Tumor-induced reprogramming of microglia to a DAM phenotype facilitates brain metastasis

Objective Brain metastasis (BrM) is a lethal complication of breast and lung cancers characterized by an immunosuppressive tumor microenvironment dominated by tumor-associated macrophages (TAM). While disease-associated microglia (DAM) are well-characterized in neurodegeneration, their role in BrM remains unexplored. This study investigates the emergence of DAM-like phenotypes and their contribution to the metastatic niche. Methods We performed a meta-analysis of single-cell RNA sequencing (scRNA-seq) data from seven independent BrM cohorts (GSE147942, GSE234832, GSE186344, GSE143423, GSE123902, GSE202371, GSE131907) to map TAM heterogeneity. To complement these descriptive findings, in vitro co-culture systems of murine (BV2) and human (HMC3) microglia with breast cancer lines (4T1/MDA-MB-231) were used to model microglial phenotypic shifts and cross-talk. Results scRNA-seq analysis revealed a prominent enrichment of DAM within the BrM niche. This microglial subset was characterized by the downregulation of homeostatic markers alongside a robust induction of SPP1, APOE, and NF-κB signaling pathways. Intercellular communication modeling via CellChat predicted that tumor-derived signals drive the transition of homeostatic microglia toward this DAM-like state, a process potentially mediated through the APP-CD74 and APOE-TREM2 axes. Reciprocally, CellChat analysis suggested that DAM interact with tumor cells via SPP1-CD44 crosstalk. In alignment with these computational predictions, in vitro assays demonstrated that tumor-conditioned microglia adopt a DAM-like phenotype—characterized by the upregulation of SPP1, APOE, and activated NF-κB signaling—which functionally drives tumor cell proliferation and migration. Pharmacological inhibition of either SPP1 or NF-κB signaling effectively attenuated these pro-migratory effects and down-regulated the expression of APP in tumor cells, indicating a reciprocal feed-forward activation loop between DAM-like microglia and metastatic cells. Finally, utilizing a BrM murine model, we confirmed that SPP1+APOE+ DAM-like microglia are profoundly enriched within the metastatic niche, where they cluster prominently within the peritumoral microenvironment. Conclusion Our findings indicate that microglia in the BrM niche exhibit a distinct, DAM-like activation profile associated with pro-metastatic traits. The predicted APP-CD74 and SPP1-CD44 signaling axes represent candidate pathways for disrupting the supportive metastatic microenvironment, warranting further in vivo functional validation.