Fig 2: Oncogenic activation of S100A9 in both Brca1-MT mice and human breast cancer patients.a Workflow of DIA-MS analysis with mammary tissues during tumorigenesis, including mammary gland tissues from 8–10 months Wild type (WTMG) and Brca1-MT (MTMG) virgin mice, tumor- adjacent mammary tissues (Tu-Adj. MG), and tumors from both Wild type (WTBT) and Brca1-MT (MTBT) mice (n = 3 mice/per genotype). b Plot of Principal Component Analysis (PCA) of samples from the same cohort of samples in (a). c Pearson correlation analysis of duplicate of each sample from the same cohort samples in a. d The patterns of increased the protein level of S100A9 during tumorigenesis with the same cohort of samples in a from DIA data analysis with Spectronaut and statistics analysis by Pearson correlation (FDR < 1, r = 0.8343, adjust p value = 0.024. The raw data are transformed by log10,the box and whisker plots summarize the normalized values, line show the SD). The data are from n = 3 biologically independent replicates. e Pearson correlation of BRCA1 and S100A9 at protein level by volcano plot from Clinical Proteomics Tumor Analysis Consortium (CPTAC) for The Cancer Genomic Atlas (TCGA) database (http://www.linkedomics.org/admin.php). f Heatmap of top 50 genes which are negatively correlated with BRCA1 at protein level from e. g Overlapping of mouse candidate gene lists from DIA analysis with top 45 genes which are negatively regulated by BRCA1 genes. h The representative images of S100A9 antibody staining on BRCA1-WT and BRCA1-MT breast cancer tissues (n = 2 in each group from PDX models) were obtained from Jackson Laboratory. i Quantifications of S100A9 positive cells per field in h, (n = 22 pictures from WT and 30 from MT in h models). j Survival outcome based on S100A9 expression of breast cancer patients (n = 4934) from Kaplan–Meier Plotter website (https://kmplot.com/analysis/index.php?p=service&cancer=breast). k, l Relative expression of S100a9 and S100a8 of mammary tissues in 4-months Brca1-WT and MT virgin mice (k) and Brca1 MT and WT mammary tumors (l) revealed by qPCR (n = 3 mice/group) and the number of dots represents biological replicates). m, n Protein levels of S100a9 and S100a8 in 4-month-old mammary tissues (m), and in Brca1 MT and WT tumors (n) as determined by western blots (n = 3 mice/group). The data are expressed as means ± SD (d, i, k, l) and P values determined by unpaired two-tailed Student’s t test (i, k, l). Scale bar: black color, 50 µM. Source data are provided as a Source data file.

Fig 3: Brca1 deficiency induces immunosuppression in mammary glands and tumors.a tSNE analysis of immune cells from wildtype (WTMG, n = 6 mice), Brca1-mutant mammary glands (MTMG, n = 6 mice), Tu-Adj. mammary tissues (MT tumor adj. MG, n = 3 mice) from Brca1-MT mice, WT breast tumor (WT BT, n = 3 mice) and breast tumor (MT BT, n = 3 mice) from Brca1-MT mice. b, c The cell populations were classified as total T-cells (CD45+CD3e+), CD4+ T cells (CD45+CD3e+CD4+ and CD8-), CD8+ T cells (CD45+CD3e+CD4-CD8+), MDSCs (CD45+CD11b+Gr1+), PMN-MDSCs (CD45+CD11b+Ly6G+Ly6C-), and M-MDSCs (CD45+CD11b+Ly6G-Ly6C+). b Quantifications of total T-cells, CD4, and CD8 cells. c Quantifications of PMN-MDSCs, M-MDSCs, and total MDSCs by FlowJo analysis (Cytof gating strategies see in Supplementary Fig. 8a, n = 3–6 mice same in a). d–g Representative images of IHC staining with antibodies of S100a9 (d) or S100a8 (f), and quantifications of S100a9 (e) or S100a8 (g), (n = 6–15 pictures from 3 mice/group, 6 pictures from WTMG, 9 from MTMG, 6 from adj.MG, 15 from WTBT, and 9 from MTBT, the number of each group same in e and g). h The strategy of isolating CD11B+/GR1+ cells from mammary gland (MG), breast tumor (BT), and spleens (SP) for RNA-sequence and CD8 + T cells suppressive assay from the mice in a. i Principal Component Analysis (PCA) analysis of RNA-sequence data of CD11B+/GR1+cells in h (n = 3 mice/group). j MDSC signature genes enrichment in spleen by GSEA analysis by comparing gene expression from spleens of Brca1-MT (MT SP) vs WT (WT SP) mice and Brca1-MT mice with breast tumor (MT-BT SP) vs MT SP. k MDSC signature genes enrichment in mammary gland (MG) and breast tumor tissues (BT) by GSEA analysis by comparing gene expression from Brca1-MT mammary glands (MT MG) vs wild-type mammary glands (WT MG), and tumors from Brca1-MT (MT BT) and WT (WT BT) mice. l Representative CFSE flow-cytometry histograms and statistics from co-culture of WT T-cells with MDSCs from spleens. m Quantifications of % of CD8 + cells in each sample in l. The T-cell were from 2 months WT mice and MDSCs from spleens of 10-month Brca1-MT mice (Brca1-MT SP), Brca1-WT tumor-bearing mice (WT-BT SP) and Brca1-MT tumor-bearing mice (Brca1-MT-BT SP). Ratio of MDSC to T cells was 1:1 (n = 3 mice/group). n Representative CFSE flow-cytometry histograms and statistics from co-culture of WT T-cells with MDSCs from MG and BT. o Quantifications of % of CD8 + cells in each sample in n. The T-cell were from 2 months WT mice and MDSCs from MG of 10-month Brca1-MT mice (Brca1-MT MG), breast tumor tissues of Brca1-WT tumor-bearing mice (WT BT) and Brca1-MT tumor-bearing mice (Brca1-MT BT). Ratio of MDSC to T cells was 1:1 (n = 3 mice/group) (FACS gating strategies see in Supplementary Fig. 8b). The data are expressed as means ± SD (b, c, e, g, m–o) and P values determined by one-way ANOVA followed by Tukey’s multiple comparisons and permutation test. Scale bar: black color, 50 µM. Source data are provided as a Source data file.

Fig 4: Positive regulation loop between S100a9 and Cxcl12 amplifies oncogenic signals in Brca1-MT epithelial cells.a The Venn diagram analysis of upregulated genes in G600, Over-Expression-S100a9 in B477 cells (B477-OE-S100a9), and down-regulated genes in G600 cells expressing sgS100A9 (G600-sgS100a9) (n = 3 biological replicates/group). b KEGG Pathway analysis with 453 common (p value was calculated by hypergeometric test with KOBAS 3.0 website). c Top 20 differentially expressed genes by comparison of the gene expression profiles of four different group cells, including B477-Ctr (B1-3), B477-OES100a9 (P1-3), G600-Ctr (G1-3), and G600-sgS100a9 (G10-1-3) (the heatmap was drawn by Morpheus website and clustered by One minus Pearson Correlation). d, e Expressions of S100a9 and Cxcl12 at mRNA level by qPCR (d) at protein levels by western blotting (e) from four group cells in c. f Protein levels of S100a9, Cxcl12 and pStat3 in OE-S100a9-EMT6 cells, sgS100a9/OE-S100a9-EMT6, and sgCxcl12/OE-S100a9-EMT6 cells. g Protein levels of S100a9, Cxcl12 and pStat3 in OE-S100a9-EMT6 and G600 cells without or with S100A9 inhibitor, Tasquinimod (Tas-50 µM) by western blotting. h Protein levels of S100a9, Cxcl12, and pStat3 in WT (B477) cells with the expression of OE-S100a9, OE-S100a9/sgCxcl12, or OE-Cxcl12 by western blotting. i, k Protein levels of Cxcl12 in B477 (i) cells with OE-S100a9 and in G600 (k) cells expressing sgS100a9 at different time courses (0–48 h) and different amounts (0–4 µg) by western blotting. j, l Protein levels of S100a9 in B477 (j) cells with OE-Cxcl12 and in G600 (l) cells expressing sgCxcl12 at different time courses (0–48 h) and different amounts (0–4 µg) by western blotting. m Protein levels of S100a9, Cxcl12, and pStat3 in G600 cells after treating with Tas and FPS-ZM1, inhibitor for RAGE receptors on cell membranes by western blotting. n Protein levels of S100a9, Cxcl12, and pStat3 in B477 cells with the expression of shBrca1 and MT G600 cells with over-expression of Brca1 (OE-Brca1). o A diagram summarizing the relationship of Brca1, S100a9, Cxcl12 and pStat3 in Brca1-MT mammary epithelial cells, and their potential interaction with the surrounding immune microenvironment, which remains elusive. The data are expressed as means ± SD (d) and P values determined by one-way ANOVA followed by Tukey’s multiple comparisons. The experiments were independently repeated three times with similar results (d–n). Source data are provided as a Source data file.

Fig 5: S100A9 blockade improves the recovery of the injured cord after SCI. (A) H&E staining for histological analysis 28 days post-OR among the different experimental groups. Scale bar, 1 mm (upper panel) and 200 µm (lower panel). (B) Relative cavity sizes were calculated by dividing the cavity volume by the spinal cord volume at the lesion site in the vertical direction. (C) BBB score evaluation for motor function after SCI among the different experimental groups. Data are presented as the mean ± SD. n=5. *P<0.05, ***P<0.001. #P<0.05. S100A9, S100 calcium-binding protein A9; SCI, spinal cord injury; post-OR, after the operation; BBB, Basso-Beattie-Bresnahan; High-ABR, high ABR dose; Low-ABR, low ABR dose; n.s., not significant.