Fig. 6: Immune activation in ESR1 mutant tumors is associated with S100A8/A9-TLR4 paracrine crosstalk.

a Expressional fold changes of immune genes from ESTIMATE (n = 141)⁷³ comparing ER+ BCK-high vs. low tumors (TCGA and METABRIC) and ESR1 WT/mutant tumors. Consistently increase, decreased or inconsistent genes in are highlighted in red, blue, and black. b BCK-high and low quantiles of ER+ tumors were further divided by the mean of S100A8 and S100A9. Immune scores were compared across all four subsets (n = 188 and 101 per group of METABRIC and TCGA) together with basal tumors (n = 328 METABRIC and n = 190 TCGA). Box plots span the upper quartile (upper limit), median (center) and lower quartile (lower limit). Whiskers extend a maximum of 1.5× IQR. Mann–Whitney U-test (two-sided) was used. c Graphical presentation of experimental strategy of d. d S100A8/9 heterodimer concentrations in plasma from patients with ESR1 WT (n = 7) and mutant (n = 11) metastatic breast cancer. Box plots span the upper quartile (upper limit), median (center) and lower quartile (lower limit). Whiskers extend a maximum of 1.5× IQR. Mann–Whitney U-test (two-sided) was utilized. This experiment was done once. e TLR4 and RAGE signature enrichments between ESR1 mutant (n = 7) and WT (n = 44) tumors. Delta GSVA score was calculated by subtracting the scores of primary tumors from the matched metastatic tumors. Box plots span the upper quartile (upper limit), median (center), and lower quartile (lower limit). Whiskers extend a maximum of 1.5× IQR. Mann–Whitney U-test (two-sided) was performed. f Violin plots showing expression of four genes by log2 normalized counts in different cell subtypes using single-cell RNA-seq data from two ER+ bone metastases. g Percent of cells expressing S100A8, S100A9, TLR4, and AGER, using single cell RNA seq data shown in f. h Immunofluorescent staining of S100A8/A9 with CD45 or EpCAM in a Y537S ESR1 mutant liver metastasis. Double positive cells are pointed out and magnified. This experiment was done once. i Percentage of S100A8/A9+ cells overlapped with EpCAM+ or CD45+ cells. Data were quantified based on six representative regions of the section. Source data are provided as a Source Data file for a, b, d–g, i.