A phenotype-based cell culture model of melanoma cells that persist as residual disease after therapies leading to recurrence

A phenotype-based cell culture model of melanoma cells that persist as residual disease after therapies leading to recurrence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A phenotype-based cell culture model of melanoma cells that persist as residual disease after therapies leading to recurrence Balraj Singh, Vanessa Sarli, Nikil Erry, Anthony Lucci This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8768856/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A crucial adaptability trait of stem-like melanoma cells that persist under all selection pressures, including therapies, is their ability to survive in deep quiescence. This ability coupled with their intrinsic ability to proliferate leads to recurrence. Here we describe an approach to modeling this trait in cell culture. A lack of glutamine proved to be a selection pressure for the highly metastatic human melanoma cell line A375SM, killing more than 99% of cells and selecting rare cells based on their ability to survive in deep quiescence. After 4 weeks, cells gradually exited quiescence and proliferated indefinitely. Interestingly, by not providing fresh glutamine-free medium at this stage, we could select rare cells that persist in deep quiescence as single cells. Alternatively, we could model deeper quiescence lasting longer than 4 weeks by increasing the severity of the selection pressure using dialyzed serum in medium. The cells selected in this manner were much more resistant to paclitaxel than was their parental cell line. We obtained similar results with the highly metastatic mouse melanoma cell line B16-BL6. Thus, our phenotype-based approach is suitable for modeling abnormal deep quiescence in melanoma that is responsible for therapy resistance, disease progression, and recurrence/metastasis. Biological sciences/Cancer Biological sciences/Cell biology resistant melanoma residual disease cancer dormancy cancer evolution abnormal quiescence model melanoma recurrence Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8768856","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":591757065,"identity":"92a3ddce-3e76-4395-8f84-da61b165dc8f","order_by":0,"name":"Balraj Singh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACAwbGBiCyYWA4wMAgQYqWNJK0AAFjw2EStJjzH257+HXH+Wi+42cP3vi4p07OnIH52McveLRYzkhsN5Y9czt35pm8ZMsZzw4bWzawJc+WweewG4xt0pJtt3M3HMgxk+Y5cCBxwwEeY2Z8TjQ4fxCk5VzuhvNvzKT/HKgjQsuBxDbJj20HcjfcANrCcIAZrIXxA36/tEkztiXnzrzxxtiy58BhY4PDbMnMeHQAQ+z4M8mfbXa5fedzDG/8OFAnZ3C8+TDjD3x6gICZB5WLIYIJMM0kaMsoGAWjYBSMKAAAK/9XMzpUXwwAAAAASUVORK5CYII=","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":true,"prefix":"","firstName":"Balraj","middleName":"","lastName":"Singh","suffix":""},{"id":591757068,"identity":"23cbf725-0763-40ac-8442-12ab8de4a247","order_by":1,"name":"Vanessa Sarli","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Vanessa","middleName":"","lastName":"Sarli","suffix":""},{"id":591757070,"identity":"dcc1bea7-55f8-4ed9-8e33-9ed234839236","order_by":2,"name":"Nikil Erry","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Nikil","middleName":"","lastName":"Erry","suffix":""},{"id":591757072,"identity":"e9667855-e66c-4409-b355-1e0cde42f771","order_by":3,"name":"Anthony Lucci","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Anthony","middleName":"","lastName":"Lucci","suffix":""}],"badges":[],"createdAt":"2026-02-02 20:54:06","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8768856/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8768856/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105707216,"identity":"ca1d6b69-d1fa-432c-95d0-124f8ccee079","added_by":"auto","created_at":"2026-03-30 07:13:47","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":768322,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptSinghetalScientificReportsFeb52026.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8768856/v1_covered_ffa7a7f3-ab15-4800-8992-80aed82d0280.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A phenotype-based cell culture model of melanoma cells that persist as residual disease after therapies leading to recurrence","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"resistant melanoma, residual disease, cancer dormancy, cancer evolution, abnormal quiescence model, melanoma recurrence","lastPublishedDoi":"10.21203/rs.3.rs-8768856/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8768856/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA crucial adaptability trait of stem-like melanoma cells that persist under all selection pressures, including therapies, is their ability to survive in deep quiescence. This ability coupled with their intrinsic ability to proliferate leads to recurrence. Here we describe an approach to modeling this trait in cell culture. A lack of glutamine proved to be a selection pressure for the highly metastatic human melanoma cell line A375SM, killing more than 99% of cells and selecting rare cells based on their ability to survive in deep quiescence. After 4 weeks, cells gradually exited quiescence and proliferated indefinitely. Interestingly, by not providing fresh glutamine-free medium at this stage, we could select rare cells that persist in deep quiescence as single cells. Alternatively, we could model deeper quiescence lasting longer than 4 weeks by increasing the severity of the selection pressure using dialyzed serum in medium. The cells selected in this manner were much more resistant to paclitaxel than was their parental cell line. We obtained similar results with the highly metastatic mouse melanoma cell line B16-BL6. 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