Development of genetically tailored orthotopic implantation hepatocellular carcinoma model in oncopigs by somatic cell CRISPR editing

Hepatocellular carcinoma (HCC) is an aggressive disease with poor prognosis, necessitating preclinical models for evaluating novel therapies. Large animal models are particularly valuable for assessing locoregional therapies, which are widely employed across HCC stages. This study aimed to develop a large animal HCC model with tailored tumor mutations. The Oncopig, a genetically engineered pig with inducible TP53^R167H and KRAS^G12D, was used in the study. Hepatocytes were isolated from Oncopigs and exposed to Cre recombinase in vitro to create HCC cells, and additional mutations were introduced by CRISPR/Cas9 knockout (KO) of PTEN and CDKN2A genes. These edits increased Oncopig HCC cell proliferation and migration. Autologous HCC cells with these CRISPR edits were implanted into Oncopigs using two approaches. Ultrasound-guided percutaneous liver injections resulted in the development of localized intrahepatic masses, while portal vein injections led to multifocal tumors that regressed over time. Tumors developed by both approaches harbored PTEN and CDKN2A KO mutations. This study demonstrates the feasibility of developing genetically tailored HCC tumors in Oncopigs using somatic cell CRISPR editing and autologous implantation, providing a valuable large animal model for in vivo therapeutic assessment.