Extracellular Vesicles 2023: Technologies, Biomarker Cargo & Diagnostics Agenda

Welcome and Introduction by Conference Co-Chairperson
Mei He, Associate Professor, University of Florida, United States of America

Welcome and Introduction by Conference Co-Chairperson
Michael Graner, Professor, Dept of Neurosurgery, University of Colorado Anschutz School of Medicine, United States of America

Extracellular Vesicle Integrins: Novel Opportunities for the Diagnosis and Therapy of Cancer
Lucia Languino, Professor of Cancer Biology, Thomas Jefferson University, United States of America

The benefits of cancer therapy mediated by small extracellular vesicles (sEVs) are: low immunogenicity, ability to infiltrate biological barriers and ‘targetability’.  Cancer cells cross-talk with the tumor microenvironment by releasing sEVs.  Our studies have revealed a role for integrins in sEVs in mediating cancer cell cross-talk with the tumor microenvironment.  After confirming the fidelity of the sEV preparations by electron microscopy, density gradient, and immunoblotting, we have determined that integrins are actively packaged into sEVs isolated from cancer cells.  sEVs mediate protein transfer of integrins to microvascular endothelial cells and increase the number of their junctions and tubules.  We have also demonstrated a cross-talk between prostate cancer cells and monocytes that affects macrophage functions and have suggested that inhibition of integrins might offer a novel immune–based therapeutic strategy in prostate cancer.  Overall, these studies show that sEVs from cancer cells may contribute to a horizontal propagation of integrin-associated phenotypes from cancer cells to the tumor microenvironment. We finally demonstrate that the sEVs circulating in plasma from prostate cancer patients contain higher levels of specific integrins, as compared to plasma sEVs from age-matched healthy subjects.  Our results suggest that specific integrins in cancer patient sEVs could be clinically useful as non-invasive biomarkers for cancer 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 subfractions on plasma of stroke and periferal 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 procipitation for EV isolation is also used for the diagnosis of CCS.

Low Earth Orbit Research Opportunities on the International Space Station
Kristin Kopperud, Program Director, Biomedicine, International Space Station National Laboratory, United States of America

Overview of the ISS National Laboratory, including its mission, role, and unique set of experimental conditions. I will also discuss some research opportunities, from past to upcoming, that are available to researchers desiring to conduct experiments in microgravity.

NanoFACs EV Sorting -- Strengths and Limitations
Terry Morgan, Professor, Oregon Health and Science University, United States of America

Extracellular Vesicles as Novel Tools in Precision Medicine: Origins, Properties, and Molecular Cargo as Diagnostic and Prognostic Cancer Biomarkers
Andrew Godwin, Professor and Division Director, Genomic Diagnostics, Founding Director, Kansas Institute for Precision Medicine, Deputy Director, KU Cancer Center, University of Kansas Medical Center, United States of America

Biomarkers are measurable biological indicators with numerous clinical applications each providing unique information about an individual's health status. The identification and development of new biomarkers is helping to advance the field of precision medicine. Biomarkers, including proteins, autoantibodies, RNA/DNA, metabolites, lipids, etc., continue to impact personalized health care, given each person’s disease has a unique pattern of biomarkers. In oncology, pathologic analysis of tumor tissue biopsies remains the gold standard for the initial diagnosis of cancer. However, liquid biopsies, which analyze tumor-derived material circulating in the bloodstream and other bodily fluids, are rapidly gaining traction in the clinic. These tests offer considerable potential in oncology, which include early detection, monitoring treatment response, and disease recurrence. Recently liquid biopsy-based biomarkers include circulating tumor cells, cell-free DNA/RNA, nanoparticles, and extracellular vesicles (EVs). Regarding the latter, EVs are showing great promise as circulating biomarkers. The International Society for Extracellular Vesicles define EVs as particles naturally released from the cell that are delimited by “a lipid bilayer and cannot replicate”. Center among EVs are nano-sized vesicles (40 to 150 nm) of endocytic origin also known as small EVs/exosomes, which are produced and released by most cell types under normal physiologic and in diseased states. sEVs carry cargo representative of their originating cell including nucleic acids, cytokines, membrane-bound receptors, and a wide assortment of other, biologically active lipids and proteins. Since sEVs/exosomes travel systemically throughout the body, efforts are underway to exploit them as potential biomarkers to detect and monitor disease states. Ways to isolate sEVs and characterize their cargo for cancer diagnostics and for monitoring early response to therapy will be discussed.

Cross Talk Between Viruses and EV Biogenesis
Fatah Kashanchi, Professor and Director of Research, Lab of Molecular Virology, George Mason University, United States of America

Most RNA and DNA viruses package and release their products (DNA, RNA, proteins) in EVs to avoid detection by the host immune system. In the current study we have addressed two main questions related to timing of the EV vs. virus release using HIV-1 as a model system, and have further found a new method of isolating viral populations that have never been described before. To date, it is not clear whether there is a timing difference between EV and virion release from infected cells. Here, we found that EVs precede the secretion of viral particles at 6 hrs post-transcriptional activation and release. This early EV release could shed light on how the contents of EVs are able to increase the susceptibility of viral infection, perhaps by priming the environment prior to viral egress or altering the progression of the cell cycle in recipient cells. We also have found that when separating EVs using differential ultracentrifugation, size exclusion chromatography or simple size filtration, there are at least three distinct sizes and functional viruses when using HIV-1 as a model system.  Validation of the data was performed utilizing blocking infectivity with broadly neutralizing antibodies or culturing the producer cell lines in the presence of anti-retroviral drugs to interfere with the production of infectious virions.  Collectively, our results show how viruses use EV pathways to release their cargo prior to virion release and the virions are very heterogenous in size, biochemical and biophysical characteristics.

Astrocytes at the Crossroads of HIV and Alzheimer’s Disease
Shilpa Buch, Professor and Senior Executive Vice Chair for Research, University of Nebraska Medical Center, United States of America

Increased life expectancy of HIV+ patients in the post anti-retroviral therapy era parallels with increased incidence of HIV-associated neurological disorders (HAND) and associated comorbidities such as Alzheimer’s Disease (AD). The persistence of HAND is thought to involve poor penetration of the antiretroviral drugs across the BBB into the CNS, leading to persistent low-level viral replication in the CNS with accumulation of cytotoxic viral proteins including Tat. Interestingly, there have been reports on Tat-mediated production of the toxic neuronal amyloid protein & the interaction with the former, leading to enhanced toxicity.  Additionally, opiates have shown to exaggerate HIV mediated neuropathogenesis in different animal models. Abundant glial cells such as the astrocytes have also garnered interest as potential contributors of amyloidosis, & this could translate into a significant added burden to the process of amyloidosis in the context of Tat & Opiates. Present study was aimed at assessing the role of astrocytes in mediating Tat/Morphine induced amyloidosis in the context of HAND & opiate use disorder. Our in vivo data showed that SIV+ macaques/HIV+ patients, exhibited region specific up-regulation of the amyloidogenic components in the brain that co-localized with GFAP positive astrocytes. In vitro findings aimed at dissecting the cell-type specificity involved in amyloidosis revealed up-regulation of AD markers – BACE-1, amyloid precursor protein (APP), ABetamoC64, p-Tau, as well as increased activity of BACE-1 in Tat-exposed human primary astrocytes (HPA). Molecular mechanism(s) underlying this process involved up-regulation of hypoxia inducible factor (HIF-1a) with a concomitant translocation to the nucleus & binding to the lncRNA BACE-1AS resulting in formation of a unique complex as confirmed by RIP and EMSA. Further, ChiP assay showed functional binding of the complex to BACE-1 promoter leading in turn, to increased expression of BACE-1 by transcriptional, post transcriptional and translational mechanisms along with increased activity leading to consequential generation of ABeta-42 protein via cleavage of APP. We next sought to assess whether morphine-stimulated astrocyte extracellular vesicles (ADEV) could shuttle the amyloid cargoes to neurons. Our results showed that morphine exposure up-regulated the release of morphine-ADEVs, carrying amyloids and that silencing HIF-1Alpha in astrocytes not only reduced the numbers of released ADEVs, but also inhibited the packaging of amyloid cargos in ADEVs. These findings were further validated in brain derived EVs (BEVs) isolated from macaques, wherein it was shown that BEVs from morphine-dependent macaques, carried varieties of amyloid cargoes including the cytokine IL-1Beta. Targeting HIF-1Alpha can thus be considered as an adjunctive therapy approach for HAND patients on cART with a comorbidity of opiate misuse.