, Center for Translational Immunology, UMC Utrecht Regenerative Medicine Center, University Medical Center Utrecht, Utrecht University, The Netherlands

Joost P.G. Sluijter, J.P.G., Department of Experimental Cardiology, University Medical Center Utrecht, Utrecht University, The Netherlands

Introduction: Regeneration of damaged heart tissue upon myocardial infarction remains a major challenge. Transplantation of cardiac progenitor cells (CPCs) has been studied as a potential regenerative therapy. Interestingly, recent studies have shown that the cardioprotective effect of CPCs is mediated by the release of extracellular vesicles (EVs). The benefits of CPC‐EVs have mostly been associated with stimulation of angiogenesis and inhibition of cell death. Although macrophages have been suggested to be key for cardiac repair, the effect of CPC‐EVs on macrophages is poorly explored.

Methods: EVs were isolated from serum‐starved CPCs by ultrafiltration followed by size exclusion chromatography. Human monocytes were isolated from the blood of healthy donors and differentiated into macrophages with M‐CSF. In addition, macrophages were stimulated with LPS + IFNy or with IL4 in order to induce an inflammatory M1 and reparative M2 phenotype, respectively. The macrophages were subsequently stimulated with CPC‐EVs and analyzed by flow cytometry, bulk RNA sequencing and confocal microscopy to assess macrophage phenotype changes.

Results: Stimulation of macrophages with CPC‐EVs enhances the expression of pro‐inflammatory markers, including CD80, while decreasing anti‐inflammatory markers, such as CD200R and CD206. In line with these findings, CPC‐EV‐stimulated macrophages adopt a morphology that reflects the inflammatory M1 macrophage. Ongoing bulk sequencing on these conditions will provide in‐depth insight on phenotype changes in these cells.

Summary/Conclusion: Our data suggest that CPC‐EVs are able to induce macrophage polarization towards an inflammatory phenotype, which might have implications for CPC‐EV treatment after myocardial infarction. This underlines an urgent need to understand the molecular mechanisms underlying the immunomodulatory effect of CPC‐EVs before moving into a clinical setting.

PS08.05. Human cardiac mesenchymal stromal cell‐derived extracellular vesicles are protective of ischemia‐reperfusion injury

Max M. Chen, MS, Graduate Institute of Biomedical Materials & Tissue Engineering, Taipei Medical University, Taiwan

Andreas Czosseck, MSc, Graduate Institute of Biomedical Materials & Tissue Engineering, Taipei Medical University, Taiwan

Chuan‐Chih Hsu, MD, Department of Surgery, Taipei Medical University Hospital, Taiwan

Annette Meeson, PhD, Biosciences Institute, Newcastle University, UK

Thomashire A. George, MbChB, International PhD Program in Biomedical Engineering, Taipei Medical University, Taiwan

Chen‐Lin Chen, School of Biomedical Engineering, Taipei Medical University

David J. Lundy, PhD, Graduate Institute of Biomedical Materials & Tissue Engineering, Taipei Medical University, Taiwan

Introduction: During myocardial infarction, cardiomyocytes (CMs) die by apoptosis in response to ischemia, leading to permanent cardiac remodeling and loss of heart function. Nanometer scale particles such as EVs are amenable to delivery by intracoronary infusion and show greater uptake and retention than live cells and a longer duration of activity than isolated growth factors and cytokines. Furthermore, their complex cargo is able to activate multiple survival pathways, preventing apoptosis and promoting endogenous regeneration. Here, we explore whether EVs from a novel population of human cardiac stromal cells (CMSCs) may protect CMs from ischemia‐reperfusion injury (IRI).

Methods: CMSCs were derived from human heart tissues and maintained under MSC conditions with 10% exosome‐depleted FBS (Gibco A2720801). Ethical approval was obtained from TMU‐JIRB (N201910027). Surface markers were characterised by flow cytometry. Bone marrow‐derived MSCs (BM‐MSCs, Lonza) were used as a control. Extracellular particles were collected by ultracentrifugation (100,000g, 16h, 4˚C) of conditioned culture media and characterised by NTA, TEM and Western Blot (WB). H9C2 rat cardiomyoblasts, as a model for CMs, were maintained in DMEM‐HG, 10% FBS, 5% CO2. To induce ischemic injury, cells were subjected to 48h hypoxia (BD AnaeroPack), 0% FBS. To mimic reperfusion injury, cells were restored to normoxia with fresh culture media ± CMSC‐EVs. CCK‐8 and flow cytometry (Annexin‐V/PI) were used to measure H9C2 viability, apoptosis and necrosis.

Results: CMSCs were found to express typical MSC markers, such as CD44, CD90, CD166 and CD105, and were negative for CD19, CD45 and CD11b. RT‐qPCR analysis revealed that these cells express pro‐regenerative, anti‐apoptotic growth factors (VEGFA, IGF‐1, FGF2, ANGPT1 etc.) at levels equal or greater than BM‐MSCs. NTA revealed particles 160.1 ± 2.0 nm diameter at significantly higher yield than BM‐MSCs. TEM showed characteristic particles with diameters 100–200 nm. WB was positive for Hsp70, CD9, CD63 and CD81.

In IRI rescue experiments, CMSC‐EVs protected H9C2 cells from 48h ischemic injury, showing CCK‐8 absorbance at 144% compared to 10% FBS (100%) and 0% FBS (‐71%). Following reperfusion injury, CMSC‐EV treated cells had viability of 76%, compared to 27.7% for 0% FBS and 53.7% for 10% FBS. Flow cytometry showed that the percentage of apoptotic cells significantly decreased from 26.2% (0% FBS) or 14.8% (10% FBS) to 10.5% (CMSC‐EVs).

Summary/Conclusion: These results clearly demonstrate the therapeutic potential of CMSC‐EVs. Further work is underway to characterise their precise cargo of CMSC‐EVs, quantify their uptake, and to examine rescue pathway induction in the target cells.

PS08.06. Impact of mesenchymal stromal/stem cell isolation strategies on the immunomodulatory activity of their extracellular vesicles

Oumaima Stambouli, Institute for Transfusion Medicine

Robin Dittrich, Institute for Transfusion Medicine, University Hospital Essen, Germany

Fabiola Nardi Bauer, Institute for Transfusion Medicine, University Hospital Essen, Germany

Tobias Tertel, Institute for Transfusion Medicine, University Hospital Essen, Germany

Peter A. Horn, Institute for Transfusion Medicine, University Hospital Essen, Germany

Bernd Giebel, Prof, Institute for Transfusion Medicine, University Hospital Essen, Germany

Introduction: Mesenchymal stromal cell (MSC) derived extracellular vesicles (EVs) are increasingly considered as therapeutic agents. In our hands, only a proportion of obtained MSC‐EV preparations revealed immunomodulatory activities, both, in murine acute Graft‐versus‐Host Disease and in a multi‐donor mixed lymphocyte reaction assay (mdMLR). According to our understanding, variations in the MSC‐EV preparations’ activities are due to the heterogeneity of their parental MSCs. Here, we aimed to study impacts of the initial growth conditions on human bone marrow (BM)‐derived MSCs and their resulting EV products.

Methods: MSCs were either raised from BM aspirates or from mononuclear cells (MNCs) harvested thereof. BM aspirates and MNCs were seeded into DMEM low media supplemented with human platelet lysate (hPL) as well as in EBM media supplemented with cytokines and hPL. Non‐adherent cells were removed after 24h and cultures continued in hPL supplemented DMEM low. Growth rates, cell surface phenotypes and the osteogenic and adipogenic differentiation capabilities of obtained MSCs were analyzed. Starting from passage 1, conditioned media (CMs) were harvested every 48 hours and stored at ‐20°C until processing. After thawing, EVs were prepared from CMs applying the PEG‐ultracentrifugation method. Obtained samples were characterized according to the MISEV criteria and by imaging flow cytometry. Their immunomodulatory activities were investigated in the mdMLR assay.

Results: The initial culture conditions affect the growth rates and phenotypes of MSCs. First results imply, aspirate‐derived MSCs grow slower, reach senescence quicker and expressed less CD59 on their EVs than those derived from MNC‐derived MSCs. MSCs initially raised in EGM revealed higher expansion capacities than MSCs directly raised in DMEM low. Although, all MSC‐EV preparations harvested from early passage CMs revealed immunomodulatory activities, they showed different impacts on macrophages, which we are currently dissecting.

Summary/Conclusion: Our preliminary data imply initial seeding strategies impact the characteristics of obtained MSCs and the immunomodulatory activity of their EV products. However, more experiments need to be performed to obtain robust data.

PS08.07. Electrospun scaffolds as novel platforms for sustained release of extracellular vesicles for cell‐free tissue engineering

Hatim Alqurashi, 1‐ University of Sheffield 2‐ King Faisal University

Stuart Hunt, School of Clinical Dentistry, University of Sheffield, Sheffield, UK

Ilida Ortega Asencio, School of Clinical Dentistry, University of Sheffield, Sheffield, UK

Daniel Lambert, School of Clinical Dentistry, University of Sheffield, Sheffield, UK

Introduction: Extracellular vesicles (EVs) are membrane‐enclosed vesicles that are secreted by cells and mediate cell"cell communication via their protein, lipid, carbohydrate, and nucleic acid (RNA, DNA) cargo. EVs are involved in a multitude of physiological processes including development, cell differentiation and angiogenesis having also been associated with tissue repair. Thus, they have been suggested to offer opportunities for the development of novel cell‐free tissue engineering (TE) approaches.

Methods: Method: EVs were isolated from the conditioned media of cells in culture using ultracentrifugation (UC) and size exclusion chromatography (SEC). After characterising the size and protein abundance of EVs, they were incorporated into polycaprolactone (PCL) electrospun scaffolds. EV‐modified scaffolds were characterized using SEM, TEM and fluorescence microscopy. The release kinetics of EVs from scaffolds was examined using nanoparticle‐tracking analysis (NTA). The influence of myofibroblast‐derived EVs on cell growth and migration was studied using scratch assays.

Results: Result: EVs were successfully isolated by UC and SEC as assessed by NTA and determination of presence of EV marker proteins (CD9, CD63 and CD81). Following different methods of functionalization of PCL scaffold, EVs were well‐incorporated and distributed within scaffolds. NTA showed slow release of 40% of EVs from the scaffolds over 21 days. Myofibroblast‐derived EVs (myo‐EV) significantly increased fibroblast cell growth and migration compared to TGFbeta 1‐treated cells and untreated controls.

Summary/Conclusion: Conclusion: Here we provide evidence that electrospun scaffolds can be functionalised with EVs and provide sustained slow release, offering an opportunity to develop novel, cell‐free and tuneable approaches to tissue engineering. We also showed that myo‐EV may have a role in wound healing, and that incorporation of myo‐EVs in electrospun scaffolds may have potential as a regenerative medicine approach.

PS08.08. Mesenchymal Stromal Cell‐derived Extracellular Vesicles restore alteration of endoplasmic reticulum stress genes in Human Corneal Endothelial Cells

Lola Buono, University of Turin, Department of Molecular Biotechnology and Health Sciences, Italy

Raffaele Nuzzi, S.C.U. Ophthalmology Unit, “City of Health and Science”

Marco De Iuliis, S.C.U. Ophthalmology Unit, “City of Health and Science”

Simona Scalabrin, S.C.U. Ophthalmology Unit, “City of Health and Science”

Benedetta Bussolati, University of Turin, Department of Molecular Biotechnology and Health Sciences, Italy

Introduction: Human Corneal Endothelial Cells (HCECs) are essential to visual function, however, since they have limited proliferative capacity in vivo, they are prone to corneal endothelial dysfunction. The aim of this work was to evaluate whether EVs derived from Mesenchymal Stromal Cells (MSC‐EVs) were able to promote regeneration of HCECs after exposing them to endoplasmic reticulum stress (ER‐stress) and the mechanisms involved.

Methods: We isolated HCECs from discarded corneas in patients undergoing corneal transplantation (n = 23 patients). Human bone marrow MSCs were obtained from Lonza, cultured and characterized. MSC‐EVs were obtained from supernatants of MSCs. We evaluated the proliferation rate, apoptosis and migration of HCECs after exposure to nutrient deprivation or Tunicamycin. We then evaluated the regulation of ER‐stress‐related genes, such as CHOP, ATF4, EIF2a, BIM, XBP1, BCLXL in presence or in absence of MSC‐EVs and compared their effect with a different source of EVs, derived from patient blood serum (SER‐EV).

Results: In the selected damage conditions, the treatment with different doses of MSC‐EVs resulted in a significantly higher proliferation rate of HCECs at all the tested concentrations of EVs and in the decrease of total apoptotic cells. Moreover, MSC‐EVs induced a faster repair of the wound after 24 hours of serum‐deprivation. At molecular level, we observed an upregulation of the ER stress‐related genes ATF4, CHOP, BIM, XBP1 during HCECs damage, and a significant down‐regulation of their expression after the treatment with MSC‐EVs in all the genes tested. SER‐EVs were able to down‐regulate significantly the expression of ATF4, but not the other genes studied.

Summary/Conclusion: Our results highlight the well‐known pro‐regenerative potential of MSC‐EVs and bring to light their ability to restore HCECs from ER‐stress induced by nutrient deprivation.

PS08.09. Epigenetic reprogramming enhances the therapeutic efficacy of osteoblast‐derived extracellular vesicles for bone regeneration

Kenny Man, University Of Birmingham

Mathieu Brunet, School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom.

Maria Fernandez‐Rhodes, School of Sport, Exercise and Health Sciences, Loughborough University, Loughborough, United Kingdom.

Soraya Williams, School of Sport, Exercise and Health Sciences, Loughborough University, Loughborough, United Kingdom.

Owen Davies, School of Sport, Exercise and Health Sciences, Loughborough University, Loughborough, United Kingdom.

Angelica Federici, Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland.

David Hoey, Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland.

Sophie C. Cox, School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom.

Introduction: For bone regeneration, there is great precedence to develop acellular technologies that circumvent limitations associated with cell‐based therapies. Extracellular vesicles (EVs) have the potential to stimulate stem cell mineralisation due to their diverse cargo offerings compared to single growth factor treatments. Regulating the cell's epigenetic function through histone deacetylase (HDAC) inhibition increases their differentiation potential. Therefore, we investigated altering osteoblasts epigenome via the HDAC inhibitor Trichostatin A (TSA) on promoting osteoblast‐derived EVs potency.

Methods: TSA effect on osteoblast epigenetic functionality and mineralisation was determined by quantifying HDAC activity and calcium deposition. EVs were isolated from untreated/TSA treated osteoblasts for 2 weeks. EV size and concentration were defined using nanoparticle tracking analysis and transmission electron microscopy. EVs microRNA expression was evaluated using microarray analysis. Osteogenic differentiation of human bone marrow stromal cells (hBMSCs) cultured with untreated (MO‐EVs)/TSA treated osteoblast‐derived EVs (TSA‐EVs) was evaluated by qPCR, biochemistry and histological analysis.

Results: TSA significantly reduced osteoblast HDAC activity and enhanced calcium deposition when compared to untreated cells. The quantity of EVs generated, in addition to their protein content and size correlated with the degree of osteoblast differentiation. TSA‐EVs accelerated hBMSCs migration and proliferation compared to MO‐EVs. Importantly, TSA‐EV treatment significantly upregulated hBMSCs osteoblast‐related gene/protein expression (ALP, Col1a, OCN) and promoted extracellular matrix mineralisation when compared to MO‐EVs. Microarray analysis revealed TSA‐EV enriched microRNAs were involved in regulating mechanisms such as “endocytosis” and “Wnt signalling pathway”.

Summary/Conclusion: Taken together, these findings demonstrate the considerable utility epigenetic reprogramming provides a novel engineering approach to enhance EVs therapeutic efficacy for bone regeneration.

PS08.10. Extracellular vesicles derived from induced pluripotent stem cells in hypoxic conditions enhance angiogenesis

André Cronemberger Andrade, Paracelsus Medical University

Martin Wolf, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI‐TReCS), Paracelsus Medical University (PMU), Salzburg, Austria

Fausto Gueths Gomes, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg

Patricia Ebner, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI‐TReCS), Paracelsus Medical University (PMU), Salzburg, Austria

Heidi Marie Binder, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI‐TReCS), Paracelsus Medical University (PMU), Salzburg, Austria

Katarina Schallmoser, Department of Transfusion Medicine and SCI‐TReCS, PMU, Salzburg, Austria

Strunk Dirk, Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI‐TReCS), Paracelsus Medical University (PMU), Salzburg, Austria

Introduction: Stem cells secrete paracrine factors including EVs that are important in cellular communication and can support the regeneration of injured tissues. Reduced oxygen conditions (hypoxia) are crucial for proliferation and self‐renewal of stem cells. As hypoxia is a key regulator in development and regeneration it may also be an important factor influencing cellular communication via EVs. Therefore we investigated whether hypoxic pre‐conditioning can impact iPSC‐EV quantity and/or quality (phenotype and cargo) & thus have an impact for EV‐based therapy.

Methods: We produced iPSC EVs using tangencial fow filtration (TFF) from iPSC conditioned media from diferent oxygen levels conditions. The EVs were quantified by Tunable Resistive Pulse Sensing (TRPS). The protein content was quantified and analyzed by immunoblotting for EV markers. In addition to check if the cells were effetely under hypoxia conditions using a hypoxia marker (pimonidazole) we also checked the HIF‐1α expression by immunoblotting. The viability of the cells cultivated under different oxygen levels conditions were checked staining with 7AAD and Annexin V. The functionality of the EVs derived from different oxygen conditions were checked by Angioassay.

Results: The method using TFF to concentrate the condition media is a good strategy to produce a large amount of EVs with good recovery. A higher pimonidazole positive signal was found in the cells under 1% oxygen compared to the cells on 5% and 18% oxygen conditions. HIF‐1α was incresed in 1% oxygen condition compared with 5%, 18% and the hypoxia mimetic chemical CoCl2 (positive control). In all conditions we didn't find any loss on viability.The EV derived from 1% oxygen increased the tube like formation compared with the other vesicles and also the soluble factors.

Summary/Conclusion: The EVs derived from iPSC preconditioned in hypoxia condition showed a enhance angiogenesis pontecial. This strategy could be an important tool in the production of particularly potent and targeted EV‐based therapeutics.

PS08.11. The immune‐regulatory properties of extracellular vesicles derived from human mesenchymal stem/stromal cells

Dimitrios Tsiapalis, School of Pharmacy and Pharmaceutical Sciences & Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland

Tobias Tertel, Institute for Transfusion Medicine, University Hospital Essen, Germany

Achilleas Floudas, Molecular Rheumatology, Trinity College Dublin, Ireland

Ursula Fearon, Molecular Rheumatology, Trinity College Dublin, Ireland

Bernd Giebel, Prof, Institute for Transfusion Medicine, University Hospital Essen, Germany

Verena Börger, Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg Essen, Essen, Germany

Lorraine O'Driscoll, School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, Trinity St. James's Cancer Institute & TRAIN‐EV Marie Skłodowska‐Curie Action‐Innovative Training Network, train‐ev.eu

Introduction: Extracellular vesicles (EVs) from human bone‐marrow mesenchymal stem/stromal cells (BM‐MSCs) are of much interest because they may exert therapeutic effects, such as modulating immune response at a site of injury. However, a reliable potency assay for evaluating immune‐modulatory effects of EVs has yet to be established. Towards achieving this, here we isolated and characterised EVs from MSCs cultured under different conditions and assessed their immuno‐regulatory properties, in term of influence on inflammatory cytokines release from CD4+ T‐cells derived from peripheral blood mononuclear cells (PMBCs) of rheumatoid arthritis (RA) patients.

Methods: BM‐MSCs were cultured in T175‐flasks in media supplemented with either human platelet lysate (hPL) or EV‐depleted FBS. After 48h, conditioned media was collected. EVs were isolated using PEG precipitation and characterised using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), immunoblotting, and flow‐imaging (Amnis ImageStream). Flow cytometry (LSRFortessa) analysed a panel of cytokines released from CD4+ T‐cells exposed to the MSC‐EVs.

Results: NTA indicated significantly more EVs/particles released when MSCs were cultured with hPL‐supplemented medium versus medium with EV‐depleted FBS. TEM revealed spherical‐shaped EVs, with both cultures. Immunoblots showed the presence of EV‐associated proteins CD9, CD63, CD81, CD105 and Syntenin‐1 and the absence of Calnexin. Supporting these findings, flow‐imaging showed increased numbers of CD9+, CD63+ and CD81+ positive sEVs when MSCs were cultured in hPL‐medium. Preliminary data demonstrated that the MSC‐EVs modulate (typically suppressing) the release of four cytokines that are associated with RA pathogenesis, i.e. TNFα, IL‐22, GM‐CSF and IL‐2.

Summary/Conclusion: These findings highlight the immuno‐regulatory potential of MSC‐EVs. Further studies validating immune‐modulatory effects of MSC‐EVs on CD4+ T‐cells are underway.

PS08.12. Comparative analysis of extracellular vesicles (EVs) derived from adipose tissue‐ and bone marrow‐ derived mesenchymal stromal cells

Cansu Gorgun, University of Genova

Maria Elisabetta Federica Palamà, Department of Experimental Medicine (DIMES), University of Genoa, Italy

Daniele Reverberi, U.O. Molecular Pathology, IRCCS Policlinico San Martino, Genoa, Italy

Roberta Tasso, University of Genova

Chiara Gentili, Department of Experimental Medicine (DIMES), University of Genoa, Italy

Introduction: Mesenchymal stromal cells (MSCs) isolated from different tissue origins already present variations in their secretory profile. Since EVs from different origin contain diverse contents and exert different functions, some studies have come up with the comparison of exosomes from adipose tissue‐ and bone marrow derived MSCs.Yet, these studies were limited for considering only one type of EV subpopulation. Considering whether an EV‐dependent function is specific to a given EV subtype, in this study, we carried out a detailed and comparative characterization of middle‐sized and small‐sized EVs released by both adipose‐tissue (AD)‐ and bone marrow(BM)‐MSCs to investigate their involvement as modulators of MSC paracrine effects.

Methods: EVs have been separated from the conditioned media of AD‐ and BM‐MSCs by serial differential centrifugations in order to collect both medium‐sized and small‐sized EVs. EVs were characterized by transmission electron microscopy, tunable resistive pulse sensing and western blot. The expression of the tetraspanin family members was evaluated using a non‐conventional flow cytometry approach. The role of both medium and small‐sized EVs derived from AD and BM in the different phases of the endochondral ossification process has been evaluated by ex vivo mouse metatarsal culture model that allows for the study of both vessel sprouting and linear bone growth.

Results: Although EVs derived from adipose tissue‐ and bone marrow derived MSCs present similar characteristics in terms of size, concentration and marker expression, they possess significant differences in their protein content that are reflected in their functional effects. The comparison of proteins with a wide range protein array showed that EVs derived AD‐MSCs contained pro‐ angiogenic factors in comparison to the BM counterpart. As a consequence, they were able to induce a significant increase in vessel sprouting. On the other hand, EVs derived from the bone marrow contained a higher amount of pro‐differentiation and chemotactic proteins in comparison to the adipose tissue MSC‐EVs, and they were able to prompt growth plate morphology. Interestingly, during these comparisons of functional effects, even though that same concentration of EVs applied, small sized EVs had a significant effect in comparison with their counterpart middle‐sized EVs.

Summary/Conclusion: In conclusion, this study highlights the importance of selecting the appropriate MSC‐source for EV‐ based therapies for targeted therapeutic applications. Indeed, in the context of endochondral ossification, EVs from both ADSCs and BMSCs have diverse functional effects. Considered these EV properties of the EVs mirroring the parental cells, further study of the synergistic effect of both MSC‐ derived EVs would be great interest.

PS08.13. Chitin‐based polysaccharides enhance the immunomodulating potential of human mesenchymal stem cell‐derived extracellular vesicles

Eun Seo Kim, Seoul National University

Katsuhiko Kida, Nissan Chemical Corporation

Jongsoo Mok, Seoul National University

Yeonwoo Seong, Seoul National University

Seo Yeon Jo, Seoul National University

Tatsuro Kanaki, Nissan Chemical Corporation

Masato Horikawa, Planning and Development Division

Kyung‐Hee Kim, National Cancer Center

Tae Min Kim, Seoul National University

Tae Sub Park,Seoul National University

Joonghoon Park, Seoul National University

Introduction: Mesenchymal stem cell (MSC) transplantation is a promising treatment in regenerative medicine. However, MSCs grown in two‐dimensional (2D) culture conditions vary significantly in cell shape from in vivo, with downregulated stemness genes and secretion of paracrine factors.

Methods: Here, we evaluated the effect of three‐dimensional (3D) culture with chitin‐based, water‐insoluble polysaccharides on the characteristics of human Wharton's jelly‐derived MSCs (hMSCs).

Results: After 3D culture of hMSCs with the chitin‐based polysaccharides, the retrieved cells showed significantly improved cell proliferation rate compared with the conventional 2D counterpart. Transcriptome analyses indicated that 3D culture enhanced gene expression involved in cell stemness, migration ability, and extracellular vesicle (EV) production. Subsequent biochemical studies showed that expression levels of OCT4, NANOG, and SSEA4 were upregulated and migration ability was increased in hMSCs grown under 3D culture conditions. In addition, EV production was significantly increased in 3D cells compared to 2D cells, and EVs from 3D cells showed to have a differentiated protein profile compared to 2D EVs. Analyses of gene and drug connectivity revealed that 2D and 3D EVs had similar functions as immunomodulators. However, 3D EVs had entirely different therapeutic profiles based on activation of the disease‐associated signaling pathways for different infectious and metabolic diseases. Transcriptome responses after cell treatment revealed that fenbendazole, an immunomodulator, formed the same cluster as 3D EVs, and 2D EVs belonged to the same cluster as vehicle control. Therefore, through the similarity analysis of the global transcriptome response, it was confirmed that 3D EVs induces a mostly similar pharmacological response to fenbendazole.

Summary/Conclusion: The pharmacological mechanisms and predicted applications based on Connectivity map analyses supported the high therapeutic potential of 3D EVs for certain conditions. Therefore, EVs from 3D cultured hMSCs using chitin‐based polysaccharides can be applied as novel therapeutic agents for treatment of immune and metabolic diseases.

PS08.14. Investigating the functionality of miRNAs in Wharton's Jelly‐derived small extracellular vesicles (sEV) and their potential role in neuro‐regeneration

Vera Tscherrig, Department for BioMedical Research (DBMR), Graduate School for Cellular and Biomedical Sciences (GCB), and Department of Obstetrics and Feto‐Maternal Medicine, Inselspital

Sophie Cottagnoud, Department of Obstetrics and Feto‐maternal Medicine, University Women's Hospital, Inselspital, Bern University Hospital, Bern, Switzerland

Valérie Haesler, Department of Obstetrics and Feto‐maternal Medicine, University Women's Hospital, Inselspital, Bern University Hospital, Bern, Switzerland

Patricia Renz, Department of Obstetrics and Feto‐maternal Medicine, University Women's Hospital, Inselspital, Bern University Hospital, Bern, SwitzerlandGraduate School for Cellular and Biomedical Sciences (GCB), University of Bern, Bern, Switzerland, and

Daniel Surbek, Department of Obstetrics and Feto‐maternal Medicine, University Women's Hospital, Inselspital, Bern University Hospital, Bern, Switzerland

Andreina Schoeberlein, Department of Obstetrics and Feto‐maternal Medicine, University Women's Hospital, Inselspital, Bern University Hospital, Bern, Switzerland

Marianne Jörger‐Messerli, Department of Obstetrics and Feto‐maternal Medicine, University Women's Hospital, Inselspital, Bern University Hospital, Bern, Switzerland

Introduction: Perinatal white matter injury (WMI) is one of the most common neurological complications of preterm birth and it is a global health problem resulting in long‐term neurodevelopmental and neurobehavioral disabilities. Until now there is no cure for perinatal WMI. Recently, our lab and others have shown promising results towards the use of mesenchymal stromal cell derived small extracellular vesicles (MSC‐sEV) as a therapeutic approach for neuronal injuries. It is known that sEV secreted by MSC carry small non‐coding RNA such as microRNAs (miRNAs). MicroRNAs might interfere with signaling pathways involved in premature WMI. Thus, we hypothesize that miRNAs, released by sEV upon uptake in their target cells, have a key function in the observed beneficial effects of MSC‐sEV.

Methods: MSC were isolated from the connective tissue of human umbilical cords, the so‐called Wharton's jelly. sEV were purified from the cells using ultracentrifugation, followed by size‐exclusion chromatography (SEC). The fractions were characterized according to the expression of sEV markers using western blot analysis and miRNAs by quantitative PCR.

Results: The SEC fractions with the highest protein content also showed positive signals for the sEV markers CD81 and CD63. No cellular contamination has been observed (no signal for GM130 or Grp94). These sEV fractions contained high amounts of miRNAs, such as miRNA 22–5p, miRNA 27b‐3p or let7b‐5p.

Summary/Conclusion: The highly abundant miRNAs in the sEV fractions target specific apoptotic or inflammatory pathways and drive oligodendrocyte differentiation. Therefore, these miRNAs might influence WMI outcomes. For better understanding of this hypothesis, the sEV fractions, containing the most abundant miRNAs, are currently tested for their functionality using dual luciferase assays.

PS08.15. Matrix‐bound vesicles within extracellular matrix in the regulation of mesenchymal stem cells differentiation in vitro

Ekaterina Novoseletskaya, Institute for regenerative medicine, Medical research and education center, Lomonosov Moscow State University

Daria Selina, Faculty of Biology, Lomonosov Moscow State University

Nataliya Basalova, Institute for regenerative medicine, Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russia

Yana Danilova, Faculty of Biology, Lomonosov Moscow State University

Olga Sokolova, Faculty of Biology, Lomonosov Moscow State University

Anastasia Efimenko, Institute for regenerative medicine, Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russia

Introduction: Extracellular matrix (ECM) regulates various cell functions, including stem cell differentiation, but little is known about the impact of matrix‐bound vesicles (MBVs) into these effects. Mesen‐chymal stem/stromal cells (MSCs) are critical contributors of tissue renewal and repair pro‐cesses due to the secretion of paracrine substances that regulate the homeostasis of tissue‐specific stem cell niches. We evaluated how MBVs within the native ECM produced by MSC could affect the behavior of stem cells.

Methods: ECM was obtained from cell sheets of immortalized MSCs by decellularization (dECM) using CHAPS and DNase type I treatment, and vesicle‐like structures in dECM were observed using scanning electron microscopy (SEM) and confirmed by transmission electron microscopy (TEM). We compared 3 approaches to isolate MBVs by treating dECM with 1) collagenase I type, 2) collagenase I type and hyaluronidase, 3) trypsin. Isolated MBVs were visualized by TEM and characterized for the presence of key exosome markers by immunoblotting. Then we explored the effects of MBVs on human adipose‐derived MSC trilineage differentiation (adipo‐genic, osteogenic or chondrogenic) by the addition of isolated MBVs or their removal from dECM by phospholipase, isopropanol, or RNase treatment.

Results: We confirmed that MBVs were comprised of MSC‐produced ECM and could be effectively iso‐lated or removed from dECM by all tested protocols. No significant effect on MSC differentia‐tion was detected after the treatment of dECM with RNase, while isopropanol treatment led to the slight decrease of MSC osteogenic and chondrogenic differentiation and isolated MBVs stimulated MSC adipogenic differentiation.

Summary/Conclusion: We assume that MSC‐produced ECM includes MBVs which could regulate the functional activity of stem cells interacted with ECM. Further research is needed to explore if MBVs in MSC‐produced ECM represent a specific class of extracellular vesicles.

PS09.01. 3D‐Printed Gelatin Methacrylate Hydrogel for Sustained Release of MSC EVs

Louis J. Born, University of Maryland

Shannon McLoughlin, University of Maryland

Bhushan Mahadik, University of Maryland

John Fisher, University of Maryland, College Park

Steven M. Jay, University of Maryland, College Park

Introduction: Complex wounds, resulting from disease, trauma, or surgical intervention, remain a major source of morbidity; thus, continued effort to develop new wound therapies is necessary. Mesenchymal stem/stromal cell (MSC) extracellular vesicles (EVs) are one avenue of investigation that have shown a therapeutic benefit in wound healing, particularly by inducing angiogenesis. However, rapid clearance of EVs from the body after a single, naked administration is one limitation hindering clinical translation. We hypothesize that a customizable gelatin methacrylate hydrogel using 3D‐printing will allow for the sustained release of MSC EVs within a therapeutic window.

Methods: Gelatin methacrylate (GelMa) hydrogels will be 3D‐printed as both homogenous and 3D‐concentric discs using MSC EV‐loaded GelMa as a bioink. Crosslinking density will be varied based on amount of LAP photoinitiator added to the GelMa solution, and ultraviolet radiation will be used to crosslink the gelatin methacylate. Hydrogels will be placed in PBS, and PBS will be collected and replenished periodically over time. EVs will be concentrated using 300kDa nanoseps and resuspended in equal volumes of PBS. EVs released will be measured using BCA and an ELISA for exosomal marker CD63. An endothelial scratch assay will be used to test for retained bioactivity of EVs.

Results: MSC EVs were successfully loaded into GelMa, 3D printed into discs, and crosslinked by UV irradiation. Initial release studies of a homogenous disc resembled a burst release profile using BCA and CD63 ELISA. EVs released from the discs within a 24h period showed retained bioactivity in a human umbilical cord endothelial cell (HUVEC) scratch assay. HUVECs treated with GelMa‐released EVs in basal media had increased gap closure compared to basal media alone (p < 0.05).

Summary/Conclusion: Release of bioactive MSC EVs from 3D‐printed GelMa is achievable and utilizing such a printing method may allow for the tailoring of release profiles for optimized wound healing.

PS09.02. Extracellular vesicles from Red blood cells Modified with gp350 protein deliver therapeutic cargo for Targeting Therapy of Burkitt lymphoma

Huiqing Xiu, Zhejiang University School of Medicine

Introduction: The feasibility of extracellular vesicles (EVs) as a drug carrier has been verified countlessly. For clinical treatment, the source of EVs is an important issue to be considered. At present, most of the sources of EVs in literature are cell lines, but cells secrete a small amount of EVs, and the EVs from which they originate may carry carcinogenic DNA and other harmful substances. A potential ideal source of EVs for therapeutic use may be human red blood cells.: 1) Red blood cells lack of nuclear DNA and mitochondrial DNA; 2) Red blood cells are the most abundant cell type in the body; 3) Red blood cells can be easily obtained from the human body and have been used safely and routinely for decades For blood transfusion. In addition, another problem with drug‐loaded treatment of EVs is its targeting. Most of the EVs that have not been modified will accumulate in the liver tissue. At present, most of the modification methods are by overexpression of the plasmid into the cell line, collecting the supernatant after the overexpression of the plasmid, and extracting EVs. However, this method is time‐consuming and labor‐intensive, and is not suitable for primary cells or cells that are difficult to overexpress. Therefore, we need to explore new ways to modify EVs.

Methods: 1. Purify gp350 protein with transmembrane segment using prokaryotic system.

2. Extract human blood, separate red blood cells, incubate overnight with calcium ionophore, and extract EVs derived from red blood cells after gradient high ionization and ultraionization.

3. Red blood cells‐derived EVs are mixed with doxorubicin and electroporated to load the drug.

4. The EVs derived from red blood cells are incubated with the purified gp350 protein with transmembrane segments and electroporated at low voltage.

Results: 1. Gp350‐modified 293T‐derived EVs can target CD21 positive tumor cells in vitro

2. The gp350 protein was successfully loaded into the red blood cell‐derived EVs membrane by low voltage electroporation

3. Gp350‐modified red blood cell‐derived EVs can target CD21 positive tumor cells, loaded with chemotherapeutic drugs have increased cytotoxicity in CD21+ tumor

4. Gp350‐modified red blood cell‐derived EVs loaded with chemotherapeutic drugs have high biosafety

Summary/Conclusion: First, we found that gp350 modified EVs can effectively target CD21 positive tumor cells. We purified the gp350 protein and successfully loaded it into the red blood cell‐derived EVs membrane by co‐incubation and low‐voltage electroporation (termed RBC‐EVs/gp350), which effectively targeted CD21 positive tumors. The RBC‐EVs/gp350 loaded with chemotherapeutic drug showed high toxicity to CD21 positive tumors and have high biosafety. In conclusion, we developed a new method for modifying extracellular vesicles.

PS09.03. Hybrid nanoconstructs for cancer therapy based on zinc oxide nanocrystals shielded by extracellular vesicles

Bianca Dumontel, Department of Applied Science & Technology, Politecnico di Torino

Francesca Susa, Department of Applied Science & Technology, Politecnico di Torino

Tania Limongi, Department of Applied Science & Technology, Politecnico di Torino

Luisa Racca, Department of Applied Science & Technology, Politecnico di Torino

Nadia Garino, Department of Applied Science and Technology, Politecnico di Torino

Doriana Debellis, Electron Microscopy Facility, Fondazione Istituto Italiano di Tecnologia Genova

Roberto Marotta, Electron Microscopy Facility, Fondazione Istituto Italiano di Tecnologia Genova

Valentina Cauda, Department of Applied Science & Technology, Politecnico di Torino

Introduction: Thanks to peculiar properties connected with their biological origin and function, extracellular vesicles (EVs) are emerging as promising tools in drug delivery. This study focuses on the encapsulation of zinc oxide nanocrystals (ZnO NCs) in cell‐derived EVs for the creation of a hybrid nanoconstruct for the treatment of cancer cells, which will combine the cytotoxic potential of ZnO nanostructures with the stability and biomimicry imparted by EVs‐shielding.

Methods: ZnO NCs synthesized by a microwave‐assisted synthesis were combined with EVs extracted form cell culture supernatants of B‐lymphocytes through a differential ultracentrifugation protocol. The encapsulation was obtained through an active loading method, based on the application of freeze‐thaw cycles followed by co‐incubation steps. The nanoconstructs were characterized by fluorescence microscopy, transmission electron microscopy, nanoparticle tracking analysis and flow‐cytometry.

Results: The encapsulation of ZnO NCs in EVs was successfully achieved, obtaining hybrid nanoconstructs characterized by promising loading efficiency. The optimization of the number of applied freeze‐thaw cycles and of the duration of the subsequent incubation steps allowed the production of loaded‐EVs characterized by a well‐preserved morphology and surface protein expression. The greater colloidal stability in biological media of the hybrid nanoconstructs with respect to pristine ZnO was also assessed.

Summary/Conclusion: In this study, we show the successful encapsulation of ZnO NCs in cell‐derived EVs for the creation of a novel therapeutic nanoconstruct. The EVs‐shielding efficiently stabilizes the ZnO NCs in biological environment and would also improve their biocompatibility and capability to interact with cells, proposing a strategy for the development of more reliable and effective tools for the treatment of cancer cells.

PS09.04. In vitro homing and targeting capabilities characterization of native and engineered lymphocytes‐derived extracellular vesicles

Francesca Susa, Department of Applied Science & Technology, Politecnico di Torino

Tania Limongi, Department of Applied Science & Technology, Politecnico di Torino

Bianca Dumontel, Department of Applied Science & Technology, Politecnico di Torino

Luisa Racca, Department of Applied Science & Technology, Politecnico di Torino

Valentina Cauda, Department of Applied Science & Technology, Politecnico di Torino

Introduction: In this study the homing and targeting capabilities of native (nEVs) and engineered (eEVs) extracellular vesicles (EVs) were investigated to develop new therapeutic/theranostic strategies. The targeting efficiency towards lymphocytes, myeloid (HL60) and lymphoid (Daudi) cell lines was evaluated by anti‐CD20 engineering the EVs.

Methods: Lymphocytes‐derived EVs were isolated through a differential ultracentrifugation protocol and characterized by nanoparticle tracking analysis, bradford assay. and flow cytometry (FC) analysis. The cytotoxic effect and the internalization capabilities of both nEVs and eEVs were tested after 24 and 48 hours treatments using WST‐1 assay, FC and fluorescence microscopy (FM).

Results: Results evidenced that EVs isolated from healthy cells are fully tolerated and characterized by having an exceptional tropism towards the parental cell line and the two tested cancer cell lines. FC and FM showed a significantly higher internalization of nEVs in lymphocytes and Daudi cells than in HL60, while, by anti‐CD20 engineering EVs, it was possible to successfully tune EVs tropism towards the target Daudi cells.

Summary/Conclusion: In this study the innate tropism of nEVs and the active heterologous targeting of eEVs were compared and eEVs showed a significant ability to selectively target the Daudi cancer cell line.

These EVs showed a high potential for the design of a new artificial or hybrid