Hepatocellular carcinoma (HCC) spans more than 780,000 new annual diagnoses, and causes 750,000 yearly mortalities. This deadly malignancy is expected to become the third leading cause of cancer death by 2030, highlighting the urgent need for new treatment strategies. While human clinical trials are the benchmark for advancing standard-of-care 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 murine studies and human clinical practice is thus of vital importance. The Oncopig Cancer Model (OCM) is a novel transgenic swine platform that recapitulates human cancer through development of site/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 results in development of phenotypically consistent porcine HCC (pHCC) cell lines which faithfully recapitulate the in vitro and in vivo features of human HCC.
Eight pHCC lines were established from primary hepatocytes isolated from resected liver specimens (median 9.4, range 4.9-15.0 g) of 4- to 8-week-old OCMs (n = 8), with a median yield of 2.5 x 106 (range 1.5-5.5 x 106) cells/g and 52% (range 30-95%) viability. At 24-hours post-isolation, porcine hepatocytes were transformed into pHCC with median 95% (range 82-98%) efficiency, and were maintained in culture for median 9 (range 6-15) passages. Morphological and behavioral phenotyping of pHCC cells performed using qualitative and quantitative assays were compared to the most widely used human HCC cell line for in vitro investigations (HepG2). Similar to human HCC, all pHCC cell lines exhibited Arginase-1 immunohistochemical positivity—indicating hepatocellular origin—and 100% purity. Eight of 8 (100%) pHCC cell lines showed RT-PCR proven transgene expression, confirming malignant transformation. Flow cytometry demonstrated a median pHCC cell cycle length of 13.0 (range 12.0-16.9) hours, similar to human HCC (15.1 hours). pHCC migration assay showed a median time to half gap closure of 6.0 (range 4.3-9.0) hours, comparable to HepG2 (3 hours). In vivo malignant capability was confirmed by subcutaneous xenograft growth in both SCID mice and Oncopigs, resulting in biopsy proven malignant masses within 7-14 days.
The results of the current work indicate that pHCC cell lines may be consistently developed from OCMs, and validates OCM pHCC as a platform which accurately replicates human cancer for translational research.