Moreover, tumors developed from HBx mice exhibited phenotypes of mixed HCC and cholangiocarcinoma (CC) within the same liver. The HCC-like tumors exhibited features of hepatocellular morphologies, whereas CC-like tumors strongly resembled the “cholangiolocellular” subtype described in humans that exhibits poorly differentiated characteristics (Fig. 3B). Staining of tumor 3-MA chemical structure sections with AFP and
CK19 confirmed that the tumors are composed of hepatocytes and cholangiocytes (Fig. 3C). Furthermore, we detected EpCAM+ tumor cells in both HCC and CC tumor tissues (Fig. 3D). The complete penetrance of both tumor types subsequent to HPC expansion suggested that tumors may derive from stem/progenitor cells and supported our hypothesis that HPCs are involved in HBx-induced tumorigenesis. As shown in Fig. 3A, consistent DDC treatment induced bilineage tumors in HBx-expressed livers. The results raised a question if tumors are derived from transformed HPCs. To identify if the bilineage tumor derived from HBx-induced HPCs, we isolated EpCAM+CD45− HPCs from HBx transgenic mice and WT control mice after 1, 2, 3, or 4 months of DCC treatment, respectively. One × 106 cells were then injected subcutaneously into NOD/SCID mice (n = 6). Eight weeks later, EpCAM+CD45− HPCs derived from all WT mice and 1, 2, or 3-month
DDC-treated HBx mice did not produce any tumors, whereas EpCAM+CD45−HPCs Sorafenib BMN 673 concentration from 4-month DDC-treated HBx mice formed tumor in four out of six mice (Fig. 4A,B). H&E staining and immunohistochemical analysis of AFP and CK19 revealed that these tumors contained mixed cell characteristics (Fig. 4C-E). EpCAM+ cells were also detected in these tumors (Fig. 4F). Therefore, these results demonstrate that chronic injury induced by DDC in the long term (at least 4 months) gradually enhanced the effect of HBx on HPCs and increased
their tumorigenicity potential. Our results have shown that HBx induced expansion of HPCs with increased expression of stemness genes and oncogenes (Fig. 2B). Importantly, HPCs isolated from premalignant HBx mice induced a subcutaneous tumor xenograft (Fig. 4A,B). The question is, what is the mechanism underlying HBx-promoted expansion and transformation of HPCs? To answer the question we analyzed the liver injury, inflammatory response, and signaling pathways during the process of HPC’s response to DCC. To determine if it was because of HBx exacerbated DDC-induced liver injury, we detected the serum alanine aminotransferase (ALT) level and found there was no difference between WT and HBx groups at any timepoint (Fig. 5A), concluding that the degree of liver damage is not associated with HPC proliferation.