additional month in order to let them reach similar size. However, even on day 71, they were significantly less vascularized. While functional vessels were observed at the center of tumors overexpressing CCL20, no functional vessels were observed at the center of the control tumors and only on the borders of these tumors. The mean6SD values of DSo2 were calculated from a region of interest containing the whole tumor, and normalized to contra-lateral muscle, pooling data from 9 mice from the PC3- CCL20.30 group and 5 mice from the PC3-mock group (four slices/mouse; Fig. 4C, p,0.001). These results suggest that high expression of CCL20 results in early neovascularization of the tumors while in control tumors, the development of necrosis was mediated by poor perfusion. Neutralization of CCL20 inhibits the CCL20 and CXCR4- dependent growth of various tumor cells in vivo Having established the role of CCL20 in cancer development in vivo, we evaluated the effect of neutralizing antibodies to human CCL20 on the growth of CCL20-and CXCR4 overexpressing PC3 cells. First, we tested the ability of anti-CCL20 antibodies to neutralize the CCL20-induced adhesion in vitro of PC3-CCL20.30 cells to collagen I. The presence of monoclonal anti-human CCL20 antibodies abolished the adhesion of PC3-CCL20.30 cells to collagen I in response to CCL20 stimulation (Fig. 4D). Next, we assessed the in vivo potential of neutralizing anti-CCL20 antibodies. PC3-CCL20.30 cells were injected subcutaneously into SCID/bg mice. Twenty-four hours after cell injection, mice started to get treatment with subcutaneous injections of anti-human CCL20 antibody or isotype control, 20 mg of antibody per injection, three times a week, during four weeks. A significant decrease in tumor growth was observed in anti-CCL20-treated mice (Fig. 4E, F). In the control group, nine out of ten mice developed subcutaneous tumors which rapidly progressed over time. In contrast, in the ani-CCL20-treated group, only five out of ten mice developed tumors, while four of the produced tumors were very small in size (0.2 cm60.2 cm or 0.1 cm60.1 cm) and did not progress over time. Moreover, histological evaluation of H&E- stained tissue sections of xenograft tumors demonstrated that neutralizing antibodies to CCL20 inhibited intensive aberrant blood vessel formation and promoted extensive necrotic tissue damage in treated tumors (data not shown). To confirm the role of CCL20 in the development and progression of CXCR4-overexpressing prostate tumors, we evaluated the effect of neutralizing antibodies to human CCL20 on the growth of CXCR4-overexpressing PC3 cells. PC3- CXCR4.5 cells were injected subcutaneously into SCID/bg mice. Injected animals were treated with the anti-human CCL20 antibody or isotype control according to the same protocol used in mice injected with CCL20-overexpressing cells. Animals treated with anti-CCL20 antibodies demonstrated a delay in tumor appearance – in the control group on day 28, 100% of animals developed visible tumors, while in the anti-CCL20-treated group, only 60% appeared with tumors on the same day. Furthermore, the treatment with anti-CCL20 antibodies inhibited the growth of CXCR4-expressing prostate tumors (Fig. 4G). It has recently been shown that CCR6 and CCL20 are significantly upregulated in CRC and colorectal liver metastases (CRLM) [27]. To further test the role of CCL20 in the development and progression of colon cancer, we evaluated the effect of neutralizing antibodies to human CCL20 on the growth of HT-29 tumor cells. HT-29 cells were injected subcutaneously into nude mice. Injected animals were treated daily with the anti- human CCL20 antibody or isotype control. Treatment with anti- CCL20 antibodies significantly inhibited the growth and invasion CCL20 and CXCR4 Interaction of HT-29 tumors (Fig. 4H). Moreover, antibodies to CCL20 inhibited the invasion of tumor cells into surrounding tissues similarly to the skin and muscle (data not shown). We therefore concluded that neutralizing antibodies to CCL20 suppressed in vivo CXCR4-dependent and independent prostate and colon tumor growth. CXCR4 and CCL20 are co expressed in human prostate cancer In order to further test the relevance of in vivo role of CCL20/ CCR6 axis in prostate cancer development, we first evaluated the expression of CCL20 and CCR6 in human prostate cancer tissues with the use of commercially available array of 52 paraffin- embedded prostate sections from patients with advanced prostate cancer. All specimens were graded using pathologic stage and the Gleason score system. The immunohistochemical staining re- vealed that the majority of tumor samples (50 out of 52, 96%) expressed the CCR6 receptor at heterogeneous levels. Whereas the ligand for CCR6, CCL20, was expressed in 34 out of 52 tumor samples (65.4%). The CCL20 and CCR6 staining were located mostly in epithelial and fibromuscular stromal cells (Fig. 5A). The majority of tumor samples that expressed CCL20 co-expressed CCR6. In contrast, normal human prostate tissue samples expressed very low levels of CCL20 and CCR6 (Fig. 5A). Out of 34 CCL20-positive sections, 16 samples were highly positive for CCL20. Average Gleason score in samples with high CCL20 expression was 8.7561.48 (n = 16) versus 7.7261.75 (n = 36) in samples with low or negative CCL20 expression (p = 0.02) (Fig. 5A9). Average stage in samples with high CCL20 expression was 3.2860.97 (n = 16) versus 3.7560.68 (n = 36) in samples with low or negative CCL20 expression (p, = 0.07) (Fig. 5A9). No prevalence in high levels of CCR6 in progressive stage IV sections was detected (Fig. 5A0). These results demonstrate that high CCL20 expression correlates with high Gleason score (e.g., tumor grade) and higher staging of the disease in this array. To further study the expression pattern of CCL20 and CCR6 in prostate tumors, we stained additional specimens from 44 primary tumors. All tissue sections were obtained from the Pathology Department of the Hadassah Hebrew University Medical Center. The average Gleason score in these patients was significantly lower than in the commercial array, 5.77 versus 8.04 respectively. Similar to the commercial array CCL20 was expressed in 70.5% of the tumors and CCR6 was expressed in 95.5% of the tumors sections. However, in contrast to the commercial array no significant correlation between CCL20 expression and Gleason score (e.g., tumor grade) and staging was found. These results suggest that CCL20 and CCR6 are commonly expressed in prostate cancer tumors; however, more studies are needed to demonstrate their diagnostic significance. In order further test the possible interaction between CXCR4 receptor and CCL20 chemokine in prostate cancer, we evaluated the coexpression of CCL20 and CXCR4 in human prostate cancer tissues with the use of commercially available additional array of 48 paraffin-embedded prostate sections from patients with advanced prostate cancer. Similar to our previous [17] and current stud and the all samples in this array expressed the CXCR4 receptor and CCL20 chemokine at heterogeneous levels. Expression level of CXCR4 and CCL20 was determined by scoring the staining intensity as low, intermediate or high. Co- expression of CXCR4 and CCL20 in the same cancer cells was observed (Fig. 5B). Moreover, similar expression level of CXCR4 and CCL20 was observed on the majority of prostate tumor samples (38 out of 48 samples, 79.5%) (Fig. 5B, 5B9). Four additional samples demonstrated close expression pattern, and PLoS ONE | www.plosone.org 8 April 2009 | Volume 4 | Issue 4 | e5125