Single-Cell Profiling Reveals a Treg-Rich, NK Cell-Depleted Immune Microenvironment in Triple-Negative Breast Cancer with High-Glucocorticoid Receptor Expression

Purpose: Analyses of patients with early-stage, treatment-naïve triple-negative breast cancer (TNBC) have demonstrated that high glucocorticoid receptor (GR) expression in primary tumors is associated with poor prognosis. We previously observed that GR-high primary TNBCs exhibited significantly increased numbers of tumor-infiltrating regulatory T cells (Tregs) compared with GR-low tumors. To further investigate GR-associated immunologic features, we leveraged imaging mass cytometry (IMC) to profile additional immune cell phenotypes and spatial architecture in GR-high versus GR-low primary TNBC. Patients and Methods: Tumor-infiltrating immune cells were profiled in formalin-fixed paraffin-embedded (FFPE) core biopsies from five untreated GR-high and four GR-low TNBC tumors using IMC with a 21-antibody panel. Regions of interest (ROI) were selected within pan-cytokeratin-positive tumor nests. Data underwent unsupervised clustering, and cell types were identified based on protein expression profiles. Analyses compared cell-type abundance and spatial interactions in GR-high versus GR-low tumors. Results: GR-high tumors exhibited significantly greater Treg infiltration within tumor nests than GR-low tumors. GR-high TNBC also showed a comparatively greater abundance of activated memory CD8+ T cells, cytotoxic CD4+ T cells, and effector memory CD4+ T cells. In contrast, GR-low tumors exhibited relatively greater representation of HLA-ABC-positive (HLA-ABC+) cancer cells as well as early-activated dendritic cells (DCs) and natural killer (NK) cells. Spatial analysis revealed that Tregs in GR-high tumors colocalized more frequently with proliferating tumor cells relative to Tregs in GR-low tumors. NK cells in GR-high tumors displayed relatively less colocalization with proliferating tumor cells. Conclusion: Compared with GR-low disease, treatment-naïve GR-high primary TNBC exhibits a more immunosuppressive tumor microenvironment characterized by greater Treg density, closer Treg-cancer cell proximity, reduced NK cell infiltration, impaired immune surveillance, and decreased abundance of HLA-ABC+ cancer cells. These findings implicate TNBC cell GR signaling as immunosuppressive, likely through mechanisms resulting in both differential immune cell enrichment and altered spatial organization.