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Graner. Extracell Vesicles Circ Nucleic Acids 2020;1:3-19 I http://dx.doi.org/10.20517/evcna.2020.08 Page 15

transthyretin (TTR) in the liver, where TTR mutations cause transthyretin amyloidosis. Their system used
100-fold less siRNA than a clinically-approved drug delivery particle. This is scalable (they have used this in
non-human primates) and appears to be immunologically safe. They also showed utility in small intestine
and kidneys, implying use for targets beyond the liver.

Lance Liotta (George Mason University, US) spoke of how to reverse tumor-induced immune suppression at
the level of the sentinel lymph node. He initiated the discussion with a question: how do tumor EVs actually
exit the solid mass of the tumor and traverse the extensive extracellular barriers to get into peripheral
circulation? They believe that this “open door” is the lymphatic system for interstitial draining. This implies
that tumor EVs in blood have likely already circulated through lymph nodes. Using tumor tissue directly,
they are able to harvest EVs from the tumor interstitial fluid and separate them by 2K, 10K, and 100K ×
g fractions. They have also developed a nanoparticle (Nanotrap) that allows for controlled affinity release
of cytokines that stimulates migration of immune cells (notably, antigen presenting cells) to remodel
the lymph node. The accompanying goal is to have the appropriate tumor EVs (those with antigens for
processing and presentation to T cells) enter the now-“hot” lymph nodes. These studies revealed that the
2K vesicles promoted both primary tumor growth as well as metastasis, while the 100K vesicles inhibited
this tumorigenicity and progression. This may be related to VEGF content of the 2K fraction, as well as the
autophagosome characteristics of that fraction; the roles of extracellular autophagosomes in cancer require
further study.

Modification of EVs is another means of improving their delivery capability. Ikuhiko Nakase (Osaka
Prefecture University, Japan) showed that one component of EVs as therapeutics is their loading with
biofunctional molecules. The other component is targeting EV delivery. EV uptake is thus critical for cargo
delivery, and internalization by endocytosis is considered a major pathway for this. However, Ikuhiko’s
group found that active induction of macropinocytosis (via cancer-related receptors and oncogenic RAS)
significantly enhances EV uptake. For instance, stimulation of EGFR enhances EV uptake by several orders
of magnitude. Further, they created a modification of the Cathelicidin antimicrobial peptide (CAMP/
CAP18), called sCAP18, that enhances cancer cell EV uptake by inducing macropinocytosis. They have also
made sCAP12-2, and stearyl-modified versions of these for insertion into EV membranes. Interestingly,
target-cell glycosaminoglycans were also required for the macropinocytotic uptake. Loading the EVs with
saporin (ribosome inactivator) resulted in efficient cell killing.

In an eye-opening talk, James Patton (Vanderbilt University, US) discussed the roles of EVs in retinal
regeneration. In retinal regeneration, the need is for quiescent stem cells to enter the cell cycle and
proliferate. Knowing that EVs from KRAS mutant cancer cells can induce KRAS wild type cells to proliferate
and invade, James’ group pursues EV-driven modes of retina regeneration. A cell type called Muller glia (MG)
in the retina is responsible for generating all the retinal cell type upon damage. In zebrafish, this process is
active in retinal regeneration; it is far less so for chicks, and even less active in mammals. Using the zebrafish
model, they attempted to drive Muller glia proliferation by treating retinas with EVs from a KRAS mutant
cell line (DKO1), which resulted in de-differentiation of some MG cells, along with some proliferation. These
outcomes prompted a large-scale screen of 59 different types of EVs to measure proliferative responses in
zebrafish eyes. They found that a rat glioma cell line, C6, provided active EVs. Further refinement of their EV
isolation techniques identified a small EV/exosome fraction that carried the biologic activity. In attempts to
boost the this activity, they found that overexpression of IL6 and ASCL1 in the C6 producer line did result
in more active exosomes. Going forward, the research group hopes to produce designer exosomes capable of
targeting MGs with high payloads of active cargo to promote retinal regeneration.

The final session of the conference was on EVs as biomarkers, conducted by hosts Julie Saugstad (Oregon
Health and Science University, US) and Saumya Das (Harvard/MGH US). David Lyden (Weill Cornell