Laboratories, Burlingame, CA). Sections were additionally blocked with 10% serum from the animal source of the appropriate corresponding secondary antibody diluted in incubation media [0.1 mol/L Tris (pH 7.5), 0.15 mol/L NaCl, 2% nonfat dry milk, and 0.1% Triton X-100] and then incubated in primary antibody overnight at 4uC. CXCL12 was detected with rabbit polyclonal antibody (1:66 dilution). Immunoreactive complexes were detected using the corresponding secondary biotin-conjugated antibodies augmented by streptavidin-horseradish peroxide and visualized by 3,39-diaminobenzidine supplied by DAKO (Carpinteria, CA). Slides were then counterstained with hematoxylin, dehydrated through a series of alcohols and xylene, and coverslipped in 50:50 xylene/Permount. Control sections were incubated with isotype- matched IgG. Localization Measurements Primary human brain endothelial cells expressing mCherry and human GBM cells were grown on Matrigel coated coverslips, as above. After treatment, cells were fixed in 70% ethanol at 220uC for 10 minutes and counterstained with DAPI. Images were acquired on a Zeiss Scope.A1 fitted with an Axiocam MRc camera and using Axiovision software (Carl Zeiss, North America (Thornwood, NY)). Distances between GBM cell nuclei and the nearest endothelial cell nucleus were measured using a vector tool in Axiovision. For U87-HBMEC or HUVEC co-localization we measured ,1000 cells in 4 separate experiments for each condition. We then used the average +/2 SEM. For Primary GBM-HBMEC co-localization cells we measured ,450 cells from 3 different patient specimens. We then used the average +/2 SEM. Growth Assays Primary GBM: After treatment, tumor cells were fixed and identified via immuno-labeling for glial fibrillary acidic protein (GFAP). Images were acquired using a Molecular Devices Image Express Micro High Content Imager at the High-Throughput Screening Core at Washington University in St. Louis. Automated image analysis using MetaExpress and AcuityExpress (Molecular Devices (Sunnyvale, CA)) was performed to count the total number of GFAP positive cells/well. Cell counts were normalized to control conditions in which GBM cells were cultured with Matrigel, but in the absence of ECs. Established GBM cell line: For U87 cells expressing firefly luciferase, cell number was evaluated using bioluminescence measurements at the Molecular Imaging Core facility, BRIGHT Institute, Washington University in St. Louis. Endothelial cells did not express luciferase activity. Cell number, as determined by direct cell counts, and bioluminescence were linearly proportional under our culture conditions (data not shown). Cells were grown in 96 well black-wall clear-bottom plates (Costar). After treatment, cells were washed into phenol red-free media and exposed to 0.15 mg/ml luciferin for 10 minutes. Bioluminescence was measured using a charge-coupled device camera-based biolumi- nescence imaging system (IVIS 50; Caliper; exposure time 1–30 s, binning 8, field of view 12, f/stop 1, open filter). Regions-of- interest (ROIs) were drawn over images of wells and biolumines- cence data, expressed as total photon flux (photons/s), were normalized to untreated controls [31,36]. TUNEL assays Terminal nucleotidyl transferase–mediated nick end labeling (TUNEL) staining of fixed cells was done by standard procedures according to the manufacturer’s directions (Roche Applied Science, Indianapolis, IN) along with nuclear counterstain. CXCL12 Mediated GBM-Endothelial Cell Interactions TUNEL-positive cells were detected under direct fluorescence microscopy. TUNEL positivity is reported as the percent of total GBM cell nuclei that were TUNEL positive. Generation of intracranial xenografts Intracranial xenografts were generated as described previously. Homozygous NCR nude mice (Taconic Farms) were anesthetized [intraperitoneal ketamine (87 mg/kg)/xylazine (13 mg/kg); Phoe- nix Pharmaceuticals], the cranium was exposed, and a small hole was made 2 mm lateral and posterior to the bregma with a size 34 inverted cone burr (Dremel). Mice were positioned in a stereotactic frame (Stoelting) and 50,000 cells in 5 mL PBS were injected through a 27-gauge needle over 1 min at 3 mm below the dura mater. The incision was closed with Vetbond (3M). Bioluminescence imaging NCR nude mice bearing intracranial xenografts of U87-Luc expressing scrambled shRNA (sc-U87-Luc) or CXCR4-specific shRNA (shCXCR4-U98-Luc) were injected with D-luciferin (150 mg/g; Biosynth) as previously described. After anesthesia using 2.5% isoflurane, mice were imaged with a charge-coupled device camera-based bioluminescence imaging system (IVIS 50; Xenogen; exposure time 1–30 s, binning 8, field of view 12, f/stop 1, open filter). Signals were displayed as photons/s/cm2/sr. Regions of interest were defined manually and images were processed using Living Image and IgorPro Software (Version 2.50) as described. Raw data were expressed as total photon flux (photons/s). Statistical Analyses All values represent the means from experiments repeated at least 3 separate times. Data were analyzed using GraphPad Prism version 4.00 (GraphPad Software) using the specific statistical tests identified in the corresponding figure legends. Potential statistical outliers were detected by application of Grubb’s test. A single animal was removed from the in vivo studies as an outlier. A second animal exhibited highly erratic bioluminescence and was also excluded. This did not alter the results. Results We previously demonstrated that systemic administration of the specific CXCR4 antagonist AMD 3100, inhibited the intracranial growth of U87 glioblastoma xenografts by increasing apoptosis and decreasing proliferation of tumor cells [14]. Both tumor cells and endothelial cells express CXCR4, and to distinguish whether tumor cell-CXCR4 function is required for tumor growth, we depleted CXCR4 by shRNA-mediated knock-down in U87 glioblastoma cells that had also been engineered to express a fusion protein of firefly luciferase and eGFP (shCXCR4-U87-Luc). Control cells were generated through expression of a scrambled shRNA (sc-U87-Luc). CXCR4 depletion was confirmed by western blot analysis (Figure 1A). Intracranial xenografts of shCXCR4-U87-Luc and sc-U87-Luc cells were generated in nude mice as described [26,31]. Bioluminescence imaging 48 hrs post- intracranial injection was similar between the two groups [mean photon flux for sc-U87-Luc: 6.786106; and for shCXCR4-U87- Luc: 7.176106] suggesting that CXCR4 was not required for tumor cell engraftment. In contrast, CXCR4 depletion in U87 cells significantly suppressed their intracranial growth over a four- week experimental period (Figure 1B). These data strongly indicate that tumor cell CXCR4 function is required for tumor growth. PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e33005