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SDIRSACR Oncology Insights

As part of the larger liquid biopsy framework, EV assays need to be framed as complementary to circulating tumor DNA
and cell-free DNA, rather than competitive technologies, because they bear cell-of-origin information in proteins, lipids,
glycans, and small RNAs not accessible from ctDNA, and ctDNA is optimized for detecting tumor fraction and tracking
genomic evolution over time (10, 11). In metastatic prostate cancer, cfDNA longitudinal tumor fraction is correlated
with metastatic burden and response to therapy and provides good utility in disease dynamics that can be augmented
by EV-based phenotyping for functional interpretation as well as increased sensitivity in multimodal algorithms (8, 11).
Contemporary syntheses emphasize that EV-mediated communication in the tumor microenvironment—e.g., stromal
determinants of resistance—is mechanistic context to biomarker alterations, especially when EV diagnostics are used
to direct adaptive therapy or to predict resistance(6, 8).
Clinical translation also requires transparency and reproducibility in reporting, and these factors are always
acknowledged as determinants of scientific transparency in both the clinical literature and in the EV field itself (40,
42). Authors should explain pre-analytics like sample type, collection tubes, handling temperatures and times, and
any clarification steps in a clear manner, with matrix-specific mitigations like uromodulin reduction and alkaline
washes explained in urine-based protocols to facilitate replication and quality benchmarking (40, 41). Isolation
parameters—operating conditions, method class, and capture chemistries—have to be reported in sufficient detail
to enable replication between platforms, and particle identity has to be validated by orthogonal sizing as well as by
canonical positive and negative markers suitable to the biofluid and method, according to EV-TRACK guidelines for the
completeness of reporting (36, 40). Analytical approaches, calibration and normalization, and validation design should
be pre-specified and, to the extent possible, blinded and consistent with the targeted clinical use; these elements
reflect current reporting templates for diagnostics and biomarker studies and strongly enhance peer review and reuse
(40, 42). Finally, as terminology is itself a moving target with subtypes and biogenesis pathways being revised, authors
have to report operational definitions and report limitations in nomenclature to ensure interpretability across studies
of differing isolation and characterization depth (44, 45).
In short, the field has long since outgrown differential ultracentrifugation and now comfortably resides on immuno-,
chemical-, microfluidic-, and nanomaterial-based platforms that increase yield, purity, and tumor specificity at rates
compatible with clinical testing, and that can be supplemented with single-particle analytics to release diagnostically
enriched subpopulations with actionable resolution (46). Transparent, EV-TRACK-aligned reporting; matrix-tailored,
versioned standard operating procedures; and prespecified plans for multimodal integration with ctDNA and clinical
variables now represent the defining steps for translating EV assays from discovery to reproducible, clinically relevant
diagnostics with the understanding that EVs report on and shape tumor biology, including response and resistance to
therapy in the tumor microenvironment (6, 8)

Conclusion
EVs have become indispensable players in cancer biology, acting not only as cellular activity waste but as dynamic
players in tumor development, metastasis, immune evasion, and drug resistance. Their ability to transport a high and
diverse payload of biomolecules—characteristic of their cells of origin—is particularly valuable for cancer diagnosis.
The enrichment of tumor-related proteins, RNAs, and lipids in EVs, as well as secretion within easily accessible body
fluids, positions them well to be effective candidates for efficient non-invasive biomarker identification and early
cancer detection. Despite having great potential, there are technical hindrances to their clinical use, particularly
with respect to standardization and scalability of EV isolation procedures. Current gold-standard techniques such as
ultracentrifugation are time-consuming, wasteful, and not favorable for repeated clinical use. While immunoaffinity-
based techniques are specific, scalability by expense and complexity is low. New high-throughput, low-cost EV isolation
technologies—e.g., utilization of single-domain antibodies—are a key next step towards unlocking the complete
diagnostic and therapeutic potential of EVs. With continued research and resolution of those technology hurdles, EVs
can revolutionize cancer diagnostics such that the early detection, enhanced patient stratification, and monitoring of
treatment response in real time can be feasible in a minimally invasive manner.

Acknowledgment: This research was supported by the Science Fund of the Republic of Serbia, Grant PRISMA No. 4747,
Project title: Advancing REversible immunocapture toward SCALablE EV purification—RESCALE-EV, the European Union
under Grant Agreement No. 101182851and the Ministry of Science, Technological Development, and Innovation of the
Republic of Serbia Agreement No. 451-03-136/2025-03/200168

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