A Platform to Establish a Working Hierarchy of Glioblastoma Multiforme Cells:

Implication on Cancer Cell-microenvironment Interaction and Response to Drugs

Abstract

Glioblastoma multiform (GBM), a grade IV glioma, is the most common and aggressive cancer in the central nervous system. Current treatment for GBM includes surgical resection, radiation, and the frontline DNA alkylating drug, temozolomide (TMZ). The current median survival for GBM patients is about 14.5 months with 5% patients surviving up to 5 years. We propose that discerning distinct subsets within heterogeneous GBM will provide avenues for research to improve new therapies. We used different methods to isolate GBM cell subsets. These include stable transfectants of GBM cell lines with a lentiviral system in which green fluorescence protein (GFP) is regulated with tandem repeats of Oct4a and Sox2 response elements. Parallel studies with a plasmid using the full-length regulatory region of Oct4a indicated reduced efficiency in separating cell subsets, relative to SORE6-GFP lentivirus. Stem cell-linked gene expressions and function studies such as ALDH1, tumorsphere and in vivo passaging of GFP hi subsets confirmed the presence of cancer stem cells (CSCs). We also studied a more efficient method that could be relevant for primary GBM cells. We selected tumorspheres by plating heterogeneous GBM cells and then serially passaged the spheres. Studies for stem cell genes indicated that this method could be used for primary GBM cells. Overall, this study provided insights into methods to isolate GBM subsets, including primary GBM cells. The advantages of the methods are discussed.