Mokoena Xolisile, Mabeta Peace, Cordier Werner, Flepisi Brian Thabile
Department of Pharmacology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Dr Savage Road, Prinshof 349-Jr, Private Bag X323, Arcadia, Pretoria, 0007, South Africa.
Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Private Bag X323, Gezina, Pretoria, 0031, South Africa.
J Neurooncol. 2025 Jan;171(2):443-453. doi: 10.1007/s11060-024-04870-5. Epub 2024 Nov 13.
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.
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.
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.
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.
胶质母细胞瘤（GBM）是一种侵袭性强且对治疗耐药的脑肿瘤，可能会影响血脑屏障（BBB）的完整性。血脑屏障是主要由内皮细胞形成的中枢神经系统的保护屏障。本研究旨在探讨GBM细胞对血脑屏障的体外作用。
采用脑内皮（bEnd.3）细胞作为血脑屏障的模型。在48小时时间点从U87 GBM细胞中提取胶质母细胞瘤条件培养基（CM），并用新鲜培养基稀释至40%。使用xCELLigence系统在处理48小时和72小时后评估48小时收集的U87-CM对bEnd.3细胞生长的影响。此外，还使用xCELLigence系统在U87和bEnd.3共培养模型中连续48小时研究bEnd.3细胞的生长情况。在处理48小时和72小时后，使用划痕迁移试验评估bEnd.3细胞的迁移情况。使用Transwell通透性连续1小时评估屏障完整性，并在48小时和72小时后使用蛋白质印迹法评估紧密连接（TJ）蛋白表达。
用U87-CM处理32小时（p < 0.05）、40小时（p < 0.01）和48小时（p < 0.001）后，bEnd.3细胞生长显著下降，而bEnd.3和U87细胞共培养在16小时（p < 0.05）、24小时（p < 0.001）、32小时（p < 0.01）、40小时（p < 0.001）和48小时（p < 0.001）后细胞生长增加。用U87-CM处理24小时（p < 0.05）和48小时（p < 0.01）后，bEnd.3细胞的迁移显著增加。与U87共培养48小时的bEnd.3细胞在15分钟和30分钟时间点的通透性显著增加（p < 0.05）。此外，用U87-CM处理的bEnd.3细胞以及与U87细胞共培养的bEnd.3细胞中，ZO-1和闭合蛋白的表达均显著增加（p < 0.05）。
目前的研究结果表明，U87细胞可能通过CM中的分泌产物以及共培养模型中的细胞间相互作用改变bEnd.3细胞的完整性。这可能有助于理解GBM影响血脑屏障的机制，从而有助于对其进行管理。
