Effect of glioblastoma cells on brain endothelial cell growth, migration, permeability, and tight junction proteins in vitro

Abstract

Introduction: Invasion of cancer cells within the central nervous system (CNS) can impact the blood-brain barrier (BBB) directly through cell invasion or indirectly through the cancer secretome. The BBB, formed mainly by endothelial cells, is a crucial protective barrier of the CNS. Glioblastoma (GBM) is the most aggressive and malignant tumour of the CNS, with high mortality and an average survival rate of 12 to 15 months with treatment. The underlying mechanisms of BBB disruption due to GBM remain unclear. To elucidate the overall effect GBM has on the BBB, this study aimed to investigate the effect of GBM-conditioned media and cells on endothelial cell growth, migration, permeability, and the expression of tight junctions. Methods: The brain endothelial cell line (bEnd.3) was used as a model of the BBB. The GBM (U87) cell line was used to collect conditioned media (CM) at 48 hours (h) and for the indirect co-culture with End.3 cells. The study used two endothelial models: mono-culture of bEnd.3 cells treated with U87-CM and an indirect co-culture of bEnd.3 cells with U87 cells. Both models were compared to a mono-culture of bEnd.3 cells untreated (negative control). The endothelial cell morphology (by phase-contrast microscopy), and the cytotoxicity of CM (using the sulforhodamine B assay), were assessed on the untreated bEnd.3 mono-culture and CM treated mono-culture of bEnd.3 cells. The following was conducted in the two models: endothelial cell migration rate (scratch migration assay), cell growth (xCELLigence real-time cell analyser system), barrier integrity (sodium fluorescein permeability), and tight junction protein expression (Western blotting). Results: The cytotoxic effects of CM on bEnd.3 cells were observed only in the U87-CM collected at 96 h. The U87-CM suppressed the growth of bEnd.3 cells. Conversely the indirect co-culture significantly increased cell growth (p<0.05). The U87-CM increased the migration of bEnd.3 cells from 10% at 24 h to 33% at 48 h compared to the negative control. The U87-CM did not alter permeability of bEnd.3 cells, whereas the indirect co-culture weakened the permeability of the barrier from 15.97 × 10-5 cm/s (negative control) to 20.96 × 10-5 cm/s (treated bEnd.3 cells). The U87-CM increased the expression levels of occludin and ZO-1 in bEnd.3 cells following 48 h, compared to the negative control. The indirect co-culture increased the expression of ZO-1 by 1.2 fold following 48 h exposure. Conclusion: The chosen U87-CM did not alter the morphology of endothelial cells and had minimal cytotoxic effect on the bEnd.3 cells. However, a real-time analysis of the bEnd.3 cell growth revealed that U87-CM suppressed cell growth, and an indirect co-culture with the U87 cells promoted cell growth. The CM maintained the integrity of the BBB and the co-culture caused BBB instability. Both U87-CM and U87 indirect co-culture can influence the growth, migration, permeability, and tight junction expression process in bEnd.3 cells. It is evident that the secreted factors play a role in BBB modulation. However, further investigations are required to understand the signalling pathways activated by the secretome in CM and indirect co-culture on bEnd.3 cells.