Extracellular Vesicles 2022: Biology, Disease & Medicine Agenda

Conference Registration, Materials Pick-Up, Morning Coffee and Tea

exRNA Analysis In Biofluids to Differentiate Diseases and Injuries of the Nervous System
Kendall Van Keuren-Jensen, Professor and Deputy Director, Translational Genomics Research Institute, United States of America

Our laboratory characterizes the exRNA cell-free and EV cargo in biofluids for the information they can provide about changes associated with disease, injury processes and progression.

Plasma Extracellular Vesicles For Cardiovascular Disease
Dominique PV de Kleijn, Professor Experimental Vascular Surgery, Professor Netherlands Heart Institute, University Medical Center Utrecht, The Netherlands, Netherlands

Cardiovascular Disease (CVD) is with the cardiovascular events of Ischemic Heart Disease and Stroke, the number 1 and 2 cause of death in the world and expect to increase especially in Asia. We use plasma extracellular vesicle (EV) protein content of vesicles from plasma sub-fractions on plasma of stroke and peripheral artery disease(PAD) patients, patients after carotid atherectomy (CEA) and patients suspected for chronic coronary syndrome (CCS). Using 25 ul of plasma, we developed an automated 96-well based protocol using sequential precipitation. Using samples of the AtheroExpress, the largest ongoing CEA biobank we try to early detect the risk of a second Major Adverse Cardiovascular Event (MACE: myocardial infarction, stroke or cardiovascular death) in PAD and CEA patients. Identification of such high risk patients is very important for possible (expensive) add-on pharmaceutical therapy or the decision to operate or not. Sequential precipitation for EV isolation is also used for the diagnosis of CCS.

Using Neuronal Enriched Extracellular Vesicles To Determine Brain Health
Lynn Pulliam, Professor of Laboratory Medicine and Medicine, University of California San Francisco, United States of America

Circulating plasma EVs can be captured by a neuronal surface protein (L1CAM) and the cargo interrogated to determine the health of neurons in real time. We have used protein arrays to look at neuronal EV cargo in people with HIV or post-COVID cognitive impairment.

Mid-Morning Coffee, Tea and Networking in the Exhibit Hall

Next generation nanoFACS with High Resolution Imaging and Custom 50um Nozzle
Terry Morgan, Professor of Pathology and Laboratory Medicine, Oregon Health and Science University, United States of America

Nanoscale flow cytometry is an emerging technology designed to image, count, and isolate cell- and size-specific extracellular vesicles (EVs). More efficient approaches to nanoscale flow sorting (nanoFACS) are required for sufficient EV yield per volume for validation studies of sorting specificity and more precise characterization EV protein and nucleic acid contents.

Digital Flow Cytometry For Single-EV Analysis
Daniel Chiu, A. Bruce Montgomery Professor of Chemistry, University of Washington, United States of America

We have developed a multi-parametric high-throughput flow-based method for the analysis of individual extracelluar vesicles (EVs), which are highly heterogeneous and comprise a diverse set of surface protein markers as well as intra-vesicular cargoes. Yet, current approaches to the study of EVs lack the necessary sensitivity and precision to fully characterize and understand the make-up and the distribution of various EV subpopulations that may be present. Digital flow cytometry provides single-fluorophore sensitivity to enable phenotyping single EVs with unprecedented precision and sensitivity.

Networking Lunch in the Exhibit Hall with Exhibitors and Conference Sponsors

Trends in EV Characterization Technologies
Jean-Luc Fraikin, CEO, Spectradyne

Methods for characterizing EVs are evolving, and new technologies are being developed that deliver size, concentration, and fluorescent phenotyping, each to a varying degree of success.  Learn about how some of these new and cutting-edge technologies work, their strengths and weaknesses, and where we at Spectradyne see the technology landscape moving next.

Paving the Way to Automation and Standardization for Scalable Isolation of EVs
Jared Lynch, Director of Business Development, IZON Science

As the potential for EV-based diagnostics and therapeutics continues to grow, so too does the need for scalable isolation and precise characterization. In this presentation, I will present how technologies and services offered by Izon Science fill this gap. I will introduce Izon’s newly released Gen 2 qEV SEC isolation product line as well as our recent work on scaling up EV isolation and purification using larger sized qEV columns with automated flow systems. Finally, you will be taken through an overview of Izon platforms and offerings, to gain a clear understanding of how Izon technologies can fit into a larger workflow.

Biomarker Co-localization Measurements (C-NTA) Using the New Particle Metrix ZetaView x30 Family
Sven Rudolf Kreutel, Chief Executive Officer, Particle Metrix GmbH and CEO, Particle Metrix Inc., USA

During the last decades, Nanoparticle Tracking Analysis (NTA) has emerged as a vital and fast characterization technology for biological nanoparticles like Extracellular Vesicles, Exosomes and Viruses. While classic NTA scatter operation feeds back particle size and total concentration, the fluorescence detection capability enables the user to gain specific biochemical information. Determination of biomarker co-localization however, is a challenging task for any analytical instrument. A new laser generation ensuring perfect overlap of the illumination volumes of individual channels paired with very fast switching times between fluorescence channels lay the foundation of co-localization nanoparticle tracking analysis (C-NTA) introduced recently by Particle Metrix. For the first time, we report results of co-localization measurements of reference material as well as real biological samples based on NTA technology on the new ZetaView® PMX-230 TWIN.

Nanoflow Cytometry: Sensitive Sizing and Phenotyping of Single EVs
Clayton Deighan, North American Sales and Applications Manager, NanoFCM

This presentation will review the principles and practice of nano flow cytometry. Covering the operational and experimental parameters important to resolving and counting single EVs. The latest updates on the technical capabilities of nano flow will be provided and multiple published examples of the data will be presented.

Mid-Afternoon Coffee and Tea Break and Networking in the Exhibit Hall

Applications for Small Particle Analysis Using High Sensitivity Flow and Imaging Flow Cytometry
Haley Pugsley, Manager and Senior Scientist, Luminex Corporation

Research in the fields of virology, microbiology, nanoparticles, and extracellular vesicles (EVs) has shown tremendous growth over the past few years. Viruses, which range in size from 17 nm to ~1.5 microns, small bacteria, nanoparticles, and small EVs such as exosomes (less than 150 nm in diameter), are all on the subcellular scale. Quantifying and characterizing small particles in a reproducible and reliable manner is challenging due to their small size. In this presentation, we will demonstrate the capabilities for small particle applications from Amnis® CellStream^® and Amnis^® ImageStream^®X Mk II. The Amnis CellStream system has the advantage of high throughput flow cytometry with higher sensitivity to small particles due to the CCD-based, time-delay-integration image capturing system. Here, we will present immunophenotyping data from EVs. The Amnis ImageStreamX Mk II that combines the quantitative power of flow cytometry with microscopy in one system has a High Gain mode to increase the sensitivity for small particles by adjusting the CCD-camera to a higher gain setting, increasing the signal obtained from the small particles while minimizing the noise. In this portion of the presentation, examples of High Gain mode using murine leukemia virus-sfGFP will be shown.

Networking Reception with Beer and Wine in the Exhibit Hall: Network with Colleagues and Engage with Exhibitors and Conference Sponsors

An Evening with Luminex -- Visit their Labs in Downtown Seattle, View Product Demonstrations, Enjoy Food & Drinks and Engage

Morning Coffee and Pastries in the Exhibit Hall

Exosome Engineering for Active Targeting and Intracellular Delivery of Therapeutic Proteins: Realization from Concept
Chulhee Choi, CEO, ILIAS Biologics Inc.

As extracellular vesicles that play an active role in intercellular communication by transferring cellular materials to recipient cells, exosome offer great potential as a natural therapeutic drug delivery vehicle. Currently, both academia and industry try to develop exosome platform-based therapeutics for disease management, some of which are already in clinical trials. An optogenetically engineered exosome system (EXPLOR®) that we previously developed was implemented for loading therapeutic cargo into exosomes which can deliver therapeutic cargos into target cells in free form. We are studying the clinical potential of ‘Exo-target®’, therapeutic exosomes with EXPLOR technology, in multiple disease areas including inflammatory diseases. NF-kB has been well accepted as master regulator for inflammation. The introduction of IkB into target cells can inhibit inflammatory responses by restraining nuclear translocation of NF-kB. Therefore, we have developed Exo-target loaded with dominant active form of IkB (Exo-srIkB). We found that Exo-srIkB treatment attenuates both local and systemic inflammation and animal mortality associated inflammatory disease including sepsis, ischemic reperfusion induced acute kidney injury and preterm birth in our preclinical studies. Especially, maternally injected Exo-srIkB could cross-over placenta barrier to deliver therapeutic cargos to fetal side, which resulted in prolonged pregnancy by more than 24 hours and additional advantages. Altogether, these results suggest therapeutic value of Exo-target for various disease by delivering API intracellularly to the target cells.

Cancer-derived Extracellular Vesicles as Early Disease Biomarkers
Andreas Möller, Associate Professor, Group Leader and Head, Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Australia

Late diagnosis of cancer is a key cause of the high cancer-associated mortality. However, a most cancer progression occurs ‘silently’, associated with no or diffuse symptoms. A non-invasive test, for example using blood, to determine that an individual has an early stage cancer would provide curative therapeutic options and presents an important unmet clinical needs. In this presentation, I will summarize the research approaches in the area of cancer-derived EVs undertaken by my group. I will discuss novel data on how liquid biopsies based on small amounts of blood can determine the presence of early stage cancer and how this can be potentially applied clinically. In detail, we derived a cancer-specific multi-protein EV marker capable of detecting the presence of 8 cancer types in individuals, including those patients with stage I. Confirming the platform independence of the biomarker, we transferred the test to a SERS-based microfluidics platform, and demonstrate the capacity to distinguish patients with benign and malignant conditions. Overall, our biomarker is capable of detecting early stage cancer in the blood of individuals and has various potential applications in reducing cancer-associated mortality.

Use of Circulating Extracellular Vesicle Protein Biomarkers to Detect Disease Earlier
Jean Lewis, Associate Director, Research, Biological Dynamics, United States of America

Circulating extracellular vesicles (EVs) contain a wealth of biomarkers and are key to numerous emerging liquid biopsy approaches; information from these EV biomarkers can be harnessed for diagnosis of many different diseases.  For example, EVs released from tumors may carry biomarkers signaling the presence and type of cancer; EVs bearing markers of neurological damage can be found in circulating blood, and in the case of infectious disease, pathogen-related markers can be released on EVs from infected blood cells.  While there are multiple innovative, emerging technologies designed to measure EV biomarker levels, many rely on multi-step procedures for prior isolation of the EVs from plasma or serum or are very low throughput.  To advance EV biomarker analysis towards true point-of-care diagnostics, we have developed a lab-on-a-chip nanoparticle isolation platform (Verita™), designed to both isolate and immobilize extracellular vesicles directly from unprocessed blood fractions onto an AC Electrokinetics (ACE) microelectrode array.  The EV-associated biomarkers can be either eluted for further analysis or quantified directly on-chip, where the entire process takes less than an hour, showing the potential for point-of-care applications.  The Verita™ platform was used to screen clinical serum or plasma samples and demonstrated feasibility for detecting various disease types.  For example, performance of an assay using ACE-purified plasma EVs from stages I-II pancreatic cancer showed a sensitivity of 83% at 99% specificity.  EVs quantified directly on-chip can be probed with biomarkers related to early cancer or Alzheimer’s disease, and can be used to detect TB infection with an AUC of 1.00.

Know Your EVs Inside Out with ExoView
Min Kyoung Sun, Field Application Scientist, Unchained Labs

Comprehensively characterizing your EVs can be a struggle even in a highly purified cell culture sample. Throw in complex biofluids, limited sample volume and rare EV subpopulations and that struggle can become a nightmare. Thankfully the ExoView R200 provides the solution! We will discuss how our proprietary ExoView chips utilize the power of affinity capture to isolate EVs from starting materials ranging from cell culture media to murine cerebral spinal fluid. All without the need for prior sample purification. Once EVs are bound to the surface of the chip, the ExoView platform can size, count and phenotype vesicles on a single particle basis, in one sample run. The R200 features 4 fluorescent channels (blue, green, red and far red) allowing individual EVs to be phenotyped by up to 5 surface or cargo proteins with single molecule sensitivity. We will cover the use of ExoView Tetraspanin kits for providing EV characterization on the basis of common exosome markers and highlight how ExoFlex chips allow full user customization to enable capture and detection of EVs via specific cell or disease markers.

Extracellular Vesicles in Cancer Progression
Lucia Languino, Professor of Cancer Biology, Thomas Jefferson University, United States of America

Cancer cells crosstalk with the tumor microenvironment (TME) by releasing small extracellular vesicles (sEVs).  sEVs are enriched in integrins, cell surface receptors for extracellular matrix proteins.  We have shown that alphaV integrins are markers for prostate cancer cell sEVs, which were isolated by density gradients, and characterized by electron microscopy, NTA and immunoblotting analysis, as per MISEV2018 guidelines.  These sEVs were also characterized for integrin and tetraspanin colocalization by ExoView analysis. We show now that some alphaV integrins promote the formation of unique, low density sEVs and bind a soluble ligand, as quantified using ExoView.  sEV integrins, upon release, are then transferred to microvascular endothelial cells and macrophages in the TME.  In functional assays using prostate cancer cells, we provide evidence that sEV integrins, upon transfer, modulate recipient cell proliferation and migration.  We also describe, as evaluated by our proteomic and immunoblotting analyses, alterations of signaling pathways in recipient microvascular endothelial cells and macrophages upon transfer of cancer cell derived sEVs.  Finally, alphaV integrins’ expression in donor cancer cells reduces sEV levels of molecules that have a tumor suppressive role, or promotes enrichment of effectors that support cancer cell differentiation into aggressive metastatic phenotypes. Overall, our studies show that sEVs from cancer cells may contribute to a horizontal propagation of integrin-activated pathways from cancer cells to the neighboring cells to promote a pro-tumorigenic TME.

Convective Transport and Binding of Extracellular Vesicles Establish Biologically Relevant Spatial Gradients
Steven C. George, Edward Teller Distinguished Professor and Chair, Department of Biomedical Engineering, University of California-Davis, United States of America

Extracellular vesicles (EVs) are small (50-150 nm diameter) composite particles secreted by cells and comprised of a lipid-based membrane surrounding an aqueous core.  The membrane and core can each incorporate a wide range of molecules (e.g., proteins, nucleic acids) that can impact cellular function; thus, EVs can impact in vivo biology, but have also generated significant excitement for their potential theranostic (therapeutic and diagnostic) applications in cancer. How EVs are transported (convection, diffusion, and binding) within the extracellular space is poorly understood.  We hypothesized that EVs are transported predominantly by convection through the interstitium and could establish a spatial gradient via binding to laminin through integrins alph3beta1 and alpha6beta1 expressed on their surface. Our early experimental studies demonstrate EV binding to a laminin-rich extracellular matrix (ECM) increases as the malignant potential of the cellular source increases (MCF10A, MCF10DCIS, MCF10CA1). Binding of the EV to the ECM generates an observable spatial gradient, which impacts the migration of M2-like differentiated macrophages.  The magnitude of the gradient is partially abrogated by blocking antibodies to alpha3beta1 and alpha6beta1 integrin subunits. Examination of EV interstitial transport will enhance our understanding of the dynamic tumor microenvironment and could present new targets for early-stage disease.

Networking Lunch, Meet Exhibitors and Engage with Colleagues

Anti-Metastatic Activity of PRR851, A Specific Inhibitor of the Nuclear Transport of Extracellular Vesicles
Aurelio Lorico, Professor of Pathology, Touro University Nevada School of Medicine, United States of America

The metastatic process is responsible for the vast majority of cancer deaths, yet most therapeutic efforts have focused on targeting and interrupting tumor growth rather than impairing metastasis. Identifying and testing new pathways to disrupt the process of metastasis is the key to developing new therapeutic strategies that improve cancer survival. We have identified and described a novel cellular pathway that allows extracellular vesicles (EVs) derived from cancer cells to transfer oncogenic cargoes into the nuclear compartment of mesenchymal stromal cells and transform their gene expression. This pathway relies on the translocation of Rab7+ late endosomes, which contain endocytosed EVs, into the nucleoplasmic reticulum (NR) of recipient stromal cells. The interaction between the two organelles; late endosomes and the nucleus, is mediated by a novel tripartite protein complex (named VOR) formed by VAMP-associated protein A (VAP-A) in the outer nuclear membrane, cytoplasmic oxysterol-binding protein -related protein-3 (ORP3) and endosomal Rab7 GTPase. Silencing VAP-A or ORP3 in recipient cells prevents late endosome entry into the NR, thereby blocking EV-mediated intercellular communication. We have designed and synthesized new chemical entities (NCEs) that inhibit VOR complex formation and nuclear transfer of EV cargoes. We have identified a lead NCE (PRR851), that reduced the incidence of metastasis in a syngeneic mouse model of malignant melanoma without apparent off-target toxicity. We will discuss the structure-activity relationship of PRR851 and its analogs and new data in experimental metastasis systems.

Glioblastoma Extracellular Vesicle Specific Peptides Inhibit EV-induced Neuronal Cytotoxicity
Xiaoli Yu, Assistant Professor, Department of Neurosurgery, University of Colorado Anschutz Medical Campus, United States of America

Glioblastoma (GBM) is the most aggressive and lethal form of brain tumors. Tumor cells release extracellular vesicles (EVs) which have been shown to play a critical role in cellular communication in the tumor micro-environment. We discovered that GBM plasma EVs were smaller in size, had no relationship between size and concentration, but showed highly significant correlation between EV concentration and plasma protein concentration. Importantly, GBM EVs purified from both plasma and tumor cell line produced IgG-mediated, complement dependent cytotoxicity in neurons. We identified high affinity phage peptides for GBM EVs by screening phage-displayed random peptide libraries. Significantly, we showed that GBM EV peptides inhibit EV-induced complement-dependent cytotoxicity in neurons in dose- and time-dependent manner. Phage peptide technologies can be used for isolation and characterization of EVs derived from brain tumors. EV specific peptides can be explored for  biomarkers and mechanisms  of brain tumor EV-induced neuronal death.

Lung Transplant Immunology Through the Exosome Lens
Billie Hwang, Transplant Immunologist, University of Washington, United States of America

Lung transplantation improves survival and quality-of-life for patients with end-stage lung disease. Clinical risk factors post-transplant including primary graft dysfunction (PGD) and acute and chronic lung allograft dysfunction (ALAD and CLAD) negatively impact the long-term benefits of transplantation and continues to be an area of research that has not been fully understood. Exosome immunology is a novel area of research that directly implicates extracellular vesicles (30-150nm) in playing a major role in a wide variety of diseases and immune responses. Despite the explosion of exosome studies in the last 5 years, their role in solid organ transplantation has been limited. In our studies, we aim to understand the role of exosomes in the donor:host immune responses and as prognostic biomarkers in clinical outcomes post-transplant including the development of acute and chronic rejection. In addition, we aim to develop a potentially novel paradigm that redefines lung transplant immunology through an exosome lens.

Mid-Afternoon Coffee and Tea Break and Networking in the Exhibit Hall

Differential Effects of APOE Genotype on Cerebrospinal Fluid Extracellular Vesicle miRNAs in Females vs. Males with Alzheimer's Disease
Julie Saugstad, Professor, Department of Anesthesiology & Perioperative Medicine, Oregon Health & Science University, United States of America

Alzheimer’s disease (AD) is the most common form of dementia and the sixth-leading cause of death in the United States. We identified and validated miRNA biomarkers for Alzheimer's disease (AD) in cerebrospinal fluid (CSF) from living donors, then showed that five of the AD miRNAs trend down in expression from controls to mild cognitive impairment to AD. Our recent studies revealed that two biological risk factors for AD, sex and APOE e4 genotype, differentially effect CSF EV miRNA levels: APOE e4 within each sex altered distinct miRNAs, and predicted gene targets and pathways of the altered miRNAs are known to contribute to neurodegeneration in AD. We are currently establishing the physical features and molecular cargo of AD CSF EVs within the context of APOE genotype and sex, with the goal to identify novel gene targets of CSF EV miRNAs that are relevant to AD pathophysiology.

Biomarker Analysis of Extracellular Vesicles (EVs) Derived from Peripheral Blood Mononuclear Cells (PBMCs) of Patients with Inflammatory Bowel Disease (IBD)
Emilia Scharrig, Post-Doctoral Fellow, Thomas Jefferson University, United States of America

IBD is a hyperinflammatory disorder that manifests with a severely compromised intestine, combined with dysregulation of the immune system. Although several factors have been associated with the disease, the pathophysiological mechanism is still not well-understood. Recent research suggests that EVs may be involved in the progression of IBD inflammation; still little is known about the contribution of immune cells-derived EVs to this pathology. To find out potential biomarkers of IBD we evaluated the protein composition of PBMCs-derived EVs from IBD patients and healthy controls. Most of the protein content (over 90%) of PBMC-derived EVs was shared among all the analyzed samples. A unique set of proteins present in IBD-EVs that has the potential to be used as biomarkers of the disease was identified. The specific content of the EVs and their variations are telling the story of a disease.

Regulation of Macrophage Polarization by Prostate Cancer-derived Extracellular Vesicles
Nicole Naranjo, PhD Candidate, Thomas Jefferson University, United States of America

Small extracellular vesicles (sEVs) mediate intercellular communication and carry cargoes that may alter the recipient cell phenotype and functionality. Our group has shown that   IFIT3 (Interferon induced protein with tetratricopeptide repeats) plays an intrinsic role as a regulator of STAT1 (Signal transducer and activator of transcription 1) expression in prostate cancer (PrCa) derived sEVs. STAT1 and IFIT3 are part of the Interferon Stimulated Genes protein family which are proteins that play key roles in macrophage polarization. To evaluate the role of PrCa sEVs in modulating the macrophage phenotype, we used CRISPR Cas9 methods to downregulate STAT1 or IFIT3 protein expression in PC3 PrCa cells (PC3-STAT1KO or PC3-IFIT3KO). Subsequently, we treated macrophages with sEVs derived from these cells and assessed surface expression of the macrophage M2 polarization markers CD163 or CD204. Our results show that CD163 and CD204 surface expression is dramatically reduced after treatment of macrophages with PC3 sEVs (mock or WT) as well as PC3 sEVs devoid of STAT1 or IFIT3. These results show that CD163 and CD204 reduction in macrophages is independent of STAT1 or IFIT3.  Furthermore, we demonstrate that the decrease of CD163 and CD204 protein levels in macrophages by PC3 sEV treatment is associated with decreased levels of their associated total RNA levels. In a parallel investigation, we performed proteomic analysis of macrophages after treatment with PC3, PC3-STAT1KO or PC3-IFIT3KO cell derived. Although polarization is independent of STAT1 or IFIT3, our Ingenuity Pathway analysis results show differential alterations of functional pathways in macrophages after treatment with PC3, PC3-STAT1KO or PC3-IFIT3KO cell derived sEVs. The functional pathways that are altered by STAT1 or IFIT3 are related to cellular movement, cellular proliferation, inflammatory response, and immune response. These results demonstrate that PrCa derived sEVs significantly alter the phenotype and protein content of macrophages, thus providing new avenues to explore the crucial contribution of PrCa derived sEVs in cancer progression.

The Polyaneuploid Cancer Cell (PACC) State Promotes Therapeutic Resistance Through Extracellular Vesicles (EVs)
Chi-Ju Kim, Postdoctoral Research Fellow, Johns Hopkins University School of Medicine, United States of America

The Johns Hopkins Cancer Ecology Center recently discovered a novel form of cancer therapeutic resistance achieved through the polyaneuploid cancer cell (PACC) state. The PACC state is triggered through a conserved evolutionary program that results in sustained polyploidization of the aneuploid genome. Cells in the PACC state are resistant to all therapies, not only the stressor that induced entry into the PACC state, and that the cell population that arises from the induced PACC state likewise shows reduced sensitivity to multiple agents. Clinically, the presence of cells in the PACC state in radical prostatectomy specimens of prostate cancer (PCa) patients increases the risk of progression. Utilizing a novel extracellular vesicle (EV) isolation platform, combined with development of a 3D imaging-based cellular EV uptake assay as well as a multiplexed EV RNA cargo profiling method, it was discovered that PACCs release significantly larger numbers of EVs. It is hypothesized that EVs contain factors (i.e., multidrug resistance proteins) that transmit resistance to adjacent cancer cells. An understanding of PACC-derived EVs and the impact of the transferred EV cargo on the phenotype of therapeutic-sensitive cells will provide critical insight into the acquisition of therapy resistance in lethal cancer.

Unique sEV sub-Populations Derived from alphaVbeta3 Integrin+ Cancer Cells
Cecilia Verrillo, Research Student, Thomas Jefferson University, United States of America

It is known that transmembrane cell adhesion receptors, such as integrins, are deregulated in cancer. The alphaVbeta3 integrin is upregulated in many cancers, is a promoter of metastasis, and is found in small extracellular vesicles (sEVs) isolated from prostate cancer (PrCa) cells.  We demonstrate that the exogenous expression of alphaVbeta3 in sEVs isolated from the PrCa cell line C4-2B results in the formation of unique, low density sEVs.  These sEVs were characterized by NTA and western blot (WB) analysis of sEV markers (Alix, TSG101, and the tetraspanins (TSPs) CD9 and CD81) and by the absence of calnexin.  These sEVs also express Kindlin-2 (K2), a positive regulator of alphaVbeta3 activity. Downregulation of K2 via CRISPR/Cas 9 in PC3 PrCa cells shows that endogenous expression of alphaVbeta3 in sEVs is unaffected when compared to CRISPR/Cas 9 control sEVs.  This result indicates that there is an alphaVbeta3 loading mechanism into sEVs that is independent of K2.  We investigated the activity of alphaVbeta3 in PC3 sEVs by utilizing fibrinogen, a molecule that specifically binds to the active conformation of alphaVbeta3.  This binding was visualized using ExoView R100, which is a platform that allows single sEV analysis.  A monoclonal antibody (mAB) to alphaVbeta3 was used to detect alphaVbeta3 levels on the surface of PC3 sEVs.  Our results show that both fibrinogen and the mAB to alphaVbeta3 display similar binding patterns; alphaVbeta3 is mainly present in sEVs captured by the CD9 TSP antibody and is less abundant in sEVs captured by CD63 and CD81 TSP antibodies.  We conclude that alphaVbeta3 may be present in its active conformation in specific CD9-positive populations of sEVs. We finally show, through WB and ExoView analysis, that programmed death-ligand 1 (PD-L1), a protein upregulated in PrCa and key player in the immune evasion of tumor cells, is present in PrCa sEVs.  Since alphaVbeta3 is known to upregulate cellular expression of PD-L1, our data suggest that alphaVbeta3 may influence the levels of PD-L1 in sEVs derived from PrCa cells.

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