Identifying Hepatocellular Carcinoma (HCC) cells with cancer stem cell-like properties.

Hepatocellular Carcinoma (HCC) is a challenging condition to treat in the clinic due to its distinct tumor phenotype, aggressive growth, and chemotherapy resistance. Cancer Stem Cells (CSCs) are a poorly differentiated subpopulation of cells within the tumor microenvironment, and role of CSCs in tumor initiation and chemotherapy resistance is not well understood in HCC. In this study, we have successfully identified HCC cells with CSC-like properties that showed resistance to Doxorubicin. Using the established serum free culture techniques, we have enriched CSC-like cells among hepatocellular cancer cells. These enriched cells exhibit all CSC-like properties i.e. self-renewal capability, anchorage independent growth with retained proliferation property and resistance to Doxorubicin. We confirmed expression of CSC surface markers (EpCAM, CD90, CD44, CD133) and functional markers (ALDH activity and dye exclusion properties) in enriched HCC-CSCs. Preliminary in vivo data in our mouse models supports that spheroid forming CSCs show higher rate of tumor proliferation compared to untreated (non-spheroid forming) HCC cells. Wnt/β- catenin pathway components were also found to be overexpressed in CSCs when compared to non spheroids. Analysis of 24 paired human specimens from HCC tissues by IHC showed higher EpCAM expression, and Western Blot analysis suggested concomitant changes in β-catenin and EpCAM levels. In conclusion: (1) we have successfully induced and identified HCC cells with CSClike properties; (2) Aberrant β-catenin up-regulation mediated Wnt/β-catenin signaling activation was observed in enriched hepatoma spheroids

Wnt/β-Catenin mediated cancer stem cell activation in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers, with variable presentation, high chemotherapy resistance and early tumor recurrence. In this dissertation, we have systematically enriched, identified and characterized HCC cancer stem cells (CSCs) from 3 different HCC cell lines (Hepa1-6, HepG2, and Hep3B) using in vitro serum-free culture method. Enriched spheroids acquired CSC properties, primarily self-renewal capability, spheroid formation ability, and drug resistance. Heterogeneous CSCs enriched from Hepa1-6 cells demonstrated higher tumor initiation and proliferation capability in vivo, compared to control HCC cells in orthotopic immunocompetent mouse model. EpCAM+ CSCs were significantly enriched within heterogeneous Hepa1-6 CSC spheroids. Consequently, we studied the fate of EpCAM+ CSCs in orthotopic immunocompetent mouse model with 3 different liver microenvironments i.e. control normal liver, steatosis (fatty liver induced), and nonalcoholic steatohepatitis (NASH). EpCAM+ CSC mediated HCC carcinogenesis was observed in NASH livers, but failed to develop in control and steatosis liver microenvironment. This is the first study evaluating CSCs in immunocompetent mouse model and demonstrated the importance of liver microenvironment for EpCAM+ CSC mediated tumor initiation. We then performed a lineage tracking study by stably incorporating copGFP or mCherry in Hepa1-6 cells by lentivirus transduction, and examined the origin of tumors in NASH liver microenvironment using orthotopic C57L/J immunocompetent mouse model. Our findings suggest that tumor growth was dose dependent and most tumor nodules arose from copGFP expressing EpCAM+ CSCs. The Wnt/β-catenin pathway components were also found to be overexpressed in CSCs when compared to control. Human HCC specimen analyses suggested concomitant changes in β-catenin and EpCAM levels. Gene analyses of step-wise spheroid formation process identified possible dedifferentiation mechanisms regulated by β-catenin in CSC spheroids. Loss of function analysis by siRNA mediated transient knockdown of β-catenin confirmed its role in spheroid enrichment and doxorubicin resistance. Canonical Wnt pathway study using chemical inhibitors identified β-catenin mediated CSC activation was regulated at nuclear level, and not at the cytoplasmic GSK3β destruction complex level. Analysis of human and mouse tumors suggested that constitutive activation of β-catenin transcription failed to respond to NOTUM, a feedback inhibitor of canonical Wnt/β-catenin signaling