Metformin enhances response to chemotherapy combined with immunotherapy in a triple negative breast cancer in vivo model

Triple-negative breast cancer (TNBC) is a clinically heterogeneous disease that is predominantly treated with chemotherapy. Although immune checkpoint inhibitors targeting the PD-1/PD-L1 axis, such as atezolizumab (ATZ), have expanded therapeutic options, their clinical benefit remains limited, emphasizing the need for strategies that improve efficacy without increasing toxicity. Metformin (MTF), a widely prescribed antidiabetic drug, has demonstrated anticancer properties and synergistic effects with chemotherapy and immunotherapy in several tumor models. This study evaluated whether adding MTF to a chemo-immunotherapy regimen could enhance outcomes in an in vivo TNBC model. In vitro experiments were performed to determine the IC50 values of MTF and doxorubicin (DOXO) in EMT6 breast cancer cells and to assess their combined effects. In vivo studies were conducted using BALB/c mice bearing subcutaneous EMT6 tumors. Treatment was initiated when tumor volumes reached 50–120 mm³ . DOXO and ATZ were administered intraperitoneally for two weeks, with DOXO given twice weekly and ATZ once weekly. ATZ was injected at least three hours after DOXO, and MTF was provided via drinking water. Tumor growth was monitored daily and compared among treatment groups. The addition of MTF enhanced the antitumor efficacy of low-dose DOXO (0.3 mg/kg) combined with ATZ, producing tumor-growth inhibition equivalent to that achieved with a 20-fold higher DOXO dose plus ATZ. After fourteen days, tumor volumes in the triplet therapy group were reduced compared with DOXO plus ATZ alone. Importantly, mice receiving the MTF-containing regimen maintained body weight, indicating no added toxicity. These findings support metformin as an adjunct to chemo-immunotherapy. • Metformin significantly enhances the antitumor efficacy of combined doxorubicin and PD-L1 blockade in a TNBC mouse model. • Addition of metformin allows low-dose doxorubicin to achieve efficacy comparable to a 20-fold higher chemotherapy dose. • The metformin-containing triplet results in significant tumor-volume reduction compared with chemo-immunotherapy alone. • Metformin does not increase treatment-related toxicity, as indicated by preserved body weight in treated mice. • Metformin may enhance chemo-immunotherapy effectiveness and reduce chemotherapy side effects in TNBC patients.