Development, Maintenance, and Characterization of Porcine Hepatocellular Carcinoma Cell Lines

S.S. Patel, S. Chaki, F.M. Thomas, A. Qazi, K.M. Schachtschneider, M. Stewart, E. Pollack, R.C. Gaba, L.B. Schook
American Association for Cancer Research Annual Meeting, March 29 - April 3 2019, Atlanta, GA

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.