Associate Professor, The College of Idaho, ID
Glioblastoma multiforme is an astrocyte-derived high grade glioma with poor prognosis. As the most common primary brain tumor in adults, it is universally fatal and has a median survival time of 15 months. The murine glioma cell line GL261 is a model system used to investigate glioma tumorigenesis and glioma cell phenotype. In culture, adherent GL261 cells can be coaxed to grow as free-floating cell aggregates (neurospheres) by supplementing the culture media with growth factors. Cells grown as neurospheres are phenotypically different from adherent cells, and if implanted into host animals will form malignant tumors. However, the underlying basis of these phenotypic differences is unclear, and whole-transcriptome data is not available for the GL261 cell type. This project uses RNA-seq to compare gene expression in GL261 cells cultured adherently or under conditions that generate neurospheres. Identification of genes that are differentially expressed will lead to a better understanding of cellular changes that permit malignant tumor growth.
Adherent Cell Culture Conditions: GL261 adherent cells were cultured in DMEM (Corning) with 10% Fetal Bovine Serum (Atlanta Biologicals) and Penicillin/Streptomycin/Glutamine (Sigma-Aldrich). Cells were incubated at 37.0°C with 5.0% CO2 in 75cm culture flasks. Cells were passaged by trypsinization and media was changed every 3-5 days.
Induction of Neurospheres: Adherent cells were trypsinized and cultured in serum free DMEM (Corning) and Penicillin/Streptomycin/Glutamine (Sigma-Aldrich) with the addition of fibroblast growth factor (FGF, 20ng/ml), epidermal growth factor (EGF, 20ng/ml), and B27 (1:50). Differentiation was induced 3-5 days after initial plating. Media and growth factors were replaced every 3-5 days thereafter. Cells were incubated at 37.0°C with 5.0% CO2 in 75cm culture flasks.
Total RNA was isolated from adherent cells and from neurospheres at 14 days post-induction and used to generate Illumina RNA-Seq libraries.
This RNA-Seq dataset was used to characterize changes to gene expression in GL261 cells (a model system for high grade glioma tumors) between two conditions: 1) adherent cells and 2) cells cultured under conditions that promote the formation of free-floating cell aggregates known as neurospheres (a more tumorigenic phenotype).
Materials are under development.
