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Page 4 of 9 Lai et al. J Cancer Metastasis Treat 2019;5:65 I http://dx.doi.org/10.20517/2394-4722.2019.011

Hence, development of EV-based liquid biopsy is hampered by two technical challenges, isolation of bona
fide lipid membrane vesicles and EV isolation from small sample volumes.

ISOLATING EVS THROUGH THEIR UNIQUE DEFINING FEATURE, LIPID MEMBRANE
The unique defining feature that differentiates EVs from other secreted cellular products is their lipid
membrane. Therefore, isolating EVs via their lipid membrane would enhance the specific enrichment of
EVs.

Although the lipid membrane of EVs originates from the secreting cells, the EV membrane is not
representative of the outer or inner cell membranes per se but rather of specific microdomains in cellular
membranes. For example, the membrane of exosomes derived from Daudi cells is enriched in lipid rafts
[24]
as evidenced by the presence of GM1 gangliosides which are enriched in lipid rafts . It was further
reported using annexin V (AV) binding studies that phosphatidylserines are exposed on the outer surface
[25]
of exosomes . However, it is not clear if exosomes with exposed phosphatidylserines are exosomes or
apoptotic bodies as membrane vesicles from healthy cell populations are likely to contain apoptotic bodies
[26]
from apoptotic cells that are inevitably present in a healthy cell population . In addition, it was also
[27]
observed that in platelets, AV binding activity was restricted mainly to microvesicles, and not exosomes .
Finally, we observed that small EVs produced by mesenchymal stem/stromal cells (MSCs) consist of at
least 3 sub-types that are distinguished by their mutually exclusive binding affinity for cholera toxin B
[28]
(CTB) chain, AV and shiga toxin B (ST) chain . The latter three proteins bind membrane lipids, GM1
gangliosides, phosphatidylserines and globotriaosylceramides, respectively. The extraction of small EVs
by these three proteins was confirmed by scanning electron microscopy images of nanosized, spherical
structures of approximately 100-200 nm. Protein analysis of the three EVs revealed an enrichment of
exosome-associated proteins such Alix, Tsg 101, CD9, CD81 in the CTB-binding EVs which were much
reduced in the AV-binding EVs and not detectable in the ST--binding EVs. RNA was detectable only in the
[28]
ST--binding EVs . It was also established through pulse-chase studies that the CTB-binding EVs have an
endosomal biogenesis, i.e., exosomes while the biogenesis of the AV- and ST-binding EVs is not known [28,29] .
In the pulse-chase studies, the MSCs were pulse-fed with either biotinylated transferrin or biotinylated
CTB and the biotinylated proteins was then “chased” to determine if the biotinylated proteins re-appear in
the culture medium as EVs.

CTB, AV and ST are multi-meric with at least five lipid-binding monomers per molecule. It was reported
that these monomers bind their lipid targets in a positive co-operative manner such that the lipid-bound
ligand is thermodynamically most stable when all the lipid-binding monomers are bound by the target
lipids [30-32] . Therefore, the binding of an EV type by any of the three ligands implies that the targeted
lipids for the ligands are exposed on the EV membrane surface and are also present in high localized
[28]
concentration to enable multimeric binding . This thermodynamic preference of each ligand to bind
multiple lipid molecules ensures that the ligand binds lipid-rich macromolecules or complexes such as EVs.

As a consequence of this preference, the lipid-binding ligand cannot be immobilized on a solid support
and must be in solution to minimize steric hindrance and facilitate ligand binding to multiple lipids
(unpublished observation, SKL). This thus minimizes the versatility in designing the procedural protocol
for isolation.

Together, the above observations suggest that EVs could be isolated through their lipid membrane via
specific membrane lipid-binding ligands as shown [Figure 1].