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Page 16 Graner. Extracell Vesicles Circ Nucleic Acids 2020;1:3-19 I http://dx.doi.org/10.20517/evcna.2020.08

Medicine, US) led the session with his group’s extensive work on extracellular vesicle and particle (EVP)
cancer biomarkers. The Lyden lab discovered exomeres, non-membranous nanoparticles of ~30nm
diameter, which are abundant at pre-metastatic niches; the small sizes of exomeres may contribute to their
wide distribution capabilities. These EVPs are among a broad range of extracellularly-released materials
possessing biologic content, including dsDNA, which their group found mostly on EV surfaces. Using
various separation techniques, including AF4 following differential ultracentrifugation, they categorize EVs
into Exo-L (90-120 nm), Exo-S (60-80 nm), and exomeres (< 50 nm), cognizant that there are also larger
vesicles. The diversity of the vesicles may relate to the diversity of local and systemic consequences of cancer.
The biodistribution of cancer EVs in vivo likely relates to surface molecules that interact with other cell
surface markers at metastatic organ sites. Their work implies that the organotropism of metastatic tumors
may be initiated by the tumor EVs, particularly relating to EV integrin content (which may differ in ratio to
the parental cell content). The overall impact of EV-driven cancer biology led them to analyze the proteomes
of EVs from > 400 human cancer samples (cell lines, tissue explants and numerous biofluids). The goals were
to define common human exosome markers from different source types (tissues, cell lines and biofluids);
to identify tumor-specific exosome biomarkers; and to classify primary tumors of unknown origins by
exosome proteome signatures. Some notable proteins include HSPA8 and CD9 present in/on many cancer
exosomes, but CD63 served mostly as a marker for murine exosomes. The group has many matched samples
of tumors, adjacent (and sometimes distant) normal tissues, and plasma, which allow for tissue explant
exosome comparisons intra-patient. The proteome profiles may represent biomarkers and therapeutic
targets, and the normal tissue serves as a means against drug targets that would affect normal tissue. Pathway
analysis derived from pancreatic cancer exosome proteomes found epithelial-mesenchymal transition
(EMT), coagulation, and TGFb signaling as highest ranking. For lung cancer, the pathways were EF2, G2M
checkpoint, and MYC targets. Across the 18 cancers in their study, certain proteins stood out as pan-cancer
markers such as THBS2, VCAN, SRRT, and TNC. For pancreatic cancer, they identified 50 exosome proteins
from patients that were not present in age/sex matched controls. Across cancers, plasma exosomes had sets
of cancer proteins, but also sets of immune proteins that were not found in healthy donors.

A new area for EV studies, EVs in cancer related fatigue (CRF), was introduced by Dilorom Sass (NIH,
National Institute of Child Health and Human Development, US). In CRF, there is an unusual sense of
physical, emotional, or cognitive tiredness related to cancer or cancer treatment. CRF affects 30%-90% of
cancer patients receiving therapy, and 30% will continue to experience such fatigue, months or even years
post-treatment. Prior work has suggested that a wide variety of cytokines may be involved. Circulating
EVs bearing cytokines could be systemic mediators and biomarkers of such inflammation. Dilorom’s study
searched for associations between levels of fatigue in men after radiation treatment for prostate cancer,
determined by questionnaire, and 45 plasma EV-associated immune markers. Hierarchical clustering of the
cytokine readouts identified two clusters. A closer analysis found that eotaxin, HSP27, IL3, IP-10, and MIP3
alpha were significantly higher in the EV samples from the fatigued cohort. These studies raise questions
in terms of measurements of plasma cytokine values: what fraction is actually measured, and are we
overlooking a valuable source of potentially protected cytokines associated with EVs?

Janusz Rak (McGill University, Canada) introduced “leukobiopsy” as a variant of our typical takes on
liquid biopsy. The successes of current liquid biopsy, biofluid-based biomarker information relevant to
disease states, are significant in some areas, far less so in others. Janusz’s group had demonstrated transfer
of the oncogenic EGFR variant, EGFRvIII (found in gliomas), from aggressive cancer cells that expressed
the protein to more indolent cells that did not express it. This converted the indolent cells into far more
aggressive tumors; the presence of EGFRvIII on vesicles in blood suggested that it could be a cancer-specific
biomarker. As they extended studies into the RAS oncogene, they noted that cells receiving EVs from RAS
mutant cells now possessed RAS protein, RNA, and DNA, implying an extraordinary passage/delivery
capacity. Using various assays, the group demonstrated that normal cells could also take up cancer vesicles,