Abstract 4251: Murine hepatocellular carcinoma stem cells express pluri-potency-associated transcription factors and are sensitive to immune-mediated apoptosis

Worldwide the incidence of hepatocellular carcinoma (HCC) resulting from hepatitis C infection and chronic inflammation is increasing. Poor prognosis HCC is characterized by over-expression of Ep-CAM and alpha-fetoprotein (AFP) which define a population of tumor-initiating cells with stem/progenitor features. Current evidence supports the view that cancer stem cells (CSCs) constitute the primary pathogenic component of both metastases and minimal residual disease, and that elimination of CSCs is required for cure. Typically cell surface markers have been used to identify CSCs, however cell surface phenotype alone does not identify CSC in all tumors from the same tissue or in all patients. Therefore there is a need to define and test more reliable markers of CSCs. Oct-4, Sox-2 and Nanog transcription factors are key regulatory factors in maintenance of pluripotency and have been demonstrated to be robust intracellular biomarkers for tissue and cancer stem cells. A GFP-based reporter driven by a promoter responsive to Nanog or Oct-4 was used to mark pluripotent tumor cells from in vitro cultured, cancer cell lines (breast cancer- 4T1.2), and from autochthonous hepatocellular carcinoma induced by hydrodynamics-based gene transfer of constituitively active Akt and β-Catenin. This tumorigenesis model allows tumor initiation by delivery of clinically relevant oncogenes whose sporadic distribution reflects spontaneous cancer. The molecular profile of primary Akt/β-Cat-induced murine HCC recapitulates that observed in poor prognosis Ep-CAM+/AFP+ human HCC. Further, in vitro functional analyses suggest that Ep-CAM and CD133 (surface markers) and Nanog and Oct-4 (transcription factors required for maintenance and regulation of pluripotency in undifferentiated cells) are CSC markers in this model of HCC. Directly ex vivo, murine HCC stem cells express abundant CD1d and MHC class II in addition to TNF-R2, lymphotoxin-β receptor (LTβR) and DR5. The functional significance of CD1d, MHC class II, TNF-R2, LTβR and DR5 expression will be assessed in future studies using in vitro apoptosis and cytotoxicity assays and transplantation at orthotypic sites prior to adoptive transfer of tumor-specific T cells. Studies of freshly isolated CSCs have demonstrated that a variety of mechanisms combine to render CSC resistant to conventional chemo- and radiotherapies. This study has combined in vitro and in vivo approaches to address whether CSC can be rendered sensitive to T cell-med