Abstract
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.
Highlights
Primary cultures of normal hepatocytes are widely used to study the biochemical/molecular events involved in cell death, proliferation and differentiation [1, 2]
Only RH cells were double positive for CD90.1/CD24, (Figure 6D), as CD90.1 was not detected in the immortalized-non tumorigenic RNT cell line (Figure 6C). These findings show that only RH cells contain tumor stem-like cells, as defined by the double expression of CD90.1 and CD24, in agreement with the results obtained from human samples [21]
Both cell lines were obtained by perfusion of rat livers exposed to the Resistant-Hepatocyte protocol, in the presence or in the absence of carcinogenic treatment, and could be maintained in culture for more than 50 passages, without signs of senescence
Summary
Primary cultures of normal hepatocytes are widely used to study the biochemical/molecular events involved in cell death, proliferation and differentiation [1, 2]. To improve the maintenance of hepatocyte function and the longevity of the cultures, several alternatives have been developed, but still several problems need to be overcome. Some conditions were identified to improve in vitro culture, including continuous medium and oxygen supply, and metabolite removal [4, 5]. Various hollow www.impactjournals.com/oncotarget fiber bioreactor systems were developed using hepatocytes of several species [6]. In these systems, cells attach to the surface of fibers or membranes and reorganize themselves into three-dimensional structures that may result in a hepatocyte microenvironment closely resembling the physiological one. The described cell culture systems are not yet standardized and cannot be transferred to other laboratories
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