Development and Characterization of a Large Animal Model of Hepatocellular Carcinoma and Liver Cirrhosis
thesisposted on 01.08.2019, 00:00 authored by Ron C Gaba
Hepatocellular carcinoma (HCC) involves more than 780,000 new annual cases, and causes 750,000 yearly mortalities. This deadly malignancy is expected to develop into the third leading cause of cancer death by 2030, highlighting the urgent need for new treatment strategies. While human clinical trials represent the standard for advancing clinical cancer therapeutics, preclinical animal models represent pivotal tools for translational investigations to develop and test novel therapeutics both in vitro and in vivo. The development of clinically relevant systems to serve as a bridge between preclinical mouse studies and human clinical practice is thus of vital importance. The Oncopig Cancer Model (OCM) is an innovative transgenic swine platform that recapitulates human cancer through development of site and cell specific tumors after Cre recombinase induced expression of heterozygous KRASG12D and TP53R167H transgenes. In this study, we tested the hypothesis that isolation and transformation of OCM hepatocytes from multiple individuals across a large OCM cohort results in development of phenotypically consistent OCM HCC that faithfully recapitulate the in vitro and in vivo features of human HCC. Fifteen OCM HCC lines were established from primary hepatocytes isolated from resected liver specimens (median 10.1, range 4.9-26.0 g) from Oncopigs (n = 15) of median age 45 (range 39-70) days, with a median yield of 2.9 x 106 (range 0.7-13.0 x 106) cells/g and 64% (range 20-97%) viability. At 24-hours post-isolation, hepatocytes were transformed into OCM HCC with median 94% (range 70-99%) transfection efficiency, and were maintained in culture. Morphological and behavioral phenotyping of OCM HCC cells performed using qualitative and quantitative assays were compared to the most widely used human HCC cell lines. Fifteen of 15 (100%) OCM HCC cell lines showed reverse transcription polymerase chain reaction (RT-PCR) proven transgene expression, confirming malignant transformation. Flow cytometry demonstrated a median OCM HCC cell cycle length of 13.6 (range 10.0-16.9) hours, similar to human HCC. Migration assay showed a median time to half gap closure of 6.0 (range 4.3-9.0) hours, faster than human HCC and suggesting a more invasive nature. Alpha fetoprotein production by OCM HCC cell lines was similar to the AFP secretion by AFP-producing human HCC cell lines (11,549 versus 10,532 ng/dL, P = 0.824), confirming potential utility of AFP as an OCM HCC biomarker, similar to human HCC. Similar to human HCC, all OCM HCC cell lines exhibited Arginase-1 immunohistochemical positivity—indicating hepatocellular origin—and median 100% (range 88-100%) purity. In vivo malignant capability was confirmed by subcutaneous xenograft growth in both SCID mice (68 tumors formed in 38 mice) and Oncopigs (34/50, 68% of injection sites), resulting in biopsy proven malignant masses within 14-21 days. An intra-hepatic tumor was also developed in one Oncopig, confirming the utility of the OCM as a translational HCC model. The results of the current work indicate that HCC cell lines may be consistently developed from OCMs and show capability for in vivo solid tumor development, and validates OCM HCC as a useful platform which accurately replicates human cancer for translational research.