When EV Dose Is Not What It Seems: Quantitative Mismatch Between Particle Number- and Protein-Based Dosing

Extracellular vesicles (EVs) are widely studied as mediators of intercellular communication, yet their quantitative characterization remains inconsistent, with EV abundance often assumed to be equivalent from particle number or bulk protein content. In this study, we systematically investigated how vesicle release rate, cellular proliferation, and tumor origin jointly shape EV-associated protein output. Using a panel of 19 human cancer-derived and non-cancer cell lines cultured under standardized conditions, we isolated small extracellular vesicle–enriched fractions and quantified particle number, EV-associated protein content, cell number, and incubation time in line with current MISEV recommendations. By integrating these measurements, we derived complementary quantitative descriptors capturing vesicle release characteristics (VRC), average protein content per vesicle (VP), and vesicular protein release characteristics (PRC). Cancer cells released substantially more EVs per cell than non-cancer cells, yet their vesicles contained significantly less protein on a per-vesicle basis. Reduced EV protein load was independently associated with both cancer origin and proliferation rate and was not explained by differences in vesicle size. Despite the elevated vesicle output, cancer-derived cells exported less total vesicular protein per cell and time unit. These findings demonstrate that particle number and protein content describe distinct and non-equivalent aspects of EV output. Our results highlight the limitations of single-metric normalization strategies and underscore the need for integrated, multidimensional reporting to enable biologically meaningful comparisons of EV preparations.