Advancing exRNA
Communication Research

The NIH Common Fund has announced new funding opportunities for advancing research into exRNA communication.

Updated guidelines on Minimal Information for Studies of Extracellular Vesicles have now been published in the Journal of Extracellular Vesicles (JEV, Taylor & Francis) as MISEV2018.

The original MISEV2014 guidelines were released in 2014 by the Board of Directors of the International Society for Extracellular Vesicles (ISEV) to provide guidance in standardization of protocols and reporting in the EV field. Accumulating more than 800 citations since its release, the MISEV2014 guidelines have achieved the aim of becoming a guiding standard for researchers. A 2016 survey of ISEV members reaffirmed the need for guidelines and recommended that they be updated regularly…but with broad community input to accommodate and shape the quickly developing field.

MISEV2018 updates the topics of nomenclature, separation, characterization, and functional analysis, integrating the contributions of over 380 ISEV members, a strong tribute to the commitment of ISEV members. A two-page checklist summarizing the main points is also included.

So what’s new? MISEV2018 recommends the use of ‘extracellular vesicle’ as the preferred generic terminology for use in publications, in part due to challenges in confirming the biogenesis mechanisms of exosomes, microvesicles, and other particles, and in part due to the vague and varied uses of other terms. Separation and concentration options are now many and diverse; researchers should pick the methods most fit for downstream purpose and, more importantly, report these clearly and accurately. The EV-TRACK database (van Deun et al., Nature Methods, 2017) is supported as a means to record these details in order to improve clarity and reproducibility. To establish presence of EVs, examples of EV-enriched markers are provided, but the need for “negative” (better: “depleted”) markers is also highlighted. MISEV2018 adds topology as a recommended form of EV characterization, for example identifying where in or on a vesicle your favorite protein or RNA resides. It also recommends functional analysis of the ‘non-EV’ fractions to confirm EV-specific function (or not!). An appreciation of EV heterogeneity is included with a reminder that ‘larger EVs matter’ and a request to explore a range of EV subtypes in functional studies. Finally, although some of the specific details contained in MISEV2018 are focused on mammalian components, it is appreciated that the guidelines are applicable to non-mammalian and non-eukaryote research.

Please contact the corresponding authors, Clotilde Théry and Kenneth Witwer with any questions or comments.

For more information on the process of writing and publishing MISEV2018, see this white paper and Witwer et al., J. Extracell. Vesicles, 2017.

References

Lotvall J, Hill AF, Hochberg F, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the international society for extracellular vesicles. J. Extracell. Vesicles. (2014) 3: 26913. doi:10.1080/20013078.2018.1535750. PMID:25536934.

Witwer KW, Soekmadji C, Hill AF, et al. Updating the MISEV minimal requirements for extracellular vesicle studies: building bridges to reproducibility. J. Extracell. Vesicles. (2017) 6: 1396823. doi:10.1080/20013078.2017.1396823. PMID:29184626.

Théry C, Kenneth W Witwer KW, Aikawa E, et al. 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. J. Extracell. Vesicles. (2018) 7: 1535750. doi:10.1080/20013078.2018.1535750.

This post originally appeared in Bioquick News.

The 2018 annual meeting of the American Society for Exosomes and Microvesicles (ASEMV) was held October 20-24 in Baltimore, hard by the water’s edge in the Baltimore Marriott Waterfront Conference Center. ASEMV president, Stephen Gould, PhD, Professor of Biological Chemistry & Co-Director, Graduate Program in Biological Chemistry, Johns Hopkins, reported a record attendance of 250 scientists from the United States and around the world (Korea, Norway, Sweden, Canada, Australia, Japan, UK, Italy, Portugal, The Netherlands) at this historically intimate and highly interactive meeting that benefits greatly from having communal meals and no overlapping sessions. The five-day meeting featured over 100 podium presentations and myriad posters. The daily consecutive sessions typically ran from 8.30 in the morning to 9.30 in the evening, and were followed by two hours of poster viewing and interaction among researchers and with sponsors. The communal meals and poster sessions offered excellent opportunities for significant interaction amongst conference participants and also for interaction between attendees and the over 20 companies (see below) that were sponsors of the meeting. Dr. Gould highlighted the key role of these sponsors in enabling this very special meeting, and noted that this year featured record sponsorship, with almost triple the number of sponsors relative to the number for last year’s meeting at Asilomar in California. This impressive increase in sponsorship is a reflection of the recent explosion of research and interest in exosomes from many quarters of medicine and science.

Among the themes of this year’s meeting was the growing appreciation for the heterogeneity of exosomes/microvesicles in terms of content, surface markers, size, and function. The similarities between exosomes and viruses were discussed in a number of talks. The brain’s use of exosomes for cell-to-cell communication within the brain, and also to communicate beyond the brain, was highlighted in multiple presentations. One of these suggested the dual promise of extracellular microRNAs in the diagnosis and pathology of Alzheimer’s disease. The role of exosomes in metastasis, carrying information from primary cancer cells to sites of future metastasis, was discussed and presented as further strong support for the century-old “Seed & Soil” hypothesis advanced originally by London surgeon Stephen Paget in 1889 (https://en.wikipedia.org/wiki/Stephen_Paget). Dr. Paget’s original article was titled “Distribution of Secondary Growths in Cancer of the Breast” (Paget, 1889).

One presentation described EVs as epigenetic mediators of systemic communication in murine experimental sepsis and another, by sepsis expert Antonio De Maio, PhD, Professor and Member of the Biomedical Sciences Program at the University of California San Diego, described how phospholipids within EVs may contribute to the activation of target cells. Dr. De Maio, a graduate of the Central University of Venezuela in Caracas, had previously been Associate Professor and Research Director for the Division of Pediatric Surgery at Johns Hopkins, where he had also led the Committee for the Recruitment of Under-Represented Minorities to Graduate Programs. At UCSD, Dr. De Maio is also Director of the Initiative to Maximize Student Diversity Program at the university. At UCSD, Dr. De Maio’s laboratory focuses on the molecular and genetic bases of the response to injury.

Two presentations on tick exosomes and two on bacterial outer membrane vesicles highlighted the broad spectrum of exosome significance throughout the kingdoms of life. An opening night presentation suggested that vesicle-cloaked virus clusters are the optimal units for inter-organismal viral transmission.

The role of exosomes in glioblastoma was the subject of multiple presentations. Janusz Rak, MD, PhD, Senior Scientist in the Child Health and Development Program, and Professor, Department of Pediatrics, McGill University, began the Sunday morning sessions with a talk on the role of EVs in the evolution of glioma-initiating cells. Quantification of cancer EV populations using super-resolution microscopy was another highlight of Sunday morning talks.

ARC is repurposed retrotransposon Gag protein that mediates intercellular RNA transfer in brain

Paul Worley, MD, Professor of Neurology at Johns Hopkins and an expert on the molecular basis of learning and memory, with a focus on cellular mechanisms that support synapse-specific plasticity, opened the Sunday evening session with a highly stimulating discussion of how the neuronal gene ARC encodes a repurposed retrotransposon Gag protein that mediates intercellular RNA transfer. Dr. Worley described evidence suggesting that Gag retroelements have been repurposed during evolution to mediate intercellular communication in the nervous system. Previous work had shown that the neuronal gene ARC is essential for long-lasting information storage in the mammalian brain and mediates various forms of synaptic plasticity. ARC has been implicated in neurodevelopmental disorders. It has been shown that ARC self-assembles into virus-like capsids that encapsulate RNA. Endogenous ARC protein is released from neurons in EVs that mediate the transfer of ARC mRNA into new target cells, where it can undergo activity-dependent translation. Purified ARC capsids are endocytosed and are able to transfer ARC mRNA into the cytoplasm of neurons. ARC exhibits similar molecular properties to retroviral Gag proteins. Evolutionary analysis has indicated that ARC is derived from a vertebrate lineage of Ty3/gypsy retrotransposons, which are also ancestors to retroviruses.

Sensational Tuesday evening

Tuesday evening featured a number of riveting presentations in a sensational session moderated by Xandra Breakefield, PhD, Professor of Neurology, Harvard Medical School, and Geneticist, Massachusetts General Hospital. A presentation on the use of machine learning-assisted histopathology to categorize large oncosomes held the audience spell-bound. Another suggested that EVs serve as delivery vehicles for LINE-1 retrotransposons.

Dr. Tushar Patel, Dean of Research at the Mayo Clinic-Jacksonville and an expert on liver cancer and liver transplants, had opened the session with a discussion of how biological nanoparticles might serve as therapeutic agents.

Other presentations in this session included ones on tools for live monitoring of exosome release from single cells, on the detection of mutant KRAS and TP53 DNA in circulating exosomes from healthy individuals and patients with pancreatic cancer, and on how an infected cell tolerates its viral pathogen using the exosomal pathway.

Beach Boys performance can’t distract ASEMV attendees

The attendees’ profound interest in exosomes was indicated on the opening evening of the meeting. At the outset, in outlining the logistics of the meeting, ASEMV president Dr. Gould explained that a late-breaking room change for Saturday night’s opening session had been occasioned by concern that the original room might be too noisy due to a performance taking place downstairs by The Beach Boys. The Beach Boys? Many thought that Dr. Gould was joking. But yes, the real Beach Boys were actually playing at a benefit event just downstairs from the ASEMV opening session, and yet, such was the audience’s interest in exosomes that no one moved. This reporter, however, could not resist checking out the iconic band after the opening ASEMV session had ended, and the photo here was taken of The Beach Boys who were indeed playing just downstairs. One of the original band members, Mike Love, was playing keyboard and singing. The Beach Boys performance was the highlight of a gala evening sponsored by Chimes (https://chimes.org/), a Baltimore-based international not-for-profit organization dedicated to assisting people with intellectual and behavioral challenges to achieve their fullest potential. It was an awesome backdrop to what would be an awesome ASEMV meeting.

Nearby International Human Virology meeting features major session on “Exosomes in health & disease”

And one further note is that the Institute for Human Virology (IHV), headed by legendary HIV virologist Dr. Robert Gallo, was holding its 20th International Meeting in the Four Seasons Hotel, right next to the Marriott where the ASEMV meeting was held.

Further indication of the exploding interest in exosomes was that the IHV meeting held a major session on exosomes this year. Titled “Exosomes in Health and Disease,” this session was listed second among nine sessions called out for special attention on the IHV meeting web page (http://www.ihv.org/ihvmeeting/). Areas of emphasis in this session ranged from cytokines in EVs to mechanisms of EVs in viral transmission.

Chairpersons of the IHV exosome session were Robert Gallo, MD, Director, Institute of Human Virology, University of Maryland School of Medicine, US, and Leonid Margolis, PhD, Senior Investigator, National Institute of Child Health and Human Development, US.

Speakers and topics included Xandra Breakefield, PhD, Professor of Neurology, Harvard Medical School / Genetist, Massachusetts General Hospital, “Extracellular Vesicle As Advance Forces in Cancer;” Dr. Margolis, “Not All Soluble Cytokines Are Soluble: Cytokines in Extracellular Vesicles Mediate Cell-Cell Communications;” Fatah Kashanchi, PhD, Former Director of Research, George Mason University, “Presence of HIV-1 RNA in Extracellular Vesicles from HIV-1 cART-Treated Cells;” Ayuko Hoshino, PhD, Instructor of Molecular Biology in Pediatrics, Weill Cornell Medical College, “Exosomal Protein Signatures: Mechanistic Insights and Biomarker Potential;” and Yoel Sadovsky, MD, Executive Director, Magee-Womens Research Institute, University of Pittsburgh, “Placental Exosomes in Maternal-Placental-Fetal Communication and Viral Resistance.”

Sponsors of ASEMV 2018 annual meeting

Sponsors of the ASEMV 2018 annual meeting included Particle Metrix, System Biosciences (SBI), iZON, Caris Life Sciences, nanoView Diagnostics, WAKO, ReNeuron, Norgen Biotek Corporation, Millipore Sigma, Beckman-Coulter, Wyatt Technology, AcouSort, Spectradyne, Fiber Cell Systems, ONI, abcam, Ceres Nano, Nanostics Precision Health, cellex, HansaBioMed Life Sciences, Lonza, and NanoTech.

Reference
Paget S. The distribution of secondary growths in cancer of the breast. The Lancet 133: 571-573. doi: 10.1016/S0140-6736(00)49915-0


Image: Kelsey Burke

This post originated as a press release from the University of Pennsylvania.

Cancerous tumors are more than a lump of cells growing out of control; they participate in active combat with the immune system for their own survival. Being able to evade the immune system is indeed a hallmark of cancer. Now, researchers from the University of Pennsylvania show that, to assist in the fight, cancer cells release biological “drones,” small vesicles called exosomes circulating in the blood and armed with the protein PD-L1, which causes T cells to tire before they have a chance to reach the tumor and do battle.

The work, published in the journal Nature (Chen et al., 2018), is a collaboration between Wei Guo of Penn’s School of Arts and Sciences and Xiaowei Xu of the Perelman School of Medicine. While primarily focused on metastatic melanoma, the team found that breast and lung cancer also release the PD-L1-carrying exosomes.

The research offers a paradigm-shifting picture of how cancers take a systemic approach to suppressing the immune system. In addition, it also points to a new way to predict which cancer patients will respond to certain checkpoint inhibitor drugs, which disrupt immune suppression to fight tumors, and a means of tracking the effectiveness of such therapies.

“Immunotherapies are life-saving for many patients with metastatic melanoma, but about 70 percent of these patients don’t respond,” says Guo, a professor of biology. “These treatments are costly and have toxic side effects, so it would be very helpful to know which patients are going to respond. Identifying a biomarker in the bloodstream could potentially help make early predictions about which patients will respond and, later on, could offer patients and their doctors a way to monitor how well their treatment is working.”

“Exosomes are tiny lipid-encapsulated vesicles with the diameter less than one-hundredth of a red blood cell. What we have found with these circulating exosomes is truly remarkable,” says Xu. “We collected blood samples from melanoma patients treated with anti-PD1 checkpoint inhibitor therapy. This type of liquid biopsy assay allows us to monitor tumor-related immune suppression with time.”

One of the most successful innovations in cancer therapy has been the use of checkpoint inhibitor drugs, which are designed to block attempts by cancer cells to suppress the immune system to allow tumors to thrive and spread. One of the primary targets for this class of drugs is PD-1, a protein on the surface of T cells. Tumor cells express PD-L1, which interacts with PD-1, effectively turning off that cell’s anti-cancer response. Blocking that interaction using checkpoint inhibitors reinvigorates T cells, allowing them to unleash their cancer-killing power on the tumor.

While it was known that cancer cells carried PD-L1 on their surface, Guo, Xu and colleagues found, in the new work, that exosomes from human melanoma cells also carried PD-L1 on their surface. Exosomal PD-L1 can directly bind to and inhibit T cell functions. Identifying the exosomal PD-L1 secreted by tumor cells provides a major update to the immune checkpoint mechanism, and offers novel insight into tumor immune evasion.

“Essentially exosomes secreted by melanoma cells are immunosuppressive.” Guo says. “We propose a model in which these exosomes act like drones to fight against T cells in circulation, even before the T cells get near to the tumor.”

Since a single tumor cell is able to secrete many copies of exosomes, the interaction between the PD-L1 exosomes and T cells provides a systemic and highly effective means to suppress anti-tumor immunity in the whole body. This may help explain why cancer patients have weakened immune systems.

Because exosomes circulate in the bloodstream, they present an accessible way of monitoring the cancer-T cell battle through a blood test, compared to a traditional, more-invasive tumor biopsy. After an acute phase of treatment, the researchers envision such a test as a way to monitor how well the drugs are keeping cancer cells in check.

By measuring pre-treatment levels of PD-L1, oncologists may be able to predict the extent of tumor burden in a patient and associate that with treatment outcome. In addition, a blood test could measure the effectiveness of a treatment. For example, levels of exosomal PD-L1 could indicate the level of T cell invigoration by immune checkpoint inhibitors.

“In the future, I think we will begin to think about cancers as a chronic disease, like diabetes,” says Guo. “And just as diabetes patients use glucometers to measure their sugar levels, it’s possible that monitoring PD-L1 and other biomarkers on the circulating exosomes could be a way for clinicians and cancer patients to keep tabs on treatment. It’s another step toward precision and personalized medicine.”

 

Reference

Chen G. et al. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature (2018) 560: 382–386. doi: 10.1038/s41586-018-0392-8 PMID: 30089911


Guo and Xu coauthored the work with Penn’s Gang Chen, Alexander C. Huang, Wei Zhang, Min Wu, Jiegang Yang, Beike Wang, Honghong Sun, Wenqun Zhong, Bin Wu, Xiaoming Liu, Lei Guan, Tin Li, Shujing Liu, Ruifeng Yang, Youtao Lu, Liyun Dong, Suzanne McGettigan, Ravi Radhakrishnan, Junhyong Kim, Youhai H. Chen, Giorgos C. Karakousis, Tara C. Gangadhar, Lynn M. Schuchter, and E. John Wherry, as well as collaborators from Wuhan University, The Wistar Institute, Xi’an Jiaotong University, the University of Texas MD Anderson Cancer Center, and the Mayo Clinic.

The research was supported by the National Institutes of Health (GM111128, GM085146, AI105343, AI108545, AI082630, and AI117950), Parker Institute for Cancer Immunotherapy, American Heart Association, Tara Miller Melanoma Foundation, University of Pennsylvania, Wistar Institute, Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, CAST Foundation, and NSFC Foundation.

Wei Guo is a professor of biology in the School of Arts and Sciences, and Xiaowei Xu is a professor of pathology and laboratory medicine and of dermatology in the Perelman School of Medicine at the University of Pennsylvania.

Wei Guo, Xiaowei Xu, and Gang Chen are listed as inventors on a patient owned by the University of Pennsylvania related to this work. Guo and Xu serve on the Scientific Advisory Board and have equities in Exo Bio, a company that has licensed the patent from the University of Pennsylvania.