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

ABSTRACT: 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.

ORGANISM(S): Sus scrofa

PROVIDER: GSE311743 | GEO | 2025/12/01

REPOSITORIES: GEO

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Project description:Background: Despite advances in therapeutics, outcomes for hepatocellular carcinoma (HCC) remain poor and there is an urgent need for efficacious systemic therapy. Unfortunately, drugs that are successful in preclinical studies often fail in the clinical setting, and we hypothesize that this is due to functional differences between primary tumors and commonly used preclinical models. In this study, we attempt to answer this question by comparing tumor morphology and gene expression profiles between primary tumors, xenografts and HCC cell lines. Methods: Hep G2 cell lines and tumor cells from patient tumor explants were subcutaneously (ectopically) injected into the flank and orthotopically into liver parenchyma of Mus Musculus SCID mice. The mice were euthanized after two weeks. RNA was extracted from the tumors, and gene expression profiling was performed using the Gene Chip Human Genome U133 Plus 2.0. Principal component analyses (PCA) and construction of dendrograms were conducted using Partek genomics suite. Results: PCA showed that the commonly used HepG2 cell line model and its xenograft counterparts were vastly different from all fresh, primary tumors. Expression profiles of primary tumors were also significantly divergent from their counterpart patient-derived xenograft (PDX) models, regardless of the site of implantation. Xenografts from the same primary tumors were more likely to cluster together regardless of site of implantation, although heat maps showed distinct differences in gene expression profiles between orthotopic and ectopic models. Conclusions: The data presented here challenges the utility of routinely used preclinical models. Models using HepG2 were vastly different from primary tumors and PDXs, suggesting that this is not clinically representative. Surprisingly, site of implantation (orthotopic versus ectopic) resulted in limited impact on gene expression profiles, and in both scenarios xenografts differed significantly from the original primary tumors, challenging the long-held notion that orthotopic PDX model is the gold standard preclinical model for HCC.

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

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