Stem cells compartment activation during heparocarcinogenesis in a HBV-transgenic model

The hepatocarcinogenesis consists of many steps and long term courses from normal to malignant tissue. Besides the various etiological factors, chronic exposure to a wide variety of substances can induce an inflammatory response and oxidative stress with consequent hepatocytes dead and regenerative phenomena. When the replicative ability of hepatocytes is impaired, the activation of the hepatic stem cell (SCs) compartment permits the recovery of the mass and functionality of the liver. The cancer stem cells (CSCs) theory postulates that cancer is composed in a hierarchy consisted of many heterogeneous cells with different grades of differentiation, in which only the CSCs are capable of initiating cancer growth. In exception of their tumor-initiating capacity, the CSCs share similar phenotypic signatures and functional properties with normal stem cells. Until now, the origin of the CSCs in hepatocellular carcinoma (HCC) is still unclear. The general objective of this project is to investigate the involvement of the SCs in the hepatocarcinogenesis, starting from early injury to development of HCC. In this study, we employed the HBV-transgenic mouse C57BL/6J-TG(ALB1HBV)44BRI/J (TG) that develops a progressive hepatic damage that well mimics the natural history of human hepatocarcinogenesis. Based on the analysis of the different SCs markers (CD34, CD133, Epcam, Krt19) expressed in hepatic tissues at different courses of the disease (from 3 months to 18 months), we observed the activation and the progressive amplification of the SCs compartment. In particular, there was a strong correlation between the expression pattern of CSCs markers and different stages of hepatic pathologies: a progressive increase of CD133, CD34 and Afp expression was noticed from normal, early cellular damage, dysplasia, and HCC, while Krt19 was decreased in HCC compared to other pathologies. We also performed the isolation and characterization of putative hepatic SCs and CSCs from both TG and their wild type counterpart C57BL/6J (WT) animals in different growth mediums. The isolated primary cells showed up-regulation of the SCs markers compared to total tissue with low or no expression of hepatocytes markers (Alb and Krt18). They had the ability to form 3D clones in matrigel after 7-10 days plating. In summary, our data indicates that this mouse model is well-corresponded with the natural history of hepatocarcinogenesis in human and can represent a good tool to study the involvement of SCs and CSCs in the progression of the disease. In addition, the possibility to isolate SCs and CSCs will be a potent tool to study the differences of developmental and oncogenic molecular pathways between normal SCs and CSCs and to develop novel CSCs-targeted therapy.