Modeling Liver Cancer Molecular and Histological Features by CRISPR Editing of Primary Porcine Hepatocytes

Preclinical models are essential for advancing therapeutic strategies in hepatocellular carcinoma (HCC), an aggressive disease with poor survival outcomes. Here, we established genetically defined HCC models by ex vivo CRISPR editing of porcine hepatocytes. Hepatocytes were isolated from Yucatan minipigs and subjected to combinations of 2-4 gene alterations, including CRISPR-mediated knockout of tumor suppressor genes (TP53, PTEN, CDKN2A, AXIN1), with or without c-myc overexpression. Edited hepatocytes were expanded in culture, and those harboring at least three gene alterations formed tumors in SCID mice that recapitulated human HCC histologically. Clonal derivatives from the cell pools generated mouse xenograft tumors with distinct liver cancer histological features. Transcriptomic profiling of the cell models and xenograft tumors revealed activation of cell cycle pathways and similarity with human HCC. Further, autologous intrahepatic implantation of edited hepatocytes into pigs produced tumors with prominent immune infiltration, establishing an immune-competent large-animal model of HCC. Collectively, this HCC modeling platform provides insights into cooperative gene alterations underlying HCC pathogenesis and enables preclinical testing of systemic and locoregional therapies in a clinically relevant large-animal context. Overall design: RNA-seq profiling of primary porcine hepatocytes, cell lines developed by subjecting the hepatocytes to gene alterations, xenograft tumors developed in SCID mice, intrahepatic pig tumors, normal pig liver tissues.