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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 \u201cATP fluorescence density\u201d 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.<\/jats:p>","DOI":"10.1021\/acsnano.6c15798","type":"journal-article","created":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T06:02:39Z","timestamp":1790834559000},"page":"27508-27525","source":"Crossref","is-referenced-by-count":0,"title":["Absolute Quantification\nof Intravesicular Adenosine\nTriphosphate in Single Extracellular Vesicles Reveals Mitochondrial\nBioenergetic Rewiring in Chemoresistance"],"prefix":"10.1021","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-4213-7303","authenticated-orcid":true,"given":"Xu","family":"Xiao","sequence":"first","affiliation":[{"name":"Xiamen University , , , ,","place":["Xiamen, Fujian, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuqing","family":"Luo","sequence":"additional","affiliation":[{"name":"Xiamen University , , , ,","place":["Xiamen, Fujian, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenxi","family":"Liu","sequence":"additional","affiliation":[{"name":"Xiamen University , , , ,","place":["Xiamen, Fujian, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haonan","family":"Di","sequence":"additional","affiliation":[{"name":"Xiamen University , , , ,","place":["Xiamen, Fujian, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunyun","family":"Hu","sequence":"additional","affiliation":[{"name":"Xiamen University , , , ,","place":["Xiamen, Fujian, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cheng","family":"Lu","sequence":"additional","affiliation":[{"name":"Xiamen University , , , ,","place":["Xiamen, Fujian, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Chen","sequence":"additional","affiliation":[{"name":"Xiamen University , , , ,","place":["Xiamen, Fujian, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaozhen","family":"Zhan","sequence":"additional","affiliation":[{"name":"Xiamen University , , , ,","place":["Xiamen, Fujian, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guoqiang","family":"Su","sequence":"additional","affiliation":[{"name":"Xiamen University , , , ,","place":["Xiamen, Fujian, China, 361003"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7482-6863","authenticated-orcid":true,"given":"Xiaomei","family":"Yan","sequence":"additional","affiliation":[{"name":"Xiamen University , , , ,","place":["Xiamen, Fujian, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,1]]},"reference":[{"issue":"4","key":"2026101310085673300_cit1","doi-asserted-by":"publisher","first-page":"274","DOI":"10.1038\/s41568-024-00786-4","article-title":"Metabolic interplays\nbetween the tumour and the host shape the tumour macroenvironment","volume":"25","author":"Altea-Manzano","year":"2025","journal-title":"Nat. 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Toxicol."},{"key":"2026101310085673300_cit7","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.drup.2014.11.002","article-title":"The modulation of\nABC transporter-mediated multidrug resistance in cancer: A review\nof the past decade","volume":"18","author":"Kathawala","year":"2015","journal-title":"Drug Resistance Updates"},{"issue":"7","key":"2026101310085673300_cit8","doi-asserted-by":"publisher","first-page":"452","DOI":"10.1038\/s41568-018-0005-8","article-title":"Revisiting\nthe role of ABC transporters in multidrug-resistant\ncancer","volume":"18","author":"Robey","year":"2018","journal-title":"Nat. Rev. Cancer"},{"issue":"1","key":"2026101310085673300_cit9","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1038\/nrclinonc.2016.60","article-title":"Cancer metabolism:\na therapeutic perspective","volume":"14","author":"Outschoorn","year":"2017","journal-title":"Nat. Rev. Clin. 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Immunol."},{"issue":"5","key":"2026101310085673300_cit19","doi-asserted-by":"publisher","first-page":"369","DOI":"10.1038\/s41580-022-00460-3","article-title":"Challenges and directions in studying cell-cell communication by\nextracellular vesicles","volume":"23","author":"van Niel","year":"2022","journal-title":"Nat. Rev. Mol. Cell\nBiol."},{"issue":"12","key":"2026101310085673300_cit20","doi-asserted-by":"publisher","first-page":"924","DOI":"10.1038\/s41571-025-01074-2","article-title":"Clinical\nrelevance of extracellular vesicles in cancer-therapeutic and diagnostic\npotential","volume":"22","author":"Greening","year":"2025","journal-title":"Nat. Rev. Clin. Oncol."},{"issue":"4","key":"2026101310085673300_cit21","doi-asserted-by":"publisher","DOI":"10.1002\/jev2.12320","article-title":"MitoEVs: A new player in multiple disease pathology\nand treatment","volume":"12","author":"Zhou","year":"2023","journal-title":"J. Extracell. 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Pharmacol."},{"issue":"1","key":"2026101310085673300_cit25","doi-asserted-by":"publisher","DOI":"10.1038\/srep20903","article-title":"ATP-containing\nvesicles in stria vascular marginal\ncell cytoplasms in neonatal rat cochlea are lysosomes","volume":"6","author":"Liu","year":"2016","journal-title":"Sci. Rep."},{"issue":"6062","key":"2026101310085673300_cit26","doi-asserted-by":"publisher","first-page":"1573","DOI":"10.1126\/science.1208347","article-title":"Autophagy-dependent anticancer immune responses induced\nby chemotherapeutic agents in mice","volume":"334","author":"Michaud","year":"2011","journal-title":"Science"},{"issue":"12","key":"2026101310085673300_cit27","doi-asserted-by":"publisher","first-page":"1741","DOI":"10.4161\/auto.21858","article-title":"ATP is released from autophagic vesicles\nto the extracellular space in a VAMP7-dependent manner","volume":"8","author":"Fader","year":"2012","journal-title":"Autophagy"},{"issue":"3","key":"2026101310085673300_cit28","doi-asserted-by":"publisher","first-page":"159","DOI":"10.1038\/s41577-022-00760-x","article-title":"Mitochondrial\ncontrol of inflammation","volume":"23","author":"Marchi","year":"2023","journal-title":"Nat. Rev. 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Chem."},{"issue":"35","key":"2026101310085673300_cit51","doi-asserted-by":"publisher","first-page":"12176","DOI":"10.1021\/ja104052c","article-title":"Size differentiation\nand absolute\nquantification of gold nanoparticles via single particle detection\nwith a laboratory-built high-sensitivity flow cytometer","volume":"132","author":"Zhu","year":"2010","journal-title":"J. Am. Chem. Soc."}],"container-title":["ACS Nano"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acsnano.6c15798","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancac3\/article-pdf\/20\/40\/27508\/68722128\/acsnano.6c15798.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancac3\/article-pdf\/20\/40\/27508\/68722128\/acsnano.6c15798.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T14:44:48Z","timestamp":1791902688000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancac3\/article\/20\/40\/27508\/5443132\/Absolute-Quantification-of-Intravesicular"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,10,1]]},"references-count":51,"journal-issue":{"issue":"40","published-online":{"date-parts":[[2026,10,1]]},"published-print":{"date-parts":[[2026,10,13]]}},"URL":"https:\/\/doi.org\/10.1021\/acsnano.6c15798","relation":{},"ISSN":["1936-0851","1936-086X"],"issn-type":[{"value":"1936-0851","type":"print"},{"value":"1936-086X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10,1]]}}}