Low Dose Cyclophosphamide Modulates Tumor Microenvironment by TGF-β Signaling Pathway

The tumor microenvironment has been recently recognized as a critical contributor to cancer progression and anticancer therapy-resistance. Cyclophosphamide (CTX) is a cytotoxic agent commonly used in clinics for the treatment of cancer. Previous reports demonstrated that CTX given at low continuous doses, known as metronomic schedule, mainly targets endothelial cells and circulating Tregs with unknown mechanisms. Here, we investigated the antitumor activity of two different metronomic schedules of CTX along with their corresponding MTD regimen and further explored their effect on immune function and tumor microenvironment. Toxicity evaluation was monitored by overall survival rate, weight loss, and histopathological analysis. A nude mouse model of Lewis lung cancer was established to assess the anti-metastatic effects of CTX in vivo. CD4⁺, CD8⁺, and CD4⁺CD25⁺FoxP3 T cells were selected by flow cytometry analysis. Low and continuous administration of CTX was able to restore immune function via increase of CD4⁺/CD8⁺ T cells and depletion of T regulatory cells, not only in circulatory and splenic compartments, but also at the tumor site. Low-dose CTX also reduced myofibroblasts, accompanied with an increased level of E-cadherin and low N-cadherin, both in the primary tumor and lung through the TGF-β pathway by the downregulated expression of TGF-β receptor 2. Our data may indicate that several other molecular mechanisms of CTX for tumor may be involved in metronomic chemotherapy, besides targeting angiogenesis and regulatory T cells.