Introduction. The glucocorticoid receptor (GR) plays a highly context-dependent role in tumorigenesis. The GR agonist dexamethasone is routinely administered as supportive therapy alongside chemotherapy to reduce anaphylactic reactions and nausea. However, recent RNA-based and preclinical studies have suggested caution when using glucocorticoids in patients with breast cancer, as GR activation has been shown to promote metastasis in mouse models. Our aim was to investigate GR mRNA and protein expression at the single-cell level and to correlate it with patient survival.

Materials and Methods. GR expression was analyzed at the single-cell level using the following approaches: i) First, confocal immunostaining was performed to examine GR expression in different cell types within triple-negative breast cancer (TNBC). ii) Findings were validated using single-cell sequencing of TNBC samples and conventional immunohistochemistry on 161 TNBC tissue samples. iii) Finally, the effect of GR activation on tumor cell migration was assessed through time-lapse imaging of single-cell tracking in vitro using MDA-MB231 and Hs578T TNBC cell lines treated with dexamethasone.

Results and Discussion. Confocal staining revealed heterogeneous, medium-to-low GR expression in TNBC tumor cells compared to cells in the tumor microenvironment (TME). High GR expression was detected in immune cells, including infiltrating T cells and macrophages. In our TNBC cohort, cytoplasmic GR staining positively correlated with Ki67 expression. Additionally, patients with high nuclear GR staining had significantly shorter progression-free survival (HR = 0.35 [0.08–1.50]; p = 0.0185). Single-cell tracking time-lapse migration experiments demonstrated that GR activation increased cell motility in a time-dependent manner. Single-cell sequencing showed a significant positive correlation between the GR signature and migration signature (R = 0.61, p < 2.2e-16). Tumor cells with higher GR encoding NR3C1 expression exhibited an increased migration signature. Fast-migrating cells displayed significantly higher expression levels of NR3C1 and its targets SGK1 and TSC22D3 compared to clusters of slow-migrating cells.

Conclusion. Glucocorticoids are essential supportive therapy in routine chemotherapy. However, our findings suggest that high nuclear GR expression is a negative prognostic marker in TNBC patients. GR activation enhances tumor cell migratory potential, which may contribute to poorer outcomes. Therefore, careful monitoring of TNBC patients with high nuclear GR expression is warranted. Funding: NRDI FK135065, Bolyai Research Fellowship of the Hungarian Academy of Sciences, National Laboratories Excellence program (under the National Tumor Biology Laboratory project (NLP17).