Cnsc-02. investigating the role of the wnt signaling transduction pathway in glioma blood-brain-barrier integrity

Abstract The WNT signaling transduction pathway has been linked to cancer stem cell self-renewal, differentiation, and blood brain barrier (BBB) development. Previous studies have shown that WNT pathway inhibition increased BBB permeability, specifically in WNT medulloblastoma. Glioblastoma, an aggressive malignant tumor with poor prognosis, has also been shown to have high WNT expression and variable BBB permeability. We hypothesize that activating WNT pathway inhibition in glioblastoma stem cells (GSCs) will inhibit glioma progression, increase BBB permeability within the tumor microenvironment, and enhance overall treatment responsiveness. We transduced primary pediatric derived glioma stem cells with overexpressing vectors for WNT inhibitors (DKK1 or WIF1). To characterize effects of DKK1 or WIF1 overexpression, we evaluated cell migration patterns via RTCA xCelligence, cell cycle progression via Annexin 5 assays, and cell viability via cell-titer glo. Brain endothelial cell integrity was evaluated with the treatment of CHIR99021, WNT pathway activator, or GSC condition media with immunoblotting for junctional protein expression (Claudin-5, Claudin-3, Occludin, ZO-1, VE-Cadherin). Verification of WNT inhibitory overexpressing GSCs for DKK1 and WIF1 was performed using RNAseq, proteomics, and immunoblotting. DKK1-GSCs demonstrated a statistically significant impairment in the migratory slope compared to empty vector GSCs. Cell cycle analysis also revealed that DKK1-GSCs were arrested in G0/1 phase while WIF1-GSCs were arrested in S/G2M phase. CHIR99021 endothelial treatment resulted in increased secretion of WNT inhibitors DKK1 and SFRP1 and resultantly, there was a decrease in junctional protein expression. Overall, WNT inhibitor overexpressing GSCs exhibited unique cellular patterns and indirectly disrupted endothelial cell integrity. Future studies will evaluate orthotopic WNT inhibitor overexpressing transplant rodent models to determine BBB integrity, drug permeability, model survival, and chemotherapeutic treatment response. By enhancing our understanding of the WNT pathway in GSCs, we anticipate understanding the use of future therapeutic WNT pathway targeting to increase chemotherapeutic responsiveness and improve glioblastoma prognosis.