CXCL12 Mediated GBM-Endothelial Cell Interactions Figure 1. Deletion of CXCR4 suppresses the growth of intracranial U87 xenografts. (A) Western blot analysis of CXCR4 expression in U87 cells infected with lentirvirus encoding a scrambled shRNA control (scRNA) or a short hairpin RNA directed against CXCR4 (CXCR4 shRNA). CXCR4 expression declined with increasing viral mulitplicity of infection (MOI). (B) Animals were injected with U87 cells expressing either control (scrambled, sc-U87-Luc) or a CXCR4 specific shRNA (shCXCR4-U87-Luc). Growth curves were derived from serial bioluminesence imaging measurements (six to eight animals per experimental group) over the four-week experimental period post tumor cell implantation. Presented are the mean Bioluminescence Ratios (photon flux week (1–4)/photon flux hr48) 6 SEM for each group. * = P,0.005 as determined by two-way ANOVA. doi:10.1371/journal.pone.0033005.g001 In vivo, CXCR4 function depends upon its ligand CXCL12. As previously described [11,14,15,17,39] and illustrated in Figure 2A, CXCL12 is localized to the endothelium of tumor- associated blood vessels in GBM. Therefore, we hypothesized that similar to normal neural stem cell niches [40], endothelial cell CXCL12 and tumor cell CXCR4 would play important roles in the biology of the GBM perivascular stem-like cell niche. Figure 2. Primary human brain endothelial cells express CXCL12 and GBM cells express CXCR4. (A) Human glioblastoma specimen immunostained for CXCL12 (brown) demonstrates expression in vascular endothelial cells. t = tumor cells, e = cross-section through tumor-associated capillary, and Scale bar = 25 mm. (B) HBMECs, in co- culture on Matrigel, express CXCL12 (red). Nuclei are counterstained blue with DAPI. Scale bar equals 25 mm. (C) Human brain micro-vascular endothelial cells cultured on Matrigel (Mat HBMEC) secrete CXCL12 into the media as determined by ELISA. Mat alone indicates results from Matrigel alone-conditioned media. N = 3. ** = p,0.005 as determined by two-tailed t-test. (D) Single cell suspensions from two different adult GBM patients (GBM1, GBM2) and cultured U87 cells express CXCR4 by western blotting. CXCR4 appears red and the actin loading control appears green. doi:10.1371/journal.pone.0033005.g002 To assess whether CXCR4 mediates direct interactions between GBM and endothelial cells we turned to an in vitro co-culture model similar to that used by others [8,16], in which primary human brain microvascular endothelial cells (HBMECs) and either U87 cells, or primary GBM cell isolates were cultured together in extracellular matrix (Matrigel). While the mouse sarcoma origin of Matrigel could limit its relevance in modeling the brain perivascular space, the primary components of Matrigel, including laminin, heparan sulfate proteoglycans, collagen IV and nidogen [41], are known to be essential components of brain germinal matrices, as well as the subendothelial cell basement membrane of the brain microvasculature [42]. The appropriate- ness of Matrigel for these studies is further supported by Matrigel’s successful application in studies of neural stem cells [43,44,45] and human brain tumor cells [6]. When cultured in standard fashion on tissue culture plastic, HBMECs grow as a monolayer in which many individual cells assume an ‘‘epithelioid’’ morphology with abundant cytoplasm surrounding a round nucleus (Figure S1A). In contrast, when plated on Matrigel, HBMECs adopt a lattice-like configuration reminiscent of a capillary network in which individual cells exhibit a more native morphology characterized by an elongated nucleus and cell body (Figure S1B). Reproducible lattice networks were not observed when HBMECs were cultured on plastic, glass, fibronectin or gelatin (data not shown). This restricted distribution of HBMECs in Matrigel better models the arrangement of HBMECs in vivo when compared to the uniform distribution of cells when HBMEC were cultured as a monolayer on plastic. To determine whether HBMECs cultured in Matrigel express CXCL12, we performed immunofluorescence labeling of fixed HBMECs (Figure 2B) and CXCL12 ELISAs on supernatants collected from HBMEC cultures (Figure 2C). We found that CXCL12 protein was present in HBMEC cells and released to the culture media. Thus, similar to native GBM vasculature, HBMEC Matrigel cultures could provide CXCL12 in a spatially restricted manner. Consistent with prior reports, U87 cells and primary GBM cell isolates express CXCR4 (Figure 2D). To ascertain whether HBMEC-derived CXCL12 would influence the behavior of GBM cells we first sought to determine whether HBMECs in this capillary-like configuration impose a PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33005