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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 17

Single EVP analysis
The growing awareness of EVP heterogeneity has revealed limitations in conventional bulk analysis
approaches. As a result, there is an increasing need for analyses focusing on EVP subpopulations and even
single EVPs, which have become more common over the past decade . Nanoparticle tracking analysis
[179]
(NTA) is a method for analyzing the size of isolated EVPs by imaging the scattered light caused by the
[180]
particles' Brownian motion . This allows for label-free determination of EVP size (ranging from 70 nm to
several hundred micrometers) and particle count in solution. Additionally, high-resolution flow cytometry
is gaining popularity . By staining surface antigens with fluorescently labeled antibodies, single EVP
[181]
characterization is possible. Furthermore, a proximity barcoding assay was recently used to analyze the
[182]
expression of individual molecules for single EVPs . This was accomplished by conjugating a DNA
barcode to an antibody that recognizes molecules expressed on the surface of EVPs, allowing for high-
resolution expression analysis.

Imaging techniques for single EVPs include not only conventional transmission electron microscopy but
also atomic force microscopy (AFM) for analyzing EVP surface structures . AFM allows for observation
[183]
at high resolutions, such as a vertical resolution of approximately 0.1 nm. Furthermore, super-resolution
fluorescence microscopy [photoactivated localization microscopy (PALM) and stochastic optical
reconstruction microscopy (STORM)] has become available in recent years and is playing an increasingly
significant role in single EVP analysis . This versatile technique can be used for various applications, such
[184]
as staining and observing surface antigens at the single EVP level and tracking EVP trafficking within cells.
As a means of analyzing the composition of single EVPs, research using laser tweezers Raman spectroscopy
(LTRS) has also been reported [185,186] . LTRS is a nondestructive method for analyzing molecular structures
based on Raman scattered light, and it can examine the composition of proteins, nucleic acids, and lipids in
single EVPs. By combining LTRS with other techniques, it has been demonstrated that tumor-derived EVPs
can be distinguished from erythrocyte and platelet-derived EVPs in plasma at the single EVP level .
[187]
Moreover, combining EVP labeling and microscopy has enabled in vivo single EVP analysis [188-191] .

To summarize, various analytical methods for single EVPs have become available over the past decade,
revealing characteristics of EVPs that were previously unknown due to the limitations of bulk analysis.
Additionally, observing and tracking the intracellular localization of specific EVPs have become useful for
exploring biogenesis. In vivo single EVP analysis has also become possible . The emergence of these
[190]
technologies has deepened our understanding of EVP heterogeneity. However, many of these technologies
are not suitable for high-throughput single EVP analysis and the development of such methods is much
needed. While proteomics and genomics analyses have been applied to bulk EVPs, single EVP “omics”
analysis remains challenging, and the development of systems capable of omics analysis with minimal input
is desirable. Thus, analysis techniques targeting single EVP will continue to advance and serve as a driving
force for future EVP research.

DISCUSSION
Over the last decade, the EVP-cancer field has experienced significant advancements that have
fundamentally changed our understanding of intercellular communication and cancer biology. With the
convergence of state-of-the-art technologies, interdisciplinary collaboration, and novel insights, the field of
EVPs will continue to undergo a paradigm shift in the next decade, unlocking new possibilities in cancer
research and treatment. Looking back, the discovery of EVPs as key players in cell-to-cell communication
has paved the way for a deeper understanding of their roles in various physiological processes and diseases,
particularly cancer progression and metastasis. Key processes involving EVPs include metastasis,
angiogenesis, immune response modulation, epithelial-to-mesenchymal transition, and PMN formation.