Pigs provide a valuable large animal model for several diseases due to their similarity with humans in anatomy, physiology, genetics and drug metabolism. We recently generated a porcine model for TP53R167H and KRASG12D driven hepatocellular carcinoma (HCC) by autologous liver implantation. Here we describe a streamlined approach for developing genetically tailored porcine HCC cells by CRISPR/Cas9 gene editing and isolation of homogenous genetically validated cell clones. The combination of CRISPR/Cas9 editing of HCC cells described herein with the orthotopic HCC model enables development of various porcine HCC models, each with a specific mutational profile. This allows modeling the effect of different driver mutation combinations on tumor progression and in vivo testing of novel targeted therapeutic approaches in a clinically relevant large animal model.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, representing the most common form of liver cancer. As HCC incidence and mortality continue to increase, there is a growing need for improved translational animal models to bridge the gap between basic HCC research and clinical practice to improve early detection and treatment strategies for this deadly disease. Recently the Oncopig cancer model—a novel transgenic swine model that recapitulates human cancer through Cre recombinase induced expression of KRASG12D and TP53R167H driver mutations—has been validated as a large animal translational model for human HCC. Due to the similar size, anatomy, physiology, immunology, genetics, and epigenetics between pigs and humans, the Oncopig has the potential to improve translation of novel diagnostic and therapeutic modalities into clinical practice. Recent studies have demonstrated the importance of tumor cells in shaping its surrounding microenvironment into one that is more proliferative, invasive, and metastatic; however, little is known about the impact of microenvironment signaling on HCC tumor biology and differential gene expression between HCC tumors and its tumor microenvironment (TME). In this study, transcriptional profiling was performed on Oncopig HCC xenograft tumors (n = 3) produced via subcutaneous injection of Oncopig HCC cells into severe combined immunodeficiency (SCID) mice. To differentiate between gene expression in the tumor and surrounding tumor microenvironment, RNA-seq reads originating from porcine (HCC tumor) and murine (microenvironment) cells were bioinformatically separated using Xenome. Principle component analysis (PCA) demonstrated clustering by group based on the expression of orthologous genes. Genes contributing to each principal component were extracted and subjected to functional analysis to identify alterations in pathway signaling between HCC cells and the microenvironment. Altered expression of genes associated with hepatic fibrosis deposition, immune response, and neo angiogenesis were observed. The results of this study provide insights into the interplay between HCC and microenvironment signaling in vivo, improving our understanding of the interplay between HCC tumor cells, the surrounding tumor microenvironment, and the impact on HCC development and progression.

No abstract

No abstract

Biomedical research of liver cancer requires effective model cell lines and animals in order to translate diagnostic and treatment strategies into clinical practice. An ideal translational model to study hepatocellular carcinoma (HCC) would be the use of a genetic pig model of HCC due to the many similarities between pigs and humans which include anatomy, physiology, metabolism and genetics. This study utilized the Oncopig Cancer Model (OCM), a transgenic pig model that develops site and cell specific tumors through Cre recombinase induced expression of KRASG12D and TP53R167H transgenes. Our objective was to develop an in vitro HCC model using primary hepatocytes isolated from Oncopig liver tissue. In order to develop Oncopig HCC cell lines, Oncopigs (n=36) underwent liver resection to remove a portion (5 to 20 grams) of their liver for hepatocyte isolation. Post isolation mean cell yield was 1.8 million cells per gram of tissue ranging from 2.2 million cells to 6.5 million cells. After incubation (24 hours post hepatocyte isolation), primary hepatocytes were transfected with adenoviral vector encoding Cre recombinase (AdCre), resulting in KRASG12D and TP53R167H expression and transformation of hepatocytes into HCC cell lines. Also, AdCre transfected cells will have GFP expression. Successful transfection rates (77% to 99% with a mean of 87.9%) were confirmed by fluorescent microscopy. Phenotypic characterization of OCM HCC cells was then performed. Cell migration assays were performed to characterize enhanced cell migration of transformed cells (mean t1/2 gap = 4 hours) and polymerase chain reaction (PCR) assays were done after several passages to confirm KRASG12D and TP53R167H gene expressions. HCC lines were injected subcutaneously in the abdomen of SCID or NSG mice (n=34). Each mouse was given 2 injections with each injection site receiving 5 million cells. Mouse injections resulted in 59 tumors (86.8% tumor growth success). Mean volume for the tumors was 285.3 mm3 ranging from 18.84 mm3 to 1766.3 mm3. Following confirmation of tumor development in SCID or NSG mice, HCC cell lines were autologously injected into Oncopigs (n=29). Each Oncopig was given 6 injections subcutaneously in the abdomen with each injection site having 10 million cells Autologous injections to Oncopigs were capable of growing tumors. These results indicate that a porcine HCC model can be created and used for further tumor biology research. Citation Format: Shovik S. Patel, Sulalita Chaki, Faith M. Thomas, Aisha Qazi, Kyle M. Schachtschneider, Matthew Stewart, Elizabeth Pollack, Ron C. Gaba, Lawrence B. Schook. Development, maintenance, and characterization of porcine hepatocellular carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3711.