Simple SummaryThe immune system plays an important role in the effectiveness of chemotherapy used in the treatment of different types of breast cancer (BC). However, this system is often deficient in most cancers, including BC. Thus, any strategy aimed to reactivate the immune system should amplify the effectiveness of treatments. The aim of our study is to test the beneficial effect of glyceryltrinitrate (GTN) a nitric oxide donor (used in the treatment of angina pectoris) when associated with doxorubicin, a chemotherapy used in the treatment of BC, especially the triple-negative type (TNBC). This work was performed using a mouse model of TNBC. We have shown that the combination of the two drugs induces greater tumor regression than the monotherapies, an effect linked to a stronger activation of the immune system correlated with a decrease in immunosuppressive immune cells. These findings suggest a therapeutic perspective in the treatment of TNBC.Abstract(1) Background: Immunosuppression is a key barrier to effective anti-cancer therapies, particularly in triple-negative breast cancer (TNBC), an aggressive and difficult to treat form of breast cancer. We investigated here whether the combination of doxorubicin, a standard chemotherapy in TNBC with glyceryltrinitrate (GTN), a nitric oxide (NO) donor, could overcome chemotherapy resistance and highlight the mechanisms involved in a mouse model of TNBC. (2) Methods: Balb/C-bearing subcutaneous 4T1 (TNBC) tumors were treated with doxorubicin (8 mg/Kg) and GTN (5 mg/kg) and monitored for tumor growth and tumor-infiltrating immune cells. The effect of treatments on MDSCs reprogramming was investigated ex vivo and in vitro. (3) Results: GTN improved the anti-tumor efficacy of doxorubicin in TNBC tumors. This combination increases the intra-tumor recruitment and activation of CD8+lymphocytes and dampens the immunosuppressive function of PMN-MDSCs PD-L1low. Mechanistically, in PMN-MDSC, the doxorubicin/GTN combination reduced STAT5 phosphorylation, while GTN +/− doxorubicin induced a ROS-dependent cleavage of STAT5 associated with a decrease in FATP2. (4) Conclusion: We have identified a new combination enhancing the immune-mediated anticancer therapy in a TNBC mouse model through the reprograming of PMN-MDSCs towards a less immunosuppressive phenotype. These findings prompt the testing of GTN combined with chemotherapies as an adjuvant in TNBC patients experiencing treatment failure.