Precision Liver Cancer Modeling by CRISPR Editing of Primary Human Hepatocytes

/Abstract Hepatocellular carcinoma (HCC), the most prevalent type of liver cancer, is an aggressive disease with limited therapeutic options and poor prognosis. Current therapies adopt a ‘one-size-fits-all’ approach that does not account for the marked HCC molecular heterogeneity observed across patients. Progress in the development of effective targeted HCC therapies has been hampered by limitations of existing cell and animal models of HCC, including the following. First, there is a lack of in vitro models that capture the molecular heterogeneity of human HCC, with lack of models representative of various frequently observed tumor gene alterations. Second, human HCC cell lines derived from tumors harbor numerous randomly accumulated passenger mutations, making it challenging to link specific mutations with therapeutic susceptibility. Furthermore, the lack of HCC large animal models with defined driver mutations hampers the ability to test locoregional therapies, which are used in 50- 60% of HCC cases but cannot be tested in mice due to size constraints. These challenges highlight the critical need for innovative in vitro and in vivo models to advance HCC therapy development. To address these limitations, our objective in this proposal is to develop a versatile modeling platform that can recapitulate HCC molecular heterogeneity through defined gene alterations. We hypothesize that HCC models can be established by introducing driver gene mutations commonly observed in human HCC into normal primary human hepatocytes (PHHs). We have validated the feasibility of this approach by inducing clinically relevant gene alterations into PHHs. Building on these findings, we will expand the platform to capture broader molecular diversity by inducing combinations of gene alterations based on published comprehensive analyses of HCC mutational landscape and driver gene mutation co-occurrence patterns. In addition, we will develop in vivo models amenable to locoregional therapy testing by orthotopic implantation of genetically defined HCC cells into immunodeficient rats with humanized immune system. The clinical relevance and similarity of these models to human HCC will be validated through comprehensive histological, transcriptional, and imaging analyses. The use of vertebrate animals is essential for the proposed studies. Mouse models are required to evaluate the in vivo tumorigenicity of the novel HCC cell lines and to characterize the histological features of the resulting tumors. In addition, rat models are necessary to enable testing of locoregional therapies, which cannot be adequately assessed in vitro or in mouse models due to anatomical and size limitations. Aligned with the mission of PAR-25-139, this work will provide the scientific community with innovative, clinically relevant cell and animal models for HCC research. The cell models will overcome limitations of available human HCC cells and serve as tools for high throughput screening, while the rat models will address the unmet need for large animal models with genetically defined HCC suitable for locoregional therapy testing. Together, these validated in vitro and in vivo models will accelerate the discovery of precision HCC therapies, ultimately improving patient outcomes. Project Number: 1R21CA292350-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Lobna Elkhadragy | Institution: UNIVERSITY OF ILLINOIS AT CHICAGO, Chicago, IL | Award Amount: $412,249 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 BTC-T (80)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11373656