C5a upregulates CD11b expression in PMN-MDSCs. (a, b) Induction of CD11b expression on PMN-MDSCs, as determined by flow cytometry analysis, obtained from the spleens (a) or tumors (b) of wild-type (WT) or C5aR-deficient (C5aR-KO) mice, after treatment with PMA or 10 nM C5a. Graphs show fold increase or decrease in the expression of CD11b in stimulated cells vs. baseline (equal to 1) in unstimulated cells from the same mice (WT or C5aR-KO). (c, d) Same analysis as described in (a,b) but for MO-MDSCs. For (a–d), bars represent mean values + SEM (n ≥ 5 mice per cohort). The significance of the induction of CD11b expression was determined using one sample t-test (*, P = 0.0232; **, P = 0.0040; ***, P = 0.0003; ****, P < 0.0001).

Lack of C5aR signaling reduces tumor growth with efficiency similar to that of Taxol treatment. (a) Tumor volumes for wild-type mice treated with C5aR antagonist (C5aRa), Taxol, or PBS (Control). “34 excised” indicates measurements of excised tumors. Horizontal lines represent mean tumor volumes for each group. The graph is representative for two independent experiments with n₁ ≥ 9 and n ₂ = 5 mice per cohort (*, P < 0.05, two-way ANOVA, Bonferroni post test). (b) Tumor volumes, as described in (a), for C5aR-deficient mice (C5aR-KO) and littermate wild-type controls (C5aR-WT) (n = 20 mice per cohort P < 0.0001, two-way ANOVA). (c) Tumor volumes, as described above, for C5aR-WT mice treated with PBS or Taxol, and C5aR-KO mice treated with PBS (n ≥ 6 mice per cohort, P = 0.004, two-way ANOVA). (d) The relative expression of C5aR in TC-1 cells, immature dendritic cells (DC), and peritoneal macrophages is shown. Data are presented as a ratio of C5aR mRNA to 10⁴ GAPDH mRNA molecules. C5aR was considered to be present if more than five copies of mRNA were detected for every 10⁴ copies of GAPDH mRNA.

Involvement of the classical pathway in the activation of complement during tumor growth. (a) Tumor volumes for C4-deficient mice (C4-KO) and littermate wild-type controls (C4-WT) measured after tumor cell injection. “24–25 excised” indicates measurements of excised tumors. Horizontal lines represent mean tumor volumes for each group. The graph is representative of two independent experiments, with n₁ ≥ 14 and n₂ = 12 mice per cohort (P < 0.0001, two-way ANOVA). (b) Tumor volumes, as described in (a), for factor B-deficient mice (Factor B-KO) and littermate wild-type controls (Factor B-WT) (n = 10 mice per cohort, P = 0.6126, two-way ANOVA). (c,d) Immunofluorescent detection of C1q, using C1q antibody (left, green fluorescence), or endothelial cells using CD31 antibody (middle, red fluorescence) in frozen sections of an end-point tumor from a wild-type mouse. The merged image (right) shows localization of C1q within the vasculature (yellow fluorescence). (d) Staining as described in (c) but using MBL antibody (left, green fluorescence) instead of C1q antibody. Colocalization is not observed in the merged image (right). For (c) and (d), images are representative of immunofluorescence studies performed on at least 5 wild-type mice, and scale bar represents 10 μm.

The migration of myeloid-derived cells into tumors is C5aR dependent. (a–e) Expression of C5aR (white areas) versus isotype controls (grey areas) on MDSCs of wild-type mice (n ≥ 5) from blood (a), spleen (b) and tumors (c–e). The same cells as shown in (d) were permeabilized before staining (e). (f) CD11b⁺ cell infiltration of tumors from control (left) or C5aR antagonist (C5aRa, right) treated mice. The white dashed line represents the tumor border. Scale bar, 30 μm. (g) Quantification of CD11b⁺ cells infiltrating tumors in relation to tumor volumes, based on (f) (n ≥ 6 mice per cohort, P = 0.0003, r = 0.7670, Pearson correlation). (h) Representative contour plot showing characteristics of CD45⁺CD11b⁺Gr-1⁺ cells from tumors from littermate wild-type (C5aR-WT) mice. R1, PMN-MDSCs; R2, MO-MDSCs. (i) The percentages of total MDSCs from tumors from C5aR-WT and C5aR-KO mice (P = 0.23, t-test). (j) Ratio of PMN-MDSCs to MO-MDSCs in the total tumor MDSC population in C5aR-WT and C5aR-KO mice (P = 0.001, t-test). (k) The percentages of CD11b⁺Gr-1⁺ MDSCs from CD45⁺ splenocytes from C5aR-WT and C5aR-KO mice. (P = 0.0024, t-test). For (i)-(k), bars represent mean values + SEM and n = 16 mice per cohort.

The anti-tumor T cell response is enhanced in mice lacking C5aR signaling. (a) CD8⁺ T cell infiltration of end-point tumor tissue in controls (left) and C5aR antagonist (C5aRa)-treated mice. Fluorescence indicates CD8 expression on infiltrating T cells. Scale bar, 30 μm. (b) Number of CD8⁺ T cells infiltrating tumors versus tumor volumes, based on immuofluorescence studies in (a), expressed as cells counted per 200× field (n ≥ 8 mice per cohort, P = 0.0180, r = −0.5653, Pearson correlation). (c) Hematoxylin and eosin-stained end-point spleen sections from littermate wild-type (C5aR-WT, left) or C5aR-deficient (C5aR-KO, right) tumor-bearing mice. Asterisks indicate areas of white pulp. (d) BrdU-positive end-point splenocytes in C5aR-WT (left) or C5aR-KO (right) mice bearing tumors. For (c) and (d), n ≥ 9 mice per cohort; scale bar, 60 μm. (e) Tumor volumes for C5aR-KO and C5aR-WT mice treated with either IgG or CD8 antibody (α-CD8). “23-24 excised” indicates measurements of excised tumors. Horizontal lines represent mean tumor volumes for each group (n ≥ 9 mice per cohort). Statistically significant differences (two-way ANOVA) were observed between: C5aR-WT + IgG vs. C5aR-KO + IgG (P = 0.0003) and C5aR-KO + IgG vs. C5aR-KO + α-CD8 (P = 0.0006).

Complement activation plays a role in tumor growth. (a, b) Immunofluorescent detection of complement cleavage products using C3 antibody (left, green fluorescence) or endothelial cells using CD31 antibody (middle, red fluorescence) in frozen sections from a wild-type mouse in an end-point tumor (a) or surrounding benign tissue (b). The merged image (right) shows the localization of complement cleavage products within the vasculature (yellow fluorescence) or in its close proximity (green fluorescence). Scale bar, 10 μm. Images are representative of immunofluorescence studies performed on at least 3 wild-type mice. (c) Tumor volumes for individual C3-deficient mice (C3-KO) and littermate wild-type controls (C3-WT) measured on various days after tumor cell injection. The last panel (25-26 excised) indicates volumes based on measurements obtained after mice were sacrificed and the tumors removed. Horizontal lines among each group of data points represent the mean tumor volume for that group. The graph is representative of three independent experiments, each with n = 10 mice per cohort (P < 0.0001 for the entire course of the experiment, two-way ANOVA).

C5a enhances the suppressive capabilities of tumor associated-MDSCs by regulating ROS and RNS production. (a) Inhibition of PHA-induced proliferation of CD3⁺ splenocytes from non-tumor-bearing wild-type mice in the presence of Gr-1⁺ MDSCs from tumors from wild-type (C5aR-WT) or C5aR-deficient (C5aR-KO) mice (n = 3 per cohort). (b) Representative histogram illustrating ROS and RNS production in MDSCs from tumors from C5aR-WT (grey area) and C5aR-KO (white area) mice. (c) Quantification of ROS and RNS production by MDSCs from tumors from C5aR-WT and C5aR-KO mice (P = 0.0210, Wilcoxon). (d) Quantification of ROS and RNS production by PMN-MDSCs and MO-MDSCs from tumors of C5aR-WT and C5aR-KO mice (*P = 0.0342, ** P = 0.0005, Wilcoxon). For (c) and (d), bars represent mean values of median fluorescence + SEM, and n ≥ 12 mice per cohort. (e) Arginase-1 expression in tumors from control and C5aR antagonist-treated (C5aRa) mice. (f) Quantification of immunoblot shown in (e) (P = 0.0844, t-test). (g) Correlation between arginase-1 expression from (f) and tumor volumes in control and C5aRa-treated mice (Control P = 0.0256, r = 0.8147 and C5aR P = 0.0105, r =0.7947, Pearson correlation). (h) Induction of ROS and RNS in PMN-MDSCs, from the spleens of wild-type (C5aR-WT) or C5aR-deficient (C5aR-KO) mice, after treatment with PMA or 10 nM C5a. Graph shows fold increase in ROS and RNS in stimulated cells vs. baseline in unstimulated cells from the same mice. (i) Same analysis as described in (h) but for MO-MDSCs. For (h) and (i), bars represent mean values + SEM and n ≥ 5 mice per cohort; *, P = 0.0382; **, P = 0.0270; ***, P = 0.0245; ****, P < 0.0092, one sample t-test.