Kras-driven heterotopic tumor development from hepatobiliary organoids.

Cancers arising from the biliary tract are refractory to conventional therapies, requiring the development of novel therapeutics. However, only a limited number of genetically engineered mouse models have been created, partly due to time-consuming work required. Besides, liver-specific gene manipulation mostly resulted in concurrent development of hepatocellular carcinoma, another type of liver cancer, and gall bladder-restricted gene targeting is still not feasible. Consequently, establishment of cancer type-specific disease modeling remains a technical challenge. To address this issue, we took an alternative cell-based approach to quickly induce tumorigenesis ex vivo. Specifically, murine primary organoids from liver and gall bladder were transduced with lentiviral vectors to reconstitute genetic alterations common in biliary tract cancers, followed by inoculation in immunodeficient mice. Whereas any single genetic alteration did not induce tumors, mutant Kras and repression of major tumor suppressors cooperated for tumor development within two months. Induced lesions varied among normal, dysplastic and papillary lesions to adenocarcinoma, recapitulating multi-step tumorigenesis even in a heterotopic situation. We further demonstrated that two putative oncogenes in intrahepatic cholangiocellular carcinoma, mutant Pik3ca and FGFR2-AHCYL1 fusion, were rather modest drivers for liver-derived organoids, probably requiring additional mutations or hepatic niche to robustly induce full-blown tumors. Thus, we showed that cancer cells could be readily generated from primary cells in the biliary tract, at least in cases where genetic factors play dominant roles. Collectively, the present study will likely contribute to gaining mechanistic insights into biliary carcinogenesis and providing valuable resources for drug discovery.