CCL20 and CXCR4 Interaction Figure 4. CCL20 regulates CXCR4 dependent and independent growth of tumor cells. (A) Effect of CCL20 stable expression on prostate tumor growth. PC3-CCL20.30, PC3-CCL20.10 and PC3-mock transfected cells (56106/mouse) were injected subcutaneously into SCID/beige mice. 48 days or 75 days following the injection, animals were sacrificed, and xenograft tumors generated by PC3-mock and PC3-CCL20.30 injected cells were 2 purified . Tumor size (cm ) was measured twice a week using caliper. Results are representative of three independent experiments with five mice in each group. Data is presented as mean6SE from five mice. (B) H&E staining of paraffin-embedded tumor tissue sections derived from PC3-mock and PC3-CCL20.30 tumors on day 48. Black arrows signify non-invasive borders of PC3-mock tumor (B,a), small blood vessel in PC3-mock tumor (B,b), aberrant blood vessels in PC3-CCL20.30 tumor (B, c,d), original magnification of 6200 is shown. (C) Vessel functionality (DSo2) was measured by fMRI. Functionality of the vasculature was tested during inhalation of air-CO2 and carbogen (95% oxygen+5% CO2) in mice implanted with PC3-mock cells or with PC3-CCL20.30 cells. DSo2 values from PC3-mock cells and PC3-CCL20.30 are shown. The mean6SD values of DSo2 were calculated from 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; p,0.001). (D) Adhesion of PC3-CCL20.30 cells to collagen I. PC3-CCL20.30 cells either umstimulated or stimulated with 50 ng/ml of CCL20 with or without co-incubation with neutralizing anti-CCL20 antibodies (10 mg/ml) were placed on collagen I-coated plates (10 mg/ml) for 30 minutes. Non-adherent cells were washed twice with cold PBS. Adherent cells were collected in 300 ml FACS buffer with 5 mM EDTA and counted by FACS. Data is presented as mean6SD from triplicates (** P,0.016). (E) PC3-CCL20.30 cells (56106/mouse) were injected subcutaneously into SCID/ beige mice. Twenty-four hours after the cell injection, mice started to get subcutaneous injections of anti-human CCL20 antibodies or isotype control antibodies, 20 mg of antibody per injection, three times a week, during four weeks. Tumor size (cm2) was measured once a week using caliper. Results are representative of two independent experiments with ten mice in each group. Data is presented as mean6SE from ten mice. (F) On day 64 following the cell injection, the experiment was terminated, animals were sacrificed; subcutaneous tumors were removed, measured and weighted. Data is presented as mean6SE from ten mice in each group (** P,0.0002). (G) PC3-CXCR4.5 cells (56106/mouse) were injected subcutaneously into SCID/beige mice. Twenty-four hours after the cell injection, mice started to get subcutaneous injections of anti-human CCL20 antibodies or isotype 2 control antibodies, 20 mg of antibody per injection, three times a week, during four weeks. Tumor size (cm ) was measured once a week using caliper. On day 55 following cell injection, the experiment was terminated; animals were sacrificed, subcutaneous tumors were removed, measured and weighted. Data is presented as mean6SE from ten mice in each group (** P,0.0027). (H) HT-29 cells (26106/mouse) were injected subcutaneously into nude mice. Twenty-four hours after cell injection, mice started to get subcutaneous injections of anti-human CCL20 antibodies or isotype control antibodies, 20 mg of antibody per injection, five times a week, during two weeks. On day 17 following cell injection, the experiment was terminated, animals were sacrificed; subcutaneous tumors were removed, measured and weighted. Data is presented as mean6SE from ten mice in each group (** P,0.0002). doi:10.1371/journal.pone.0005125.g004 in mice implanted with PC3-mock cells or with PC3-CCL20.30 cells. Interestingly, MRI analysis showed that DSo2 values from PC3-CCL20.30 tumors were significantly higher (Fig. 4C). DSo2 maps derived on day 41 showed enhanced tumor vascularity in tumors produced by PC3-CCL20.30 vs. control tumors that had very low DSo2 values. We left the control tumors to grow for an PLoS ONE | www.plosone.org 7 April 2009 | Volume 4 | Issue 4 | e5125