Sphere-forming culture enriches liver cancer stem cells and reveals Stearoyl-CoA desaturase 1 as a potential therapeutic target

Backgrounds: The role of sphere-forming culture in enriching subpopulations with stem-cell properties in hepatocellular carcinoma (HCC) is unclear. The present study investigates its value in enriching Cancer Stem Cells (CSCs) subpopulations and the mechanism by which HCC CSCs are maintained.

Methods: HCC cell lines and fresh primary tumor cells were cultured in serum-free and ultra-low attachment conditions to allow formation of HCC spheres. In vitro and in vivo experiments were performed to evaluate CSC characteristics. Expression levels of CSC-related genes were assessed by qRT-PCR and the correlation between sphere formation and clinical characteristics was investigated. Finally, gene expression profiling was performed to explore the molecular mechanism underlying HCC CSC maintenance.

Results: We found that both cell lines and primary tumor cells formed spheres. HCC spheres possessed the capacity for self-renewal, proliferation, drug resistance, and contained different subpopulations of CSCs. Of interest, 500 sphere-forming Huh7 cells or 200 primary tumor cells could generate tumors in immunodeficient Animals. Sphere formation correlated with size, multiple tumors, satellite lesions, and advanced stage. Further investigation identified that the PPARα-SCD1 axis plays an important role in maintenance of the CSC properties of HCC sphere cells by promoting nuclear accumulation of β-catenin. Inhibition of SCD1 interfered with sphere formation, down-regulated expression of CSC-related markers, and reduced β-catenin nuclear accumulation.

Conclusions: Sphere-forming culture can effectively enrich subpopulations with stem-cell properties, which are maintained through activation of the PPARα-SCD1 axis. Therefore, we suggest that targeting the SCD1-related CSC machinery might provide a novel insight into HCC treatment.