Figure 5. U87 localization to the peri-endothelial space is blocked by the CXCR4 antagonist AMD 3100. (A) GFP-expressing U87 cells (green) are localized to mCherry-expressing HBMECs (red). (B) Treatment of parallel co-cultures as in A with AMD 3100 results in failure of U87 cells (green) to make consistent contact with endothelial cells (red). Scale bar equals 25 microns. (C) The CXCR4 antagonist AMD 3100 redistributes U87 cells, decreasing the number of cells within the nearest proximity to HBMECs and increasing the number of cells at greater distance. * = p,0.05 as determined by two-way ANOVA. (D) AMD 3100 increased the mean distance between U87 cells and HBMECs but had no effect on the mean distance between U87 cells and HUVECs. * = p,0.05 as determined by two-tailed t-test. doi:10.1371/journal.pone.0033005.g005 localized within 20 microns of an endothelial cell in the untreated control condition (Figure 6A). Treatment with AMD3100 (2.5 ng/ml, 24 h) resulted in a modest, but consistent and significant (P,0.005, two-way ANOVA), change in GBM cell distribution. Notably there was a decrease in the proportion of cells within 20 microns of an endothelial cell (77+/20.33% vs. 69+/23%) and a commensurate increase in the proportion of GBM cells at greater distances from the peri-endothelial cell domain. The small but reproducible effect of CXCR4 antagonism on GBM cell localization suggests that the CXCL12-CXCR4 pathway participates in localization of primary GBM cells to the peri-endothelial cell domain but does not account for all of the chemo-localization effect of endothelial cells on GBM cells. Among the implications of peri-endothelial localization is the potential for trophic interactions between endothelial cells and GBM cells. To evaluate this possibility, U87 cells or primary GBM specimens were added to HBMEC networks and cell number was measured using bioluminescence imaging (for U87) or the number of GFAP positive cells/well (for primary GBM) and quantified using a computer-controlled epi-fluorescence microscope and automated image analysis. Consistent with endothelial cells providing trophic support for GBM cells, co-culture of either U87 or primary GBM cell isolates with HBMECs in Matrigel resulted in increased GBM cell number (Figure S5A–B). The magnitude of the trophic effect ranged between 1.5 and greater than twenty fold for the different primary GBM specimens when compared to GBM cells grown on Matrigel alone. To determine whether this trophic effect was mediated by CXCL12, we generated lentivirus-encoding shRNA directed CXCL12 Mediated GBM-Endothelial Cell Interactions Figure 6. The chemotactic and trophic effect of the peri- endothelial space on primary GBMs is blocked by pharmaco- logic or genetic inhibition of the CXCL12-CXCR4 pathway. (A) The distance between GFAP positive GBM cells and mCherry fluorescent protein-expressing HBMECs in co-culture was measured as in Figure 3. Error bars represent SEM from three independent experiments involving three different GBM isolates. Approximately 100 GBM (GFAP positive) cells were counted in each condition. Nearly 80% of the GFAP positive GBM cells in the culture were within 20 microns of an endothelial cell. Treatment with AMD3100 resulted in a reduction in the proportion of GBM cells in closest proximity to endothelial cells and an increase in the proportion of cells at greater distances. P,0.005 by two- way ANOVA. (B) Primary GBM cells co-cultured with HBMECs expressing a scrambled shRNA (sh-RNA-sc) show increased growth after 72 hours in culture, as measured by number of GFAP-positive tumor cells, compared to growth on Matrigel alone. The trophic effect of HBMECs on primary GBM cells is abrogated upon depletion of CXCL12 expression. N = 3, * = P,0.005 as determined by one-way ANOVA with Dunnett’s post-test for multiple comparisons. doi:10.1371/journal.pone.0033005.g006 against CXCL12, or encoding a scrambled shRNA control. CXCL12 mRNA and protein expression was reduced in primary HBMECs infected with either of two shRNAs directed against CXCL12, but not in HBMECs infected with lentivirus encoding the scrambled shRNA control (Figures S4 and S6). Hairpin shRNA-L12-1 reduced CXCL12 expression to a significantly lower level than shRNA-L12-2 and thus, only shRNA-L12-1 was used for subsequent evaluations of the effect of reduced endothelial cell CXCL12 expression on primary GBM growth. Targeted reduction of CXCL12 expression with lentivirus encoding shRNA had no effect on endothelial lattice formation in PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e33005