Simple SummaryThe complement system is activated within the tumor microenvironment and its role in cancer development has raised much attention over the last decade. The aim of our study was to investigate the effects of the C3a anaphylatoxin on tumor cells (B16/F0 melanoma cell line) and two cell components of the tumor microenvironment: macrophages (Raw 264.7 Blue cell line) and mesenchymal stem cells (3T3-L1-like cell line). We showed that C3a plays a crucial role in the tumor microenvironment and may influence the tumor’s fate by modulating the expression of cytokines (including IL-10, TGFβ1), chemokines, Cox-2, and HO-1, and upregulating the oxidative stress response. We also demonstrated that C3a regulates VEGF expression, suggesting a role of the C3a/C3aR axis in angiogenesis. Our results provide novel insights into tumorigenesis and open new therapeutic avenues (C3aR antagonists) in cancer therapy.AbstractThe complement system plays a crucial role in cancer development. Our study investigated the role of C3a anaphylatoxin on the tumor microenvironment. Our models consisted of mesenchymal stem cells (MSC-like, 3T3-L1), macrophages (Raw 264.7 Blue, (RB)) and tumor cells (melanoma B16/F0). Recombinant mouse (Mo) C3a (rC3a) was produced in CHO cells transfected with a Mo-IL10-signal peptide-Mo C3a plasmid construct. The effects of rC3a, IFN-γ, TGF-β1, and LPS were tested on the expression of C3, C3aR, PI3K, cytokines, chemokines, transcription factors, antioxidant defense mechanisms, angiogenesis and macrophage polarization (M1/M2). 3T3-L1 expressed the highest levels of C3, while C3aR was expressed more by RB. Interestingly, expression of C3/3T3-L1 and C3aR/RB was markedly upregulated by IFN-γ. rC3a was found to upregulate the expression of anti-inflammatory cytokines (IL-10) on 3T3-L1 and TGF-β1 on RB. rC3a also upregulated the expression of pro-inflammatory cytokines in RB. The expression of CCL-5 increased in 3T3-L1 in response to rC3a. On RB, rC3a did not alter M1/M2 polarization but upregulated the expression of antioxidant defense genes, HO-1, and VEGF. C3/C3a produced mainly by MSC may play a critical role in TME remodeling by stimulating both anti-inflammatory and proangiogenic activities of tumor stromal cells.