Transposon-based oncogene integration in Abcb4(Mdr2)-/- mice recapitulates high susceptibility to cholangiocarcinoma
Pinzhu Huang1, Guangyan Wei2, Jesse D Kirkpatrick3
1Division of Gastroenterology and Hepatology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; Department of Colon and Rectum Surgery, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
Background & Aims:
Cholangiocarcinoma (CCA) is a dreaded complication of primary sclerosing cholangitis (PSC) that is difficult to diagnose and associated with high mortality. A lack of animal models of CCA recapitulating the hepatic microenvironment of sclerosing cholangitis has hindered the development of novel treatments. Herein, we sought to develop a mouse model of PSC-associated CCA.
Methods:
Ten-week-old Mdr2-/- mice with congenital PSC-like disease, and healthy wild-type littermates were subjected to either modified retrograde biliary instillation or hydrodynamic tail vein injection of a sleeping beauty transposon-transposase plasmid system with activated AKT (myr-AKT) and Yap (YapS127A) proto-oncogenes (SB AKT/YAP1). The role of TGFβ was interrogated via ALK5 inhibitor (SB-525334) administration. Tumor phenotype, burden and desmoplastic reaction were analyzed histologically and via RNA sequencing.
Results:
While SB AKT/YAP1 plasmids administered via retrograde biliary injection caused tumors in Mdr2-/-, only 26.67% (4/15) of these tumors were CCA. Alternatively, hydrodynamic tail vein injection of SB AKT/YAP1 resulted in robust tumorigenesis in all fibrotic Mdr2-/- mice with high CCA burden compared to healthy mice. Tumors phenotypically resembled human CCA, expressed multiple CCA (but not hepatocellular carcinoma) markers, and exhibited a profound desmoplastic reaction. RNA sequencing analysis revealed profound transcriptional changes in CCA evolving in a PSC-like context, with specific alterations in multiple immune pathways. Pharmacological TGFβ inhibition led to enhanced immune cell tumor infiltration, reduced tumor burden and suppressed desmoplastic collagen accumulation compared to placebo.
Conclusion:
We established a new high-fidelity cholangiocarcinoma model in mice, termed SB CCA.Mdr2-/-, which recapitulates the increased susceptibility to CCA in the setting of biliary injury and fibrosis observed in PSC. Through transcriptomics and pharmacological studies, we show dysregulation of multiple immune pathways and TGFβ signaling as potential drivers of CCA in a PSC-like microenvironment.
Impact And Implications:
Animal models for primary sclerosing cholangitis (PSC)-related cholangiocarcinoma (PSC-CCA) are lacking. Thus, we have developed and characterized a new mouse model of PSC-CCA, termed SB CCA.Mdr2-/-, which features reliable tumor induction on a PSC-like background of biliary injury and fibrosis. Global gene expression alterations were identified and standardized tools, including automated whole slide image analysis methodology for tumor burden and feature analysis, were established to enable systematic research into PSC-CCA biology and formal preclinical drug testing.
