CCL20 regulates CXCR4 dependent and independent growth of tumor cells.

<p>(A) Effect of CCL20 stable expression on prostate tumor growth. PC3-CCL20.30, PC3-CCL20.10 and PC3-mock transfected cells (5×10⁶/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 purified . Tumor size (cm²) was measured twice a week using caliper. Results are representative of three independent experiments with five mice in each group. Data is presented as mean±SE 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 ×200 is shown. (C) Vessel functionality (Δ<i>S</i>o₂) 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. Δ<i>S</i>o₂ values from PC3-mock cells and PC3-CCL20.30 are shown. The mean±SD values of Δ<i>S</i>o₂ 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 µg/ml) were placed on collagen I-coated plates (10 µg/ml) for 30 minutes. Non-adherent cells were washed twice with cold PBS. Adherent cells were collected in 300 µl FACS buffer with 5 mM EDTA and counted by FACS. Data is presented as mean±SD from triplicates (** <i>P</i><0.016). (E) PC3-CCL20.30 cells (5×10⁶/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 µg 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 mean±SE 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 mean±SE from ten mice in each group (** <i>P</i><0.0002). (G) PC3-CXCR4.5 cells (5×10⁶/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 µg of antibody per injection, three times a week, during four weeks. Tumor size (cm²) 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 mean±SE from ten mice in each group (** <i>P</i><0.0027). (H) HT-29 cells (2×10⁶/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 µg 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 mean±SE from ten mice in each group (** <i>P</i><0.0002).