Complement 5a Enhances Hepatic Metastases of Colon Cancer via Monocyte Chemoattractant Protein-1-mediated Inflammatory Cell Infiltration

Abstract

Complement 5a (C5a), a potent immune mediator generated by complement activation, promotes tumor growth; however, its role in tumor metastasis remains unclear. We demonstrate that C5a contributes to tumor metastases by modulating tumor inflammation in hepatic metastases of colon cancer. Colon cancer cell lines generate C5a under serum-free conditions, and C5a levels increase over time in a murine syngeneic colon cancer hepatic metastasis model. Furthermore, in the absence of C5a receptor or upon pharmacological inhibition of C5a production with an anti-C5 monoclonal antibody, tumor metastasis is severely impaired. A lack of C5a receptor in colon cancer metastatic foci reduces the infiltration of macrophages, neutrophils, and dendritic cells, and the role for C5a receptor on these cells were further verified by bone marrow transplantation experiments. Moreover, C5a signaling increases the expression of the chemokine monocyte chemoattractant protein-1 and the anti-inflammatory molecules arginase-1, interleukin 10, and transforming growth factor β, but is inversely correlated with the expression of pro-inflammatory molecules, which suggests a mechanism for the role of C5a in the inflammatory microenvironment required for tumor metastasis. Our results indicate a new and potentially promising therapeutic application of complement C5a inhibitor for the treatment of malignant tumors.

Keywords: Chemokine; Complement; Complement 5a; Macrophage; Monocyte Chemoattractant Protein-1; Tumor Metastasis; Tumor Microenvironment.

FIGURE 1.

C5a production by colon cancer cell lines and plasma from mice with hepatic metastasis of colon cancer. A, generation of C5a and C3a by colon cancer SL4 cells after 48 h incubation in serum-free medium. C5a and C3a levels were determined by ELISA. Data represent the mean ± S.E. from three independent experiments. *, p < 0.05 versus time 0. B, generation of C5a by mice colon cancer SL4 or CT26 cells after 48 h incubation in serum-free medium. C, generation of C5a by human colon cancer HCT116 or SW480 cells after 48 h incubation in serum-free medium. D, plasma C5a levels over a time course after intrasplenic injection of SL4 colon carcinoma cells and non-transformed NIH3T3 cells (5 × 10⁵) into mice.

FIGURE 2.

C5a originating from SL4 cells facilitates tumor metastasis of colon cancer. A, gross tumor of colon cancer in spleen 14 days after intrasplenic injection of SL4 colon carcinoma cells (5 × 10⁵) and SL4 sh-C5 cells into mice (n = 4). The mean ± S.E. of C5 mRNA expression in SL4 cells and SL4 sh-C5 cells are quantified by real-time PCR (right panel); **, p < 0.01 versus sh-cont. B, the mean ± S.E. of tumor weights and tumor area % of liver tissue are quantified (bottom panel); *, p < 0.05; ***, p < 0.01 versus sh-Cont. B, gross hepatic metastases of colon cancer 14 days after intrasplenic injection of SL4 colon carcinoma cells (5 × 10⁵). Mice were pretreated with control antibody (Cont IgG) or C5 antibody (C5 Ab) (n = 4). The mean ± S.E. of tumor weights (upper panel) and tumor area % of liver tissue (bottom panel) are shown. ***, p < 0.001; *, p < 0.05 versus control antibody.

FIGURE 3.

Host C5a receptor deficiency inhibits tumor metastasis. A, gross hepatic metastases of colon cancer 14 days after intrasplenic injection of SL4 colon carcinoma cells (5 × 10⁵) into wild-type (WT) and C5aR-deficient mice (upper panel) (n = 5). H&E staining shows metastatic foci of liver colon cancer 14 days after intrasplenic injection of SL4 colon carcinoma cells (5 × 10⁵) into WT and C5aR^−/− mice (n = 5) (lower panel). The mean ± S.E. of tumor weights (upper panel) and tumor area % of liver tissue (lower panel) is quantified. **, p < 0.01 versus WT. B, immunohistochemical analysis of Ki-67 expression after injection of SL4 colon carcinoma cells into WT and C5aR^−/− mice is shown (n = 6). C, TUNNEL staining shows apoptosis of liver colon cancer 1 day after intrasplenic injection of SL4 (5 × 10⁵) into WT and C5aR^−/− mice (n = 4).

FIGURE 4.

C5aR deficiency decreases inflammatory cell infiltration in hepatic metastases of SL4 colon cancer. A, CD4⁺T lymphocytes (CD45⁺CD3⁺CD4⁺) and CD8⁺ T lymphocytes (CD45⁺CD3⁺CD8⁺) (left panel); and monocytes (CD45⁺CD11b⁺), neutrophils (CD45⁺Ly6G⁺), and dendritic cells (CD45⁺CD11C⁺) (right panel) were quantified by flow cytometry analyses of blood from WT and C5aR^−/− mice. Data represent the mean ± S.E. for n = 5 mice per group. B, macrophages (CD45⁺CD11b⁺), neutrophils (CD45⁺Ly6G⁺), and dendritic cells (CD45⁺CD11C⁺) were detected by flow cytometry analyses of hepatic metastases of SL4 colon cancer in WT and C5aR-deficient mice. Data represent the mean ± S.E. for n = 5 mice per group. *, p < 0.05; **, p < 0.01 versus WT mice.

FIGURE 5.

C5aR protein expression in tumor infiltrating macrophages and neutrophils. A, double-color immunofluorescence analyses of macrophages and C5aR expression in metastatic foci in the liver from WT and C5aR^−/− mice. The sections were immunostained using a combination of anti-F4/80 and anti-C5aR antibodies. Bars = 25 μm. B, double-color immunofluorescence analyses of neutrophils and C5aR expression in metastatic foci in the liver from WT and C5aR^−/− mice. The sections were immunostained using a combination of anti-Ly6G and anti-C5aR antibodies. Bars = 50 μm.

FIGURE 6.

Bone marrow of C5aR-deficient mice confers a reduced size of hepatic metastases of colon cancer. A, gross hepatic metastases of colon cancer in bone marrow chimeric mice. WT or C5aR-deficient (KO) mice were grafted with either WT or KO bone marrow cells. Tumor samples were collected 14 days after intrasplenic injection of SL4 colon carcinoma cells (5 × 10⁵) (n = 4). B, quantification of tumor weights of colon cancer from various bone marrow chimeric mice (mean ± S.E. of 4 mice in each group). *, p < 0.05; **, p < 0.01 versus WT.

FIGURE 7.

Effect of C5a on macrophage chemokine release in SL4 cells. A, mRNA levels of MCP-1, MIP-1α, MIP-1β, MIP-2, or RANTES were detected by real-time PCR from hepatic metastases of SL4 cells in WT or C5aR^−/− mice. Results represent mean ± S.E. of expression normalized to GAPDH and versus WT mice. **, p < 0.01 versus WT. B, protein levels of chemokines, including keratinocyte chemoattractant, MCP-1, MIP-1α, MIP-1β, or RANTES were detected by CBA assay of macrophages or neutrophils from WT and C5aR^−/− mice pretreated with PBS or 50 nm recombinant C5a, subtracted from the basal level at time 0. Data represent mean ± S.E. *, p < 0.05 versus WT. C, protein levels of phospho-Akt, phospho-p38, phosphor-p42/44, total Akt, total p38, total p42/44, or actin were detected by Western blot of macrophages from WT and C5aR^−/− mice pretreated with PBS or 50 nm recombinant C5a (left panel); protein levels of MCP-1 were detected by CBA assay of macrophages from WT and C5aR^−/− mice pretreated with MK-2206 (1 μm) for 30 min, then treated with PBS or 50 nm recombinant C5a (right panel). Data represent mean ± S.E. **, p < 0.01; *, p < 0.05 versus WT control.

FIGURE 8.

C5a receptor deficiency inhibits tumor immunosuppressive capacity in metastases of colon cancer. A, mRNA levels of NOS, IL-23, IL-1β, or IL-12p40 were measured by real-time PCR of hepatic metastases of SL4 colon cancer in WT and C5aR-deficient mice. B, mRNA levels of arginase 1, TGF-β, IL-10, or IL-12p35 were measured by real-time PCR of hepatic metastases of SL4 cells in WT and C5aR-deficient mice. Data represent the mean ± S.E. for n = 5 mice per group. *, p < 0.05 versus WT mice. C, invasion assay of SL4 cells were performed with macrophages from WT and C5aR^−/− mice pretreated with PBS or 50 nm recombinant C5a. Data represent mean ± S.E. *, p < 0.05 versus WT control.