Lung mesenchymal stromal cells influenced by Th2 cytokines mobilize neutrophils and facilitate metastasis by producing complement C3

Pre-metastatic niche formation is critical for the colonization of disseminated cancer cells in distant organs. Here we find that lung mesenchymal stromal cells (LMSCs) at pre-metastatic stage possess potent metastasis-promoting activity. RNA-seq reveals an upregulation of complement 3 (C3) in those LMSCs. C3 is found to promote neutrophil recruitment and the formation of neutrophil extracellular traps (NETs), which facilitate cancer cell metastasis to the lungs. C3 expression in LMSCs is induced and sustained by Th2 cytokines in a STAT6-dependent manner. LMSCs-driven lung metastasis is abolished in Th1-skewing Stat6-deficient mice. Blockade of IL-4 by antibody also attenuates LMSCs-driven cancer metastasis to the lungs. Consistently, metastasis is greatly enhanced in Th2-skewing T-bet-deficient mice or in nude mice adoptively transferred with T-bet-deficient T cells. Increased C3 levels are also detected in breast cancer patients. Our results suggest that targeting the Th2-STAT6-C3-NETs cascade may reduce breast cancer metastasis to the lungs.

(B) 4T1 cells were co-injected with SCR or shC3 PM-LMSCs at a 5:1 ratio (5 x 10 4 4T1 cells and 1 x 10 4 LMSCs) into BALB/c mice. Lung tissues were stained with anti-Ly6G for the identification of neutrophils, which were also analyzed by flow cytometry on day 14. n = 5, 4, 5 mice. The scale bar represents 25 μm.
(C) Neutrophils from WT and C3aR -/mice were isolated from bone marrow, and the rest of the nucleated bone marrow cells were considered as Non-Neu cells. All cells were detected for C3aR at the mRNA level.
(D) Neutrophils from WT mice were isolated from bone marrow, and the rest of the nucleated bone marrow cells were considered as Non-Neu cells. All cells were detected for C3aR at the protein level. n = 3 mice each.
(E) Recombinant murine C3a (mC3a) recruites neutrophils to the lungs. mC3a (1.25 mg/kg) or PBS was injected into BALB/c mice respectively. The mice were euthanized for the examination of neutrophils in the lungs by flow cytometry. n = 2 mice each. (E) mC3a induced neutrophils to express NETs associated genes. Neutrophils (1 x 10 6 ) were treated with mC3a (1 μg/ml) for 24 hr and examined for the expression of Pad4 and Mpo genes. n = 3 independent experiments.
(F) Immunofluorescence of H3-cit. H3-cit (red) signals were increased in lung tissues in mC3a-treated mice in comparison to control. The scale bar represents 25 μm. The images were representative of those generated from three mice each group.
All the data are presented as mean values +/-SD. ns, not significant; p < 0.05, significant, using a one-way ANOVA with Sidak post-test for Supplementary Fig. 4A; using an unpaired, two-tailed, Student's t-test for Supplementary Fig. 4C 4T1 cells and LMSCs were intravenously co-injected into BALB/c or nude mice at a 5:1 ratio (5 x 10 4 4T1 cells and 1 x 10 4 LMSCs). After 14 days, lung metastatic nodules were counted. n = 5 mice each.
(E) Immunofluorescence of H3-cit. LMSCs or PBS was injected into wild type, T-bet -/or Stat6 -/mice respectively. The mice were euthanized for the examination of NETs in the lungs 24 hr after injection of LMSCs. n = 3 mice in each group. Scale bar represents 50 μm.
All the data are presented as mean values +/-SD. ns, not significant; p < 0.05, significant, using a one-way ANOVA with Sidak post-test. Source data are provided