Genetic lineage tracing analysis of the cell of origin of hepatotoxin‐induced liver tumors in mice

The expression of biliary/progenitor markers by hepatocellular carcinoma (HCC) is often associated with poor prognosis and stem cell-like behaviors of tumor cells. Hepatocellular adenomas (HCAs) also often express biliary/progenitor markers and frequently act as precursor lesions for HCC. However, the cell of origin of HCA and HCC that expresses these markers remains unclear. Therefore, to evaluate if mature hepatocytes give rise to HCA and HCC tumors and to understand the molecular pathways involved in tumorigenesis, we lineage-labeled hepatocytes by injecting adeno-associated virus containing thyroxine-binding globulin promoter-driven causes recombination (AAV-TBG-Cre) into RosaYFP mice. Yellow fluorescent protein (YFP) was present in >96% of hepatocytes before exposure to carcinogens. We treated AAV-TBG-Cre; RosaYFP mice with diethylnitrosamine (DEN), followed by multiple injections of carbon tetrachloride to induce carcinogenesis and fibrosis and found that HCA and HCC nodules were YFP+ lineage-labeled; positive for osteopontin, SRY (sex determining region Y)-box 9, and epithelial cell adhesion molecule; and enriched for transcripts of biliary/progenitor markers such as prominin 1, Cd44, and delta-like 1 homolog. Next, we performed the converse experiment and lineage-labeled forkhead box protein L1(Foxl1)-positive hepatic progenitor cells simultaneously with exposure to carcinogens. None of the tumor nodules expressed YFP, indicating that Foxl1-expressing cells are not the origin for hepatotoxin-induced liver tumors. We confirmed that HCA and HCC cells are derived from mature hepatocytes and not from Foxl1-Cre-marked cells in a second model of toxin-induced hepatic neoplasia, using DEN and 3,3′,5,5′-tetrachloro-1,4-bis(pyridyloxy)benzene (TCPOBOP). Conclusion: Hepatocytes are the cell of origin of HCA and HCC in DEN/carbon tetrachloride and DEN/TCPOBOP induced liver tumors. (Hepatology 2016;64:1163-1177)