Development and External Validation of an M2 Macrophage-Related Prognostic Signature in Breast Cancer Using...

Tumor-associated macrophages (TAMs), particularly the M2-like phenotype, are central regulators of breast cancer progression; however, clinically applicable models capturing their functional heterogeneity and biological mechanisms remain limited.

We integrated single-cell RNA sequencing, spatial transcriptomics, and bulk RNA-seq data to construct an M2 macrophage-centered gene landscape. A machine learning framework based on 10-fold cross-validation was used to develop a prognostic signature in the TCGA-BRCA cohort (training set), with external validation in the independent GSE96058 cohort. Immune infiltration, genomic instability, pathway activity, and cell-cell communication were analyzed. KLF4 was examined in breast cancer cells and macrophages using qRT-PCR, Western blotting, siRNA-mediated knockdown, and a Transwell co-culture system.

We identified a 16-gene M2 macrophage-related signature (M2MMS) that stratified patients into subgroups with distinct survival outcomes in both cohorts. High-risk tumors showed M2 macrophage enrichment, an immunosuppressive microenvironment, elevated genomic instability, and activation of E2F and MYC proliferative programs. Cell-cell communication analysis identified MDK-NCL as a dominant tumor-TME signaling axis. KLF4 was upregulated in breast cancer cells and in macrophages after co-culture, and silencing KLF4 attenuated CD206 induction, indicating that KLF4 is functionally involved in tumor cell-induced M2-like polarization in vitro.

The M2MMS links M2 macrophage-driven immune remodeling to breast cancer prognosis and provides a basis for risk stratification and mechanistic investigation. KLF4 represents a regulatory node in tumor-macrophage interaction in vitro; its role in vivo and the clinical utility of the M2MMS require further validation.