of glioblastoma [3]. The details of this specialization have recently been suggested to include maintenance of a stem cell-like phenotype in glioblastoma cells localized to this region [6]. The stem-like phenotype includes increased DNA repair capacity, increased efflux pump expression and growth in response to endothelial cell-derived factor(s) [7,8,9,10]. Thus, the peri-vascular space is predicted to contribute to tumor growth and therapeutic resistance. It is therefore imperative to understand the biology of this space and to develop systems in which to screen for drugs that can disrupt its function(s). The vasculature of GBM is distinguished from normal brain vasculature by levels of CXCL12 expression. In extensive studies of malignant neural and astrocytic tumors, we and others found increased CXCL12 expression in the endothelium of tumor- associated blood vessels. [11,12,13,14,15,16,17]. The functional significance of increased endothelial expression of CXCL12 in GBMs remains to be fully defined. CXCL12 plays multiple roles during normal development and tissue homeostasis both inside and outside the central nervous system. Prominent among these roles is the regulation of progenitor cell localization to germinal niches [18,19,20,21,22,23,24], the regulation of progenitor cell proliferation [22,25,26], survival [27] and differentiation [28], as well as the regulation of immune cell chemotaxis and activation [29]. Relevant to brain tumors, endothelial cell CXCL12 could directly regulate tumor growth and spread, promote angiogenesis and/or regulate immune response to tumors. CXCR4, a CXCL12 receptor, is expressed on brain tumor cells and high levels of expression have negative prognostic significance [30]. These findings suggest that the CXCL12/CXCR4 pathway functions in tumor growth and/or therapeutic resistance. The anti-tumor effects of AMD3100 and AMD3465, competitive antagonists of CXCR4 activation also indicate that CXCL12 regulation of tumor growth involves activation of tumor cell CXCR4 [14,31,32]. In intracranial xenograft models of GBM and medulloblastoma we demonstrated that systemic delivery of AMD3100 or AMD3465 had significant anti-tumor activity against established xenografts. Within 48 hours of the initiation of AMD3100 treatment, tumor cells exhibited decreased prolifer- ation and increased apoptosis indicating that AMD3100 disrupted a critical direct effect of CXCL12 on tumor growth [14]. Recently, using a similar U87 intracranial xenograft model of GBM, AMD3100 was shown to prevent tumor recurrence after radiation therapy through inhibition of mesenchymal progenitor cell recruitment for vasculogenesis [33]. Together, these studies underscore the potential complexity of CXCL12 effects on brain tumor growth and the possibility for multiple mechanisms of CXCR4 antagonist action. In addition to CXCR4, CXCL12 also binds to a second G-protein coupled receptor CXCR7, which has been recently characterized. CXCR7 is shown to be expressed and functional in a number of glioma cell lines, although its exact role in tumorigenesis is still unclear [34], [35]. In order to better define the roles of CXCL12 and its receptors in functions of the GBM perivascular niche we investigated the interactions between human brain microvascular endothelial cells and GBM cells when the two cell types were cultured together. In this system, where there is no vasculogenesis and uniform oxygenation, we demonstrate that CXCL12 is essential for chemo-attracting GBM cells to the peri-endothelial cell space and mediating a direct trophic effect of endothelial cells on GBM cells. Endothelial cell effects on GBM cell growth were blocked by both the specific CXCR4 antagonist AMD3100 and knock-down of endothelial cell CXCL12 expression with short hairpin interfering RNA (shRNA). In contrast, the CXCR7 antagonists CCX733 and CCX773 had no effect on CXCL12-induced CXCL12 Mediated GBM-Endothelial Cell Interactions growth. These data indicate that targeting the CXCL12/CXCR4 pathway could abrogate a specialized trophic function of GBM- associated vasculature that contributes to brain tumor growth. These data further suggest that therapeutic regimens that combine antagonism of both perivascular niche formation and function could have synergistic effects on tumor growth and recurrence. Materials and Methods Chemicals, Reagents, and Antibodies All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. All tissue culture reagents and media were obtained from Invitrogen (Carlsbad, CA) unless otherwise indicated. A construct containing mCherry cDNA was the kind gift of Dr. Roger Y. Tsien (University of California, San Diego, CA). Antibodies utilized in this study were: PECAM/CD31 (Santa Cruz Biotechnology, Santa Cruz, CA), GFAP (Sigma- Aldrich), CXCR4 monoclonal (R&D Systems, Minneapolis, MN), CXCR4 polyclonal (Leinco, St. Louis, MO), CXCL12 (Peprotech, Rock Hill, NJ), b-actin (Sigma-Aldrich) and immunoglobulin (IgG) isotype controls (Jackson Immuno-Research, West Grove, PA). AMD3100 was purchased from Sigma-Aldrich. CCX733 was obtained from Chemocentryx (Mountain View, CA). Cell Culture Primary Human GBM cells. Primary tumor specimens from three adult patients and one pediatric patient with GBM were retrieved from the Children’s Discovery Institute Pediatric Brain Tumor Bank at Washington University School of Medicine in accordance with an Institutional Review Board–approved protocol for human research. Freshly resected tumors specimens were kept in cold low calcium artificial cerebrospinal fluid (low Ca++ aCSF: 124 mM NaCl, 5 mM KCl, 3.2 mM MgCl2, 0.1 mM CaCl2, 26 mM NaHCO3, 10 mM D-glucose). Samples were minced and placed in dissociation media (25 U/mL collagenase, 150 ug/mL hyaluronidase, trypsin, 0.2 mg/mL kynurenic acid, 1% penicillin/streptomycin in low Ca++ aCSF). Single cells were pelleted and washed in aCSF three times, then resuspended in tumor sphere media (TSM), (Dulbecco’s Modified Eagle Media Nutrient Mix F-12 (DMEM/F-12, Invitrogen) supplemented with Glutamax (Invitrogen), 20 ng/mL epithelial growth factor (EGF, Sigma-Aldrich), 20 ng/mL basic fibroblastic growth factor (bFGF, Chemicon (Billerica, MA)), 1% penicillin/streptomycin, and 16B- 27 Serum-Free Supplement (Invitrogen). Primary Human Endothelial Cells. Primary human brain microvascular endothelial cells (HBMEC) were obtained from ScienCell, Carlsbad, CA). Primary human umbilical vein endothelial cells (HUVEC) were obtained from American Type Culture Collection (ATCC, Manassas, VA). For certain experiments endothelial cells were engineered to express mCherry fluorescent protein via lentiviral infection by viral particles that were produced by the Viral Vectors Core Facility of The Hope Center for Neurological Diseases at Washington University School of Medicine followed by sorting for expression with a MoFLO high speed cell sorter. Expression vectors used in viral production contained a transgene for mCherry fluorescent protein under a CMV promoter. ECs were used between passages 3–8 and maintained in endothelial cell growth media (EGM-2MV (Lonza, Basel, Switzerland)) on gelatin-coated dishes. Established Human GBM cell line. U87 cells were originally obtained from ATTC and were engineered at low passage (,5) to express a fusion protein of firefly luciferase and enhanced green fluorescent protein (eGFP) driven by the human ubiquitin C promoter after transduction with a lentivirus (FUW- PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e33005