Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression (original) (raw)

Data availability

All data are present in the main text and/or in the supplementary information. RNA-seq datasets are available in the NCBI Gene Expression Omnibus under accession number GSE141692. Data related to human sarcoma were downloaded from the cBioportal platform (http://www.cbioportal.org/study/summary?id=sarc_tcga). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

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Acknowledgements

We thank F. S. Colombo for cell sorting experiments, J. Cibella for technical help in RNA-seq experiments and F. Grizzi for technical help in immunohistochemistry experiments. The contributions of Fondazione Cariplo (Contract no. 2016-0568 to E.M.); Ministero della Salute (RF-2013-02355470 to C.G.); the European Commission (ERC project PHII-669415), Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) (PRIN 2015YYKPNN and 20174T7NXL) and Associazione Italiana Ricerca sul Cancro (AIRC IG-19014 to A.M. and AIRC IG-21714 to C.G.) and National Institutes of Health (AI068730 to J.D.L) are gratefully acknowledged. E.M. has been the recipient of a fellowship from Fondazione Umberto Veronesi (FUV, 2016–2017). We acknowledge CINECA and ELIXIR for the availability of high-performance computing resources and support.

Author information

Authors and Affiliations

  1. IRCCS Humanitas Research Hospital, Milan, Italy
    Elena Magrini, Sabrina Di Marco, Sarah N. Mapelli, Chiara Perucchini, Roberta Carriero, Andrea Ponzetta, Piergiuseppe Colombo, Ferdinando Cananzi, Domenico Supino, Clelia Peano, Antonio Inforzato, Sebastien Jaillon, Andrea Doni, Alberto Mantovani & Cecilia Garlanda
  2. Department of Biomedical Sciences, Humanitas University, Milan, Italy
    Fabio Pasqualini, Alessia Donato, Ferdinando Cananzi, Domenico Supino, Antonio Inforzato, Sebastien Jaillon, Alberto Mantovani & Cecilia Garlanda
  3. The William Harvey Research Institute, Queen Mary University of London, London, UK
    Maria de la Luz Guevara Lopez & Alberto Mantovani
  4. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
    Edimara S. Reis & John D. Lambris
  5. Institute of Genetic and Biomedical Research, UoS Milan, National Research Council, Rozzano (Milan), Italy
    Clelia Peano

Authors

  1. Elena Magrini
  2. Sabrina Di Marco
  3. Sarah N. Mapelli
  4. Chiara Perucchini
  5. Fabio Pasqualini
  6. Alessia Donato
  7. Maria de la Luz Guevara Lopez
  8. Roberta Carriero
  9. Andrea Ponzetta
  10. Piergiuseppe Colombo
  11. Ferdinando Cananzi
  12. Domenico Supino
  13. Edimara S. Reis
  14. Clelia Peano
  15. Antonio Inforzato
  16. Sebastien Jaillon
  17. Andrea Doni
  18. John D. Lambris
  19. Alberto Mantovani
  20. Cecilia Garlanda

Contributions

E.M. designed and conducted the experiments and drafted the manuscript. S.D.M. contributed to in vitro and in vivo experiments. C. Perucchini, M.G.L., A.P. and D.S. provided technological support for in vivo experiments. F.P., P.C. and F.C. contributed to histological analysis. C. Peano performed preparation of samples for RNA-seq analysis. S.N.M., A. Donato and R.C. analyzed RNA-seq and human TCGA data. A.I., S.J. and A. Doni contributed to the experimental design and data analysis. E.S.R. and J.D.L. provided gene-targeted animals and contributed to the experimental design and data analysis. A.M. and C.G. contributed to the experimental design and the supervision of the study and wrote and revised the manuscript.

Corresponding authors

Correspondence toAlberto Mantovani or Cecilia Garlanda.

Ethics declarations

Competing interests

All authors except for J.D.L. declare no competing interests. J.D.L. is the founder of Amyndas Pharmaceuticals, which is developing complement inhibitors for therapeutic purposes and is the inventor of patents or patent applications that describe the use of complement inhibitors for therapeutic purposes, some of which are developed by Amyndas Pharmaceuticals. J.D.L. is also the inventor of the compstatin technology licensed to Apellis Pharmaceuticals (4(1MeW)7W/POT-4/APL-1 and PEGylated derivatives such as APL-2/pegcetacoplan).

Additional information

Peer review information Nature Cancer thanks Wolf Fridman, David Kirsch and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

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: 100 µm. 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).

Source data

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 10 µg/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).

Source data

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 Fig. 4d,j and Extended Data Fig. 4b. Gate of F4/80+CD206+ cells corresponds to FACS data panels of F4/80+CD206+/Aqua− (%) of Fig. 4f,h,k and Extended Data Fig. 4b. The expression of selected M1 markers (CD11c, MHC II, CD80 and CD86), reported as MFI in FACS data panels of Fig. 4g,i,l and Extended Data Fig. 4a,b, is gated on F4/80+ cells. Gate of Ly6C+ cells corresponds to FACS data panels of Ly6C+/Aqua- (%) of Fig. 4e,j and Extended Data Fig. 4a,b.

Extended Data Fig. 4 Effect of C3 and C3aR deficiency on TAMs and T cells in i.m. 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 i.m. 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).

Source data

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 CD8+CD44+CD62L− T cells in MN/MCA1 sarcomas.

Representative FACS plots showing the gating strategy for FACS analysis of CD4+, CD8+ and CD8+CD44+CD62L− T cells in MN/MCA1 tumor samples. Gate of CD4+ cells corresponds to FACS data panels of CD4+/Aqua− (%) of Fig. 6a,g and Extended Data Fig. 4d. Gate of CD8+ cells corresponds to FACS data panels of CD8+/Aqua− (%) of Fig. 6e,g and Extended Data Fig. 4d. Gate of CD8+CD44+CD62L− cells (upper right panel) corresponds to FACS data panels of CD8+CD44+CD62L−/Aqua− (%) of Fig. 6f,g and Extended Data Fig. 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: 100 µm. 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).

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Magrini, E., Di Marco, S., Mapelli, S.N. et al. Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression.Nat Cancer 2, 218–232 (2021). https://doi.org/10.1038/s43018-021-00173-0

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