Integrated bulk and single-cell transcriptome analysis reveals the impact of cyclophosphamide on tumor microenvironment in triple-negative breast cancer models

Triple-negative breast cancer (TNBC) remains a formidable clinical challenge. Although preclinical investigations utilizing TNBC mouse models have demonstrated that intermittent cyclophosphamide (CTX) treatment yields superior anti-tumor effects, partly through immune stimulation, mechanisms by which CTX modulates the tumor microenvironment (TME) are not yet fully understood. In this study, we assessed the impact of optimal (140 mg/kg every 6 days, C140) and suboptimal (100 mg/kg every 6 days, C100) CTX treatment regimens on 4T1 tumors, a widely utilized orthotopic TNBC mouse model. While the C100 regimen was better tolerated than the C140 regimen, it was less effective in inhibiting tumor growth. Through integrated bulk and single-cell RNA sequencing of tumor tissues, we discovered that CTX treatment not only inhibited cancer cell proliferation and epithelial-mesenchymal transition but also remodeled the TME by recruiting and reprogramming various immune cells, thereby enhancing the anticancer immune response in a dose-dependent manner. Although the C140 regimen demonstrated significant tumor suppression, it also induced notable extracellular matrix remodeling and led to the accumulation of cancer-associated fibroblasts within the tumor microenvironment. Furthermore, the C140 regimen caused considerable exhaustion of CD8+ T cells and was linked to increased expression of various immune checkpoint molecules, including PD-L1/PD-L2, CD39, and CD200. These effects may compromise therapeutic efficacy and potentially contribute to the development of drug resistance. Overall, our findings elucidate both cancer-cell-intrinsic and tumor microenvironment-dependent mechanisms of CTX action at both local and systemic levels, highlighting potential targets for optimizing chemoimmunotherapy in TNBC.