LAUSR

The incidence of liver cancer is growing worldwide, and it is estimated that by 2025, over 1 million individuals will be affected by liver cancer every year. Hepatocellular carcinoma (HCC) accounts for about 90% of primary liver cancer cases and is one of the leading causes of death from cancer worldwide. Establishing animal models that faithfully replicate the human disease is critical to improve our understanding of HCC pathogenesis and to test new therapeutic strategies. Here, we describe a novel human HCC model using genetically modified human hepatocytes in a humanized liver mouse. Primary human hepatocytes (PHH) were modified ex vivo through lentivirus or Lipid Nanoparticle (LNP)-mediated delivery of CRISPR/Cas9 components, resulting in over-expression of oncogenes or inactivation of tumor suppressor genes. These genetically engineered PHH were engrafted into the livers of FSRG (FAH−/−, SIRPahu/hu, RAG2−/−, IL2Rg−/−) mice, developed by Regeneron VelociGene technology, in which mouse liver parenchyma can be replaced with human hepatocytes. Tumor formation was validated in livers engrafted with human hepatocytes after different combinations of genetic modification, including over-expression of cMYC plus HRASv12, and cMYC over-expression together with p53 KO. Tumor progression could be monitored by measuring human Alpha Fetoprotein (AFP) in serum of engrafted mice. Liver tumors were positive for fumarylacetoacetate hydrolase (FAH) and human Asialoglycoprotein Receptor 1 (ASGR1), confirming their human origin, as well as the proliferation marker, Ki67, and Glypican-3 (GPC3), which is frequently over-expressed in HCC and a potential therapeutic target. In these studies, mice were engrafted with a mix of lentivirus-transduced and non-transduced hepatocytes, resulting in humanized livers with both normal and transformed human hepatocytes, providing an ideal system to recapitulate neoplastic transformation and clonal expansion of human hepatocytes carrying oncogenic mutations as well as to test the specificity of novel therapies. By combining genetic manipulation of human hepatocytes with the FSRG humanized liver mouse platform, we have established a flexible system to model clinically relevant combinations of HCC mutations for both new target discovery and therapeutic testing. Citation Format: Marisa Carbonaro, Ciro Bonetti, Hui Huang, Kehui Wang, Mathieu Desclaux, Guochun Gong, John C. Lin, Christopher Daly, Gavin Thurston, Zhe Li. Development of novel human hepatocellular carcinoma models using genetically modified human hepatocytes in FSRG humanized liver mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 35.