Investigation of pancreatic tumorigenesis by derivation of organoids, their immortalization, and genetic modification in vitro

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Pancreatic ductal adenocarcinoma (PDAC) is one of the most dreadful diseases of the human body with dismal prognosis. This is ultimately based on two aspects: late detection and poorly effective therapies. The recent two decades yielded great knowledge in the molecular profile and pathogenesis of PDAC. Pancreatic tumorigenesis is regarded to be a stepwise process. Alterations accumulating in driver genes (KRAS, CDKN2A, TP53, SMAD4) aside with other genetic disturbances lead to invasive ductal epithelium. In vitro models of pancreatic precursor lesions are scarce, which prevents new insights into the complex molecular and pathogenic processes inflicted by these specific alterations. Modern genome engineering (CRISPR/Cas9) facilitates accurate genetic modification of cells. Therefore, we attempt to introduce all four driver gene alterations sequentially into normal, unmodified pancreatic epithelial duct cells. This will derive novel pancreatic cancer precursor cell lines harboring specific genetic modifications. In addition, we will modify pancreatic duct epithelium with the immortalization vector hTERT to establish a new cell line of normal duct epithelium. Both approaches will be conducted utilizing three-dimensional culture conditions (organoids). Resulting cells will be further examined and thoroughly characterized. Those cell lines are desperately needed to investigate the effects of genetic alterations in the malignant phenotype of invasive duct epithelium and its precursors in vitro. Better fundamental knowledge of pathway perturbations affected by these and other genetic changes may reveal better targets for early detection and therapy.