Doxycycline modulation of the CD39/CD73–adenosine axis to enhance CD8⁺ T cell and M1 macrophage immunity in patient-derived breast cancer samples.

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Background: The CD39/CD73–adenosine axis functions as a metabolic immune checkpoint in breast cancer by converting extracellular ATP into immunosuppressive adenosine. This pathway promotes M2-like macrophage polarization, suppresses cytotoxic CD8⁺ T-cell activity, enhances regulatory T-cell (Treg) expansion, and facilitates tumor progression. Doxycycline, a widely used antibiotic, has emerging immunomodulatory properties; however, its role in modulating adenosine-mediated immune suppression in human breast cancer remains undefined. This study evaluated the immunometabolism effects of doxycycline across triple-negative (TNBC), HER2⁺, and ER/PR⁺ breast cancer patients. Methods: Peripheral blood mononuclear cells (PBMCs) and matched tumor biopsies were collected from patients across breast cancer subtypes. PBMCs were stimulated with tumor-conditioned media and treated with doxycycline or adenosine-pathway inhibitors (ARL67156, AMPCP). Flow cytometry assessed CD39⁺/CD73⁺ Tregs, CD8⁺ T-cell activation, IFN-γ production, and macrophage phenotypes (M1/M2). Tumor biopsies underwent gene expression and immunohistochemical analysis for CD39, CD73, ENTPD1, and NT5E. Neutrophil-to-lymphocyte ratio (NLR) was correlated with CD39/CD73 expression. Results: Doxycycline significantly reduced CD39 and CD73 expression in tumor-conditioned and patient-derived PBMCs. Treatment decreased CD4⁺FoxP3⁺CD39⁺ Tregs while enhancing CD8⁺ T-cell activation and IFN-γ secretion. Doxycycline promoted M1 macrophage polarization and reduced M2-like phenotypes. These immunomodulatory effects were most pronounced in TNBC samples, consistent with elevated baseline CD39/CD73 expression in TNBC tissues. Combination treatment with ARL67156 further enhanced CD8⁺ cytotoxic responses and M1 polarization. Tumor biopsy analyses confirmed suppression of CD39/CD73 and ENTPD1/NT5E expression following doxycycline exposure. Elevated NLR positively correlated with CD39/CD73 levels, particularly in TNBC patients. Conclusions: Doxycycline acts as a low-cost metabolic immune-checkpoint modulator capable of reversing adenosine-driven immunosuppression in breast cancer, especially TNBC. These findings support further clinical investigation of doxycycline as a complementary immunotherapeutic strategy.