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Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the second cause of cancer-related death. Search for genes/proteins whose expression can discriminate between normal and neoplastic liver is fundamental for diagnostic, prognostic and therapeutic purposes. Currently, the most used in vitro hepatocyte models to study molecular alterations underlying transformation include primary hepatocytes and transformed cell lines. However, each of these models presents limitations. Here we describe the isolation and characterization of two rat hepatocyte cell lines as tools to study liver carcinogenesis. Long-term stable cell lines were obtained from a HCC-bearing rat exposed to the Resistant-Hepatocyte protocol (RH cells) and from a rat subjected to the same model in the absence of carcinogenic treatment, thus not developing HCCs (RNT cells). The presence of several markers identified the hepatocytic origin of both cell lines and confirmed their purity. Although morphologically similar to normal primary hepatocytes, RNT cells were able to survive and grow in monolayer culture for months and were not tumorigenic in vivo. On the contrary, RH cells displayed tumor-initiating cell markers, formed numerous colonies in soft agar and spheroids when grown in 3D and were highly tumorigenic and metastatic after injection into syngeneic rats and immunocompromised mice. Moreover, RNT gene expression profile was similar to normal liver, while that of RH resembled HCC. In conclusion, the two cell lines here described represent a useful tool to investigate the molecular changes underlying hepatocyte transformation and to experimentally demonstrate their role in HCC development.

A long term, non-tumorigenic rat hepatocyte cell line and its malignant counterpart, as tools to study hepatocarcinogenesis