Longitudinal and continuous label-free monitoring of glioblastoma patient-derived tumor spheroid treatment response using quantitative oblique back illumination microscopy

Longitudinal and continuous label-free monitoring of glioblastoma patient-derived tumor spheroid treatment response using quantitative oblique back illumination microscopy | 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 Longitudinal and continuous label-free monitoring of glioblastoma patient-derived tumor spheroid treatment response using quantitative oblique back illumination microscopy Caroline Serafini, Amin Davarzani, Dan Cappabianca, Zhenmin Li, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6529836/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Jan, 2026 Read the published version in Optica → Version 1 posted You are reading this latest preprint version Abstract Glioblastoma (GBM) is an aggressive brain tumor with limited treatment options and poor patient survival, underscoring the need for novel therapeutic strategies and improved preclinical models. Patient-derived tumor spheroids (PDTSs) offer a physiologically relevant in vitro platform for evaluating treatments such as chimeric antigen receptor (CAR) T cell therapy, chemotherapy, and radiation. However, significant challenges remain in monitoring the complex three-dimensional (3D) microenvironment of the GBM PDTSs. Current imaging techniques used for this purpose are primarily endpoint analyses which lack critical real-time, non-invasive capabilities that ultimately preclude longitudinal and continuous monitoring. In this study, we introduce quantitative Oblique Back-illumination Microscopy (qOBM) as a novel, label-free, and non-invasive imaging approach for longitudinal and continuous, high-resolution monitoring of GBM PDTSs during treatment. qOBM enables real-time visualization of cellular processes, including apoptosis, cell migration, and T cell-mediated cytotoxicity by leveraging tomographic refractive index-based quantitative imaging. We construct a compact qOBM system that fits within common incubators and apply it to study the effects of radiation, chemotherapy, and immunotherapy on three patient-derived GBM cell lines, extracting both static and dynamic image features over a 72-hour treatment period. Additionally, we develop machine learning models to predict spheroid viability and cytotoxicity, demonstrating the potential of qOBM to enhance treatment evaluation. Our findings establish qOBM as a powerful tool for longitudinal and continuous spheroid monitoring, offering a non-destructive, high-resolution alternative to conventional endpoint assays and improving the evaluation of preclinical treatments for GBM. Biological sciences/Cell biology/Cellular imaging Biological sciences/Cancer/Cancer imaging Health sciences/Medical research/Experimental models of disease Health sciences/Medical research/Preclinical research Biological sciences/Immunology/Immune cell death Phase microscopy Label-free imaging Cellular dynamics Tissue dynamics Glioblastoma Spheroids Full Text Additional Declarations Yes there is potential Competing Interest. K.S. is a member of the scientific advisory boards of Bharat Biotech and Andson Biotech. Supplementary Files scaledSV4.mp4 Supplemental Video 4 scaledSV1.mp4 Supplemental Video 1 scaledSV9.mp4 Supplemental Video 9 scaledSV3.mp4 Supplemental Video 3 scaledSV2.mp4 Supplemental Video 2 scaledSV12.mp4 Supplemental Video 12 scaledSV7.mp4 Supplemental Video 7 scaledSV10.mp4 Supplemental Video 10 scaledSV8.mp4 Supplemental Video 8 scaledSV11.mp4 Supplemental Video 11 scaledSV6.mp4 Supplemental Video 6 scaledSV5.mp4 Supplemental Video 5 Scaledsv13576FurtherCompressed.mp4 Supplemental Video 13 Cite Share Download PDF Status: Published Journal Publication published 26 Jan, 2026 Read the published version in Optica → 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. 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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-6529836","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":449174926,"identity":"3ff9584b-6dfb-4ac0-9fb1-208b100d7751","order_by":0,"name":"Caroline 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microscopy","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"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":"Phase microscopy, Label-free imaging, Cellular dynamics, Tissue dynamics, Glioblastoma, Spheroids","lastPublishedDoi":"10.21203/rs.3.rs-6529836/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6529836/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Glioblastoma (GBM) is an aggressive brain tumor with limited treatment options and poor patient survival, underscoring the need for novel therapeutic strategies and improved preclinical models. Patient-derived tumor spheroids (PDTSs) offer a physiologically relevant in vitro platform for evaluating treatments such as chimeric antigen receptor (CAR) T cell therapy, chemotherapy, and radiation. However, significant challenges remain in monitoring the complex three-dimensional (3D) microenvironment of the GBM PDTSs. Current imaging techniques used for this purpose are primarily endpoint analyses which lack critical real-time, non-invasive capabilities that ultimately preclude longitudinal and continuous monitoring. In this study, we introduce quantitative Oblique Back-illumination Microscopy (qOBM) as a novel, label-free, and non-invasive imaging approach for longitudinal and continuous, high-resolution monitoring of GBM PDTSs during treatment. qOBM enables real-time visualization of cellular processes, including apoptosis, cell migration, and T cell-mediated cytotoxicity by leveraging tomographic refractive index-based quantitative imaging. We construct a compact qOBM system that fits within common incubators and apply it to study the effects of radiation, chemotherapy, and immunotherapy on three patient-derived GBM cell lines, extracting both static and dynamic image features over a 72-hour treatment period. Additionally, we develop machine learning models to predict spheroid viability and cytotoxicity, demonstrating the potential of qOBM to enhance treatment evaluation. Our findings establish qOBM as a powerful tool for longitudinal and continuous spheroid monitoring, offering a non-destructive, high-resolution alternative to conventional endpoint assays and improving the evaluation of preclinical treatments for GBM.","manuscriptTitle":"Longitudinal and continuous label-free monitoring of glioblastoma patient-derived tumor spheroid treatment response using quantitative oblique back illumination microscopy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-30 11:37:31","doi":"10.21203/rs.3.rs-6529836/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","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}}],"origin":"","ownerIdentity":"9cd4443d-0583-417f-9b22-9f6e240f9cb7","owner":[],"postedDate":"April 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47797951,"name":"Biological sciences/Cell biology/Cellular imaging"},{"id":47797952,"name":"Biological sciences/Cancer/Cancer imaging"},{"id":47797953,"name":"Health sciences/Medical research/Experimental models of disease"},{"id":47797954,"name":"Health sciences/Medical research/Preclinical research"},{"id":47797955,"name":"Biological sciences/Immunology/Immune cell death"}],"tags":[],"updatedAt":"2026-03-02T14:55:24+00:00","versionOfRecord":{"articleIdentity":"rs-6529836","link":"https://doi.org/10.1364/OPTICA.574860","journal":{"identity":"optica","isVorOnly":true,"title":"Optica"},"publishedOn":"2026-01-27 00:00:00","publishedOnDateReadable":"January 27th, 2026"},"versionCreatedAt":"2025-04-30 11:37:31","video":"","vorDoi":"10.1364/OPTICA.574860","vorDoiUrl":"https://doi.org/10.1364/OPTICA.574860","workflowStages":[]},"version":"v1","identity":"rs-6529836","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6529836","identity":"rs-6529836","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}