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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

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Abstract

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.

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Acknowledgments

We thank Ms. Mayumi Sagara of the BBB Laboratory, PharmaCo-Cell Company Ltd., and Mr. Ken Izawa and Mr. Daisuke Watanabe of the Sano Drug Group for their contributions. This study was supported in part by Grant-in-Aid for Scientific Research (#23592094 to K.H. and #23592095 to K.U.) from Ministry of Education, Culture, Sports, Science and Technology of Japan.

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We have nothing to disclose in terms of financial support or relationships that may pose a conflict of interest.

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Authors and Affiliations

  1. Department of Neurosurgery, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan

    Keisuke Toyoda, Kenta Ujifuku, Kensaku Kamada, Kentaro Hayashi, Takayuki Matsuo & Izumi Nagata

  2. Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki, 852-8523, Japan

    Kunihiko Tanaka, Shinsuke Nakagawa, Dinh Ha Duy Thuy & Masami Niwa

  3. BBB Laboratory, PharmaCo-Cell Company Ltd., 1-43 Dejima, Nagasaki, 850-0862, Japan

    Shinsuke Nakagawa, Dinh Ha Duy Thuy & Masami Niwa

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  1. Keisuke Toyoda
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Correspondence to Kunihiko Tanaka.

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Toyoda, K., Tanaka, K., Nakagawa, S. et al. 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. Cell Mol Neurobiol 33, 489–501 (2013). https://doi.org/10.1007/s10571-013-9913-z

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