LEVERAGING ORGANOID TECHNOLOGIES TO CHARACTERIZE DYNAMIC TUMOR CELL STATES DRIVING TREATMENT RESISTANCE IN PANCREATIC CANCER

A. Papargyriou^1-6, M. Najajreh^1-4, D.P. Cook⁷, C.H. Maurer^1,2, S. Bärthel⁸, P. Putze⁸, H.A. Messal⁹, S.K. Ravichandran^1-3, T. Richter¹⁰, M. Knolle¹¹, T. Metzler¹², A.R. Shastri^1,2,3, R. Öllinger¹³, J. Jasper^1,2,3, L. Schmidleitner^1,2,3, C.Schneeweis⁸, H. Ishikawa-Ankerhold¹⁴, T. Engleitner¹³, S. Dobiasch¹⁵, I. Heid, M.D. Luecken¹⁶, K. Steiger¹², G. Kaissis¹¹, J. Van Rheenen⁹, F.J. Theis¹⁰, D. Saur⁸, R. Rad¹³, M. Reichert^{1,2,3,4,5,17,18} | ¹Translational Pancreatic Cancer Research Center, Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Germany; ²Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Germany; ³Center for Functional Protein Assemblies, Technical University of Munich, Garching, Germany; ⁴Center for Organoid Systems (COS), Technical University of Munich, Garching, Germany; ⁵Bavarian Cancer Research Center (BZKF), Munich, Germany; ⁶Institute of Stem Cell Research, Helmholtz Center Munich, Neuherberg, Germany; ⁷University of Ottawa, Faculty of Medicine, Department of Cellular and Molecular Medicine, Ottawa, ON, Canada; ⁸Chair for Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, Germany; ⁹Division of Molecular Pathology, Oncode Institute, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands. ¹⁰Institute of Computational Biology, Helmholtz Center Munich, Neuherberg, Germany; ¹¹Institute of Diagnostic and Interventional Radiology, Klinikum rechts der Isar München, Technical University of Munich, Germany; ¹²Comparative Experimental Pathology, Institut für Allgemeine Pathologie und Pathologische Anatomie, School of Medicine, Technical University of Munich, Germany; ¹³Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Germany; ¹⁴Department of Medicine I, University Hospital of the LudwigMaximilians-University Munich, Germany; ¹⁵Department of Radiation Oncology, Technical University of Munich, Germany; ¹⁶Institute of Lung Health and Immunity (LHI), Helmholtz Munich, Comprehensive Pneumology Center (CPC-M), Germany; Member of the German Center for Lung Research (DZL); ¹⁷German Cancer Consortium (DKTK), Munich, Germany; ¹⁸Munich Institute of Biomedical Engineering (MIBE), Technical University of Munich, Germany

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a pronounced inter- and intra-tumoral heterogeneity, which fuels chemoresistance and contributes to high mortality rates. Previously, we have developed a branched organoid system embedded in collagen matrices that robustly recapitulates the phenotypic heterogeneity seen in both murine and human PDAC^1,2. These organoids display complex, self-organized branching morphogenesis and give rise to distinct, spatially ordered tumor cell populations that reflect their underlying molecular profiles and differentiation states. Importantly, we show that the observed heterogeneity is not random but governed by defined transcriptional programs, particularly epithelial-to-mesenchymal plasticity, that drive the emergence of discrete tumor-cell states. Using integrated phenotypic and transcriptomic profiling, we map this diversity to specific biological functions in vivo, demonstrating that each organoid phenotype corresponds to a tumor-cell state with unique metastatic potential, and therapeutic vulnerabilities. Moreover, we identify dynamic, treatment-induced phenotype reprogramming events that are targetable, paving the way for rational design of state-specific therapeutic interventions. Building on our findings, we are now focusing on elucidating intra-organoid heterogeneitysuch as tip–trunk hierarchies at the single-cell level, to uncover the mechanisms of self-organization and to determine how distinct organoid phenotypes and subpopulations contribute to liver metastasis and treatment resistance. In summary, we have established a scalable and mechanistically informative organoid platform that enables in vitro modeling of PDAC heterogeneity. This system provides a framework for dissecting the tumor cell–intrinsic drivers of phenotypic plasticity and for developing phenotype-guided treatment strategies aimed at overcoming resistance and improving patient outcomes.