{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,25]],"date-time":"2026-07-25T03:45:19Z","timestamp":1784951119226,"version":"3.55.0"},"reference-count":48,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2025,5,27]],"date-time":"2025-05-27T00:00:00Z","timestamp":1748304000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","award":["RGPIN-2018-04233"],"award-info":[{"award-number":["RGPIN-2018-04233"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010571","name":"R\u00e9seau en Bio-Imagerie du Quebec","doi-asserted-by":"publisher","award":["Recruitment Scholarship"],"award-info":[{"award-number":["Recruitment Scholarship"]}],"id":[{"id":"10.13039\/100010571","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100022425","name":"Institut Du Cancer de Montr\u00e9al","doi-asserted-by":"publisher","award":["Canderel Scholarship"],"award-info":[{"award-number":["Canderel Scholarship"]}],"id":[{"id":"10.13039\/501100022425","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100005242","name":"Universit\u00e9 de Montr\u00e9al","doi-asserted-by":"publisher","award":["Student Scholarship"],"award-info":[{"award-number":["Student Scholarship"]}],"id":[{"id":"10.13039\/501100005242","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    It is known that ultrasound-targeted microbubble cavitation (UTMC) can induce vasodilation. This image-guided spatially targeted approach is called provascular therapy when used as a radiotherapy sensitizer in radiation oncology. Extracellular adenosine-5\u2019-triphosphate (eATP), which plays an important role in vascular tone regulation, is released by cells following UTMC, possibly through sonoporation (formation of temporary and non-deadly pores in the cell membrane) and\/or cell death. Herein, we were interested in quantifying UTMC-mediated ATP released\n                    <jats:italic>in vitro<\/jats:italic>\n                    using a microfluidics-based model and study its relationship with cell fate to better understand and improve bioeffects induced by UTMC. Lipid microbubbles (MB, Definity\u00ae), luciferin-luciferase (LL \u2013 for eATP quantification), and propidium iodide (PI \u2013 poration tracer) were flowed over HUVEC cells cultured in a microfluidic device. Ultrasound at 1 MHz, varying in pressure (peak negative pressure: 300, 400 kPa) and length (10, 100, 1000 cycles) were applied to the chip. The LL chemiluminescent signal after the ultrasound pulse was acquired with an EMCCD camera to characterize ATP release kinetics. Then, a viability assay was performed with calcein-AM. An in-house MATLAB program pairing eATP kinetics with PI\/calcein data was used to classify cells into three categories (sonoporated, dead, and untreated). Within the testing conditions, a single UTMC pulse caused between 4% and 55% PI-positive (PI+) cells in the ultrasound-treated area. Amongst PI+ cells, we generally found more dead cells than sonoporated cells, except for milder pulses (300 kPa; 10 and 100 cycles). The analysis of individual responses of ATP release demonstrated that dead cells released more ATP (up to 22.4\u2009\u00b1\u200912.2\u2009fmol\/cell) than sonoporated cells (6.8\u2009\u00b1\u20093.4\u2009fmol\/cell) and at a faster release rate which peaked at 4s. This study showed that sonoporation plays a significant role in UTMC-mediated ATP release, advancing our understanding of UTMC\u2019s potential use as a radiosensitizer in solid tumors.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0319318","type":"journal-article","created":{"date-parts":[[2025,5,27]],"date-time":"2025-05-27T13:40:37Z","timestamp":1748353237000},"page":"e0319318","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["Single cell level analysis of ATP release kinetics and cell fate following ultrasound targeted microbubble cavitation using microscopy techniques"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-2287-4870","authenticated-orcid":true,"given":"Marie","family":"Amate","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ju Jing","family":"Tan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francis","family":"Boudreault","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryszard","family":"Grygorczyk","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Gervais","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9325-017X","authenticated-orcid":true,"given":"Fran\u00e7ois T. 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and the possible implications for radiotherapy","volume":"9","author":"RH Thomlinson","year":"1955","journal-title":"Br J Cancer"},{"key":"pone.0319318.ref004","doi-asserted-by":"crossref","first-page":"94","DOI":"10.3389\/fphar.2012.00094","article-title":"Targeting tumor perfusion and oxygenation to improve the outcome of anticancer therapy","volume":"3","author":"BF Jordan","year":"2012","journal-title":"Front Pharmacol"},{"issue":"4","key":"pone.0319318.ref005","doi-asserted-by":"crossref","DOI":"10.1161\/CIRCIMAGING.114.002979","article-title":"Augmentation of limb perfusion and reversal of tissue ischemia produced by ultrasound-mediated microbubble cavitation","volume":"8","author":"JT Belcik","year":"2015","journal-title":"Circ Cardiovasc Imaging"},{"issue":"13","key":"pone.0319318.ref006","doi-asserted-by":"crossref","first-page":"1240","DOI":"10.1161\/CIRCULATIONAHA.116.024826","article-title":"Augmentation of Muscle Blood Flow by Ultrasound Cavitation Is Mediated by ATP and 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Rev"},{"issue":"36","key":"pone.0319318.ref019","doi-asserted-by":"crossref","first-page":"9983","DOI":"10.1073\/pnas.1606915113","article-title":"Biophysical insight into mechanisms of sonoporation","volume":"113","author":"B Helfield","year":"2016","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"8","key":"pone.0319318.ref020","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1016\/0007-1226(94)90136-8","article-title":"New insights into the local regulation of blood flow by perivascular nerves and endothelium","volume":"47","author":"G Burnstock","year":"1994","journal-title":"Br J Plast Surg"},{"issue":"1","key":"pone.0319318.ref021","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1124\/pr.58.1.5","article-title":"Pathophysiology and therapeutic potential of purinergic signaling","volume":"58","author":"G Burnstock","year":"2006","journal-title":"Pharmacol 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A multiplexed microfluidic and microscopy study of vasodilation signaling pathways using microbubble and ultrasound therapy. In: 2020 IEEE International Ultrasonics Symposium (IUS). IEEE. 2020. p. 1\u20134. doi: 10.1109\/ius46767.2020.9251762","DOI":"10.1109\/IUS46767.2020.9251762"},{"issue":"7","key":"pone.0319318.ref025","first-page":"700","article-title":"Lytic Release of Cellular ATP: Physiological Relevance and Therapeutic Applications","volume":"11","author":"R Grygorczyk","year":"2021","journal-title":"Life (Basel)"},{"issue":"6","key":"pone.0319318.ref026","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1016\/j.ultrasmedbio.2011.02.013","article-title":"Nonspherical shape oscillations of coated microbubbles in contact with a wall","volume":"37","author":"HJ Vos","year":"2011","journal-title":"Ultrasound Med Biol"},{"key":"pone.0319318.ref027","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1016\/j.addr.2014.03.003","article-title":"Acoustic behavior of microbubbles and implications for drug delivery","volume":"72","author":"K Kooiman","year":"2014","journal-title":"Adv Drug Deliv Rev"},{"issue":"12","key":"pone.0319318.ref028","doi-asserted-by":"crossref","first-page":"2393","DOI":"10.1016\/j.ultrasmedbio.2013.08.003","article-title":"Membrane perforation and recovery dynamics in microbubble-mediated sonoporation","volume":"39","author":"Y Hu","year":"2013","journal-title":"Ultrasound Med Biol"},{"key":"pone.0319318.ref029","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/j.jconrel.2016.07.037","article-title":"Viability of endothelial cells after ultrasound-mediated sonoporation: Influence of targeting, oscillation, and displacement of microbubbles","volume":"238","author":"T van Rooij","year":"2016","journal-title":"J Control Release"},{"key":"pone.0319318.ref030","doi-asserted-by":"crossref","first-page":"426","DOI":"10.1016\/j.jconrel.2020.03.038","article-title":"Opening of endothelial cell-cell contacts due to sonoporation","volume":"322","author":"I Beekers","year":"2020","journal-title":"J Control Release"},{"issue":"26","key":"pone.0319318.ref031","doi-asserted-by":"crossref","first-page":"23867","DOI":"10.1074\/jbc.M101500200","article-title":"Brefeldin A block of integrin-dependent mechanosensitive ATP release from Xenopus oocytes reveals a novel mechanism of mechanotransduction","volume":"276","author":"R Maroto","year":"2001","journal-title":"J Biol Chem"},{"issue":"1","key":"pone.0319318.ref032","article-title":"Type 2 secretory cells are primary source of ATP release in mechanically stretched lung alveolar cells","volume":"318","author":"JJ Tan","year":"2020","journal-title":"Am J Physiol Lung Cell Mol Physiol"},{"issue":"5","key":"pone.0319318.ref033","doi-asserted-by":"crossref","first-page":"1195","DOI":"10.1113\/jphysiol.2012.244145","article-title":"Imaging and characterization of stretch-induced ATP release from alveolar A549 cells","volume":"591","author":"R Grygorczyk","year":"2013","journal-title":"J Physiol"},{"key":"pone.0319318.ref034","first-page":"5514","article-title":"The ATP permeability of pannexin 1 channels in a heterologous system and in mammalian taste cells is dispensable","volume":"125","author":"RA Romanov","year":"2012","journal-title":"J Cell Sci"},{"issue":"8","key":"pone.0319318.ref035","doi-asserted-by":"crossref","DOI":"10.1016\/j.echo.2020.03.016","article-title":"Flow Augmentation in the Myocardium by Ultrasound Cavitation of Microbubbles: Role of Shear-Mediated Purinergic Signaling","volume":"33","author":"F Moccetti","year":"2020","journal-title":"J Am Soc Echocardiogr"},{"issue":"1","key":"pone.0319318.ref036","doi-asserted-by":"crossref","first-page":"5161","DOI":"10.1038\/s41598-021-84341-3","article-title":"Sonoporation generates downstream cellular impact after membrane resealing","volume":"11","author":"X Duan","year":"2021","journal-title":"Sci Rep"},{"issue":"3","key":"pone.0319318.ref037","article-title":"Wide field of view quantitative imaging of cellular ATP release","volume":"317","author":"JJ Tan","year":"2019","journal-title":"Am J Physiol Cell Physiol"},{"issue":"7","key":"pone.0319318.ref038","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: an open-source platform for biological-image analysis","volume":"9","author":"J Schindelin","year":"2012","journal-title":"Nat Methods"},{"issue":"21","key":"pone.0319318.ref039","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1080\/14786442808674769","article-title":"XXVII. A brief account of microscopical observations made in the months of June, July and August 1827, on the particles contained in the pollen of plants; and on the general existence of active molecules in organic and inorganic bodies","volume":"4","author":"R Brown","year":"1828","journal-title":"The Philosophical Magazine"},{"key":"pone.0319318.ref040","doi-asserted-by":"crossref","first-page":"21","DOI":"10.24033\/asens.476","article-title":"Th\u00e9orie de la sp\u00e9culation","volume":"17","author":"L Bachelier","year":"1900","journal-title":"Ann Sci \u00c9cole Norm Sup"},{"issue":"8","key":"pone.0319318.ref041","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1002\/andp.19053220806","article-title":"\u00dcber die von der molekularkinetischen Theorie der W\u00e4rme geforderte Bewegung von in ruhenden Fl\u00fcssigkeiten suspendierten Teilchen","volume":"322","author":"A Einstein","year":"1905","journal-title":"Annalen der Physik"},{"issue":"2","key":"pone.0319318.ref042","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/0304-4165(96)00053-0","article-title":"The effects of temperature, pH, and magnesium on the diffusion coefficient of ATP in solutions of physiological ionic strength","volume":"1291","author":"MJ Hubley","year":"1996","journal-title":"Biochim Biophys Acta"},{"issue":"10","key":"pone.0319318.ref043","first-page":"1835","article-title":"Intracellular ATP levels determine cell death fate by apoptosis or necrosis","volume":"57","author":"Y Eguchi","year":"1997","journal-title":"Cancer Res"},{"key":"pone.0319318.ref044","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1016\/S0005-2736(03)00210-4","article-title":"Extracellular ATP as a signaling molecule for epithelial cells","volume":"1615","author":"EM Schwiebert","year":"2003","journal-title":"Biochim Biophys Acta"},{"issue":"3","key":"pone.0319318.ref045","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1007\/s11302-012-9305-8","article-title":"ATP synthesis and storage","volume":"8","author":"M Bonora","year":"2012","journal-title":"Purinergic Signal"},{"issue":"11","key":"pone.0319318.ref046","first-page":"1166","article-title":"Intracellular ATP Concentration and Implication for Cellular Evolution","volume":"10","author":"JV Greiner","year":"2021","journal-title":"Biology (Basel)"},{"issue":"9","key":"pone.0319318.ref047","doi-asserted-by":"crossref","first-page":"1593","DOI":"10.1016\/j.bpj.2022.04.007","article-title":"Propidium uptake and ATP release in A549 cells share similar transport mechanisms","volume":"121","author":"F Boudreault","year":"2022","journal-title":"Biophys J"},{"issue":"6","key":"pone.0319318.ref048","doi-asserted-by":"crossref","first-page":"872","DOI":"10.1097\/00005344-199606000-00015","article-title":"ATP-stimulated release of ATP by human endothelial cells","volume":"27","author":"P Bodin","year":"1996","journal-title":"J Cardiovasc Pharmacol"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0319318","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,5,27]],"date-time":"2025-05-27T13:40:51Z","timestamp":1748353251000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0319318"}},"subtitle":[],"editor":[{"given":"David M.","family":"Ojcius","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,5,27]]},"references-count":48,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2025,5,27]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0319318","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2025.02.02.636151","asserted-by":"object"}]},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,5,27]]}}}