Primary GBM cells were also not distributed randomly within the culture dish but instead were clustered around endothelial cells (Figure 3D). The distance between individual endothelial cells and greater than 500 GBM cells, derived from independent experiments utilizing three different primary isolates, was measured. Nearly 80% of GBM cells were localized within 20 mm of an endothelial cell. The non-random nature of this distribution indicated that primary GBM cells were also co- localized with HBMECs. Prior studies have shown that CXCR4 activation induces chemotaxis and survival in U87 cells [14]. Given the recognized role that the CXCL12-CXCR4 pathway plays in chemo- attraction of multiple cell types (lymphocytes, neurons, tumor cells [47]), we evaluated the hypothesis that GBM cells are localized to the peri-endothelial cell domain in a CXCL12- dependent fashion. We first cultured U87 cells with human umbilical vein endothelial cells (HUVECs), which form similar capillary-like networks in Matrigel but express significantly lower levels of CXCL12 mRNA (Figure 4A) and protein (Figure S4A) compared to HBMECs. Consistent with a dose-dependent CXCL12 effect on U87 localization to the peri-endothelial cell space, U87 cells were distributed in a random fashion within the HUVEC lattice with fewer U87 cells in close apposition to HUVECs compared to HBMECs (compare Figure 4B to Figure 3C). Consequently the mean distance between U87 and HUVECs was significantly higher compared to that of HBMECs (Figure 4C). To determine whether CXCL12 expression was sufficient to endow HUVECs with a localizing effect similar to HBMECs, we engineered HUVECs to express CXCL12. HUVEC-CXCL12 cells release comparable levels of CXCL12 CXCL12 Mediated GBM-Endothelial Cell Interactions into the culture media as HBMECs (Figure S4A). Co-culture of U87s with HUVEC-CXCL12 cells resulted in localization of U87cells to the peri-endothelial cell space (Figure S4B, C). The mean distance between U87 and HUVEC-CXCL12 cells was similar to that of HBMECs (Figure 4D). These data indicate that endothelial cell secretion of CXCL12 is sufficient to induce the localization of CXCR4-expressing GBM cells. To determine whether CXCR4 activation was necessary for the co-localization of GBM cells with endothelial cells we treated U87- HBMEC cultures with AMD3100. This resulted in the dispersal of U87 cells from the peri-endothelial cell domain (Figure 5A and 5B). AMD3100 reduced the fraction of U87 cells within 40 microns of an endothelial cell (Figure 5C), and the mean distance between U87 and HBMECs was increased from 40 to nearly 60 mm in these experiments (Figure 5D). The resulting distribution of U87 cells in AMD3100 treated cultures was no longer different from the random distribution. As an additional control, we evaluated the effects of AMD3100 on the mean distance between U87 and HUVECs and found that in the absence of CXCL12 secretion, AMD3100 had no effect on U87 cell distribution (Figure 5D). Together, the above data suggested that endothelial cell-derived CXCL12 induced chemo-localization of U87 cells to the peri-endothelial cell domain in a CXCR4 dependent fashion. We next sought to determine whether inhibiting CXCR4 would also alter the localization of primary GBM cells to the peri- endothelial cell space. We treated three different primary HBMEC-GBM co-cultures with AMD3100 and performed the same localization analysis as described above. Similar to the results presented in Figure 2, nearly 80% of primary GBM cells were Figure 4. U87 localization to the peri-endothelial space is correlated with CXCL12 expression. (A) Quantitative PCR reveals that HUVECs express significantly lower levels of CXCL12 than HBMECs. N = 3, ** = p,0.005 as determined by two-tailed t-test. (B) The distance between greater than 1000 eGFP-expressing U87 cells and mCherry fluorescent protein-expressing HUVECs in co-culture was measured and the distribution was plotted as the percentage of total cells in 20 micron increments (filled squares). Approximately 30% of the total U87 cells in the culture were within 40 microns of an endothelial cell (compare to Figure 3B). This distribution does not differ from a theoretical plot of a random distribution of cells (open circles) (C) A statistically significant difference existed between the mean distance of U87 cells to HBMECs versus the mean difference between U87 and HUVECs. * = p,0.05 as determined by two-tailed t-test for the means of four separate experiments involving approximately 330 measurements per endothelial co-culture per experiment. (D) When cultured with HUVECs that are engineered to over-express CXCL12, U87s show a similar co-localization pattern (as seen with HBMECs) consistent with a significant reduction in mean distance when compared to co-cultures with control HUVECs. * = p,0.05 as determined by two-tailed t-test for the means of two separate experiments involving approximately 300 measurements per endothelial co-culture per experiment. doi:10.1371/journal.pone.0033005.g004 PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33005