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Asao et al. Extracell Vesicles Circ Nucleic Acids 2023;4:461-85 https://dx.doi.org/10.20517/evcna.2023.37 Page 3

Figure 1. Overview of extracellular vesicles and particle (EVP)-focused fields of study in cancer research. Intercellular communication:
EVPs mediate intercellular communication between cancer, stromal, and immune cells by transferring various biomolecules to nearby
as well as distant organs, resulting in both local and systemic effects. Cancer-derived EVPs stimulate angiogenesis, promote tumor
growth, and suppress antitumor immunity, enhancing tumor invasion and metastatic potential. Heterogeneity: EVPs are highly diverse
and new populations are constantly being discovered. The classification of new EVP populations requires multifaceted evaluation,
including size, composition, membrane surface markers, and origin. Technology: various methods for separating EVPs, including
differential ultracentrifugation, size exclusion chromatography, immunoaffinity capture, and asymmetric flow field flow fractionation,
are available and they have their own advantages and disadvantages. Furthermore, analyses at the single EVP level, such as
nanoparticle tracking analysis, high-sensitivity flow cytometry, high-resolution microscopy, as well as in vivo imaging, are now
introduced. Biomarkers: EVPs in bodily fluids show potential as non-invasive biomarkers for early diagnosis, treatment response, and
prognosis of cancer. Their constituent proteins, nucleic acids, lipids, and metabolites can be isolated and analyzed by mass
spectrometry and next-generation sequencing. Treatment: EVPs can be used therapeutically by administering EVPs isolated from
specific cells, loading various biomolecules such as anticancer or molecularly targeted drugs, or by engineering EVPs to enhance
function. Strategies that block the production of tumor-derived EVPs or block the interaction of EVPs with target cells are also being
investigated. EMT: epithelial-mesenchymal transition; ECM: extracellular matrix; SMAP: supramolecular attack particles; ARMM:
arrestin domain-containing protein 1 -mediated microvesicles; dUC: differential ultracentrifugation; SEC: size exclusion
chromatography; IAC: immunoaffinity capture; AF4: asymmetric flow field flow fractionation; NTA: nanoparticle tracking analysis.

involved in the regulation of systemic metabolism. More recently, another small extracellular vesicle
subpopulation has been described, the small ectosomes, approximately 120 nm size particles that bud from
[8]
the plasma membrane . In contrast to exosomes, which express CD63 but little CD9, ectosomes express
CD9 and CD81 but little CD63. This finding not only challenges the conventional assumption that
tetraspanins (CD9, CD63, CD81) serve as ubiquitous surface markers for exosomes, but also highlights the
need to redefine our understanding of ectosomes as a small EVP subset.

The complexity of EVP subpopulations and classification is constantly evolving, as many other subsets have
been defined based on size, function and cargo, or the cell type/organ or even species they derive from.
Arrestin domain-containing protein 1 (ARRDC1)-mediated MVs (ARMMs) are small vesicles (40-100 nm)
that bud directly from the plasma membrane in an ARRDC1- and tumor susceptibility 101 (TSG101)-