The Effects of Y-27632 on the Propagation of Glioblastoma Stem Cells

Glioblastoma is the deadliest form of brain cancer. Patients diagnosed with glioblastoma have poor prognosis, and their median survival rate is approximately one year. Recent research shows evidence for a specialized subpopulation of glioblastoma cells called glioblastoma stem cells (GSCs). GSCs have the ability to self renew and differentiate into the heterogeneous tumor cells that constitute the entire tumor. GSCs are also thought to be resistant to current treatment techniques such as chemotherapy and radiation. For these reasons, the study of GSCs is an area of extreme interest in current glioblastoma research; however, very few GSCs exist in glioblastoma cell lines grown in conventional in vitro conditions. When glioblastoma cells are grown in serum-free media, they form tumorspheres that imitate the in vivo tumor niche and are enriched for GSCs. We have shown that the addition of the Rho-Kinase (ROCK) inhibitor Y-27632 to serum-free, cancer stem cell media increases the total number of cells and the number of GSCs that can be grown in vitro. Y-27632 has been implicated in suppressing apoptosis, and it was hypothesized that Y-27632 would increase the number of glioblastoma stem cells (GSCs) grown in vitro and improve cloning efficiencies. Indeed, our data showed that supplementing the GSC media with Y-27632 inhibits apoptosis. Furthermore, we demonstrated that Y-27632 positively affects the cells’ ability to form spheres using size analysis and limiting dilution assays. We also characterized the expression of known stem cell markers in cells grown with the inhibitor using various molecular and cell biology techniques. Our data therefore showed that Y-27632 has an overall positive effect on tumorsphere formation and is poised to have a promising application for cancer stem cell culture in the future.