158 Controlling Glioblastoma Invasion In Vivo With CD97

Abstract

INTRODUCTION: Increased levels of adhesion G Protein-Coupled Receptor (GPCR) CD97 have been detected in multiple metastatic cancers. Glioblastoma (GBM), a Grade-IV Glioma, also overexpress this receptor, and its levels positively correlate with more aggressive GBM invasion. METHODS: CD97 consists of two distinct domains: the large extracellular fragment interacting with ECM and other cell surface receptors and the GPCR core that activates heterotrimeric G Proteins G12/13. To investigate the mechanism of action of CD97, we generated a CD97 knockdown (shCD97) and overexpression (CD97-WT) constructs. Additionally, we generated two functional mutants: a constitutively active mutant lacking the N-Terminus (dNTF) and dominant-negative mutant (H436A) that cannot activate G-Proteins downstream. Glioma Stem Cells (GSC) were transduced by control and CD97 lentiviral constructs and implanted into mouse brains. Once tumors formed the brains were harvested, sliced, and immunostained to evaluate invasion and proliferation. Comparing mutants to control, dunnett’s multiple comparisons test was used for statistical analysis. RESULTS: Similar to CD97-WT, we found that both dNTF and H436A, increase tumor volume (p < 0.05). In contrast, the shCD97 mutant decreased tumor size (p < 0.05). Interestingly, only dNTF significantly increased tumor cell proliferation (p < 0.005), yet CD97-WT and H436A affected proliferation of non-tumor cells in the tumor bulk, indicating that this process requires the N-Terminus Fragment. In addition, over-expression of CD97-WT dramatically increases tumor invasion along myelinated fibers of corpus collosum, and tumor spread in the brain tissue (p < 0.05). Yet, the H436A mutant produced large, compact, and non-invasive tumors with sharp borders (p < 0.05) indicating that cell migration requires activation of G12/13. Surprisingly, immunostaining revealed that CD97 knockdown cells displayed invasive phenotype. CONCLUSIONS: Our results demonstrate the dual role CD97 plays in GBM invasion and proliferation, alluding to the molecular mechanisms that can potentially be targeted in GBM treatment.