STEM-05. GLIOBLASTOMA NEUROSPHERES DISPLAY STEMNESS CHARACTERISTICS AND UPREGULATION OF THE VEGF-A PATHWAY

Abstract

Abstract INTRODUCTION Glioma stem cells (GSC) constitute a subpopulation of tumor cells with stem-cell like properties and are believed to enhance glioblastoma’s ability to grow and confer resistance against chemoradiotherapy. It has been shown that non-adherent three-dimensional (3D) growth conditions facilitate neurosphere formation and enable isolation of GSCs in vitro. This can serve as an invaluable tool to better understand the role of GSCs in glioblastoma pathogenesis. OBJECTIVE We aimed to evaluate differentially expressed genes in non-adherent versus adherent conditions using primary tumor glioblastoma cell lines; and to identify alterations in various signaling pathways that are paramount to their growth and maintenance. Furthermore, we targeted the EMP2-HIF1α axis, a proposed regulator of VEGF expression, to determine its effects on cell viability. METHODS Three primary tumor glioblastoma cell lines were generated and cultured under adherent or non-adherent growth conditions. Sequencing was performed on the Illumina HiSeq3000 system, and gene counts were normalized by TMM. A pair-wise statistical analysis of over 20,000 genes was performed between the two conditions. A p-value < 0.05 was considered statistically significant. RESULTS After 72 hours of growth under 3D conditions, primary glioblastoma cells show an increase in stem-like properties, measured via an upregulation of SOX2 and nestin. RNA sequencing demonstrated a significant increase in expression of the following transcripts in 3D conditions compared to those grown on adherent plates: VEGF-A, EGFR, PDGFR-A, FGFR3, and TGFB2. Treatment with anti-EMP2 monoclonal antibodies significantly reduced cellular proliferation. CONCLUSION Our results demonstrate that neurospheres upregulate growth factor pathways to enable their survival in 3D conditions, suggesting an inherently different genetic landscape between differentiated tumor cells and GSCs. Future studies will investigate the cellular mechanisms responsible for upregulation of these genes; and to ultimately design more effective targeted therapeutic agents.