Spatially resolved GR-TGFβ co-activation underlies cell-type-specific signalling in triple-negative breast cancer

Abstract Background The clinical relevance of glucocorticoid receptor (GR) activation in triple-negative breast cancer (TNBC) remains controversial. While preclinical studies link GR activity to tumour progression, clinical data are inconsistent, despite the routine use of synthetic glucocorticoids as supportive therapy during chemotherapy. This paradox suggests that GR signalling may be highly context- and cell-type-specific within the tumour microenvironment. Understanding the spatial organisation and molecular partners of glucocorticoid receptor signalling is therefore essential to clarify its functional and clinical impact. Methods We comprehensively characterised spatially resolved GR expression and signalling across malignant, stromal, and immune compartments in human triple-negative breast cancer specimens. GR protein expression was assessed by immunohistochemistry, while spatial transcriptomics was used to resolve compartment-specific transcriptional programs. Stereological confocal microscopy enabled quantitative analysis of GR and transforming growth factor beta receptor 2 (TGFBR2) co-expression at single-cell resolution in a spatial distribution. These spatial data were integrated with bulk and single-cell RNA sequencing datasets to define signalling states, ligand dependence, and pathway interactions. Intercellular communication analyses were applied to identify signalling at tumour–immune interfaces. Functional relevance was assessed using in vitro migration assays and transcriptome sequencing following glucocorticoid stimulation with or without TGFBR2 inhibition. Associations with clinical outcome were evaluated using survival analyses of pathway-correlated gene signatures. Results GR expression was enriched within the tumour microenvironment and associated with shared gene programs across tumour and stromal populations, prominently involving TGFβ pathway components. Stereological protein analyses revealed increased GR activation at invasive tumour edges and adjacent normal epithelium, accompanied by marked co-localisation with TGFBR2. Spatial transcriptomics demonstrated predominantly ligand-independent GR activation in tumour cells, whereas tumour-infiltrating immune cells displayed ligand-dependent signalling linked to local corticosteroid metabolism. Intercellular communication analyses identified enhanced GR-TGFβ signalling at the tumour–immune interface. Functionally, glucocorticoid-induced tumour cell migration was abolished by TGFBR2 inhibition. Clinically, GR–correlated and glucocorticoid-induced TGFβ gene signatures were associated with reduced overall survival. Conclusions These findings reveal spatially regulated, cell-type-specific GR-TGFβ co-activation in TNBC. This signalling axis underlies pro-migratory tumour behaviour and adverse clinical outcome, identifying a potential prognostic marker and therapeutic vulnerability with important implications for glucocorticoid use in cancer treatment.