TNBC Spatial Transcriptomic Analysis across Clinical States Reveals Subtype-Specific Networks and Immunosuppressive Niches

Breast cancer is a heterogeneous disease composed of distinct molecular subtypes that influence prognosis and treatment response, with subtype discordance between primary and metastatic tumors contributing to therapeutic failure. Using GeoMx spatial transcriptomics, we profiled nonmetastatic primary tumors, metastatic primary tumors, and lymph node (LN) metastases to characterize transcriptional and immune-spatial changes during disease progression. Comparative analysis revealed strong tumor microenvironment reprogramming across clinical states. In stromal regions of metastatic tumors, we observed enrichment of extracellular matrix remodeling, tumor-associated macrophage activity, and epithelial-to-mesenchymal transition-related pathways. In epithelial compartments, nonmetastatic and LN tumors showed a secretory-proliferative program, whereas primary metastatic tumors were enriched for antigen presentation and interferon (IFN) signaling. Immune deconvolution demonstrated distinct immune landscapes, with nonmetastatic tumors enriched for innate immune cells, primary metastatic tumors showing increased T- and B-cell infiltration, and LN metastases exhibiting an immunosuppressive microenvironment dominated by M2-like macrophages and memory B cells (P < 0.05). Subtype analysis revealed frequent triple-negative breast cancer (TNBC) subtype switching during LN metastasis, with 70% of cases transitioning from nonbasal subtypes to unspecified or immunomodulatory subtypes, both associated with poorer prognosis (HR = 1.8; 95% CI, 1.2-2.9). Spatial immune profiling further identified "excluded" and "ignored" tumors lacking T-cell infiltration, whereas "inflamed" tumors retained active cytotoxic immune responses. Integrating molecular subtype, immune infiltration, and spatial architecture, we developed a novel framework termed immuno-spatial molecular subtypes (ISMS), which captures TNBC heterogeneity and immune-spatial plasticity across disease progression.<h4>Significance</h4>TNBC progression is poorly understood, particularly how tumor and immune ecosystems evolve. By applying spatial transcriptomics on tissues, we uncover that nonmetastatic tumors displayed metabolic programs and a more coordinated immune-proliferation balance. Metastatic primary showed stronger IFN and antigen-presentation programs, whereas LN metastases showed an immune-excluded niche. Our discovery that immune phenotypes diverge from canonical subtypes led to development of ISMS, a framework that captures TNBC plasticity more accurately than subtype alone.