Fig. 5

S1P3 antagonists strengthen the blood–tumor barrier in vitro. a An in vitro model of the BBB was constructed with confluent brain endothelial cells on the luminal side of a coated porous membrane, brain pericytes on the abluminal side of the membrane, and astrocytes in the bottom of the abluminal side of the chamber. To create a blood–tumor barrier (BTB) model, spheres of 231-BR tumor cells were added to the bottom of the chambers. Barrier integrity was quantified by transendothelial electrical resistance (TEER), or diffusion of a drug (doxorubicin) from the luminal to abluminal side. Cells were cultured to confluence for 4 days, at which time antagonists (b) (EX26: 1 nM, JTE-013: 17 μM, TY: 2 μM, CAY10444: 10 μM, and CYM50358: 25 nM) or vehicle were added. In BBBs and BTBs, both S1P3 antagonists selectively showed increased TEER, indicative of a tighter BBB (c) and BTB (d) (n = 5). TY-52156 and CAY10444 decreased permeability of 100 µg mL⁻¹ doxorubicin significantly in BBB (e) and BTB (f) models (n = 5). g After 24 h post-treatment with TY-52156 and CAY10444, the membranes containing brain endothelial cells were removed and stained for VE-cadherin (red) (white arrows). Nuclei were DAPI stained (blue). Increased staining and membranous localization was observed with TY-52156 and CAY10444 (scale bar = 20 μm). h The effect of vehicle, 2 µM TY-52156 or 10 μM CAY10444 on BBB TEER in cultures with or without astrocytes. Significant TEER elevation by TY-52156 and CAY10444 was dependent on the presence of astrocytes (n = 4). For (c–f) and (h), Kruskal–Wallis test and Dunn’s multiple comparison test were performed. Graphs represent the median and the error bars the 95% confidence interval, from independent transwell BBB/BTB cultures (n) from multiple experiments. *P < 0.05, **P < 0.01, ns non-significant