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

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.