Results

Inhibition of Tumor Development in CCR5−/− Mice
The effect of CCR5 in tumor development was investigated using CCR5+/+ and CCR5−/− mice. B16-F0 melanoma cells were inoculated subcutaneously into CCR5+/+ and CCR5−/− mice (n = 20). Tumor growth was monitored for 31 days. Tumor growth in the CCR5−/− mice was significantly lower than that in the CCR5+/+ mice. The tumor growth in the CCR5−/− mice was reduced to 19.1%, whereas the tumor growth in the CCR5+/+ mice was only reduced to 24.3%, in both tumor weight (Figure 1A and 1B) and tumor volume, respectively (Figure 1A and 1C). These results correlated with mice survival rates. There was a significant difference in survival rates between CCR5−/− mice (75% of starting time) and CCR5+/+ mice (15%) at the end of the experiment (Figure 1D). There was a significant difference in survival rates between CCR5−/− mice and CCR5+/+ mice. At the end of the experiment, 75% of the CCR5−/− mice survived while only 15% of the CCR5+/+ mice survived. Subcutaneous tumors were harvested from the sacrificed mice 31 days after inoculation. The immunohistochemical analysis of tumor sections, stained with H&E and for proliferation antigen PCNA, revealed greater inhibition of tumor cell growth in CCR5−/− mice. The histologic findings after H&E staining indicated that the tumor tissues of the CCR5−/− mice, but not those of the CCR5+/+ mice, contained large areas of necrosis. PCNA reactive cells were significantly reduced in the CCR5−/− mice (Figure 1E).
10.1371/journal.pone.0033747.g001 Figure 1  Inhibition of tumor development in CCR5−/− mice.
A–C, Tumor images, volumes, and weights. B16 melanoma cells (5×105 cells/mouse) were inoculated s.c. into CCR5+/+ mice and CCR5−/− mice (n = 20). Tumor volumes were measured every day and tumor weights were measured at study termination (Day 31). The CCR5−/− mice had a significant reduction in tumor growth and volume as compared to CCR5+/+ mice. The results are expressed as mean ± SD. * indicates significant difference from CCR5+/+ mice (P<0.05). D, The CCR5−/− mice demonstrated significantly higher survival rates compared to CCR5+/+ mice (by Log-rank Test). E. Tumor sections were analyzed by H&E stain and expression of proliferating cell nuclear antigen (PCNA) by immunohistochemistry. Data are means ± S.D. of four experimental animals. * indicates significant difference from CCR5+/+ mice (P<0.05). Scale bar indicates 50 m.

Decrease of NF-κB Activity in Tumor Tissue of CCR5−/− Mice
The activation of NF-κB plays a critical role in cancer cell survival, especially melanoma cancer cells and in CCR5 expression. To evaluate whether the tumor inhibition was related with the inactivation of NF-κB in melanoma, the DNA binding activity of NF-κB was determined by electromobility shift assay (EMSA) in melanoma tissue. The constitutively activated DNA binding activity was reduced in the CCR5−/− mice tumor tissue (Figure 2A). Moreover, the translocation of p50 and p-p65 into the nucleus through the inhibition of phosphorylation of IκB was also prevented in the tumor tissues of CCR5−/− mice (Figure 2B). Immunofluorescence analysis also confirmed that the intensity of nuclear staining for p50 had decreased in the tumor tissues of CCR5−/− mice (Figure 2C).
10.1371/journal.pone.0033747.g002 Figure 2  Decrease of NF-κB activity in tumor tissue of CCR5−/− mice.
A, The DNA binding activity of NF-κB was determined in the nuclear extracts of the CCR5−/− mice and CCR5+/+ mice tumor tissues by EMSA described in material and methods. B, Expression of p50 and p65 phosphorylation in nuclear extracts (NE), and IκB and IκB phosphorylation in the cytosol (CE) determined by Western blotting. C, Immunolfluorescence analysis of p50 confirmed that the intensities of nuclear staining for p50 were decreased in the tumor tissues of CCR5−/− mice. Data are means ± S.D. of four experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05). Scale bar indicates 50 µm.

Induction of Apoptotic Cell Death and Inhibition of the Expression of Anti-apoptotic Proteins in CCR5−/− Mice
To see whether inhibition of NF-κB activity results in the induction of apoptotic cell death and expression of NF-κB target apoptotic cell death regulatory protein, the levels of apoptotic cell death and expression of apoptotic cell death regulatory proteins were assessed. Apoptotic cell death in the tumor sections were evaluated in the CCR5+/+ and CCR5−/− mice that were inoculated with melanoma cells. TUNEL staining revealed a higher frequency of apoptotic cell death in the tumor tissues of the CCR5−/− mice than that of the CCR5+/+ mice (Figure 3A). Immunohistochemical and immunofluorescence showed that high levels of cleaved caspase-3 and Bax were found more frequently in the CCR5−/− mice than in the CCR5+/+ mice (Figure 3B and 3C). Consistent with the immunohistochemical data, Western blot analysis of tumor proteins showed greater increased levels of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, whereas the levels of Bcl-2 and C-IAP1 were significantly decreased in CCR5−/− mice tissues (Figure 3D).
10.1371/journal.pone.0033747.g003 Figure 3  Induction of apoptotic cell death in tumor tissues of CCR5 −/− mice.
A, Apoptotic cells in tumor sections were examined by fluorescence microscopy after DAPI and TUNEL staining as described in material and methods. The apoptotic index was determined as the number of DAPI-stained, TUNEL-positive cells that were counted. Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice. Scale bar indicates 50 µm. B and C, Apoptotic protein (cleaved caspase-3 and Bax) in tumor sections were detected by immunofluorescence assay. The reactive cell number was determined as the number of DAPI-stained, Specific antibody (cleaved caspase-3 and Bax)-positive cells that were counted. Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice. Scale bar indicates 50 µm. D, The expression of apoptotic proteins was detected by Western blotting using specific antibodies; cleaved caspase-3, cleaved caspase-9, cleaved PARP, Bax, Bcl-2 and c-IAP1 in the tumor tissues. The β-actin protein was used as an internal control. Each band is representative of three independent experimental results. Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).

Upregulation of IL-1Ra in the Tumor and Spleen of CCR5−/− Mice
To investigate the difference in the cytokine levels between the tumor and spleen tissues of the CCR5+/+ and CCR5−/− mice, we conducted a cytokine array assay using a Mouse Proteome Array (Figure 4A). Among 40 tested cytokines, the levels of IL-1Ra, MIG, MIP-1α increased about 686, 9 and 25-fold respectively, in CCR5−/− mice compared to CCR5+/+ mice. Using this technique, no other cytokines were found to be different between the CCR5−/− mice and the CCR5+/+ mice (Figure 4B). Similar altered cytokine levels were found in the spleens of CCR5−/− mice (Figure 4C). Since the IL-1Ra level was the most significantly elevated, the expression of IL-1Ra in tumor and spleen tissues was analyzed by immunofluorescence and western blotting. The expression of IL-1Ra was upregulated in tumor and spleen tissues of the CCR5−/− mice (Figure 4D-G).
10.1371/journal.pone.0033747.g004 Figure 4  Upregulation of IL-1Ra in the tumor and spleen of CCR5−/− mice.
A, Figure 4A indicates mouse cytokine array panel coordinates. Nitrocellulose membranes contain 40 different anti-cytokine antibodies printed in duplicate. B, Mouse cytokine array panel indicate the cytokine expression difference in tumor tissues of CCR5+/+ mice and CCR5−/− mice, especially IL-1Ra. Representative blot from three independent experiments is shown. Positive controls show the manufacturer’s internal positive control samples on the membrane. C, Protein immune-arrays were performed using Mouse cytokine array in spleen tissues. There were differences in cytokines between CCR5+/+ mice and CCR5−/− mice, especially IL-1Ra. Representative blot from three independent experiments is shown. Positive controls show the manufacturer’s internal positive control samples on the membrane. D and E, Immunolfluorescence analysis was used to determine the expression levels of IL-1Ra in tumor and spleen tissues. The reactive cell number was determined as the number of DAPI-stained, IL-1Ra antibody-positive cells that were counted. Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice. Scale bar indicates 50 µm. F and G, Expression of IL-1Ra was analyzed by western blotting in tumor and spleen tissues. Each band is representative of three independent experimental results. Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).

Induction of CD8 T cells and NK Cell Infiltration into Tumor and Spleen of CCR5−/− Mice
To investigate whether the inhibition of tumor growth in CCR5−/− mice is related to tumor-specific immune responses, we analyzed the distribution patterns of CD8+ cytotoxic T cell and CD57+ Natural Killer cells in tumor and spleen tissues. By staining the tumor sections from CCR5−/− mice, we found that there was a significant influx of CD8+ cells into the tumor, as well as an increase in the number of infiltrating NK cells (Figure 5A). CD8+ T cells and CD57+ NK cells were spread diffusely throughout the entire sections. To further investigate the differences in the numbers of CD8+ T cells and CD57+ NK cells between CCR5+/+ mice and CCR5−/− mice in immunity-related organ, we also analyzed the CD8 and CD57 reactive cell number in spleen tissues. The number of CD8+ T cells and CD57+ NK cells were higher in the spleen of the CCR5−/− mice than in the spleen of the CCR5+/+ mice (Figure 5B). These data suggest that by promoting the infiltration of CD8 T cells and NK cells, which are potent cytotoxic effectors for tumors, could play a role in an anti-tumor effect in CCR5−/− mice.
10.1371/journal.pone.0033747.g005 Figure 5  Induction of the infiltration of CD8+ T cells and NK cells into tumor, and increase in spleen of CCR5−/− mice.
A and B, Immunolfluorescence analysis was used to determine the expression levels of CD8 (CD8+ cytotoxic T-cell surface marker) and CD57 (NK cell marker) in tumor and spleen sections. The images shown are representative of three separate experiments performed in triplicate. Scale bars indicate 50 µm (A) and 100 µm (B). C and D, Analysis of lymphocyte phenotypes. The tumor and spleen tissues were separated at study termination (Day 31). Flow cytometry analysis was performed using FACSAria flow cytometry, and represent data were shown. Data are means ± S.D. of four experimental animals. To analyze the cell phenotype change in CCR5−/− mice and CCR5+/+ mice by inoculation of the melanoma cell, lymphocytes were isolated from the tumor and spleen tissues. The B, CD3 T, CD4 T, CD 8 T and NK cell portion of CCR5−/− mice tumor tissue were 7.2%, 48.2%, 16.9%, 32.1% and 7.8%, respectively, compared to 2.9%, 11.1%, 2.3%, 0.6% and 3.1% of CCR5+/+ mice (Figure 5C). The lymphocytes that infiltrated the tumor tissue of the CCR5−/− mice also had higher levels of CD19, CD3, CD4, CD8 and CD57 when compared to the CCR5+/+ mice. The B, CD3 T, CD4 T, CD 8 T and NK cell portion of the CCR5−/− mice spleen were 61.3%, 33.4%, 17.6%, 9.2% and 0.6%, respectively compared to 38.3%, 5.9%, 4.0%, 1.0% and 3.9% of the CCR5+/+ mice (Figure 5D). These data suggest that by promoting the infiltration of CD8 T cells and NK cells, which are potent cytotoxic effectors for tumors, could play a role in the inhibition of tumor growth in CCR5−/− mice.