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Page 208 Tutanov et al. Extracell Vesicles Circ Nucleic Acids 2023;4:195-217 https://dx.doi.org/10.20517/evcna.2023.17

[121]
immune evasion, angiogenesis, proliferation, migration, and invasion . In contrast to DPEP1, mechanistic
studies regarding CD73-positive EVs in cancer are more established, including a report that CD73 is
[122]
enzymatically active in B cells, although few studies are directly related to CRC . Cancer-derived CD73-
positive vesicles have been implicated in T-cell suppression, angiogenesis, resistance to anti-PD-1 therapy,
and suppression of T-cell clonal expansion [19,123-125] . In a recent report, EVs isolated from a CRC cell line,
DLD-1, as well as two other cancer lines, H292 and OvCAR3, were found to be enriched for CD73
compared to the cells themselves . A bispecific antibody to CD73 and EpCAM not only reduced the CD73
[126]
activity of these cancer-derived EVs, but also inhibited the effects of CD73 on reducing T-cell proliferation
and rescued the anti-tumor properties of T-cells . While a number of clinical trials have been conducted
[126]
or are under way for inhibition of CD73, the full potential of therapeutics might not be reached without
+
understanding how CD73 EVs contribute to cancer progression [119,127] .
We began to consider CD73-containing EVs as a biomarker for CRC when we discovered that they were
enriched with other GPI-APs in classical exosomes isolated from CRC cells . We found that CD73, like
[13]
DPEP1, was α2, 6-sialylated and that CD73 immunoreactivity was increased in CRC tissue compared to
[13]
normal colonic epithelium . CRC patients generally had more CD73 in sEVs isolated from plasma than
normal individuals, and, more broadly, CD73 was enriched in sEVs from a variety of different cancer cell
lines, strengthening its utility as a pan-cancer biomarker . Future studies along this line of investigation
[13]
might involve measuring the ability of CD73-positive EVs released from CRC tumors to produce adenosine
and testing the efficacy of CD73 inhibitors against circulating EVs, as systemic immunosuppression might
affect metastatic ability that could not be achieved by only tumoral CD73.

CEACAM5/CEACAM6
CEACAM5, typically referred to as CEA, is a commonly used CRC biomarker. Both CEACAM5 and
CEACAM6 are GPI-APs in the carcinoembryonic antigen (CEA) family, so called because they were
originally believed to be expressed in fetal development, absent in healthy adults, but expression
reappearing as cancers revert to an onco-fetal state [128,129] . Functionally, CEACAM5 reportedly acts as an
adhesion molecule, as well as having roles in inhibiting apoptosis, cell polarization, and differentiation in
CRC cells, as well as increasing metastatic ability [130-133] . CEACAM6, sometimes referred to as CD66c, has
similar roles, with reports highlighting its role in CRC growth and immunosuppression of T cells; its
expression as assessed by IHC in tissue is touted as a poor prognosis marker in CRC [129,131-135] .

Monitoring plasma CEA levels following surgical resection of the tumor continues to be used as a
biomarker for CRC recurrence, although its reliability is questioned . The field has pivoted to
[136]
multimarker analysis to increase the reliability and sensitivity of CEA’s diagnostic and prognostic value,
including immune population ratios, cytokines, or other cancer antigens, with some improvement in
predicting recurrence or disease presence [137-140] . While measuring CEA levels alone in the plasma or in
combination with other markers has been a focus for increasing biomarker accuracy and sensitivity, the EV
community has begun to see merit in isolating EV-bound CEA proteins for diagnostic and prognostic value.
CEACAM6 was detected from a CRC line, LIM1215, in EVs, and has been reported to be a general marker
[141]
for neutrophil-derived EVs . One study has shown that exosomal CEA from plasma increased sensitivity
and specificity for predicting distant metastasis, while another has shown that CEA microvesicle levels
+
could distinguish between benign polyps and CRC [142,143] . Along with using EV isolation to increase the
predictive value of CEA as a biomarker, groups have been combining non-EV bound CEA levels in
combination with other biomarkers that are present on EVs or found other markers that have higher
sensitivity. TSPAN1-positive exosomes by CD63 capture were found to have higher sensitivity in detection
of CRC than plasma CEA levels alone . CPNE3 combined with CEA on EVs was a superior diagnostic
[144]
biomarker for CRC than either protein alone . Other exosomal components or molecules associated with
[145]