Abstract
The blood-tumor barrier (BTB) is the primary site of nutrient and drug transport to tumor cells such as malignant gliomas. Yet, signaling pathways and factors influencing BTB permeability are poorly understood. Previous studies demonstrate the role of WNT/β-catenin signaling in establishing and fortifying blood-brain barrier integrity in a non-diseased state. Additionally, WNT proteins are highly expressed in gliomas and their surrounding vasculature. Thus, we propose inhibition of WNT/β-catenin signaling at the brain endothelium of malignant glioma can impair BTB integrity to enhance permeability for select cytotoxic agents. We used immortalized mouse brain endothelial cells (bEnd.3), akin to brain tumor endothelium, treated for 24 hours with WNT inhibitors (ICG-001, IWR-1, and LGK974). Inhibition of WNT/β-catenin signaling was confirmed by gene expression of transcription factors (Tcf4 and Birc5). Cell viability was confirmed by CellTiter Glo®. Brain endothelial cell-cell interaction was evaluated by cell impedance and resistance via the Agilent xCELLigence and ABP TEER24 systems. Using qPCR and flow cytometry, we observed changes in expression and function of Abcb1 and Abcg2 transporters. Using an in vitro BTB (bEnd.3 cells and mouse H3.3WT/K27 glioma cells) we evaluated the effect of WNT inhibition on permeability and glioma viability. We found that all the inhibitors downregulated Tcf4 and Birc5 in brain endothelium dose-dependently. Viability with inhibitors demonstrated an IC50 of 28μM for ICG-001, and 42μM for both IWR-1 and LGK974. Endothelial cell-cell interaction was transiently decreased by approximately 50% with all inhibitors at 30 minutes; increasing closer to baseline after 2-4hrs. All WNT inhibitors dose-dependently decreased Abcg2 transporter expression and function. While In vitro BTB studies are ongoing, preliminary findings demonstrate increasing permeability of BTB amongst H3.3K27 glioma cells. Our results demonstrate potential of WNT inhibitors to modulate BTB integrity and drug efflux function. More studies are warranted to explore WNT/β-catenin signaling inhibition on BTB in vivo.
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