Spatial transcriptomics resolves ductal <i>TREM2</i> ⁺ and stromal <i>FOLR2</i> ⁺ macrophages in the normal human breast at microanatomical resolution

<jats:p> Macrophage identities are coupled to specialized roles through their local niches of residence. In the breast, macrophages contribute to epithelial development and re-modeling, immune surveillance, and angiogenesis. These diverse functions imply a spatial organization of distinct transcriptional states. Yet, how this heterogeneity is spatially arranged in the normal human breast at microanatomical resolution remains largely uncharacterized. Here, we leveraged single cell RNA-sequencing and Xenium <jats:italic>in situ</jats:italic> to delineate two discrete macrophage populations. We identify a <jats:italic>TREM2</jats:italic> <jats:sup>+</jats:sup> population intercalated between basal-myoepithelial cells and analogous to previously identified ductal niche macrophages in the mouse mammary gland. <jats:italic>FOLR2</jats:italic> <jats:sup>+</jats:sup> macrophages broadly distribute across the interlobular stroma, and unbiased niche analyses further resolved a periepithelial subpopulation that localized to the intralobular stroma. Spatially weighted communication inference highlights differentially enriched signaling patterns between <jats:italic>TREM2</jats:italic> <jats:sup>+</jats:sup> and <jats:italic>FOLR2</jats:italic> <jats:sup>+</jats:sup> macrophage subsets with their surrounding microenvironment. Orthogonal spatial co-expression analyses of ligand-receptor pairs converged on <jats:italic>CX3CL1-CX3CR1</jats:italic> , in which <jats:italic>TREM2</jats:italic> <jats:sup>+</jats:sup> macrophages interact with the neighboring epithelia. Collectively, these findings show that macrophage heterogeneity in the normal human breast is organized across conserved transcriptional and microanatomical axes. </jats:p>