Fig 2: Abnormal intratumoral blood vessels in AT/RT xenografts. (A) Confocal microscopy images of a sagittal section of the brain of a mouse carrying a VUMC-AT/RT-01 xenograft, stained for the endothelial cell marker CD31, showing reduced density but increased diameter of intratumoral blood vessels. (B) Confocal microscopy image of the brain of a mouse carrying a VUMC-AT/RT-01 xenograft, stained for the blood–brain barrier endothelial cell markers GLUT1 and CLDN5, showing reduced GLUT1 expression in intratumoral blood vessels. (C) Confocal microscopy image of the brain of a mouse carrying a VUMC-AT/RT-01 xenograft, stained for the tight junction protein CLDN5, showing disorganization of tight junctions between endothelial cells of intratumoral blood vessels. (D) Quantification of vessel density, as determined by area of tissue staining positive for CD31, in VUMC-AT/RT-01 xenografts compared with normal brain. ***P < 0.001 (Mann–Whitney U-test). (E) Quantification of vessel diameter of intratumoral blood vessels in VUMC-AT/RT-01 xenografts compared with blood vessels in normal brain tissue. **P < 0.01, ****P < 0.0001 (Dunnett’s multiple comparisons test).

Fig 3: F4L5.13 promotes endothelial cell barrier function Expression of endothelial junction protein ZO-1 and stabilization of ßcatenin in bEnd.3 cells treated with 30 nM F4L5.13 or 30 nM recombinant NDP for the indicated times. Blots are representative of five different experiments. Histogram represents the ratio of ZO-1 and ßcatenin levels over ß-Tubulin measured by densitometry of five independent experiments. Data are presented as mean ± SEM. Significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test (*P < 0.05 as compared with NT).Immunofluorescence of ZO-1, CLDN3, and CLDN5 localization at bEnd.3 cell junctions. bEnd.3 cells were treated or not with 30 nM F4L5.13 or 30 nM Norrin (NDP) with or without 100 ng/ml VEGF for 1 h. ZO-1 is shown in green, CLDN3/5 in red, and DAPI in blue. Scale bars: 15 µm.Quantification of ZO-1, CLDN3, and CLDN5 fluorescence intensity. Each column represents 40 measurements of fluorescence intensity per condition (y-axis). Data are presented as mean ± SEM. Significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test (*P < 0.05 as compared to VEGF treatment).Transendothelial permeability assay quantifying the passage of FITC-dextran through a monolayer of bEnd.3 cells. Passage of FITC-dextran was measured after exposure of bEnd.3 cells to 100 ng/ml VEGF, 30 nM F4L5.13 or both or pretreated with VEGF for 1 h before treating with F4L5.13 for 1 h. Data are presented as mean ± SD, n = 5 independent experiments. Significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test (*P < 0.05 as compared to VEGF treatment).

Fig 4: Stabilizing beta-catenin in ECs switches the area postrema and choroid plexus vasculatures to a BBB-like state.(A,B) Coronal sections showing the area postrema (A; demarcated by white dots and abbreviated AP) and choroid plexus (B; abbreviated CP) from a control mouse (left panels) and from a mouse with EC-specific deletion of Ctnnb1 exon 3 (right panels), immunostained as indicated. In the area postrema and choroid plexus, ECs in the control are CLDN5-/PLVAP+/LEF1-, and ECs with deletion of Ctnnb1 exon 3 are CLDN5+/PLVAP-/LEF1+. ECs in the surrounding brain parenchyma are CLDN5+/PLVAP-/LEF1+. In the right panels of the bottom 2 rows of images, arrows point to LEF1 in EC nuclei within the area postrema and choroid plexus with deletion of Ctnnb1 exon 3. Arrowheads in the bottom 2 rows of images point to LEF1 in EC nuclei in the adjacent brain parenchyma, which are unaffected by deletion of Ctnnb1 exon 3. The efficiency of CreER-mediated recombination in ECs is >90% in the area postrema and ~80–90% in the choroid plexus, as judged by the conversion of EC markers. Scale bars, 100 µm.

Fig 5: Abnormal intratumoral blood vessels in AT/RT tumor tissue. Immunohistochemical staining of GLUT1 and CLDN5 in tumor and normal brain tissue from the VUMC-AT/RT-01 patient showing a loss of GLUT1 in endothelial cells of preexisting blood vessels in the tumor, and a loss of both GLUT1 and CLDN5 in endothelial cells in areas of microvascular proliferation.