Fig. 1: Tumor-associated PreNeu predominantly infiltrate highly proliferative ER⁺ breast cancers.

a UMAP plots derived from PhenoGraph cluster analyses of concatenated low proliferative (n = 3) and highly proliferative (n = 3) breast cancer biopsies gated on CD45⁺ cells. Cluster 1: CD11b⁺, CD33⁺, CD66b⁺, CD15⁺, CD163⁻, LOX-1⁻; Cluster 2: CD11b⁺, CD33⁺, CD66b⁺, CD15⁺, CD163⁻, LOX-1⁺; Cluster 3: CD11b⁻, CD33⁻, CD66b⁺, CD15⁻, CD163⁻, Lox-1⁺; Cluster 4: CD11b⁻, CD33⁻, CD66b⁻, CD15⁻, CD163⁻, LOX-1⁻. b Bar graph displaying the frequency of the four clusters in normal tissues (NT, n = 3), low proliferative ER⁺ breast cancer biopsies (Low proliferative ER⁺ BC; n = 48) and highly proliferative breast cancer biopsies (Highly proliferative ER⁺ BC; n = 15). c UMAP plot derived from BD Rhapsody scRNA-seq cluster analysis of the neutrophil compartment in a highly proliferative ER+ tumor (n = 1). d Metascape analysis of enriched gene pathways of cluster C neutrophil compared to clusters A and B. Saturation of red color is proportional to p value significance. e Upregulated genes (FDR < 0.01; log2FoldChange > 1) in PreNeu compared to LOX-1⁺ neutrophils sorted from highly proliferative ER⁺ breast cancer biopsies resulting from single-cell differential expression analysis (Smartseq-2). Significantly upregulated genes are organized into a protein-protein interaction network generated using String Database. The dimension of the nodes is proportional to the highest statistical significance (represented as −10xlog10FDR). Saturation of red color is proportional to increased gene expression in PreNeu compared to LOX-1⁺ neutrophils (represented as log2FoldChange). Yellow color indicates the manually selected genes. Highlighted in yellow are genes belonging to the magnified subnetwork shown on the right of the figure panel. f UMAP plot derived from BD Rhapsody scRNA-seq data displaying the enrichment score of the PreNeu signature derived from scSMART-seq analysis based on significantly upregulated genes. g Pseudotime trajectory embedded in the UMAP plot with cluster C as the starting point of the trajectory (green line). h Bar graph showing the percentage of Ki67 positive cells in the different populations of neutrophils (from n = 3 patients). Data are represented as mean ± SEM. Statistical analyses (one-way analyses of variance, ANOVA, multiple comparison test). i Bar graph displaying the percentage of cluster 3 cells within CD45⁺ cells in normal tissues (NT, n = 3), low proliferative (LP, n = 48), or highly proliferative (HP, n = 15) ER⁺ breast cancer biopsies. Each symbol represents an individual patient. Data are represented as mean ± SEM. Statistical analyses (one-way analyses of variance, ANOVA, multiple comparison test). j Cluster 3 relative abundance on CD45+ cells stratified based on tumor grade (n = 9 for grade 1, n = 40 for grade 2, n = 13 for grade 3). Data are represented in violin plots with median (bold line) and quartile (regular lines). Statistical analyses (Brown-Forsythe and Welch ANOVA tests). k cluster 3 to cluster 2 ratio in NT (n = 3), LP (n = 48), and HP (n = 15) ER⁺ breast cancer biopsies. Each symbol represents an individual patient. Data are represented as mean ± SEM. Statistical analyses (one-way analyses of variance, ANOVA, multiple comparison test). l Bar graph displays the percentage of cluster 2 within CD45⁺ cells in NT (n = 3), LP (n = 48), and HP (n = 15) ER⁺ breast cancer biopsies. Each symbol represents an individual patient. Data are represented as mean ± SEM. Statistical analyses (one-way analyses of variance, ANOVA, multiple comparison test). Source data are provided as a Source data file.