Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression

Abstract

Complement has emerged as a component of tumor promoting inflammation. We conducted a systematic assessment of the role of complement activation and effector pathways in sarcomas. C3^-/-, MBL1/2^-/- and C4^-/- mice showed reduced susceptibility to 3-methylcholanthrene sarcomagenesis and transplanted sarcomas, whereas C1q and factor B deficiency had marginal effects. Complement 3a receptor (C3aR), but not C5aR1 and C5aR2, deficiency mirrored the phenotype of C3^-/- mice. C3 and C3aR deficiency were associated with reduced accumulation and functional skewing of tumor-associated macrophages, increased T cell activation and response to anti-PD-1 therapy. Transcriptional profiling of sarcoma infiltrating macrophages and monocytes revealed the enrichment of MHC II-dependent antigen presentation pathway in C3-deficient cells. In patients, C3aR expression correlated with a macrophage population signature and C3 deficiency-associated signatures predicted better clinical outcome. These results suggest that the lectin pathway and C3a/C3aR axis are key components of complement and macrophage-mediated sarcoma promotion and immunosuppression.

Keywords: C3; C3a receptor (C3aR); Complement; cancer; cancer-related inflammation; immunotherapy; lectin pathway; macrophages; mannose binding lectin (MBL); sarcoma.

Extended Data Fig. 1:. The lectin pathway and the C3aR promote 3-MCA-induced sarcomagenesis and FS6 sarcoma growth.

(a-h) 3-MCA-induced sarcoma growth curves (left panels) or mean tumor volume (± SEM, right panels) in C3^−/− (a, n=20 wt, n=18 ko), MBL1/2^−/− (b, n=10 wt, n=14 ko), C4^−/− (c, n=10 mice in each group), C1q^−/− (d, n=7 wt, n=8 ko), fB^−/− (e, n=9 wt, n=12 ko), C5aR1^−/− (f, n=10 wt, n=19 ko), C5aR2^−/− (g, n=14 wt, n=10 ko) and C3aR^−/− (h, n=14 wt, n=16 ko) mice. Experiment of panel d was performed using wt littermates. (i) Representative H&E staining of 3-MCA tumor tissues of wt, C3^−/−, MBL1/2^−/−, C4^−/− and C3aR^−/− mice. One experiment performed, three representative fields have been acquired for each group (n=4 mice in each group). Scale bar: 100μm. (j-q) FS6 tumor incidences in C3^−/− (j, n= 4 wt, n=8 ko); MBL1/2^−/− (k, n=10 wt, n=9 ko); C4^-/ (l, n=11 wt, n=10 ko); C3aR^−/− (m, n=15 wt, n=12 ko); C1q^−/− (n, n=15 wt, n=8 ko); fB^−/− (o, n=9 wt, n=4 ko); C5aR1^−/− (p, n=12 wt, n=5 ko) and C5aR2^−/− (q, n=12 wt, n=8 ko) mice. (r-u) FS6 tumor volumes (mean ± SEM) (r, t) and incidences (s, u) in MBL1/2^±/− (r-s, n=12 he, n=8 ko) and C3aR^±/− (t-u, n=21 he, n=12 ko) littermates. One representative experiment out of three (a), two (b, d, e, f, j and k) or one (c, g, h, l, n, p, q, r, s, t and u) performed is shown. o, m: two pooled experiments. The same wt mice were used simultaneously as control mice of experiments of panels p, q and one out of the two pooled experiments of panel m. Two-tailed Mann Whitney test (r) or unpaired two-tailed Student’s t test (t). Exact p values are reported, two-tailed Wilcoxon matched-pairs signed rank test (j-q, s and u).

Extended Data Fig. 2:. Complement deposition on sarcoma cells.

(a) Flow cytometry analysis of C3b, iC3b, C3c deposition on cells derived from peritoneal washes of wt mice incubated with heat inactivated (HI) or normal serum. Representative FACS plot from a single mouse (right panel) and quantification of C3b, iC3b, C3c-positive cells on total cells is shown (left panel) (n=6 different mice, mean ± SEM). (b) Representative FACS plots showing the staining with digoxigenin-labeled DSA, GNA, MAA and SNA lectins or anti-digoxigenin alone in 3-MCA-derived sarcoma cells treated with 10μg/ml tunicamycin (red) or vehicle (blu). (c-d) Flow cytometry analysis of C3b, iC3b, C3c deposition on 3-MCA-derived (c) and MN/MCA1 (d) sarcoma cells incubated with normal serum from wt and MBL1/2^−/− [n=7 (c) and n=5 (d) independent experiments], C1q^−/− [n=6 (c) and n=5 (d) independent experiments], C4^−/− [n=3 (c and d) independent experiments] or fB^−/− [n=3 (c) and n=2 (d) independent experiments] (mean ± SEM). For some experiments the same normal wt serum was used as control of different ko sera. Exact p values are reported. Two-tailed Mann Whitney test (c, C4^−/− serum) or unpaired two-tailed Student’s t test (c, excluding C4^−/− serum, and d).

Extended Data Fig. 3:. Gating strategy for FACS analysis of TAMs and monocytes in MN/MCA1 sarcomas.

Representative FACS plots showing the gating strategy for FACS analysis of TAMs and monocytes in MN/MCA1 tumor samples. Gate of F4/80+ cells corresponds to FACS data panels of F4/80⁺/Aqua⁻ (%) of Figures 4d, 4j and Extended Data Figure 4b. Gate of F4/80⁺ CD206⁺ cells corresponds to FACS data panels of F4/80⁺ CD206⁺/Aqua⁻ (%) of Figures 4f, 4h, 4k and Extended Data Figure 4b. The expression of selected M1 markers (CD11c, MHC II, CD80 and CD86), reported as MFI in FACS data panels of Figures 4g, 4i, 4l and Extended Data Figure 4a–b, is gated on F4/80⁺ cells. Gate of Ly6C⁺ cells corresponds to FACS data panels of Ly6C⁺/Aqua⁻ (%) of Figures 4e, 4j and Extended Data Figure 4a–b.

Extended Data Fig. 4:. Effect of C3 and C3aR deficiency on TAMs and T cells in im injected MN/MCA1 and FS6 models.

(a) Analysis by FACS of M2 macrophage frequency (F4/80⁺CD206⁺) and of selected M1 marker (CD11c, MHC II, measured as MFI) expression gated on total macrophages (F4/80⁺) in wt and C3^−/− mice sacrificed 27 days after im MN/MCA1 tumor cell injection (n=6 wt mice, n=5 ko mice, mean ± SEM). (b) Analysis by FACS of monocyte (Ly6C⁺), macrophage (F4/80⁺), M2 macrophage frequency (F4/80⁺CD206⁺) and of CD86 (measured as MFI) expression gated on total macrophages (F4/80⁺) in wt and C3aR^−/− mice sacrificed 34 days after FS6 tumor cell injection (n=9 wt mice, n=4 ko mice; mean ± SEM). (c) Quantitation of vessel density, vascular area and vascular coverage by pericytes in 3-MCA-derived tumors of wt and C3^−/− mice (n=7 mice in each group; mean ± SEM). (d) Frequency of CD3⁺CD4⁺, CD3⁺CD8⁺ and activated effector/effector memory T cells (CD8⁺CD44⁺CD62L⁻) in wt and C3aR^−/− mice sacrificed 34 days after FS6 tumor cell injection (n=9 wt mice, n=4 ko mice; mean ± SEM). a-d: one experiment performed. Exact p values are reported, unpaired two-tailed Student’s t test or two-tailed Mann Whitney test (a-d).

Extended Data Fig. 5:. Transcriptional profiling analysis of sarcoma infiltrating monocytes.

(a) Heatmap showing the first 1000 more differentially expressed genes in wt (blue) versus C3-deficient (red) monocytes. (b) PC analysis of RNA expression of macrophages and monocytes. X and Y axes represent the first and the second PC, respectively. (c) Number of differentially expressed mouse genes and human orthologues in tumor infiltrating leukocytes.

Extended Data Fig. 6:. Gating strategy for FACS analysis of CD4⁺, CD8⁺ and CD44⁺CD62L⁻CD8⁺ T cells in MN/MCA1 sarcomas.

Representative FACS plots showing the gating strategy for FACS analysis of CD4⁺, CD8⁺ and CD44⁺ CD62L⁻ CD8⁺ T cells in MN/MCA1 tumor samples. Gate of CD4⁺ cells corresponds to FACS data panels of CD4⁺/Aqua⁻ (%) of Figures 6a, 6g and Extended Data Figure 4d. Gate of CD8⁺ cells corresponds to FACS data panels of CD8⁺/Aqua⁻ (%) of Figures 6e, 6g and Extended Data Figure 4d. Gate of CD44⁺CD62L⁻CD8+ cells (upper right panel) corresponds to FACS data panels of CD8⁺ CD44⁺ CD62L⁻/Aqua⁻ (%) of Figures 6f, 6g and Extended Data Figure 4d.

Extended Data Fig. 7:. Complement activation and prognostic significance of C3aR expression in UPS patients.

(a-d) Representative magnification images (20X) of immunostaining analysis for C1q (a), C4d (b), C3c (c) and C3aR (d) in UPS tissue sections. One experiment performed (n=19 patients), 10 representative fields have been acquired for each patient. Representative images for patients showing negative (0% IRA, left panel) or positive (>0% IRA, central and right panels) staining for C3aR expression (d). Scale bar: 100μm. (e-f) Kaplan-Meier survival curves representing the DFS (e) and the metastasis-free survival (f) for patients showing negative (n=5 patients) or positive (n=14 patients) staining for C3aR expression. Exact p value of Log-rank test for survival curves, Hazard ratio (HR) and confidence intervals (CI) are indicated in the figures (e, f).

Figure 1.. The lectin pathway and the C3aR promote 3-MCA-induced sarcomagenesis

(a-h) 3-MCA-induced sarcoma incidence in C3^−/− (a, n=20 wt, n=18 ko), MBL1/2^−/− (b, n=10 wt, n=14 ko), C4^−/− (c, n=10 mice in each group), C1q^−/− (d, n=7 wt, n= 8 ko), fB^−/− (e, n=9 wt, n=12 ko), C5aR1^−/− (f, n=10 wt, n=19 ko), C5aR2^−/− (g, n=14 wt, n=10 ko) and C3aR^−/− (h, n=14 wt, n=16 ko) mice. One representative experiment out of three (a), two (b, d, e and f) or one (c, g and h) performed is shown. Experiment of panel d was performed using wt littermates. Exact p values are reported, two-tailed Wilcoxon matched-pairs signed rank test.

Figure 2.. The lectin pathway and the C3aR promote transplanted sarcoma growth

(a-p) MN/MCA1 and FS6 tumor volumes (mean ± SEM) in C3^−/− (a, n=5 mice in each group) and (b, n=14 wt, n=8 ko); C4^−/− (c, n=10 wt, n=9 ko) and (d, n=11 wt, n=10 ko); C1q^−/− (e, n=9 mice in each group) and (f, n=15 wt, n=8 ko); fB^−/− (g, n=6 wt; n=4 ko) and (h, n=9 wt, n=4 ko); MBL1/2^−/− (i, n=7 mice in each group) and (j, n=10 wt, n=9 ko); C3aR^−/− (k, n=9 wt, n=9 he, n=8 ko) and (l, n=15 wt, n=12 ko); C5aR1^−/− (m, n=7 wt, n=6 ko) and (n, n=12 wt, n=5 ko); C5aR2^−/− (o, n=7 wt, n=4 ko) and (p, n=12 wt, n=8 ko) mice. (q) Primary tumor volume and number of lung metastasis 27 days after MN/MCA1 tumor cell im injection in wt and C3^−/− (mean ± SEM, n=10 wt, n=7 ko) mice. One representative experiment out of 15 (a), 8 (k), two (b, c, g, i, j, m and q) or one (d, f, n, o and p) performed is shown. e: Four pooled experiments performed with co-housed wt and heterozygous littermates. h, l: two pooled experiments. k: one out of 8 experiments was performed with co-housed littermates. The same wt mice were used simultaneously as control mice of experiments of panels i and m or for panels n, p and one out of the two pooled experiments of panel l. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Exact p values at the day of sacrifice are: a: p=0.0003; b: p=0.0014; c: p=0.025; d: p=0.0033; e: p=0.0218; j: p=0.0008; k: p<0.0001; l: p=0.0215; q left: p=0.0097; q right: p=0.0104. Unpaired two-tailed Student’s t test (a: 16 and 21 days; c: 18 and 21 days; e; f; g; h; k: 16 days; m; o; q: 20 and 26 days) or two-tailed Mann Whitney test (a: 12 and 19 days; c: 15 days; d; i; j; k: 19 and 21 days; l; n; p; q: 24 days).

Figure 3.. Complement deposition occurs on sarcoma cells

(a) Representative images of immunofluorescence analysis of C3 cleavage products (C3b, iC3b, C3c) deposition, vascular cells (CD31⁺ cells) and immune cells (CD45⁺ cells) in wt and C3^−/−-derived MN/MCA1 tumor tissues. Scale bar: 100μm. One out of two experiment performed (n=4 mice in each group), two-five or six fields have been acquired for each mouse in the two experiments. (b) Quantitation of C3 cleavage products (C3b, iC3b, C3c) deposition in wt and C3^−/−-derived tumor tissues (n=4 mice per group). Each dot represents the mean of fluorescence intensity (MFI) of one field; six fields have been acquired for each mouse. (c, d) Local C3a (c) and C5a (d) levels at different time points upon MN/MCA1 tumor cell injection in wt and C3^−/− mice (n=3 wt mice at 14 days, n=9 wt mice at 21 days and 9 ko mice at 21 days). b-d: Mean ± SEM is shown. (e) Flow cytometric analysis of C3 deposition on 3-MCA-derived, FS6 and MN/MCA1 sarcoma cells incubated with heat inactivated (HI) or normal serum. Representative FACS plot (left panels) and quantification of C3b, iC3b, C3c-positive cells on total cells are shown (right panels) (n=10 independent experiments for one 3-MCA-derived cell line, n=4 independent experiments for FS6 cells, n=8 independent experiments for MN/MCA1 cells, mean ± SEM). (f) Flow cytometric analysis of C3 deposition on sarcoma cells incubated with normal serum upon treatment with 10μg/ml tunicamycin or vehicle overnight. Representative FACS plots (left panels) and quantification of C3b, iC3b, C3c-positive cells (right panels) are shown (n=5 independent experiments for 3-MCA derived cells, n=4 independent experiments for FS6 cells, n=5 independent experiments for MN/MCA1 cells, mean ± SEM). Exact p values are reported. Unpaired two-tailed Student’s t test (b; e; f: for MN/MCA1; d) or two-tailed Mann Whitney test (c; e; f: for 3-MCA1 and FS6).

Figure 4.. C3 and C3aR deficiency are associated with an M1-like TAM phenotype

(a) C3aR mRNA expression in MN/MCA1 (left panel) (n=3 independent experiments) or 3-MCA derived (right panel) (n=9 independent experiments) tumor cells and in leukocytes sorted from MN/MCA1 (left panel) or 3-MCA-induced (right panel) tumors of wt mice (n=5 or 6 mice; mean ± SEM). (b) Immunofluorescence analysis of C3aR expression in wt peritoneal macrophages, used as positive control, and MN/MCA1 tumor cells. One experiment performed with two technical replicates for each cell culture, two-three fields have been acquired for each replicate. Scale bar: 10μm. (c) Immunofluorescence analysis of C3aR expression in tumor infiltrating macrophages (Iba-1+ cells) in wt derived MN/MCA1 tumor tissues. One experiment performed (n=5 mice), two-three fields have been acquired for each mouse. Scale bar: 50μm. (d, e) Analysis by FACS of macrophage (F4/80+) (d) and monocyte (Ly6C+) (e) frequency among living cells (Aqua⁻) in MN/MCA1 tumors in wt and C3^−/− (n=9 wt, n=8 ko) or C3aR1^−/− (n=6 wt, n=8 ko) mice sacrificed 21 days after tumor cell injection. (f-i) Analysis by FACS of M2 macrophage frequency (F4/80⁺/CD206⁺) (f, h) and of the expression of selected M1 markers (CD11c, MHC II, CD80 and CD86) gated on total macrophages (F4/80⁺) (g, i) in wt and C3^−/− (f, g, n=9 wt, n=8 ko) or C3aR^−/− (h, i, n=6 wt, n=8 ko) mice sacrificed 21 days after tumor cell injection. (j-l) Frequency of macrophages (F4/80⁺) and monocytes (Ly6C⁺) (j), M2 macrophages (F4/80⁺/CD206⁺) (k) and expression of M1 markers (CD11c, MHC II, CD80 and CD86) gated on total macrophages (F4/80⁺) (l) in MN/MCA1 tumors in wt and C3aR^−/− (n=15 in each group) mice sacrificed at similar tumor volume (2cm³). d and e left panels, f and g: two experiments performed; d and e right panels, h and i: three experiments performed; j and l: one experiment performed. d-l: mean is shown. Exact p values are reported, unpaired two-tailed Student’s t test (d; e; g: CD11c, CD80 and CD86 panels; h; i; j: F4/80⁺ panel; l:CD11c, MHCII and CD86 panels) or two-tailed Mann Whitney test (f, g: MHCII panel; j: Ly6C⁺ panel; k; l: CD80 panel).

Figure 5.. Transcriptional profile analysis of sarcoma infiltrating macrophages and monocytes

(a) Tree diagram for cluster analysis of FACS-sorted MN/MCA1 tumor-infiltrating macrophages and monocytes of wt (blue) and C3^−/− (red) samples (n=4 mice per group). (b) Heatmap showing the first 1000 more differentially expressed genes in wt vs C3^−/− macrophages. (c-d) Principal component (PC) analysis of RNA expression of macrophages (c) and monocytes (d). X and Y axes represent the first and the second PC, respectively. (e-f) Volcano plot showing differentially expressed genes (absolute log2FC > 1, adjusted p-value < 0.05) up-regulated (right) or down-regulated (left) in wt vs C3^−/− macrophages (e) and monocytes (f). Differential expression analyses are based on DESeq2 algorithm, which implies linear models fitting of the negative binomial distribution for each gene followed by two-sided Wald tests (null hypothesis of no difference between groups). Multiple experiment correction is applied within adjusted p-values. (g-h) Enrichment Functional Analysis using Ingenuity Pathway Analysis of differentially expressed genes (red: up-regulated, green: down-regulated) in wt vs C3^−/− macrophages (g) and monocytes (h) showing modulation of MHC class II complex. The intensity of the color indicates the degree of up- or down-regulation. Solid or broken lines indicate direct or indirect relationship, respectively. (i) Heatmap representing the expression level of genes involved in the MHC class II complex pathway in wt (blue) versus C3^−/− (red) macrophages and monocytes.

Figure 6.. Effect of C3 and C3aR deficiency on T lymphocytes and response to immunotherapy

(a) FACS analysis of helper T cell (CD3⁺/CD4⁺) frequency in MN/MCA1 tumors (left, n=9 wt mice, n=8 C3^−/− mice; right, n=6 wt mice, n=7 C3aR^−/− mice). (b) Analysis by RT-PCR of selected Th1 markers of CD4⁺ T cells sorted from MN/MCA1 tumors (mean ± SEM; n=5 mice per group). (c-e) Frequency of CD4⁺Tbet⁺ and CD4⁺Eomes⁺ (c, n=9 wt mice, n=7 C3^−/− mice; d, n=7 mice in each group), CD3⁺CD8⁺ (e, left, n=9 wt mice, n=8 C3^−/− mice; right, n=6 wt mice, n=7 C3aR^−/− mice) and activated effector/effector memory T cells (CD8⁺CD44⁺CD62L⁻) (f, left, n=9 wt mice, n=8 C3^−/− mice; right, n=6 wt mice, n=7 C3aR^−/− mice) in MN/MCA1 tumors. (g) CD3⁺CD4⁺, CD3⁺CD8⁺ and activated effector/effector memory T cell (CD8⁺/CD44⁺/CD62L⁻) frequency in MN/MCA1 tumors of mice sacrificed at similar tumor volume (2cm³) (n=15 mice in each group). a-g: mean is shown. (h) Local IFNγ in MN-MCA1 tumors (mean ± SEM; n=28 wt, n=16 C3^−/−, n=9 C3aR^−/− mice from 4 pooled experiments). (i, j) MN/MCA1 primary tumor volume in mice treated with anti-CD8 (n=12 wt mice + ctrl IgG, n=9 wt mice + anti-CD8, n=11 ko mice + ctrl IgG, n=9 ko mice + anti-CD8) (i), anti-IFNγ (n=10 wt mice + ctrl IgG, n=10 wt or ko mice + anti- IFNγ, n=9 ko mice + ctrl IgG) (j) or ctrl IgG. (k) Primary tumor volume after MN/MCA1 sc injection in mice (n=9 mice in each group) treated with anti-PD-1 or ctrl IgG. (l) MN/MCA1 primary tumor volume and lung metastasis in mice treated with anti-PD-1 or ctrl IgG, (n=6 mice in each group). (m, n) Primary tumor volume after MN/MCA1 (n=6 mice vehicle + ctrl IgG, n=7 mice vehicle + anti-PD1, n=9 mice C3aRa + ctrl IgG, n=8 mice C3aRa + anti-PD1) (m) or FS6 (n=8 mice vehicle + ctrl IgG, n=9 mice vehicle or C3aRa + anti-PD1, n=8 mice C3aRa + ctrl IgG) (n) sc injection in wt mice treated with C3aRa or vehicle, and anti-PD-1 or ctrl IgG. i-n: mean ± SEM is shown. a, e and f left panels: three experiments performed; a, e and f right panels: four experiments performed; c, d: two experiments performed; b, g, j, k, l, m and n: one experiment performed; i: two pooled experiments. Exact p values are reported, unpaired two-tailed Student’s t test (b: for Stat4, Eomes, Ifng and Tnfa; c: left panel; d; e; f: right panel; g; h) or two-tailed Mann Whitney test (a; b: for Tbet and Il2; c: right panel; f: left panel), Kruskal Wallis [p=0.0008 (i), p=0.0042 (k), p=0.0111 (l left panel), p=0.0033 (l right panel), p=0.0009 (m) and p=0.0194 (n left panel)] or Ordinary one-way Anova (p<0.0001) (j) with unpaired two-tailed Student’s t test or two-tailed Mann Whitney U-test as post-hoc tests (i-n) and two-tailed Wilcoxon matched-pairs signed rank test (n, right panel).

Figure 7.. Prognostic significance of C3 deficiency-associated signatures in sarcoma patients

(a-b) Correlation plot between C3AR1 expression level and macrophage (a) or monocyte (b) quantification scores. Blue line: linear model interpolating curve; darker bands: linear model confidence intervals (n = 263 patients). (c-d) Kaplan-Meier survival curves of TCGA sarcoma patients divided based on the median enrichment scores of C3 deficiency-associated signatures of macrophages (n=60 increasing genes) (c) and monocytes (n=85 increasing genes) (d) (n=262 patients in total, n = 131 patients in each group, the patient corresponding to the median value has been excluded by the analysis). Shaded areas of Kaplan-Meier survival curves represent 95% upper and lower confidence intervals. (e-f) Correlation plot between C3 deficiency-associated signature of macrophages (e) or monocytes (f) and a M1-like macrophage signature (n=263 patients). Blue line: linear model interpolating curve; darker bands: linear model confidence intervals. (a-f) Exact p value of two-sided Log-rank test (for survival curves) and correlation test (Spearman correlation for scatterplots) are reported.