Physical Selection of Murine Astrocytes to Study Intra-tumoral Heterogeneity in Glioblastoma Multiforme

Glioblastoma multiforme is one of the most aggressive tumors, with an extremely low five-year survival rate of 10%. Vast inter- and intra-tumoral heterogeneity in GBM tumors leads to aggressive phenotypes and poor prognosis in most patients. One source of heterogeneity stems from variance in adhesion strength of cells to extracellular matrix, which leads to differences in migration and invasion. We hypothesized that a cell line with a set of mutations that are common in GBM will have a subpopulation of cells that will drive invasion based on their adhesion strength. In order to identify and characterize the cells that lead to aggressive GBM outcomes, we optimized and utilized a parallel plate flow chamber assay to expose wtEGFR murine astrocytes to shear stress and sort the population based on adhesion strength. Sorted populations were described either as weakly or strongly adherent and analyzed through various downstream assays such as the spinning disk shear assay to quantify adhesion strength and immunofluorescence staining to elucidate focal adhesion formation. While phenotypic differences between sorted populations and unsorted control populations were not easily reproduced between replicates at 48 hours post exposure to shear stress, differences between the populations were more easily identified 16 hours post exposure to shear stress. This indicated that phenotypic differences that are revealed as a result of physical selection are not permanent and that the underlying mechanisms of these differences need to be uncovered