media for 24 hours. Scale bar equals 50 mm. (B) HBMECs plated at similar density to (A), 30 minutes after establishing a Matrigel layer within the culture dish, become organized into a capillary- like network. Scale bar equals 100 mm. (TIF) Figure S2 Localization of GBM cells to the Perivascular space. (A) Algorithm for calculating random distribution of tumor cells within HBMEC lattice. The lattice-work is assumed to contain concentric circles whose fractional area is calculated as shown. Fractional area is graphed as a function of distance from endothelial cells (ECs) to determine how a random distribution of tumor cells would appear. Red and gray bars on graph correspond to red and gray donuts in cartoon. (B) A low magnification image of an HBMEC lattice. (C) Tumor cells (green) localize to HBMEC (red). (D) The distance between tumor cell nuclei and the nearest endothelial cell body was measured using Axiovision software (Zeiss). (TIF) Figure S3 Primary GBM cells make direct contacts with HBMECs in vitro. (A) Twenty-four hours after establishing a capillary-like network of mCherry-expressing HBMECs in Ma- trigel, primary GBM cell isolates collected from three different patients were added to the culture. 24 hours later, GFAP-positive GBM cells were seen in physical contact with HBMECs. (B) A GFAP positive GBM cell (green) extends a process to contact an HBMEC (*). Also note the GFAP- negative GBM-derived cells, identified by nuclear DAPI (blue) staining only, at a distance from the mCherry expressing HBMEC cells. Scale bar = 50 mm in panel A and 25 mm in panel D. (TIF) Figure S4 Manipulation of CXCL12 expression regu- lates endothelial cell function. (A) The amount of CXCL12 secreted into the media by HUVECs (control or CXCL12 over- expressing) and HBMECs (control or infected with CXCL12 shRNAs) were quantified using ELISA. CXCL12 over-expression of HUVECs significantly increased the amount of chemokine secreted to the media (*p,0.05, as determined by one-way ANOVA and Newman-Keuls multiple comparison test). Super- natant from HBMECs contained significantly higher amounts of CXCL12 compared to HUVECs (#p,0.05, as determined by one-way ANOVA and Newman-Keuls multiple comparison test). CXCL12 knockdown reduced supernatant CXCL12 levels in HBMECs. (B) U87 cells (expressing eGFP) preferentially coloca- lize with CXCL12 over-expressing but not control (C) HUVECs (expressing mCherry). Scale bar = 500 mm. (TIF) Figure S5 Primary HBMEC exert a trophic effect on primary GBM cells that is blocked by AMD3100. (A) The trophic effect of endothelial cells on luciferase-expressing U87 cells was measured in four separate experiments by bioluminescence imaging (BLI) of U87 luciferase activity after 3 days in co-culture. References 1. Nelson CM, Bissell MJ (2006) Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu Rev Cell Dev Biol 22: 287–309. 2. Tlsty TD, Coussens LM (2006) Tumor stroma and regulation of cancer development. Annu Rev Pathol 1: 119–150. 3. Scherer HJ (1938) Structural development in gliomas. American Journal of Cancer 34: 333–351. 4. Spence AM, Muzi M, Graham MM, O’Sullivan F, Krohn KA, et al. (1998) Glucose metabolism in human malignant gliomas measured quantitatively with PET, 1-[C-11]glucose and FDG: analysis of the FDG lumped constant. J Nucl Med 39: 440–448. CXCL12 Mediated GBM-Endothelial Cell Interactions In the absence of HBMECs, AMD3100 has no effect on total cell number as measured by BLI. Co-culture with endothelial cells results in a significant increase in BLI and this effect was blocked by both AMD3100. P,0.05 as determined by one-way ANOVA with Dunnett’s post-test for multiple comparisons. (B) Co-culture with HBMEC in Matrigel (Mat) stimulates primary adult GBM cell growth at 72 hours. The trophic effect is inhibited by AMD3100 (AMD). Shown are the means and SEM of values normalized from triplicate cultures involving a single primary GBM isolate. ** = P,0.005 as determined by one-way ANOVA with Dunnett’s post-test for multiple comparisons. Similar results were obtained with two other primary GBM cell isolates. (C) TUNEL assay in parallel primary GBM- endothelial cell co- cultures as those described in (B) indicates that treatment with AMD3100 increased GBM apoptosis (n = 3). (TIF) Figure S6 Lentiviruses encoding shRNA targeting CXCL12 (shRNA-L12-1, 2) decrease CXCL12 mRNA levels in primary HBMEC cells relative to HBMECs infected with lentivirus encoding a scrambled control shRNA (shRNA-sc). * = P,0.05 as determined by two-way Student’s T-test, n = 3. (TIF) Figure S7 Trophic effects of endothelial cell-derived CXCL12 are mediated through CXCR4. (A) CXCR7 expression in U87 GBM cells grown in co-culture with HBMECs (top panels) or monoculture (bottom panels) was evaluated by immunohistochemistry. IgG controls are shown on the left and specific CXCR7 immunolocalization is on the right. U87 cells express GFP and appear green. HBMECs exhibit a membranous pattern of CXCR7 expression (arrow). In contrast, U87 cells exhibit little or no CXCR7 expression. Scale bar = 50 mM. (B) U87 cell growth was also measured in the presence of CXCR7 antagonists, CCX773 and CCX771, or the inactive control compound CCX704. The effects of CCX733 and CCX771 were indistinguishable from the control compound CCX704. (TIF) Acknowledgments The authors would like to thank Dr Amy Barone and Jayne Marassa for technical assistance and Dr David Wilson and Nicole Warrington for critical reading of the manuscript. The authors would also like to thank Dr Mark Penfold of ChemoCentryx for kindly providing the CXCR7 antagonists. Author Contributions Conceived and designed the experiments: SR RS JBR. Performed the experiments: SR RS EJC BMW EJ TS. Analyzed the data: SR RS EJC EJ DP-W JBR. Contributed reagents/materials/analysis tools: JL. Wrote the paper: SR RS JBR. 5. Rampling R, Cruickshank G, Lewis AD, Fitzsimmons SA, Workman P (1994) Direct measurement of pO2 distribution and bioreductive enzymes in human malignant brain tumors. Int J Radiat Oncol Biol Phys 29: 427–431. 6. Calabrese C, Poppleton H, Kocak M, Hogg TL, Fuller C, et al. (2007) A perivascular niche for brain tumor stem cells. Cancer Cell 11: 69–82. 7. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, et al. (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444: 756–760. 8. Borovski T, Verhoeff JJ, ten Cate R, Cameron K, de Vries NA, et al. (2009) Tumor microvasculature supports proliferation and expansion of glioma- propagating cells. Int J Cancer 125: 1222–1230. PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e33005