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Tutanov et al. Extracell Vesicles Circ Nucleic Acids 2023;4:195-217 https://dx.doi.org/10.20517/evcna.2023.17 Page 209

EVs that increased diagnostic accuracy when combined with CEA include miR-150-5p, lnRCA CRNDE-h,
and CAT1 [146,147] . We utilized FAVS to flow sort EVs double-positive for CEA and DPEP1 from the plasma
[13]
of CRC patients as a means of detecting EVs released from the cancer rather than other sources .
CEACAM5 was present in exomeres and supermeres isolated from CRC cell lines and detected in the
plasma from CRC patients but not from healthy controls . Interestingly, EVs from bacteria can affect the
[13]
release of CEA. Exposure of EVs isolated from Lactobacillus rhamnosus but not from other bacteria to CRC
cell lines increased CEA levels and inhibited cell proliferation . Understanding how the microbiome and
[148]
associated EVs might affect both CRC progression and detection is an emerging field of study.

GPC1
Glypican-1, encoded by the gene GPC1, is another GPI-AP that we identified as being secreted by a wide
variety of cancer cell lines, as well as primary kidney epithelial cells, with a marked enrichment in exomeres
[13]
and supermeres in comparison to sEVs . Glypican-1 can modulate signaling pathways by binding to
growth factors, and it has been reported to regulate TGF-β signaling to increase proliferation and migration
while inhibiting apoptosis in CRC cells [149,150] . Glypican-1 has been reported to be a cancer exosomal marker,
in contrast to our finding that they are mostly associated with nanoparticles . In a CRC context,
[151]
overexpression of Snail, a transcription factor involved in EMT, has been reported to increase the
presentation of glypican-1 on CRC EVs . Glypican-1 has been shown to be increased in EVs isolated from
[152]
CRC tumor tissue and plasma in comparison to normal controls and was regulated by miRNAs, leading to
reduced secretion in an EV-bound form . The discrepancy between our results showing that glypican-1 is
[153]
enriched in nanoparticles, not sEVs, highlights the ever-evolving EV field and the importance of continually
improving methods of isolation and parsing subsets .
[13]

Challenges and Future Directions
Previous findings from our laboratory, and others, have reported the enrichment of GPI-APs in CRC
sEVs [5,13,154] . The presence of GPI-APs in sEVs dates back to the early 1990s, at which time several GPI-APs,
including AChE, DAF, MIRL, and LFA-3, were identified in human reticulocyte sEVs . Since then, GPI-
[155]
APs have become recognized as part of the standard repertoire of sEVs, and functional roles assigned to
them [78,156,157] . For instance, NKG2D-ligands including GPI-APs such as ULBP1, ULBP2, and ULBP3 have
been reported to display two main mechanisms of release: shedding by metalloproteases and recruitment to
sEVs. Expression of these proteins is induced by stress and signals for immune activation, and they can also
[158]
serve as immune decoys when released by tumor cells . The enrichment of lipid rafts and sphingomyelins
in sEVs make them a perfect carrier for GPI-Aps, as lipid-based sorting is a main mechanism for GPI-AP
delivery to the cell surface [7,159,160] . GPI-APs can be incorporated into early endosomes that will eventually
mature into multivesicular bodies (MVBs) or as newly synthesized GPI-APs that can be directly trafficked
from the Golgi to MVBs . While there are multiple ways that GPI-APs can be packaged into
[161]
exosomes [78,161] , it is believed that the majority of GPI-APs leaving cells via exosomes start their journey at
the cell surface. Furthermore, this protein class has been associated with exosome biogenesis; for example,
the abundance and activity of the GPI-AP, tetherin, affects the ability of exosomes to detach from the donor
cell membrane and adhere to the recipient cell membrane. In tetherin knockout cells, there was a reported
decrease in vesicles that remain attached to the plasma membrane clustered at its surface after MVB fusion
with the plasma membrane and an increase in exosomes discharged in the medium. This phenotype could
[162]
be rescued by wild-type tetherin but not tetherin lacking its GPI anchor .

Going forward, the forms and compartments in which extracellular GPI-APs are found will be informative.
Although there is limited published information on GPI-APs in exomeres and supermeres due to their
recent discovery, GPI-APs are transported in various non-vesicular structures in the extracellular space,
both as particles and multimers. The absence of an EV membrane dictates the need for other ways of