Initial Contact of Glioblastoma Cells with Existing Normal Brain Endothelial Cells Strengthen the Barrier Function via Fibroblast Growth Factor 2 Secretion: A New In Vitro Blood–Brain Barrier Model

• DOI: 10.1007/s10571-013-9913-z
• Publication Date: 2013-02-05T06:45:00Z
• Authors: Keisuke Toyoda; Kunihiko Tanaka; Shinsuke Nakagawa; Dinh Ha Duy Thuy; Kenta Ujifuku; Kensaku Kamada; Kentaro Hayashi; Takayuki Matsuo; Izumi Nagata; Masami Niwa
• Keywords: Vascular Endothelial Growth Factor A; 0301 basic medicine; Lung Neoplasms; Brain Neoplasms; Cell Membrane; Green Fluorescent Proteins; Endothelial Cells; Cell Communication; Models, Biological; Rats; Tight Junctions; 3. Good health; 03 medical and health sciences; Blood-Brain Barrier; Cell Movement; Cell Line, Tumor; Culture Media, Conditioned; Electric Impedance; Animals; Humans; Fibroblast Growth Factor 2; Rats, Wistar; Glioblastoma

Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood-brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.