Introduction: Hepatocellular carcinoma (HCC) is the most common primary liver cancer, and predominantly presents at an advanced stage at which most patients are not candidates for curative surgery. Currently available therapeutic options yield an overall survival of advanced HCC patients not exceeding one year. Hence, there is a critical need to develop novel therapeutic strategies for HCC which necessitates the availability of effective animal models for preclinical assessments. As cancer treatments move toward targeted therapeutics based on genetic alterations in the tumor, modeling the role driver mutations play in tumorigenesis and therapeutic susceptibility is essential. Here, we aim to develop an integrated rapid approach for generating porcine HCC models representative of mutational profiles observed clinically. Pigs represent an ideal animal disease model due to their similar anatomy, physiology, metabolism, immunology, and genetics compared with humans. Importantly, in contrast to murine models, the size of the pigs permits utilization of devices and procedures used clinically in humans.
Methods: Porcine HCC cells were developed from an Oncopig Cancer Model (OCM), a transgenic pig that recapitulates human cancer through induced expression of TP53R167H and KRASG12D, as described previously [1,2]. Generated OCM HCC cells were autologously injected into 6 subcutaneous (SQ) sites in an Oncopig with alcohol-induced liver cirrhosis. Two weeks later, the SQ tumors were excised and engrafted intrahepatically in the same animal, and tumor progression was monitored by ultrasound and CT scan. To generate HCC tumors with different mutational profiles, the OCM HCC cells were subject to CRISPR-Cas9 mediated editing of several genes including KRASG12D, TP53R167H, and ARID1A, a tumor-suppressor gene mutated in 10-15% HCC. Gene editing was performed by transfection of a ribonucleoprotein (RNP) complex comprising a fluorescently-labeled gRNA targeting the gene of interest and recombinant Cas9 nuclease, and was analyzed by Illumina sequencing. Enrichment of edited cells was accomplished by sorting of transfected fluorescent cells by FACS and single cells were plated in 96-well plates.
Results: OCM HCC cells and SQ tumors recapitulated histopathological characteristics of human HCC similar to previously described [2]. Five of 6 SQ sites (>80%) developed 1-2 cm tumors within 2 weeks. Engraftment of SQ tumor fragments into the cirrhotic Oncopig liver resulted in a 1 cm tumor within 4 weeks, and was histologically characterized as HCC. CRISPR-Cas9 mediated gene-editing efficiencies ranged from 11.8-16.3% for KRASG12D, TP53R167H, and ARID1A, and comprised small insertions or deletions (INDELs) near predicted Cas9 cleavage sites. FACS increased the fraction of edited cells, as the RNP transfection efficiency into OCM HCC cells was 17-25%. Single cell clones were expanded and screened for gene editing by Sanger sequencing.
Conclusions: The OCM can be used for development of orthotopic autologous HCC tumors in a cirrhotic liver microenvironment, and represents a novel and promising large animal model for HCC. Successful editing of KRASG12D, TP53R167H and/or oncogenes or tumor suppressor genes in OCM HCC cells enables the generation of genetically-defined HCC tumors similar to those detected clinically. Hence, the OCM can be used as a versatile platform for generating HCC models that can serve as tools for testing innovative precision medicine approaches, and for investigating the contribution of distinct driver mutations on tumor progression and treatment susceptibility.