Blood-tumor barrier in focus - investigation of glioblastoma-induced effects on the blood-brain barrier.

Abstract

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 168hours (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 24h 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.