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

ABSTRACT 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.