Single-cell profiling reveals distinct populations of tumor-associated macrophages and metastatic tumor cells in breast cancer brain metastasis

Abstract Breast cancer brain metastasis (BCBM) is a major cause of breast cancer-related mortality, but the molecular mechanisms underlying its progression remain poorly understood. Here, we profiled the tumor immune microenvironment of BCBM at single-cell resolution and identified candidate regulators associated with brain metastatic progression. Single-cell RNA sequencing (scRNA-seq) was performed on brain metastatic tissue, adjacent tumor tissue, cerebrospinal fluid, and circulating tumor cells (CTCs) from seven patients, and bulk DNA sequencing was conducted on primary breast tumors, peripheral blood, and brain metastases from 47 patients. Analysis of 131,880 single cells identified 63 distinct cell clusters, including 12 tumor-associated macrophage (TAM) subtypes and 5 metastatic tumor cell (MTC) subtypes. Circulating TAMs displayed an M1-like inflammatory phenotype, whereas tissue-resident TAMs were predominantly M2-like. MTCs exhibited substantial transcriptional heterogeneity, and a neuro-related subtype showed adaptive upregulation of neuronal signaling pathways. Brain metastases also harbored a higher mutational burden than primary tumors, with recurrent mutations in GABRB3 and NRXN1 associated with poorer patient survival. Functional experiments further showed that loss of GABRB3 or NRXN1 impaired tumor growth and brain colonization in xenograft models. These findings nominate GABRB3 and NRXN1 as candidate regulators of brain metastatic fitness in BCBM and support their further evaluation as biomarkers and therapeutic targets.