胶质母细胞瘤（GBM）是成人中最常见的恶性原发性脑肿瘤，其特点是治疗选择有限、频繁复发和诊断后生存期短。到目前为止，现有的疗法和治疗方法都没有被证明是有效的治愈方法。能够模拟 GBM 体内病理生理学的预测性人类血肿瘤屏障 (BTB) 测试系统的可用性将引起临床前研究的极大兴趣。在这里，我们提出了一种新的 BTB 体外测试系统，结合了 GBM 球体和源自人诱导多能干细胞 (hiPSC) 的 BBB 模型。我们将 hiPSC 来源的脑毛细血管内皮样细胞 (iBCEC) 与 GBM 共培养源自 U87-MG 和 U373-MG 细胞系的球体，采用基于细胞培养插入物的格式。对培养 168 小时 (h) 的球体进行监测，表征 GBM 特异性标记物表达，并用标准化疗药物进行处理，以区分 2D 单一培养物和 3D 球体之间的抑制效果。 GBM 诱导的 iBCEC 屏障完整性变化通过测量跨内皮电阻 (TEER)、紧密连接 (TJ) 蛋白 claudin-5 和 occludin 以及葡萄糖转运蛋白 1 (Glut-1) 的免疫细胞化学染色来验证。 GBM 诱导的血管内皮生长因子 (VEGF) 分泌也得到了量化。仅共培养 24 小时后，TJ 蛋白、occludin 和 claudin-5 的表达减少以及 iBCEC 中屏障的显着破坏，证实了我们的假设。 TEER 从 1313±±265 Ω*cm2 减少至 712±±299 Ω*cm2 (iBCECs   U87-MG) 和 762±±316 Ω*cm2 (iBCECs   U373-MG)。此外，与 2D 培养物相比，3D 球体在体外对标准 GBM 化疗药物表现出更强的抵抗力。我们证明了简化、稳健的体外 BTB 测试系统的建立，具有在临床前治疗筛选和研究 GBM 引起的病理变化方面的潜在应用在 BBB。© 2024。作者。

Glioblastoma (GBM) is the most prevalent, malignant, primary brain tumor in adults, characterized by limited treatment options, frequent relapse, and short survival after diagnosis. Until now, none of the existing therapy and treatment approaches have proven to be an effective cure. The availability of predictive human blood-tumor barrier (BTB) test systems that can mimic in-vivo pathophysiology of GBM would be of great interest in preclinical research. Here, we present the establishment of a new BTB in-vitro test system combining GBM spheroids and BBB models derived from human induced pluripotent stem cells (hiPSCs).We co-cultured hiPSC-derived brain capillary endothelial-like cells (iBCECs) with GBM spheroids derived from U87-MG and U373-MG cell lines in a cell culture insert-based format. Spheroids were monitored over 168 hours (h) of culture, characterized for GBM-specific marker expression and treated with standard chemotherapeutics to distinguish inhibitory effects between 2D mono-culture and 3D spheroids. GBM-induced changes on iBCECs barrier integrity were verified via measurement of transendothelial electrical resistance (TEER), immunocytochemical staining of tight junction (TJ) proteins claudin-5 and occludin as well as the glucose transporter-1 (Glut-1). GBM-induced secretion of vascular endothelial growth factor (VEGF) was additionally quantified.Our hypothesis was validated by reduced expression of TJ proteins, occludin and claudin-5 together with significant barrier breakdown in iBCECs after only 24 h of co-culture, demonstrated by reduction in TEER from 1313 ± 265 Ω*cm2 to 712 ± 299 Ω*cm2 (iBCECs + U87-MG) and 762 ± 316 Ω*cm2 (iBCECs + U373-MG). Furthermore, 3D spheroids show more resistance to standard GBM chemotherapeutics in-vitro compared to 2D cultures.We demonstrate the establishment of a simplified, robust in-vitro BTB test system, with potential application in preclinical therapeutic screening and in studying GBM-induced pathological changes at the BBB.© 2024. The Author(s).