Figure 3. Perivascular glioma cell co-option causes a breakdown of the blood-brain barrier.

The tracer, Evans blue, permeates into the brain parenchyma in tumor-bearing (n=3 animals) (bottom), but not in sham mice 3 weeks post surgery (n=3 animals) (top), Scale 5 mm. (a). Immunofluorescence of CD31 and intravenously injected tracers, tetramethylrhodamine-albumin (TRITC-alb, white) or -cadaverine (TRITC-cad, white), and implanted eGFP-expressing human glioma cells (D54) allow analysis of tracer leakage in relation to tumor burden. The large MW 70 kDa TRITC-albumin (b), as well as the small MW 950 Da TRITC-cadaverine (d) can be found outside the vasculature (arrows) (b-f, Suppl. Fig.6). Note that leaked TRITC-cadaverine is taken up by nearby neurons (arrows) (d,e). No extravasation was seen in the absence of tumor cells (c,f). To assess extravasation of the tracer Cascade blue (MW 10 kDa), Aldh1l1-eGFP-scid (eGFP) immunodeficient mice, which were previously implanted with TdTomato-expressing human glioma cells (D54), were retro-orbitally injected with Alexa Fluor 633 hydrazide dye (633 hyd) and Cascade blue (g,h). Accumulation of Cascade blue occurs in the brain's parenchyma indicating breakdown of the blood-brain barrier (BBB), where perivascular astrocytes have been displaced by glioma cell co-option (g). Vessels lacking tumor cells do not show extravasation of the dye (h). Immunohistochemistry for the TJ proteins zonula occludens-1 (ZO-1) (i) and claudin-5 (k) show that these protein are lost from endothelial cells labeled with CD31 where tumor cells are present (enlarged panel, arrows), but colocalize with endothelial cells in tissue that has not been co-opted. Quantification of ZO-1 shows reduction of vessel coverage in areas co-opted by glioma cells. 8.86 ± 0.64% vessel area is covered by ZO-1 in control images, whereas this number was reduced to 5.1 ± 1.11% in areas where vessels were coopted by tumor cells (n=9 images each for control and tumor-covered vessels from 3 animals, paired t-test directly comparing control and tumor images in the same slice, p=0.037, error bars refer to SEM) (j). Black font was used to describe white labels in confocal images. Scale 20 μm. Statistical data: b,c n=10 animals; d-f n=7 animals; g n=2 animals; h n=4 animals; i,k n=6 animals.