2026-10-14T10:07:22Z https://api.figshare.com/v2/oai

oai:figshare.com:article/34039048 2026-10-01T05:14:15Z category_1 category_4 category_7 category_19 category_21 category_873 category_128 category_133 portal_63 item_type_6 month_year_10_2026

Absolute Quantification of Intravesicular Adenosine Triphosphate in Single Extracellular Vesicles Reveals Mitochondrial Bioenergetic Rewiring in Chemoresistance Xu Xiao (314389) Yuqing Luo (6598292) Chenxi Liu (286325) Haonan Di (12980576) Yunyun Hu (380121) Cheng Lu (57646) Hao Chen (5190) Xiaozhen Zhan (19208336) Guoqiang Su (4744521) Xiaomei Yan (325329) Biophysics Biochemistry Medicine Pharmacology Biotechnology Chemical Sciences not elsewhere classified Hematology Plant Biology sup >+</ sup quantitative metabolite analysis providing direct evidence particle adenosine triphosphate mediated metabolic communication loaded liposome standards generalizable analytical framework fundamental analytical gap cellular metabolic states bioenergetic analyses trace thereby enabling liposome intravesicular adenosine triphosphate chemosensitive cancer cells calibrated absolute measurement cellular atp pool thereby expanding chemoresistant cells vesicular cargo vesicle level targeted screen targeted metabolomics spatial coupling mitochondrial energetics mechanistic understanding laser platform investigating ev higher proportion flow cytometry ev biogenesis drug efflux constructed dual broader utility absolute quantification 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. 2026-10-01T00:00:00Z Text Journal contribution 10.1021/acsnano.6c15798.s001 https://figshare.com/articles/journal_contribution/Absolute_Quantification_of_Intravesicular_Adenosine_Triphosphate_in_Single_Extracellular_Vesicles_Reveals_Mitochondrial_Bioenergetic_Rewiring_in_Chemoresistance/34039048 CC BY-NC 4.0