Glioblastoma cells alter brain endothelial cell homeostasis and tight junction protein expression in vitro

Abstract

Background: Glioblastoma (GBM) is an aggressive therapy-resistant brain tumour that may impacts the integrity of the blood–brain barrier (BBB). The BBB is a protective barrier of the central nervous system formed mainly by endothelial cells. This study aimed to investigate the in vitro effect of GBM cells on the BBB. Methods: Brain endothelial (bEnd.3) cells were used as a model of the BBB. Glioblastoma-conditioned media (CM) was extracted at the 48-h (h) time-point from the U87 GBM cells and diluted to 40% with fresh media. The effect of the U87-CM collected at 48 h on bEnd.3 cell growth was evaluated following 48 and 72 h of treatment using the xCELLigence system. Additionally, bEnd.3 cell growth was also investigated in a U87 and bEnd.3 co-culture model continuously for 48 h using the xCELLigence system. The migration of bEnd.3 cells was assessed following 48 and 72 h using the migration scratch assay. The barrier integrity was evaluated continuously for 1 h using the transwell permeability, and the tight junction (TJ) protein expression was evaluated using Western blot assay following 48 and 72 h. Results: There was a significant decrease in bEnd.3 cell growth following 32 h (p < 0.05), 40 h (p < 0.01), and 48 h (p < 0.001) of treatment with U87-CM, while co-culturing of bEnd.3 and U87 cells increased cell growth following 16 h (p < 0.05), 24 h (p < 0.001), 32 h (p < 0.01), 40 h (p < 0.001), and 48 h (p < 0.001). The migration of bEnd.3 cells significantly increased following both 24 (p < 0.05) and 48 h (p < 0.01) of treatment with U87-CM. The permeability of bEnd.3 cells co-cultured with U87 for 48 h was significantly increased (p < 0.05) at the 15- and 30-min time points. Furthermore, the expression of ZO-1 and occludin was significantly increased (p < 0.05) in both bEnd.3 cells treated with U87-CM as well as bEnd.3 cells co-cultured with U87 cells. Conclusion: The current findings suggest that U87 cells alter the integrity of bEnd.3 cells possibly through the secretomes in the CM and through cell–cell interactions in co-culture models. This may assist in the understanding of the mechanisms by which GBM affects the BBB, which may aid in the management thereof.