Background and Aims:
Hepatocellular carcinoma (HCC) represents the most frequent primary liver malignancy and is one of the leading causes of cancer related death worldwide [L.Kulik, Gastro. 2018]. HCC evolves in a long-term process with accumulation of (epi)genetic alterations that lead to the activation of oncogenic pathways. We aimed to derive hepatoma cells (HC) from a novel mouse model for HCC, reflecting chronic liver damage and fibrosis together with inherited and acquired genetic mutations to enable rapid anti-HCC in vivo drug screening.
Method and Results:
FVB Mdr2 knockout (KO) mice were injected with diethylnitrosamine (DEN, 10 µg/g body weight) at 3 weeks of age and exposed to phenobarbital (0.05%) in their drinking water. 8 months old HCC bearing mice were sacrificed and macroscopic lesions (measuring up to 5 mm) were carefully separated from non-tumorous tissue, minced and transferred into supplemented Dulbecco's modified Eagle's medium. After 8 months of subculturing, HC were harvested and transcript levels for HCC markers were quantified by qPCR. Compared to RNA extracted from livers of healthy FVB mice, the HC exhibited a high expression (> 100-fold) of alpha fetoprotein (AFP) and epithelial cell adhesion molecule (EpCAM), while transcript levels of the HCC suppressor gene HNF4a were significantly downregulated. HE-staining revealed all signs for malignancy, such as a high nucleus/cytoplasm ratio with hypertrophy and a high number of mitoses with atypical mitotic forms. Downregulation of E-cadherin compared to control hepatocytes indicated epithelial-mesenchymal transition of the HC. In a first testing, Sorafenib attenuated HC proliferation in a dose-dependent manner with an IC50 value of ˜7µM as demonstrated before for the HCC cell line HepG2.
Conclusion:
We have established a new HC line from our syngeneic Mdr2-/- HCC mouse model that replicates major features of human HCC. The cells display histologic signs of malignancy and characteristic HCC tumor markers as determined on the transcript and protein level. Sorafenib showed a dose-dependent antiproliferative effect on these HC. Conclusively, the HC are a promising cell model for ex vivo rapid screening of drug candidates, to be validated in the in vivo model and finally in patients.
