Extended Data Fig. 9: Spatial transcriptomic analysis of immune-epithelial co-occurrence in mammary glands from young adult mice (related to Fig. 7).

(a-c) Identification and annotation of spatial transcriptomics (ST) spots (shown as 1.7x size) within young mammary gland tissues (n = 2; 3 M #ST1 and 3 M ST#2) (a). UMAP visualization of ST spots, colored by ST clusters type (b). Each ST spot is classified as epithelial-enriched, immune-enriched, or stromal-enriched ST, using the expression of selected marker genes (c), and colored accordingly. (d) Proportion of ST spots expressing markers for indicated immune cell types in tissues from young adult (3 M) and older (18 M) mice, normalized to the total ST spot number for each sample (n = 2 per age group; boxplots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, replicates). (e) Expression of indicated immune marker genes in ST spots in each tissue (n = 2) after excluding the lymph node. Dot plot shows the fraction of ST spots expressing the marker gene per ST cluster (epithelial-enriched, immune-enriched, or stromal-enriched) colored by mean expression. (f) Expression of indicated immune marker genes in ST spots in mammary tissues (n = 2) from young adult (3 M) and older (18 M) mice after excluding the lymph node. Proportions are normalized to the total ST spot number expressing indicated immune marker genes for each sample (n = 2 per age group). (g) Permutation tests comparing immune marker expression profiles in mammary tissues from old (Fig. 7c) and young adult (Extended Data Fig. 9e) to random sampling (n = 10,000). Significance is calculated as a Z-score (n = 2 per age group; boxplots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, replicates).