Fig. 4

C3aR signaling promotes the pro-meatastatic function of CAF. a CAFs were sorted by Flow cytometry as PDGFRa⁺F4/80⁻ cells of 4 T1 tumor tissues from WT or C3aR^−/− mice. Immunofluorescence analysis of C3aR expression in WT and C3aR^−/− CAF. b The migratory properties of 4 T1 cells cultured with WT and C3aR^−/− CAFs detected in scratch assays (*P < 0.05). c-d The migration and invasive capability of 4 T1/EO771 tumor cells co-cultured with WT CAFs and C3aR^−/− CAFs (*P < 0.05). CAF obtained from 4 T1 tumor-bearing WT mice were stimulated with rmC3a(0.5μg/ml) for 24 h, the expression of α-SMA was analyzed by immunofluorescence (e) and western blotting assay (f). The software ImageJ was used to qualify the signal intensities of the western blot, and the ratio of α-SMA/β-actin is presented. g Quantitative PCR analysis of mRNA level of CAF markers (PDGFRA, FAP, ACTA2) and functional cytokines (TGF-β1, HGF, VEGFA) in treated or untreated CAFs was performed. h-i 4 T1 cells were co-injected with CAFs derived from WT or C3aR^−/− mice in the mammary fat pad. The number of 4 T1 lung metastasis tumor burden was calculated after 28 days. Data are expressed as the mean ± SEM. (*p < 0.05,**p < 0.01,***p < 0.001)