Systematic Single-Cell Dissection of Cell Cycle and TGFβ-Induced State Transitions Underlying Gemcitabine Resistance in 3D Pancreatic Tumor Tissue Models

Systematic Single-Cell Dissection of Cell Cycle and TGFβ-Induced State Transitions Underlying Gemcitabine Resistance in 3D Pancreatic Tumor Tissue Models | 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 Research Article Systematic Single-Cell Dissection of Cell Cycle and TGFβ-Induced State Transitions Underlying Gemcitabine Resistance in 3D Pancreatic Tumor Tissue Models Maryam Almasi, Jesús Guillermo Nieves Pereira, Thomas Dandekar, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7825090/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 Background: Pancreatic ductal adenocarcinoma (PDAC) is highly lethal due to late diagnosis and rapid resistance to chemotherapy (e.g. Gemcitabine, GEM) with poor outcome. We investigate resistance mechanisms in a 3D tissue model. Methods: We established a three dimensional PANC-1 model on a decellularized porcine intestinal tissue matrix, recapitulating patient-like features. Niche-specific drug response and resistance induction after TGF-β1 stimulation motivated single-cell RNA sequencing (scRNA-seq) and systems biological analysis comparing GEM treatment with or without TGF-β1 stimulation. Results: Moderate GEM resistance was associated with E2F1, mTOR, and checkpoint signaling, including upregulation of CDK1, AURKA, TPX2, TOP2A, and BIRC5. TGF-β1 promoted additional resistance through EMT, KRAS signaling, glycolysis, and hypoxia pathways, with early EMT drivers followed by late induction of SPOCK1, MBOAT2, COL5A1, ADAMTS6, THBS1, and FN1. Decision level and trajectory analysis resolved G1–S progression and TGF-β1–induced EMT and revealed enrichment of GEM-surviving cells along a unique bottleneck centered on the CDK1–CDKN1A (p21) axis. This "S-phase persistence" state represented proliferative drive (CDK1–Wee1) counterbalanced by TGF-β1/SMAD3 checkpoint signaling, producing a poised but arrested phenotype. Hybrid EMT/S-phase–persistent cells co-expressed EMT markers and cell-cycle regulators (e.g., RRM1/2, MYBL2, CLSPN, DTL), conferring replication-stress tolerance and hallmarks of resistance. Conclusions: Our study maps the dynamic emergence of GEM resistance in PDAC at single-cell resolution. Beyond PDAC, this work underscores the value of matrix-based 3D scRNA-seq models and provides a generalizable framework for dissecting cancer resistance. Pancreatic ductal adenocarcinoma 3D tumor model cell state landscape dynamic network analysis single-cell RNA sequencing TGF-β1 signalling Gemcitabine cell cycle epithelial–mesenchymal transition Full Text Additional Declarations No competing interests reported. Supplementary Files 20251009SupplPANC3DscRNAsub.docx SupplementaryTables.xlsx 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7825090","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":545256871,"identity":"6eb423b3-7efa-4a56-8ac8-2c98b2dc420c","order_by":0,"name":"Maryam Almasi","email":"","orcid":"","institution":"University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Almasi","suffix":""},{"id":545256873,"identity":"38516be5-33bb-4df7-8abc-1a6a600758eb","order_by":1,"name":"Jesús Guillermo Nieves Pereira","email":"","orcid":"","institution":"University Hospital 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single-cell RNA sequencing, TGF-β1 signalling, Gemcitabine, cell cycle, epithelial–mesenchymal transition","lastPublishedDoi":"10.21203/rs.3.rs-7825090/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7825090/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e\u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) is highly lethal due to late diagnosis and rapid resistance to chemotherapy (e.g. Gemcitabine, GEM) with poor outcome. We investigate resistance mechanisms in a 3D tissue model.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e\u003cp\u003eWe established a three dimensional PANC-1 model on a decellularized porcine intestinal tissue matrix, recapitulating patient-like features. Niche-specific drug response and resistance induction after TGF-β1 stimulation motivated single-cell RNA sequencing (scRNA-seq) and systems biological analysis comparing GEM treatment with or without TGF-β1 stimulation.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e\u003cp\u003eModerate GEM resistance was associated with E2F1, mTOR, and checkpoint signaling, including upregulation of CDK1, AURKA, TPX2, TOP2A, and BIRC5. TGF-β1 promoted additional resistance through EMT, KRAS signaling, glycolysis, and hypoxia pathways, with early EMT drivers followed by late induction of SPOCK1, MBOAT2, COL5A1, ADAMTS6, THBS1, and FN1. Decision level and trajectory analysis resolved G1\u0026ndash;S progression and TGF-β1\u0026ndash;induced EMT and revealed enrichment of GEM-surviving cells along a unique bottleneck centered on the CDK1\u0026ndash;CDKN1A (p21) axis. This \"S-phase persistence\" state represented proliferative drive (CDK1\u0026ndash;Wee1) counterbalanced by TGF-β1/SMAD3 checkpoint signaling, producing a poised but arrested phenotype. Hybrid EMT/S-phase\u0026ndash;persistent cells co-expressed EMT markers and cell-cycle regulators (e.g., RRM1/2, MYBL2, CLSPN, DTL), conferring replication-stress tolerance and hallmarks of resistance.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e\u003cp\u003eOur study maps the dynamic emergence of GEM resistance in PDAC at single-cell resolution. Beyond PDAC, this work underscores the value of matrix-based 3D scRNA-seq models and provides a generalizable framework for dissecting cancer resistance.\u003c/p\u003e","manuscriptTitle":"Systematic Single-Cell Dissection of Cell Cycle and TGFβ-Induced State Transitions Underlying Gemcitabine Resistance in 3D Pancreatic Tumor Tissue Models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 22:41:02","doi":"10.21203/rs.3.rs-7825090/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":"51b3f410-c637-42d8-8d45-42709b569477","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-16T15:09:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-25 22:41:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7825090","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7825090","identity":"rs-7825090","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}