Immunogenic potential of medium-dose intermittent cyclophosphamide therapy in murine triple-negative breast cancer tumor lines 4T1 and E0771
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Triple-negative breast cancer (TNBC) is characterized by poor prognosis, increased tumor aggression and typically occurs in younger women. Chemotherapy drugs are currently used to treat TNBC, however research into novel therapies is essential to improve patient outcomes. Immunogenic chemotherapy uses cytotoxic drugs to induce an immune stimulatory form of cell death, known as immunogenic cell death (ICD). ICD stimulates innate and adaptive immune responses to the tumor and can result in tumor ablation and anti-tumor immunity. Production of type-1 interferons has sometimes been associated with ICD, and these cytokines and their downstream genes are known to be involved in immune cell attraction and activation. Here we investigate the immunogenic potential of two TNBC cell lines, E0771 and 4T1, using cyclophosphamide, an established ICD-inducing chemotherapeutic drug. Strong induction of type-1 IFN signaling to downstream genes was induced in both cell lines by treatment with the chemically activated cyclophosphamide derivative, 4-hydroperoxycyclophosphamide (4HC). Antibody treatment against IFNα/β receptor 1 partially blocked some of these 4HC-induced gene responses, which suggested that 4HC activates additional pathways to induce IFN-stimulated genes. Additionally, 4HC-treated cells secreted soluble factors capable of inducing these genes in drug-free cells. 4T1 and E0771 tumors implanted orthotopically into syngeneic mice were treated with cyclophosphamide on a 6-day medium-dose intermittent schedule. Tumor responses were distinct between cell lines. Type-1 IFN gene responses were induced by cyclophosphamide in 4T1 tumors, but the responses were weak in E0771 tumors. Nevertheless, E0771 tumors rapidly regressed after two cycles of cyclophosphamide treatment with strong tumor infiltration by cytotoxic T-cells, natural killer cells, macrophages and dendritic cells. In contrast, 4T1 tumors entered growth stasis after two treatment cycles that lasted through 7 cycles, with weaker immune cell infiltration that lacked natural killer cells. IFN-stimulated gene induction in E0771 tumors peaked 3 days after the first cyclophosphamide treatment and was followed by immune cell infiltration starting 3 days later. Additionally, in E0771 tumors, expression of Foxp3, an immunosuppressive regulatory T-cell biomarker, decreased by 70% within 3 days of cyclophosphamide treatment. Thus, cyclophosphamide given on a metronomic schedule may be efficacious for treating some TNBC tumors, with striking differences between tumor models in the activation of type-1 IFN signaling, immune cell recruitment and tumor regression responses.