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“Join the the R-EV-olution!” Highlights from 3rd Annual Mid-Atlantic Extracellular Vesicle (EV) Scientific Symposium

(BY RACHEL DERITA, PhD,Thomas Jefferson University, Department of Cancer Biology). The field of extracellular vesicles (EVs) is expanding rapidly and this was never more evident than when a group of prominent leaders in the field, and scientists more recently entering the field, all came to The Wistar Institute in Philadelphia, Pennsylvania for the 3rd Annual Mid-Atlantic Extracellular Vesicle Scientific Symposium on February 26, 2019. The Symposium provided an opportunity for veteran EV researchers from different backgrounds, and scientists who have more recently entered the field, to both present their research and network with each other to exchange insights on this exciting and accelerating field that seems more important in more different areas almost every week. The Symposium began with a talk by Kenneth Witwer, PhD, Associate Professor of Molecular and Comparative Pathobiology at Johns Hopkins University, and a Co-Chair of the Symposium, who is also Executive Chair of the International Society of Extracellular Vesicles (ISEV). Recently, Dr. Witwer and Clotilde Thery, PhD, INSERM Director of Research at the Institut Curie, and over 380 other contributing ISEV members, published the “Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018) in the Journal of Extracellular Vesicles. This is an update to guidelines first published in 2014 in response to a need for increased methodologic understanding and rigor in the EV field. Dr. Witwer highlighted some of the most important updates made to MISEV since 2014. The most prominent of these was in the nomenclature of EVs. There are many different types of EVs that are secreted from the cell, such as microvesicles that bud directly from the membrane and exosomes, which are derived from the endosomal system inside the cell and are, on average, smaller than microvesicles. The terms used for these EVs have been inconsistent and it was declared in MISEV 2018 that the best term to use, in most cases, is “EV,” unless specific sub-cellular origin can be demonstrated. Most commonly used characterization methods for EVs are currently not specific enough to distinguish “exosomes” from “microvesicles” completely, thus the term “EV” is suggested. The term “extracellular particle” is suggested for those samples that are known to be extracellular in nature, but that have not been proven to be vesicular.

The next major topics covered by Dr. Witwer involved EV isolation and characterization methods. The overarching theme he stressed is that there are a vast number of isolation methods, and you should choose your method(s) to best fit your downstream analytical needs. For example, should you use a method that gives a higher yield, but is less specific, or the other way around?

MISEV 2018 also highlights that there is a need for better “negative” markers to show purity of EVs, as markers that have previously been thought to be “negative” in EVs (such as the endoplasmic reticulum protein calnexin) are now being found associated with EVs in certain conditions.

Single EV analysis is also recommended in the form of nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). For functional analyses involving EVs, attendees were warned against labeling the vesicles with fluorescent membrane dyes due to the ability of the dye to aggregate to itself and provide false signals. For the complete MISEV 2018 article published in the Journal of Extracellular Vesicles, use this link (https://www.tandfonline.com/doi/full/10.1080/20013078.2018.1535750).

Another speaker early in the Symposium was Jan Lotvall, MD, PhD, Professor/Chief Physician (Asthma, Allergy, Inflammation, Eosinophils) at the University of Gothenburg in Sweden. He is also the founder and first president of the ISEV. As a veteran EV researcher, Dr. Lotvall spoke on the importance of considering EV heterogeneity of size, morphology, and cargo, even from a single cell type. He demonstrated how these parameters are altered based on isolation methods (for example, 10,000 x g versus 100,000 x g ultracentrifugation of EV source biofluid and low-density versus high-density EVs separated on a density gradient). A highlight of his recent work is identification of a subset of small EVs (approximately less than 150 nm in diameter) that contain the mitochondrial inner membrane proteins MTCO2 and COX6, which are more abundant in melanoma patients than in healthy individuals.

Dr. Lotvall was not the only one to speak about mitochondria-derived cargo in EVs. Michael Paulaitis, PhD, Professor of Ophthalmology at Johns Hopkins University, demonstrated that there are mitochondrial microRNAs inside EVs from rential pigment epithelial cells, and that these microRNAs are increased during mitochondria stress.

In a model of age-related macular degeneration, he showed that certain candidate microRNAs, upregulated in EVs during stress, targeted proteins related to cellular health and energy production, such as the tumor suppressor PTEN and the mitochondrial membrane protein COX III.

There were many talks about research related to developing EVs as biomarkers for disease. There have been some preliminary successes in finding EV-associated proteins or nucleic acids that can be analyzed to identify disease, but there are difficulties in reaching a standard rigorous enough for the clinic.

Some preliminary successes include work by Jonni Moore, PhD, Professor of Pathology & Laboratory Medicine at the Hospital of the University of Pennsylvania, Perlman School of Medicine, who is a veteran expert in the field of flow cytometry. Using cytometry on EVs, Dr. Moore was able to help predict cardiovascular disease in patients who have recently undergone non-cardiac vascular surgery.

David Speicher, PhD, Professor of Biochemistry & Biophysics at the Wistar Institute, and a Co-Chair of the Symposium, highlighted the difficulties in proceeding from pre-clinical to clinical levels of rigor in the detection of biomarkers, and also demonstrated the importance of determining whether your biomarker candidate of interest is inside the EV, in the EV membrane, attached to the outside of the EV, or completely outside the EV.

Uta Erdbrügger, MD, a nephrologist in the Department of Medicine in the University of Virginia Health System spoke about the many methodological considerations that need to be made when analyzing EVs and potential biomarkers from urine. Highlights of her presentation included the consequences of various pre-treatments done to urine before EV isolation and her report that, while some are beneficial, others can harm the EVs.

Another highlight was Dr. Erdbrügger’s use of hydrostatic filtration dialysis after ultracentrifugation to obtain superior results for the isolation of pure EVs and the enrichment of certain protein markers compared relative to the results that can be obtained with previous standard EV isolation techniques.

George Daaboul, President & CEO of NanoView Biosciences, has created a technology to analyze multiple EV surface antigens on single vesicles using antibody targeting and fluorescent signals. This technology can help biomarker studies move forward by providing the ability to study the EV surface in a multiplex fashion using very small volumes of starting biofluid.

In addition to work describing the use of EV-based biomarkers to identify and diagnose disease, there are also studies that show EVs are useful in tracking patient response to therapy. In August 2018, Wei Guo, PhD, Professor of Biology at the University of Pennsylvania, published an article in Nature (https://www.nature.com/articles/s41586-018-0392-8) reporting results showing that exosomal PD-L1 levels in melanoma patients can be used to track response to anti-PD-1 cancer therapy, and to stratify patients who respond to this therapy and those who do not. This is significant, because, despite the fact that anti-PD-1 immunotherapy has been shown successful in re-activating the immune system against cancer, it only works on about 20-30% of patients and there is a clinical need to be able to predict who will respond and who will not.

Prashanth Vallabhajosyula, MD, Associate Professor of Surgery at the University of Pennsylvania, has also shown that EVs can be used to detect early signs of organ transplant rejection .

In addition to the use of EVs as biomarkers, there was also significant work presented on specific EV-associated molecules implicated in EV biogenesis and secretion and functional effects on recipient cells. Andy Hoffman, DVM, Dean of the University of Pennsylvania Veterinary School, and a Co-Chair of the Symposium, showed the importance of the molecule TGFβ in mesenchymal stem cell-derived EVs for EV-mediated modulation of immune cell behavior.

My own principal investigator (PI), Lucia Languino, PhD, Professor in the Department of Cancer Biology at Thomas Jefferson University and a Co-Chair of the Symposium, showed our laboratory’s work on the importance of integrins on prostate cancer EVs for their modulation of the tumor microenvironment, and My Mahoney, PhD, Associate Professor in the Department of Dermatology & Cutaneous Biology at Thomas Jefferson, demonstrated that the cell adhesion and cell signaling molecule desmoglein 2 is in EVs and modulates recipient cell proliferation and the amount of EVs secreted from cells.

Additionally, Norman Haughy, PhD, Professor of Neurology and Vice Chair for Research in the Department of Neurology at Johns Hopkins University, implicated certain EV microRNAs in the destructive effect that astrocyte-derived EVs can have on recipient neuron cells.

The last major topic of EV science presented at this Symposium had to do with aging. Nicole Noren Hooten, PhD, Staff Scientist at the National Institute of Aging, presented work on potential EV-based markers of aging, as well as work identifying alterations in EV cargo, function, and concentration in patients with diabetes mellitus.

Overall, this Symposium highlighted the significant work coming from EV researchers in the Mid-Atlantic Region of the United States, and the outstanding questions that still remain.

For example, when looking for biomarkers in EVs, correctly purifying and examining the correct subset of EVs remains a challenge. Many factors need to be considered, such as pre-treatment of the biofluid to get rid of contaminants without disrupting EVs, and the isolation method to correctly purify the subset of EVs that will highlight a particular biomarker.

In all studies, it is agreed that, due to the vast number of technical options, EV isolation and characterization methodology must be determined by the particular disease being investigated and the scientific question being asked.

Each EV is its own treasure trove of biological information and the sky continues to be the only true limit to how EV research can progress, both in clinical and basic science arenas.

Many thanks go to the organizers/Co-Chairs of this Symposium: Andrew M. Hoffman, DVM, DVSc, University of Pennsylvania; Lucia R. Languino, PhD, Thomas Jefferson University; David W. Speicher, PhD, The Wistar Institute; and Kenneth W. Witwer, PhD, Johns Hopkins University

To see more of the published research of the scientists mentioned in this article, please visit http://www.Pubmed.org.

[MISEV 2018 article "Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018): A Position Statement of the International Society for Extracellular Vesicles and Update of the MISEV2014 Guidelines"] [Symposium Announcement and List of Speakers]