Abstract 6497: Modeling HCC molecular and histological features by CRISPR editing of primary hepatocytes

Abstract Background: Hepatocellular carcinoma (HCC) is an aggressive disease with poor response to current therapies, highlighting the need for more effective therapeutic strategies. Preclinical HCC models are necessary tools for assessment of novel therapies. Pigs offer a valuable disease model due to their similarities with humans and their large size which allows testing clinically relevant procedures. Here, we aimed to develop HCC cell and animal models by precise gene manipulation of primary porcine hepatocytes. Materials and Methods: Hepatocytes were isolated from minipigs (n=4) and subjected to CRISPR knockout (KO) of tumor suppressor genes, with or without oncogene overexpression (OE). Gene alteration combinations included KO of TP53, PTEN, and/or CDKN2A, with or without c-myc overexpression. Gene alterations were validated by Sanger sequencing, PCR, and Western blotting. The developed cell lines were injected subcutaneously into severe combined immunodeficient (SCID) mice to confirm their tumorigenicity and assess histological features. Cells with TP53KO; PTENKO; CDKN2AKO; c-mycOE (TPKM) were then autologously injected into pigs (n=2) by percutaneous ultrasound-guided liver parenchymal injection and tumor development was monitored by ultrasound and CT imaging. Tumors from mice and pigs were analyzed by histological and transcriptional analyses. Results: The induced gene alterations in porcine hepatocytes resulted in the successful development and propagation of four distinct cell lines, all validated for the intended modifications. Injection of these cells into SCID mice resulted in tumor development confirming in vivo tumorigenicity. Histological analysis of mouse xenograft tumors identified epithelial neoplasms with multiphenotypic cells, displaying moderate to poor differentiation, and closely mimicking human liver cancer. In pigs, autologous injection of TPKM cells resulted in intrahepatic mass formation within 3 weeks. Histological analysis of the porcine masses revealed neoplastic cells with positive Arginase staining, negative CK19 staining, and intratumoral immune cell infiltration. Transcriptional analysis of the mouse and pig tumors showed activation of pathways associated with cancer cell proliferation and hepatic fibrosis. Conclusion: This study successfully establishes genetically defined HCC models with histological and molecular features resembling human HCC by CRISPR editing of primary porcine hepatocytes. These innovative HCC cell models provide valuable tools for drug screening and mechanistic studies, and the porcine model is a promising tool that harbors defined tumor driver mutations and is suitable for locoregional therapy testing. Together, these novel HCC cell and animal models offer promising avenues for preclinical evaluation of innovative therapeutic strategies. Citation Format: Lobna Elkhadragy, Olayinka G. David, Caitlyn C. Castillo, Isadora Andre Rosa Lopes1, Hannah Liu, Luke R. Jordan, Luke N. Redlon, Grace Guzman, Jonathan P. Samuelson, Lawrence B. Schook, Amaia Lujambio, Kyle M. Schachtschneider, Ron C. Gaba. Modeling HCC molecular and histological features by CRISPR editing of primary hepatocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6497.