Extended Data Fig. 6: The role of the CCR1-CCL9 axis in regulating splenic neutrophils during 4T1 tumor progression.

(a) Chemokine receptor expression in neutrophils from RNA-seq data; n = 5, mean ± SD. (b) The expression of CCR1, CXCR2, and CXCR4 in splenic CD45⁺Ly6G⁺ and CD45⁺Ly6G⁻ cells of 4T1 tumor bearing mice. (c) CXCR2 expression on CXCR4^lowCCR1^high and CXCR4^highCCR1^low neutrophils. (d) CCR1 expression on Ly6G⁺c-Kit⁺ neutrophils of tumor bearing mice. Histogram shows CCR1 expression in splenic Ly6G⁺ or Ly6G⁺c-Kit⁺ neutrophils of mice bearing 4T1 tumor. (e) scRNA-seq data from GSE139125 were downloaded and analyzed following the guideline of Seurat. Featured genes from subpopulation 2 with CCR1 high expression were used for KEGG enrichment analysis. (f) Chemokine expression in the spleen of healthy and 4T1 tumor-bearing mice; n = 4 mice, mean ± SD. (g) Ccl6 expression in the spleen of healthy (n = 3), 4T1-bearing mice (n = 4) or tumor bearing mice with neutrophil depletion (α-Ly6G, n = 4); mean ± SEM. (h) Relative Ccl9 expression in the bone, spleen, and draining lymph nodes (LN) of 4T1 tumor bearing mice and healthy controls; n = 6 mice, mean ± SEM. (i) Transwell assay showing the role of CCL9 in neutrophil chemotaxis; n = 3, mean ± SD. (j) Neutrophil distribution in the spleen of 4T1 tumor-bearing mice with or without CCL9 neutralizing antibody administration (Left) and statistical analysis of the distance of neutrophils to lymphoid nodules in the spleen (Right); n = 37–51 fields from 4 individual mice, mean ± SD. (k) Cleaved CCL9 stimulates HL60 proliferation. Cleaved CCL9 was over-expressed in 293T cells; n = 6, mean ± SD. (l) The expressions of C/EBPα, C/EBPβ, and MYC (m) in HL60 cells treated with cleaved CCL9. Data are representative of at least two independent [(d), (f) - (l)] experiments. All replicates are biological replicates. P values were determined by unpaired two-tailed Student’s t-test [(f) – (k)].

Source data