Fig 2: Chronic psychological stress mobilizes spleen-derived MDSCs into the lung in a CXCR2-dependent manner. A Representative pictures and growth curves of tumors in CXCR2WT and CXCR2KO mice under CUMS (n = 6). B The fluorescence intensity of lung metastases in CXCR2WT and CXCR2KO mice under CUMS (n = 3). C Representative lung images in HE staining for 2–4 weeks in CXCR2WT and CXCR2KO mice bearing breast cancer under CUMS (black circle indicates the metastatic lesions of the lung) (n = 3). D Representative immunofluorescence images for PMN formation for 2–4 weeks in the lungs of CXCR2WT and CXCR2KO mice bearing breast cancer under CUMS (CK19 labeled cancer cells, Gr-1 and CD11b labeled MDSCs, the scale bars indicate 10 µm). E MDSCs population in the lung, peripheral blood, and spleen of CXCR2WT and CXCR2KO mice bearing breast cancer under CUMS. F Flow diagram for establishing CXCR2WT/KO bone marrow chimeric mice and follow-up detection. CXCR2WT/WT mice that underwent sham surgical operation served as control (upper panel). Representative immunofluorescence images for the recruitment of exogenous mCherry-labeled MDSCs to the lung and PMN formation in the lung (lower panel). G Flow diagram for establishing CXCR2KO/WT bone marrow chimeric mice and follow-up detection. CXCR2KO/KO mice that underwent sham surgical operation served as control (upper panel). Representative immunofluorescence images for the recruitment of exogenous mCherry-labeled MDSCs to the lung and PMN formation in the lung (lower panel). Data are represented as Mean ± SD. For statistical analysis, repeated measures analysis of variance (A) and unpaired t-tests (E) were applied. *P < 0.05, #P < 0.01

Fig 3: CXCL1/CXCR2 signaling regulates the proliferation, motility, and immunosuppressive activity of spleen-derived MDSCs. A MDSCs proliferation under different treatments for 24 h was determined by EdU assay. MDSCs motility was assessed using the Transwell chamber. B Proliferation and motility of spleen-derived MDSCs from CXCR2 knockout mice (CXCR2KO) or CXCR2 wild-type mice (CXCR2WT) were detected with or without CXCL1 (10 ng/ml) treatment for 24 h. C The proliferation of CD8+ T cells in the MDSCs (from CXCR2WT or CXCR2KO mice) co-culture system was detected by CFSE assay. D-E The granzyme B and perforin levels were measured in the MDSCs (from CXCR2WT or CXCR2KO mice) and CD8+ T cells co-culture system. F MDSCs were untreated or pretreated with CXCL1 (10 ng/ml) for 24 h. The capability of MDSCs to induce 4T1 migration was detected in the transwell co-culture system. G Flow diagram for 4T1 cell apoptosis detection (upper panel). MDSCs were untreated or pretreated with CXCL1 (10 ng/ml) for 24 h. MDSCs were then co-cultured with 4T1 and CD8+ T cells (CD8+T: MDSCs: 4T1 = 5: 2.5: 1) for 24 h. The 4T1 cells were collected and further distinguished by CD45− gating. The ability of MDSCs inhibiting CD8+ T-induced apoptosis in 4T1 cells was measured using AnnexinV and 7-ADD labeling (lower panel). Data are represented as Mean ± SD. For statistical analysis, unpaired t-tests were applied. *P < 0.05, #P < 0.01