Abstract 533: Astrocytes enhance the migration potential of glioblastoma stem-like cells

Abstract

Abstract Glioblastoma (GBM) cells in situ are characterized as highly invasive and migratory: properties considered a barrier to successful treatment. The mechanisms mediating the migration of GBM cells have yet to be clearly defined. Moreover, migration has not been described using GBM stem-like cells (GSCs), which data suggest play a critical role in GBM development and maintenance. Therefore, we have defined the migration potential of human CD133+ GSCs grown under in vitro conditions and in an attempt to simulate the GBM microenvironment have determined the influence of astrocytes. For these studies we used GSCs engineered to express GFP; migration was determined at 48h after seeding cells into the upper chamber of transwell Boyden chambers. Initially, GSCs were seeded into Boyden-Chambers in which the upper chamber was coated with poly-L-lysine (PLL). Under these conditions GSCs migrated from the top chamber and could be detected in the bottom chamber. When human astrocytes were plated in the bottom chamber the percentage of GSCs migrating through the top chamber increased approximately 3-fold; the GSCs were detectable on top of the astrocyte monolayer. These data suggest that astrocytes attract GSCs enhancing their migratory activity. To begin to address the processes mediating the increase in GSC migratory behavior in the presence of astrocytes, co-cultures of astrocytes and GSCs were maintained for 72h, the GFP expressing GSCs were then separated using FACS and subjected to microarray gene expression analysis. After MAS 5.0 analysis, the effects of the co-culture conditions on GSC gene expression were determined by dividing the expression levels from co-cultured GSCs by those obtained from sorted GSCs not cultured in the presence of astrocytes. Affymetrix IDs with a >2.0 fold-change were uploaded into WebGestalt software (http://bioinfo.vanderbilt.edu/webgestalt/) and KEGG pathway analysis (p<0.05) was preformed. The KEGG pathways identified for the co-cultured GSCs included a number pertaining to cell migration such as Focal Adhesion (p=0.0001), Cell Adhesion Molecules (p=0.0002) and ECM-receptor interaction (p=0.0006). Enriched genes within the KEGG pathways included integrins (ITGs), neural adhesion molecules (NCAMs), laminin (LAM), collagens (COL) and Tenascine (TNC). Additional genes associated with cell migration and whose expression was modified at least 2-fold in GSC co-cultured with astrocytes were connective tissue growth factor (CTGF), fibroblast growth factors (FGF), insulin-like growth factor (IGF), CD44 and bone morphogenetic proteins (BMP). These results are consistent with an astrocyte-mediated increase in the GSC expression of genes whose protein products directly and/or indirectly regulate cell migration. Moreover, these data provide additional support for a critical role of normal brain cells in the regulation of GBM biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 533. doi:10.1158/1538-7445.AM2011-533