Flow cytometric analysis In order to characterize the expression levels of chemokine receptors CXCR4 and CCR6 on cancer cell lines, the cells were stained with human specific direct-labeled antibodies and analyzed by FACScalibur (Becton Dickinson Immunocytometry Systems), using CellQuest software. For CXCR4 expression analysis, anti- human CXCR4 monoclonal antibody, clone 12G5 (R&D Systems, Minneapolis, MN) or polyclonal anti-N-terminus anti- body (Chemicon International, Temecula, CA, USA) were used. For CCR6, anti-human CCR6 monoclonal antibody, clone 53103.11 (R&D Systems, Minneapolis, MN) was used. RNA interference The Dicer-substrate siRNA duplexes of CXCR4 (NCBI accession number NM_003467): sense 59 - UAAAAUCUUC- CUGCCCACCAUCUAC - 39, antisense 59 – GUAGAUGGU- GGGCAGGAAGAUUUUAUU - 39 were purchased from IDT, Coralville, IA. The non-specific siRNA duplexes were used as a control. PC3-CXCR4.5 cells were transfected with 200 nmol/L siRNA in serum-free medium using Oligofectamine reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Establishment of tumor xenografts Nude and SCID/beige mice (C.B -17/IcrHsd-SCID-bg) were maintained under defined flora conditions at the Hebrew University Pathogen-Free Animal Facillity. All experiments were approved by the Animal Care Committee of the Hebrew University. Prostate cancer cell lines (PC3, PC3-CXCR4.5, PC3- CCL20.30 and PC3-CCL20.10) and HT 29 cells were grown to 80% confluence, harvested, and injected subcutaneously in the flank of mice (56106/mouse). Once palpable, tumors were measured and tumor size (width6length) was calculated. For the neutralizing experiments, mice were treated with subcutaneous injections of monoclonal anti-human CCL20 antibody (MAB360, R&D Systems, Minneapolis, MN) or control IgG1 antibody. At the end of the experiments, animals were sacrificed, tumors were harvested, measured and weighted. MRI analysis of tumor growth and blood vessel functionality and maturation were done as previously described (18). Immunohistochemistry and scoring Two different commercial prostate tumor tissue microarrays were used: CA2 array included 52 prostate cancer tissue sections and CA3 array included 48 prostate cancer tissue sections (SuperBioChip Lab). In addition tissue samples of primary References 1. Zlotnik A, Yoshie O (2000) Chemokines: a new classification system and their role in immunity. Immunity 12: 121–127. 2. Balkwill F (2003) Chemokine biology in cancer. Semin Immunol 15: 49–55. 3. Murphy PM (2001) Chemokines and the molecular basis of cancer metastasis. N Engl J Med 345: 833–835. 4. Balkwill F (2004) The significance of cancer cell expression of the chemokine receptor CXCR4. Semin Cancer Biol 14: 171–179. 5. 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Formalin-fixed, paraffin-embed- ded tissue samples were initially dewaxed, rehydrated, treated with EDTA buffer and blocked with CAS blocking reagent (Zymed Laboratories, San Francisco, CA, USA) for 30 minutes in room temperature. Samples were then incubated overnight at 4uC in a humidified chamber with anti-human CCL20 polyclonal antibody (PeproTech EC, London, UK), diluted to final concentration 20 mg/ml, or alternatively with anti-human CCR6 monoclonal antibody (R&D Systems, Minneapolis, MN) diluted to final concentration 10 mg/ml, or with anti-human CXCR4 monoclonal antibody, clone 12G5 (R&D Systems, Minneapolis, MN) diluted to final concentration 10 mg/ml. Next, the sections stained for CCL20 were incubated with diluted 1:1000 biotinylated goat-anti- rabbit antibody (Jackson ImmunoResearch), for 30 minutes at room temperature and thereafter with horseradish peroxidase - conjugated streptavidin (Zymed Laboratories, San Francisco, CA, USA) for 30 minutes at room temperature. The sections stained for CCR6 or CXCR4 were incubated with secondary anti-mouse horseradish peroxidase-conjugated antibody (DakoCytomation, Glostrup, Denmark) for 30 minutes at room temperature. 3- amino-9-ethylcarbazole (AEC) was used for color development, and sections were counterstained with hematoxylin. Analysis of CCL20 and CCR6 expression was determined by scoring the staining intensity as negative, weak or strong by two independent investigators. Scoring was performed blindly, without knowledge of overall Gleason score or tumor pathologic stage. Statistical analysis Data are presented as means6SD or 6SE. Statistical comparison of means was performed by a two-tailed unpaired Student’s t test. Differences with a P,0.05 were determined as statistically significant. Acknowledgments We thank for Dr. Mario Baras (Hadassah Hospital) for statistical analysis; and Mery Clausen (Gene Therapy Institute, Hadassah Hospital) for technical assistance. 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