mRNA profiles of tumor infiltrating macrophage and monocyte derived from C3 wild-type and knock-out mice, subjected to sarcoma transplantable model

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. C3 wild-type and C3 knock-out mice were injected with mycoplasma-free MN/MCA1 cells (0.5 milion/100 µl PBS sc into the right flank). Tumors were collected 21 days after tumor cell injection and washed once with physiologic solution. Single-cell suspension were obtained by mechanical and enzymatic dissociation in 0.1 mg/ml Type IV Collagenase for 1h at +37°C. Monocytes (CD45+, CD11b+, Ly6G-, CD11c-, F4/80-, Ly6C+) and macrophages (CD45+, CD11b+, Ly6G-, CD11c-, Ly6C-, F4/80+) were sorted on a FACSAria cell sorter. RNA was purified from cells with the Single Cell RNA Purification Kit and sequenced with Illumina NextSeq 500.