Targeting epithelial plasticity in pancreatic cancer

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Dynamic cell fate decisions regulated by cellular plasticity in response to altered environmental cues including treatment-imposed pressure are critical for tumors to adapt. Such plastic cellular behaviors drive tumor initiation, delamination of tumor cells from the primary site and metastatic outgrowth. Furthermore, plasticity contributes to tumor heterogeneity as well as resistance to therapy. We have recently generated a novel PDAC organoid morphogenesis system which allows us to capture tumor plasticity and heterogeneity in a dynamic fashion during tumor organoid morphogenesis. Interestingly, PDAC cells from distinct molecular clusters reproducibly generate complex three-dimensional structures that allow to infer the molecular subtype. In this project, we will exploit the organoid morphogenesis assay to identify the molecular programs driving phenotypic heterogeneity in PDAC. In WP 1, we will acquire unique insight into inter-organoid heterogeneity by transcriptional profiling of the entire spectrum of branched organoid phenotypes. At the same time, we will dissect intra-organoid heterogeneity using single cell profiling with spatial resolution. In WP 2, we aim to elucidate the molecular dynamics and mechanics in pancreatic cancer organoid development by quantification and dynamic modelling of the growth process of PDAC organoids using live cell imaging in combination with labeled regulators of the cytoskeletal machinery. In addition, in WP 3 we will quantify involved strains and stresses during branching morphogenesis by applying localized cuts in the organoid-adjacent matrix using UV lasers resulting in relaxation which allows us in turn to determine the tension within the structures. Lastly, in WP 4, we will determine the functional role of cellular plasticity by perturbation of drivers regulating inter- and intra-organoid phenotypic heterogeneity. This will be achieved by a drug screen using pre-clinical and clinical drugs in phenotypically mapped PDAC organoids to identify heterogeneity altering drugs. Drugs that limit phenotypic heterogeneity will be tested in a large panel of PDAC patient-derived organoids (PDOs) in combination with first-line poly-chemotherapy. Overall, we propose an innovative and interdisciplinary strategy to identify and perturbate molecular mechanisms driving cellular plasticity and heterogeneity. The final outcome will be a rational strategy to understand the molecular and biophysical mechanism driving organoid heterogeneity and plasticity as well as to intervene pharmacologically phenotypic diversity at defined developmental phases of organoids morphogenesis derived from distinct PDAC subtypes. Both phenomena are considered key regulators of resistance to chemotherapy and metastatic dissemination, particularly in PDAC.