Extended Data Fig. 1: Analysis of TAMs and astrocytes in BCBM.

a Whole mount brightfield and fluorescent microscopy images of metastatic brains (Met1-3) used to generate the scRNA-seq dataset described in Fig. 1d. Metastatic lesions are GFP⁺ (green). Results are representative of a single experiment. Scale bar = 50 mm. b IF staining shows IBA1⁺ cells (red) in normal human brain and three resected patient BCBM tumors. Insets show cell morphology, exhibiting evenly spaced, ramified microglia in normal human brain contrasting heavily infiltrated ameboid microglia in BCBM patients. Results are representative of a single experiment. Scale bar = 50 μm. c Representative FACS plots show gating for single, live (Sytox negative) myeloid cells (CD45⁺CD11b⁺), astrocytes (CD45⁻ASCA2⁺) and 231BR cells (CD45⁻GFP⁺) isolated for scRNA-seq. d Identification of mouse and human cells by the frequency of reads that align to the mm10 mouse genome. Cutoffs used to identify mouse cells (>0.875 aligned, n = 51,418 cells), human cells (<0.05 aligned, n = 7336 cells) and doublets (0.05–0.875 aligned, n = 913 cells) are shown. e Violin plots show cell distributions for key quality control metrics pre- and post- filtering and removal of poor quality cells. Cells were removed that displayed <500 or >2000 genes (nFeature_RNA), or >10% of genes mapped to the mitochondrial genome (percent mito genes). f Bar chart shows the frequency of cells contributed by each mouse that localize to each cell type in Fig. 1f. g tSNE plot shows astrocytes colored by control or metastatic condition. h Volcano plot shows genes differentially expressed (n = 6,542) between astrocytes from control and metastatic brains determined by Wilcoxon rank sum test, (p < 0.01). See Supplementary Table 2 for full list. Select genes with an absolute value average natural logFC >0.35 are colored and labeled. The y-axis represents the -log10 of Bonferroni corrected P values, and the x-axis represents average natural logFC between conditions.