Fig. 4. C3 promotes NET-mediated metastasis.

A H3-cit, a marker for NETs, was reduced in C3aR^−/− neutrophils. Proteins from wild-type or C3aR^−/− neutrophils were detected using western blotting analysis. The WB images were representative of three independent experiments. B Representative immunofluorescence images showing the H3-cit staining (red) of lung tissues from WT-lung of FVB mice or A, PM, and metastatic lung of MMTV-PyMT mice. Scale bar represents 25 μm. The images were representative of those generated from three mice each group. C PM-LMSCs and M-LMSCs induced neutrophils to form NETs. Neutrophils (1 × 10⁶) were co-cultured with LMSCs from MMTV-PyMT mice at different tumor stages for 24 h and were examined for H3-cit. Left, representative images of immunofluorescence staining. Scale bar represents 10 μm. Right, increased H3-cit revealed by western blotting analysis. The IF images were representative of those generated from three independent experiments. WB was done twice. D NET formation in the lungs of tumor-bearing mice required C3. 4T1 cells were co-injected with WT or C3^−/− LMSCs into BALB/c mice. Lung tissue sections were stained anti-MPO. Scale bar represents 50 μm. The images were representative of those generated from three mice each group. E Knockdown of C3 in LMSCs reduced NETs in co-cultured neutrophils. Neutrophils (1 × 10⁶) were co-cultured with SCR or shC3 PM-LMSCs for 24 h and were examined for H3-cit. Scale bar represents 25 μm. Right, decreased H3-cit expression revealed by western blotting analysis. The images were representative of three independent experiments. WB was repeated once. F mC3a induced NET formation. Neutrophils (1 × 10⁶) were treated with recombinant mC3a (1 μg/ml) for 24 h and examined for H3-cit. Scale bar represents 10 μm. Med was the cultured medium. The images were representative of three independent experiments. WB was done twice. G DNase I abolished the metastasis-promoting activity of LMSCs. After LMSCs and 4T1 cells were co-injected into BALB/c mice, DNase I (400 U per mouse) was administered i.v. on days 1, 4, 7, and 10, and lung metastatic nodules were counted on day 14. n = 4 mice each. Data are presented as mean values ± SD. ns, not significant; p < 0.05, significant, using a one-way ANOVA with Sidak’s post test. Source data are provided as a Source Data file for Fig. 4A, C, E–G.