Figure 1. Glioma cells can associate with blood vessels of all sizes and types.

Immunofluorescence of CD31 (PECAM) and eGFP-expressing human glioma cells (D54) (a) or patient-derived xenograft lines GBM22 (b) and GBM14 (c) implanted in the cerebrum of immunodeficient mice highlighting the high number of invading glioma cells found along the vasculature. To be scored “vessel-associated” we required overlap of pixels of the endothelial cell (red) and the tumor cell (green) label in 1 to 2 um single planes of confocal images. Only a minority of cells is not found along the vasculature (d-f, arrow heads). Perivascular glioma cells can associate with capillaries (<7 μm) (g), penetrating arterioles or venules (7-35 μm) (h), and large arteries or veins (>35 μm) (i). Quantitative analysis based on 1634 cells in 5 random sections from each slice and 3 slices from 4 different animals (j). Immunofluorescence of Alexa Fluor 633 hydrazide dye (633 hyd, white) and eGFP-expressing astrocytes (Aldh1l1-eGFP) or human glioma cells (D54) implanted in the cerebrum of immunodeficient mice allowing to distinguish arterioles/arteries from venules/veins and capillaries (k-m). Black font was used to describe white labels in confocal images. Quantification of 75 sections from 4 tumor-bearing animals show that glioma cells associate with every type of vessel (j). In vivo visualization of eGFP-expressing human- glioma cells implanted in the cerebrum of immunodeficient mice found invading along the vasculature outlined by tetramethylrhodamine-dextran (TRITC-dex). Arterioles show divergent and venules show convergent blood flow at branch points (arrows in k,o,p). Perivascular glioma cells can associate with capillaries (n), penetrating arterioles (o), penetrating venules (p). Scale, 20 μm. Statistical data: a,d n=4 animals; b n=5 animals; c n=7 animals; f quantification for n=4 D54 animals, n=3 GBM22 and GBM14 animals; g-m n=9 animals; n-p n=34 animals.