Extended Data Fig. 3: Transcriptional analysis of splenic neutrophils.
From: Glycolytic neutrophils accrued in the spleen compromise anti-tumour T cell immunity in breast cancer
(a) Neutrophils were isolated from the blood, bone marrow, lung, spleen, and primary tumor of mice bearing 4T1 tumor and analyzed by flow cytometry. The purity of neutrophils was detected before and after cell sorting. Neutrophils are pooled from 6 individual mice. (b) KEGG enrichment analysis of differentially expressed genes in neutrophils from the lungs or tumor versus spleen. (c) Heatmap showing the expression of immunoregulating factors in neutrophils from different tissues. (“N1” neutrophil markers: Fas, CCl3, Nos2; “N2” neutrophil markers: Ccl2, Arg2, Mrc1). (d) Highly purified T cells and neutrophils were isolated from the spleen of 4T1 tumor-bearing mice and subjected to metabolomics analysis. Differential metabolites in neutrophils and T cells were used for enrichment analysis by an online toolkit at https://www.metaboanalyst.ca/. (e) Pan T cells were isolated from naïve mice and co-cultured with splenic neutrophils from 4T1 tumor-bearing mice at a 2:1 ratio, in the presence of titrated glucose concentrations. After anti-CD3/CD28 stimulation for 3 days, CCR7 expression was determined by flow cytometry. (f) Comparison of total ROS production by neutrophils from the bone marrow, spleen, and blood with or without PMA stimulation. Data are representatives of two [(e) - (f)] independent experiments.
