Fig 1: Tumor neutrophil ferroptosis was related to a distinctive subset of CD4 T cells enriched in chemoresistant tumors. A, Uniform Manifold Approximation and Projection (UMAP) visualization of tumor-infiltrating CD4 T cells from five chemosensitive and five chemoresistant breast cancers by scRNA-seq. B, Three-dimensional UMAP plot of CD4 T cells colored by chemosensitivity. C, The proportions of different subpopulations of CD4 T cells in sensitive and resistant tumors. D, Heatmap displaying scaled expression of discriminating genes for each cluster of CD4 T cells in scRNA-seq data. E, Heatmap for IL1B expression in CD4 TILs from nine chemosensitive and nine chemoresistant patients by bulk RNA-seq. F, Representative flow cytometry for IL1β and CXCL3 expression in chemosensitive and chemoresistant tumor-infiltrating CD4 T cells. G, Cell death ratio of peripheral neutrophils pretreated with the inhibitors for apoptosis, necrosis, or ferroptosis and cocultured with IL1β+CXCL3+-d or IL1β+CXCL3+ CD4 T cells. H, Representative flow cytometric images for cellular lipid peroxidation of peripheral neutrophils cocultured with different CD4 T cells. I, Proliferation of tumor-specific CTLs upon exposure to peripheral neutrophils pretreated with IL1β+CXCL3+ or IL1β+CXCL3+-d CD4 T cells. J, Representative immunofluorescence images (left) and quantification (right) for IL1β and CXCL3 expression in CD4 T cells in chemosensitive and chemoresistant breast cancer sections. Arrows, IL1β+CXCL3+ CD4 T cells. Scale bar, 50 μm. K, Correlation between IL1β+CXCL3+ CD4 T cells and ferroptotic neutrophils in breast tumor specimens. Results are represented as mean ± SD of n = 5 (A–D and G–I), n = 9 (E and F), or n = 468 (J) different patients; for K, n = 468 different patients; ***, P < 0.001 by Student t test (F), two-sided one-way ANOVA with the Tukey test (G, I, and J) or two-tailed Pearson correlation coefficient test (K). Quantification is shown in Supplementary Figs. S4 and S5 (F and H).
Fig 2: Cross-talk between neutrophils and Fer-CD4 T cells maintained extensive neutrophil ferroptosis. A, Gene Ontology (GO) terms associated with upregulated genes of C2_IL1B cluster in scRNA-seq. B, Gene set enrichment analysis of bulk RNA-seq revealed enrichment of neutrophil chemotaxis genes in chemoresistant CD4 TILs. C, Scheme of chemotaxis assays with Boyden transwell chambers. D, Representative images (left) and quantification (right) of chemotaxis assays for peripheral neutrophils toward CM of different CD4 TILs. Scale bar, 100 μm. E, Migration tracks of neutrophils in μ-slide chemotaxis experiments toward CM of non–Fer-CD4 or Fer-CD4 T cells. F, Representative fluorescent images of cytoskeleton staining (left) and quantification (right) of filopodium-like protrusions (FLP) of peripheral neutrophils in the presence of CM from non–Fer-CD4 or Fer-CD4 T cells. Scale bar, 5 μm. G, Ex vivo tumor slice migration assays for the recruitment of CFSE-labeled neutrophils into breast tumor slices with high or low density of Fer-CD4 T cells. Scale bar, 100 μm. H, Dot plot for the expression of various chemokines in C2_IL1B cells in scRNA-seq. I, ELISA for CXCL3, IL8, and S100A9 release of non–Fer-CD4 or Fer-CD4 T cells. J and K, Migration (J) and filopodium-like protrusions (K) of peripheral neutrophils in the presence of CM from Fer-CD4 T cells and different antibodies. L, Cell death of peripheral neutrophils in the presence of CM of Fer-CD4 T cells and different antibodies categorized by location (top or bottom chamber of transwell system). M, CXCL3 and IL1β protein level of naïve CD4 T cells by immunoblotting after coculturing with control or ferroptotic neutrophils, which were generated by engagement with non–Fer-CD4 or Fer-CD4 T cells, respectively. Results are represented as mean ± SD of n = 7 (D) or n = 5 (F and I–M) or n = 6 (G). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-sided one-way ANOVA with the Tukey test (D, F, and J–L) or Student t test (G, I, and M). Representative images are shown in Supplementary Fig. S7 (J and K).
Supplier Page from Biorbyt for Human CXCL3 ELISA Kit
Application Notes: Detection Wavelength: 450 nm