Transmigration of Neural Stem Cells across the Blood Brain Barrier Induced by Glioma Cells

<div><p>Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB) was evaluated in an <i>in vitro</i> model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs) cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM) from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE₂, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.</p></div

HGF, VEGF and the lack of EGF in glioma C6 CM induce NSCs transmigration across RBMECs.

<p>A) The amount of HGF and VEGF determined by ELISA is higher in glioma C6 CM than in astrocyte CM, while the opposite is found for EGF. IP of HGF, VEGF or EGF reduces the amount of the growth factor present in CM. HGF: N = 4, F(3,12) = 26.54; VEGF: N = 3, F(3,8) = 20.98; EGF: N = 3, F(3,8) = 44.52; **P<0.01, ***P<0.001, ns = not significant; as assessed by one-way ANOVA followed by Bonferroni's post hoc test. B) ReNcells CX transmigration assay across RBMECs done by placing in the basal compartment of the Millicell, DMEM with HGF or EGF; or DMEM with neutralizing antibodies against HGF, VEGF or EGF; or astrocyte or glioma C6 CM with or without neutralizing antibodies against HGF, VEGF and EGF. Control corresponds to the transmigration induced by glioma C6 CM without the neutralizing antibodies against HGF (upper panel), VEGF (middle panel) and EGF (lower panel), and is considered as 100% transmigration. HGF: N = 3, F(6,14) = 124.7; VEGF: N = 3, F(4,10) = 275.5; EGF: N = 3, F(7,16) = 30.77; **P<0.01, ***P<0.001, ns = not significant; as assessed by one-way ANOVA followed by Bonferroni's post hoc test. C) RBMECs cultured for 8 h in DMEM with HGF and EGF have a TEER similar to that obtained with astrocyte CM. IP of VEGF reverses the decrease in TEER induced by glioma C6 CM whereas IP of EGF from astrocyte CM decreases TEER in a manner similar to that obtained with glioma C6 CM. HGF: N = 3, F(2,16) = 5.668; **P<0.01 with respect to astrocytes CM; VEGF: N = 3, F(2,20) = 14.64; *P<0.05 with respect to astrocytes CM; #P<0.05 with respect to glioma C6 CM; EGF: N = 3, F(3,30) = 23.07; *P<0.05, **P<0.01, ***P<0.001 with respect to astrocytes CM; ^#P<0.05, ^##P<0.01, ^###P<0.001 with respect to glioma C6 CM; ⁺P<0.05, ⁺⁺P<0.01, ⁺⁺⁺P<0.001 with respect to astrocytes CM + α-EGF; as assessed by one-way ANOVA followed by Bonferroni's post hoc test.</p

Glioma C6 CM induces transmigration of NSCs across RBMECs.

<p>A) Transmigration of ReNcells CX across RBMECs induced by astrocyte or glioma C6 CM. Light microscopy image of toluidine blue stained cells on the basal surface of a filter and scheme illustrating each assay. B) Graphed data. N = 3, F(1,12) = 343.8; *** p<0.001, ns = not significant; as assessed by two-way ANOVA followed by Bonferroni's post hoc test. C) In an 8 h transmigration assay, a similar degree of ReNcells CX transmigration is attained with glioma C6 cells or glioma C6 CM placed in the basal compartment. N = 10, F(3,36) = 60.74; ***P<0.001, ns = not significant; as assessed by one-way ANOVA followed by Bonferroni's post hoc test. D) Representative light microscopy image of toluidine blue stained cells on the basal surface of the filter and scheme illustrating each assay.</p

Zonulin present in glioma C6 CM, opens the BBB and favors ReNcells CX transmigration.

<p>A) Zonulin is detected by Western blot in glioma C6 CM and not in astrocyte CM. B) ReNcells CX transmigration assay across RBMECs done by placing in the basal compartment of the Millicell, astrocyte or glioma C6 CM with or without zonulin. N = 6, F(3,20) = 228.2; *** p<0.001; as assessed by one-way ANOVA followed by Bonferroni's post hoc test. C) Elimination of zonulin from glioma C6 CM reverses the decrease in TEER exerted by glioma C6 CM. N = 3, F(3,30) = 5.981; **P<0.01, ***P<0.001 with respect to glioma C6 CM; as assessed by two-way ANOVA followed by Bonferroni's post hoc test.</p

Glioma C6 CM opens the BBB of RBMECs.

<p>A) TEER of RBMECs in the conditions illustrated in the scheme. N = 3, F(3,30) = 19.97; *P<0.05, **P<0.01, ***P<0.001, with respect to 1; ^#P<0.05, ^##P<0.01, ^###P<0.001, with respect to 3; as assessed by two-way ANOVA followed by Bonferroni's post hoc test. B) Immunofluorescence localization of claudin-5 and occludin in RBMECs incubated with astrocytes or glioma C6 CM. Arrow, continuous cell border staining; arrowheads, holes appearing at cell borders. C) Scanning electron micrograph of ReNcells CX moving across a hole in the endothelial monolayer.</p

The presence of PGE₂ and the lack of EGF in glioma C6 CM promote a leakier BBB.

<p>A) Quantitation of PGE₂ by ELISA in astrocyte CM and in glioma C6 CM derived from cells treated or not with COX-2 inhibitor NS398. N = 3, F(2,6) = 161.9; **P<0.01, ***P<0.001; as assessed by one-way ANOVA followed by Bonferroni's post hoc test. B) TEER of RBMECs cultures incubated in the basal compartment with astrocytes or glioma C6 CM derived from control or NS398 treated cells. N = 3, F(2,20) = 53.56; *P<0.05, **P<0.01 with respect to astrocytes CM; ^##P<0.01, ^###P<0.001 with respect to glioma C6 CM; as assessed by two-way ANOVA followed by Bonferroni's post hoc test. C) Quantitative analysis of cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10 present in astrocyte and glioma C6 CM. N = 3, df = 2, as assessed by Student's t-test.</p

Expression of TJ proteins in transmigrating cells favors their passage across RBMECs.

<p>A) Analysis by Western blots of the expression of occludin, claudins 1 to 5 and CRTAM in NIH-3T3 and L-fibroblasts, ReNcells CX and RBMECs. B) The transmigration of NIH-3T3 and L-fibroblasts across RBMECs is independent of the CM present in the basal compartment, but is significantly higher in NIH-3T3 fibroblasts that express claudin-2 than in L-fibroblast that do not express TJ proteins. N = 3, F(1,6) = 232.22, ***P<0.001; F(2,8) = 0.0895, ns = not significant; as assessed by two-way ANOVA followed by Bonferroni's post hoc test. C) Exogenous expression of occludin in L-fibroblasts enhances their transmigration across RBMECs monolayers. N = 9, t = 6.411, df = 8; ***P<0.001 as assessed by Student's t-test. D) Competing CRTAM mediated adhesion with soluble human CRTAM (CRTAM-Fc) added to the upper compartment of a Millicell insert, reduces transmigration of ReNcells CX. N = 4, F(2,6) = 73.86; ***P<0.001, ns = not significant; as assessed by one-way ANOVA followed by Bonferroni's post hoc test.</p