spatial organization to the culture milieu. U87-Luc cells were added to a preformed HBMEC network in which the endothelial cells had been engineered to express mCherry fluorescent protein. U87 cells appeared to localize to the peri-endothelial cell space and to make direct contact with endothelial cells (Figure 3A). Co- localization of most U87 cells to the HBMECs occurred within the first 24 hours and the mean distance between tumor and endothelial cells did not significantly change beyond that time (Figure 3B). Hence, for all subsequent co-localization experi- ments measurements were performed after 24 hrs of addition of tumor cells. To more quantitatively evaluate this co-localization we analyzed the data as follows. The spaces within the lattice-work approximate circular elements. We determined that the mean radius of these elements was 155 mm. By calculating the fractional area of concentric circles within each space, we were able to derive a relation between distance and cell number for a theoretical random distribution of cells within the lattice-work (Figure S2A). By measuring the mean distance between eGFP-expressing U87 cells CXCL12 Mediated GBM-Endothelial Cell Interactions and mCherry-expressing HBMECs (Figure S2A–D), we found that U87 cells were not distributed randomly within the culture dish but instead were clustered around endothelial cells. Approximately 50% of U87 cells were localized within 40 mm of an endothelial cell with approximately 30% of U87 nuclei within 20 mm of an endothelial cell, including cells in physical contact with the endothelium (Figure 2C). The non-random nature of this distribution indicated that U87 cells were co-localized with HBMECs. To determine whether primary GBM cells would also localize to the peri-endothelial cell space, we added primary GBM cells from patient specimens to pre-formed HBMEC lattice/tubule structures. Primary GBM tumor cells were identified by their expression of the standard histopathological marker for astrocy- toma (glioma) cells, GFAP [46]. We found that primary GBM cells also co-localized with the endothelial cells (Figure S3A) and made direct cell-to-cell contacts via their processes (Figure S3B). Figure 3. U87 and primary GBM cells localize to the peri-endothelial domain when plated with HBMECs on Matrigel. (A) Twenty-four hours after establishing a capillary-like network of mcherry-expressing HBMECs in Matrigel, eGFP-expressing U87 cells were added to the culture. Within 24 hours U87 cells were seen in physical contact with HBMECs. Scale bar = 50 microns. (B) The mean distances between U87 cells (500 to 800 cells) and HBMECs were calculated at different time points after the addition of the tumor cells to the HBMEC networks. There was a significant increase in co-localization (reduction in mean distance) within 24 hrs, which was maintained over a 72 hr period. * = p,0.05 as determined by one way ANOVA for the means of three separate experiments involving 500–800 measurements per experiment. (C) The distance between approximately 1000 eGFP-expressing U87 cells and mCherry fluorescent protein-expressing HBMECs in co-culture (24 hrs) was measured and the distribution was plotted as the percentage of total cells in 20 micron increments (black triangles). More than 50% of the total U87 cells in the culture were within 40 microns of an endothelial cell. A theoretical plot of a random distribution of cells is shown (open circles). (D) The distance between GFAP positive GBM cells and mCherry fluorescent protein-expressing HBMECs in co-culture were measured and the distribution was plotted as the percentage of total cells in 20 micron increments (black triangles). Error bars represent SEM from three independent experiments involving three different GBM isolates. Approximately 500 GBM (GFAP positive) cells were counted. Nearly 80% of the GFAP positive GBM cells in the culture were within 20 microns of an endothelial cell. A theoretical plot of a random distribution of cells is shown (open circles). doi:10.1371/journal.pone.0033005.g003 PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e33005