inhalation isofluorane (Abbot Laboratories, England) followed by an intraperitoneal injection of 225–240 mg/kg AvertinH solution and then maintained on isofluorane by inhalation throughout the procedure. The head was shaved, then the mice were immobilized in a CunninghamTM Mouse/Neonatal Rat Adaptor (Stoelting, Wood Dale, IL) stereotactic apparatus fitted into an E15600 Lab Standard Stereotactic Instrument (Stoelting), then scrubbed with 1% povidone-iodine. A 10 mm skin incision was made along the midline. The tip of a 31G K inch needle mounted on a Hamilton syringe (Hamilton, Reno, NV) served as the reference point. A 1 mm burr-hole was drilled into the skull, 1 mm anterior to and 2 mm to the right of the bregma. Daoy cells (2.56105 in 2.5 mL) were injected 3 mm deep to the bregma, corresponding to the center of the right caudate nucleus over 5 minutes. The needle was left in place for 3 minutes, to avoid tumor cell extrusion, and then withdrawn over 5 minutes. Ten days after tumor cell injection, animals received 26106 eGFP.Firefly-luciferase-expressing T lymphocytes in 2.5 mL at the same tumor coordinates in the contralateral caudate nucleus (Bregma +1 mm anterior; +2 mm to the left of the midline, +3 mm deep). The incision was closed with 2–3 interrupted 7.0 EthiconH sutures (Ethicon, Inc. Somerville, NJ). A subcutaneous injection of 0.03–0.1 mg/kg buprenorphine (BuprenexH RBH, Hull, England) was given for pain control. The primary medulloblastoma xenograft model was described previ- ously. [48] Bioluminescence imaging Isofluorane anesthetized animals were imaged using the IVISH system (IVIS, Xenogen Corp., Alameda, CA) 10 minutes after 150 mg/kg D-luciferin (Xenogen) was injected intraperitoneally. [49] The photons emitted from luciferase-expressing cells within the animal body and transmitted through the tissue were quantified using "Living Image", a software program provided by the same manufacturer. A pseudo-color image representing light intensity (blue least intense and red most intense) was generated and superimposed over the grayscale reference image. Animals were imaged after injections daily. They were regularly examined for any neurological deficits, weight loss or signs of stress and euthanized according to pre-set criteria, in accordance the Baylor College of Medicine’s Center for Comparative Medicine guidelines. Immunohistochemistry (IHC) Mice were euthanized by CO2 inhalation and fixed with intra- cardiac perfusion of 4% paraformaldehyde. The brain tissue was post-fixed overnight and embedded in paraffin, and histology was performed on 10-mm serial horizontal sections. Tissue sections were stained by a standard hematoxylin and eosin technique. Human CD3 expression in T lymphocytes was detected by human CD immunohistochemistry. The same methodology was used to stain primary medulloblastoma samples. Transwell migration assays T lymphocyte migration towards tumor chemokines was measured in vitro through the use of a transwell migration assay. Firefly luciferase expressing T lymphocytes (16105 in 100 mL serum-free media) were placed in the top insert chamber of 4mm transwell migration plate (Corning Inc., Corning, NY). The lower chamber was filled with 600 mL of either control medium, supernatant from various cell lines, or medium conditioned with a specific chemokine. After a 3 hour incubation at 37uC, the medium from the lower chamber was collected and biolumines- cence was measured for 30 seconds after 15 second incubation Tumor/Endothelium Crosstalk Attracts T Cells with 150 mg D-luciferin (3010 Luminometer, BD Biosciences, San jose, CA). Analysis of chemokine production The detection and quantification of chemokine production from tumor cells and endothelial cells was done using a cytokine array kit (Human and Murine Proteome ProfilerTM array, R&D Systems, Minneapolis, MN). Supernatant from tumor cells alone, endothelial cells alone, and both cell lines in co-culture were collected at various time points and tested for 36 different chemokines per the manufacturer’s instructions. Supernatant from the co-culture of endothelial cell lines and tumor cell lines were collected at various time points and tested by cytokine-specific ELISA per the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Flowcytometry For all flowcytometric analyses, a FACScalibur instrument (BD, Becton Dickinson, Mountain View, CA) and CellQuest software (BD) were used. Data analysis was done on .10,000 events; in all cases negative controls included isotype antibodies. Cells were washed once with PBS containing 2% FBS and 0.1% sodium azide (Sigma; FACS buffer) prior to addition of antibodies. After 15 to 30 minutes of incubation at 4oC in the dark the cells were washed once and fixed in 0.5% paraformaldehyde/FACS buffer prior to analysis. T lymphocytes were analyzed with anti-CD8 FITC, -CD4 PE, and -CD3 PerCP as well as the chemokine receptors, CXCR1, CXCR2, CXCR3, CXCR4, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7 and CD74. All monoclonal antibodies were obtained from BD Biosciences, Palo Alto, CA. Generation of knockdown medulloblastoma lines MIF knockdown (KD) and IL-8-KD Daoy cells were generated by transducing Daoy cells with RD114 pseudotyped retroviral vectors encoding shRNAs for MIF or IL8 (pRS shRNA; 4 shRNAs per target gene; Origene; Rockville, MD). Transduced cells were selected with puromycin, and MIF or IL-8 production was measured by ELISA (R&D Systems, Minneapolis, MN). Daoy cell lines with the greatest decreased of MIF (81% of controls) or IL-8 (78% of controls) production were used in subsequent experi- ments. RNA extraction and analysis RNA was isolated from explanted Daoy and KD Daoy xenografts. Total RNA concentrations were measured using the Nanodrop 1000 spectrophotometer (Nanodrop Scientific, Wil- mington, DE). RNA integrity was checked in electrophoresis using 2% agarose gel with 6% formaldehyde. The human and murine chemokine and chemokine receptor arrays were performed by SABiosciences Inc (Frederick, MD) using PAHS-022 (human) and PAMM-022 (mouse) chemokine and receptor arrays. Statistical analysis For the bioluminescence experiments, intensity signals were log- transformed and summarized using mean 6 SD at baseline and multiple subsequent time points for each group of mice. Changes in intensity of signal from baseline at each time point were calculated and compared using paired t-tests or Wilcoxon signed- ranks test. Supporting Information Figure S1 A protein profiler system was used to characterize the chemokine pattern specific to Daoy PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e20267