Single-Mitochondrion ATP Profiling Directs Discovery of Targetable OXPHOS Dependency in Cancers

Mitochondrial adenosine triphosphate (mitoATP) serves as the primary bioenergetic currency for Oxidative Phosphorylation (OXPHOS)-driven malignancies, yet its precise organelle-level quantification remains challenging due to mitochondrial heterogeneity and cytosolic interference. Herein, we report MitoATP-nFCM, a nano-flow cytometry platform enabling single-mitochondrion ATP measurement via simultaneous fluorescence and side scatter detection. We uncover 1.7-1.9-fold higher ATP levels in isolated mitochondria from breast (MCF-7, MDA-MB-231) and colon (HCT-15, HCT-116) Cancer cells than in their normal counterparts. Single-organelle analysis further reveals coordinated metabolic reprogramming in Cancer mitochondria, featuring elevated membrane potential, increased ATP Synthase expression, and reduced Hexokinase 2 levels, demonstrating their OXPHOS-dominant bioenergetic phenotype that contrasts with classical Warburg-effect expectations. Furthermore, we establish a screening strategy to identify highly potent cancer-selective inhibitors targeting Mitochondrial Metabolism. We find that bedaquiline (ATP Synthase Inhibitor) outperforms oligomycin A in specificity, VLX600 (electron transport chain inhibitor) shows superior selectivity to rotenone/metformin, and CPI-613 (tricarboxylic acid cycle blocker) surpasses other Glutaminase inhibitors. MitoATP-nFCM establishes a quantitative single-organelle platform that profiles elevated mitoATP levels in Cancer cells and enables precision screening of OXPHOS-targeting inhibitors.