Abstract
Abstract BACKGROUND Glioblastoma (GBM) patient survival and therapy response is greatly hindered by the presence of invasive glioma stem cells (GSC) and the blood-brain barrier (BBB) which limits effective drug delivery. WNT/beta-catenin signaling is important in the development and maintenance of the BBB by mediating transcription of growth factors, multidrug resistance proteins, and junctional proteins. In WNT-subtype medulloblastoma, activating mutations of beta-catenin lead to reciprocal secretion of WNT antagonists such as WIF1 and DKK1 into the tumor microenvironment. These WNT antagonists can act upon the surrounding endothelium and induce a leaky BBB. Therefore, we hypothesize that pharmacological inhibition of WNT/beta-catenin signaling in brain endothelial cells will decrease BBB integrity, enabling enhanced paracellular drug delivery to infiltrative GSCs. METHODS We recapitulated the WNT-medulloblastoma phenotype in GBM by activating WNT/beta-catenin signaling in primary human GSCs, inducing secretion of downstream WNT antagonists. Conditioned-media (CM) from GSCs was then applied to human brain microvascular endothelial cells (HBMEC) to indirectly inhibit WNT signaling. Additionally, we directly inhibited WNT/beta-catenin signaling in HBMECs with the small molecule inhibitor ICG-001. Endothelial cell-cell interaction was measured by electrical impedance using the ACEA xCELLigence system. Fenestration and junctional expression were evaluated by immunoblotting and immunofluorescence. RESULTS ICG-001 or WNT-GSC-CM, but not control GSC-CM, upregulated fenestration related protein, PLVAP, and downregulated junctional proteins claudin-5, ZO-1, and VE-Cadherin in HBMECs. Endothelial cell-cell interaction was transiently decreased by ICG-001 or WNT-GSC-CM. Pre-clinical studies are underway to evaluate the functional impact of WNT/beta-catenin inhibition on BBB integrity and permeability in rodent glioma models. Altogether, these results support targeting WNT/beta-catenin signaling in brain endothelial cells to enhance drug delivery to CNS tumors. CONCLUSION Modulation of intratumoral Wnt/beta-catenin signaling, particularly in highly resistant GSCs, may enhance chemotherapy drug delivery, potentially expanding the drug portfolio and improving the prognosis of GBM.
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