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Page 210 Tutanov et al. Extracell Vesicles Circ Nucleic Acids 2023;4:195-217 https://dx.doi.org/10.20517/evcna.2023.17

shielding the GPI anchor from the aqueous milieu, such as embedment in phospho (mono- or bi-)layers
surrounding non-vesicular lipid-filled particles (like surfactant-like particles, milk fat globules, nodal
vesicular particles, lipoprotein-like particles), oligomerization or multimerization without the use of
additional constituents, or assembly into heterometric structures with specific carrier or scaffolding
proteins [78,163-165] . Lipid-based sorting appears to indicate various methods for sorting GPI-APs into EVs, for
both releases with an intact GPI anchor, as well as cleavage of the GPI anchor. Future lipidomic analysis of
exomeres and supermeres might offer insights into their ability to include intact GPI-APs based on the lipid
content. There are, however, other ways of secreting GPI-APs aside from having an intact GPI anchor
inasmuch the anchor can be cleaved off both intracellularly and extracellularly. Intracellular cleavage might
favor incorporation into exomeres and supermeres as they are released particles with other proteins
reminiscent of stress granules and this pathway might inform the ways these nanoparticles form and
mature. In contrast, extracellular cleavage could inform EVP interplay in the circulation, which is especially
important in the context of the tumor microenvironment. Indeed, some of the GPI-APs that have been
identified in sEVs and supermeres, such as PLAUR and CEACAM5, have been shown to be cleaved by
GPLD1 [32,92-94] . Furthermore, the effects of extracellular cleavage of GPI-APs by GPI-PLD, which is abundant
in serum, and the accessibility for cleavage provided by the inclusion of GPI-APs in different types of EVPs,
[166]
are important when considering specific GPI-APs as biomarkers .

Another area where the study of EVPs and GPI-APs can complement each other is the apical versus
basolateral sorting of GPI-APs. The impact of oligomerization, missorting, and differential sorting of
different forms of the same protein (isoforms, glycosylated forms) can inform the biogenesis of EVPs and,
in turn, increase our understanding of the underlying mechanisms of apical/basolateral sorting of GPI-APs.
The heterogeneity of exosomes, and specifically heterogeneity between apically and basolaterally secreted
exosomes from polarized cells, has been discussed in the literature. Some authors claim that no less than
[167]
30% of the total protein cargo is different between the two fractions . Moreover, protein cargo in the
apical EV fraction has been reported to be more homogeneous compared to its basolateral counterpart .
[3]
Moreover, as stated above, we have been able to isolate and sort individual exosomes with characteristic
apical and basolateral cargo, which provides a unique opportunity to understand mechanisms of biogenesis
in the context of loss of polarity [Figure 6]. Some of the proteins implicated in the apical sorting of GPI-
APs, such as flotillins and annexins, are accepted as characteristic sEV marker proteins , but have been
[168]
shown to facilitate ESCRT-independent exosome pathway (flotillins ), and are marker proteins of EVs
[169]
[2]
distinct from classical exosomes (annexins A1 and A2 ).
Along with providing insights into the biogenesis of EVPs and the secretion patterns of a cell, the study of
GPI-APs allows for assigning functions to extracellular carriers based on cargo. Our lab has previously
shown that the cargo, ACE2, on sEVs and exomeres can act as a decoy for SARS-CoV-2 virus . Assigning
[118]
function to an extracellular particle based on its cargo can be translated to the CRC field in relation to GPI-
APs, as we have identified a number of GPI-APs present in sEVs, exomeres, and supermeres [Table 2]. One
striking example is that CD73 is enriched in classical exosomes . As discussed above, CD73-positive EVs
[13]
that aid in adenosine production can alter the TME toward an immunosuppressive state . It will be
[126]
interesting to determine the functions of the newly discovered supermeres and exomeres based on their
cargo .
[13]

In conclusion, with the rapid evolution of the EVP field and the emergence of new extracellular particles
that offer the ability to allocate cargo previously thought to be in EVs to their proper carrier, approaches
taking into account specific protein families like GPI-APs abundant in EVs might offer some help in
overcoming EVs notorious heterogeneity. Moreover, studying these proteins in the context of exosomes,