Abstract
Background Brain metastases (BrM) remain a major cause of mortality in breast cancer (BC), yet the spatial organization and molecular circuitry of the metastatic immune microenvironment are poorly defined.
Methods To address this gap, we integrated high-plex imaging mass cytometry (IMC) performed on human primary breast tumors (n=20 regions of interests (ROIs)) and human brain-metastasis tissues (n=40 ROIs) with publicly available datasets, including single-cell RNA sequencing (scRNA-seq) from BC (n=10) and BrM (n=12) and spatial transcriptomic (ST) data from BC (n=7) and BrM (n=1), enabling single-cell resolution of tissue architecture, functional states, and intercellular signaling.
Results IMC resolved nine major cell classes and diverse epithelial, myeloid, and T-cell subtypes, and revealed a striking shift in macrophage polarization: CD163−CD11b− macrophages were markedly depleted in brain metastases, whereas CD163+ subsets persisted. Spatial analysis demonstrated the loss of the immune-permissive CN1 neighborhood in brain metastases, which is enriched in memory T cells, B cells, and dendritic cells, and the expansion of the immunosuppressive CN9 niche in brain metastases, containing CD163+ macrophages, invasion-like epithelial cells, and exhausted T cells. ScRNA-seq integration corroborated these findings by refining the annotation of major immune and stromal lineages and confirming CD163 expression patterns across macrophage subsets. ST deconvolution further reproduced these CN1-like and CN9-like domains in situ, validating their anatomical organization across BC and BrM. Ligand-receptor inference highlighted specific inhibitory pathways—including programmed cell death protein 1/programmed death-ligand 1, growth arrest-specific 6-Tyro3, Axl, and Mer receptor tyrosine kinase family, nectin cell adhesion molecule 2 (NECTIN2)-T-cell immunoreceptor with Ig and ITIM domains, and prostaglandin E2 (PGE2)-prostaglandin E2 receptor 4—that are associated with immune evasion and metastatic growth. Functional validation in an in vivo brain metastasis model further supported the therapeutic relevance of targeting these immunoregulatory pathways.
Conclusions Together, these findings show a shift from permissive to suppressive immune niches, accompanied by pronounced macrophage reprogramming, as central features of BC adaptation to the brain. This spatially resolved framework provides mechanistic insight into the poor responsiveness of brain metastases to current immunotherapies and identifies defined inhibitory ligand-receptor axes as actionable targets for combination immunotherapy.
Authors and Affiliations
Jiale Zhu¹˒², Junjie Ye¹, Yulin Ma¹, Zhirong Lin¹˒³, Yupeng Wen¹, Jianpeng Sheng⁴, and Mei Yang¹˒⁵
¹ Department of Breast Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China
² Guangdong Cardiovascular Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China
³ Guangdong Provincial Biomedical Engineering Technology Research Center for Cardiovascular Disease, Zhujiang Hospital, Guangzhou, Guangdong, China
⁴ Chinese Institutes for Medical Research, Beijing, China
⁵ Department of Breast Cancer, Cancer Centre, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, China