Absolute Quantification of Intravesicular Adenosine Triphosphate in Single Extracellular Vesicles Reveals Mitochondrial Bioenergetic Rewiring in Chemoresistance.

The inability to quantify specific metabolites within individual extracellular vesicles (EVs) represents a fundamental analytical gap, precluding a mechanistic understanding of how cellular metabolic states are encoded into vesicular cargo. Here, we introduce single-particle adenosine triphosphate (ATP) quantification by nano-flow cytometry (SPAQ-nFCM), a laboratory-constructed dual-laser platform that overcomes particle size heterogeneity by integrating ATP-loaded liposome standards with a size-corrected "ATP fluorescence density" metric, thereby enabling liposome-calibrated absolute measurement of intravesicular ATP. Applying SPAQ-nFCM, we find that compared to chemosensitive cancer cells, chemoresistant cancer cells secrete EVs highly enriched in ATP, as evidenced by both a higher proportion of ATP-positive EVs (ATP+ EVs) and an elevated intravesicular ATP content per EV. Targeted metabolomics and bioenergetic analyses trace this to a profound metabolic reprogramming toward enhanced mitochondrial oxidative phosphorylation, thereby expanding the cellular ATP pool in chemoresistant cells. Strikingly, single-particle multiparametric analysis reveals a distinct, ATP-enriched P-glycoprotein (P-gp)-positive EV subpopulation, providing direct evidence for the spatial coupling of mitochondrial energetics to drug efflux. Moreover, we demonstrate that ATP+ EVs predominantly exhibit high expression of the classical tetraspanin marker CD63. A targeted screen further identifies conserved molecular regulators (Rab27a/b, VPS35, DRP1) governing ATP+ EV biogenesis. Thus, SPAQ-nFCM provides a generalizable analytical framework for quantitative metabolite analysis at the single-vesicle level. The link between ATP-enriched EVs and chemoresistance further underscores the broader utility of SPAQ-nFCM for investigating EV-mediated metabolic communication.