Intermittent cyclophosphamide therapy exerts distinct cancer cell-intrinsic and stromal cell-dependent effects in a murine model of triple-negative breast cancer

Triple-negative breast cancer (TNBC) poses a formidable clinical challenge. Previous studies have suggested that intermittent cyclophosphamide (CTX) treatment can produce significant anti-tumor effects while minimizing adverse side effects; however, the underlying mechanisms are only partially understood. In this study, we established a therapeutic model of intermittent CTX treatment in a murine orthotopic model of TNBC (4T1) and investigated its impact on the tumor microenvironment (TME) in vivo. High-dose (HD) and low-dose (LD) intermittent CTX regimens were administered. While the LD regimen was better tolerated, it was less effective in inhibiting tumor growth. Integrated bulk and single-cell RNA sequencing of tumor tissues revealed that CTX treatment not only inhibited cancer cell proliferation but also remodeled the TME by recruiting and reprogramming various immune cells, thereby enhancing the anti-tumor immune response in a dose-dependent manner. Our findings elucidate both cancer cell-intrinsic and stroma-dependent mechanisms of CTX action, highlighting potential targets for overcoming the translational limitations of intermittent CTX regimens in TNBC chemo-immunotherapy.