Low-Dose Cyclophosphamide Enhances the Tumoricidal Effects of 5-Day Spacing Stereotactic Ablative Radiotherapy by Boosting Antitumor Immunity

Abstract

Purpose This study aimed to investigate the potential role of low-dose cyclophosphamide (Cy) as a radiosensitizer by evaluating its impact on the immune response and the abscopal effect of stereotactic ablative radiotherapythrough preclinical models. Materials and Methods CT26 tumors (immunologically hot) and 4T1 tumors (immunologically cold), grown in immunocompetent BALB/c and immunodeficient BALB/c-nude mice, were irradiated with 20 Gy in two fractions with 5-day spacing followed by intraperitoneal injections of 9 mg/kg Cy every 3 days. Immunological changes in CT26 tumors caused by the treatments were assessed using flow cytometry. Changes in the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha) in tumors were also assessed. Splenocytes and bone marrow-derived dendritic cells (DCs) were exposed to various concentrations of Cy to assess T cell proliferation and DC differentiation. Results The combination of Cy with radiotherapy (RT+Cy) significantly suppressed tumor growth compared to RT alone in immunocompetent mice, while that effect was not observed in immunodeficient mice. Additionally, RT+Cy effectively induced abscopal effects in hot and cold tumors, with increased CD8+T cells in blood and tumors. Significantly higher expression levels of granzyme B, interferon gamma, and tumor necrosis factor alpha were observed in RT+Cy group compared to the RT alone group. In vitro data indicated that low-dose Cy promotes DC differentiation. Low-dose Cy suppressed the radiation-induced upregulation of HIF-1 alpha in the tumors. Conclusion Low-dose Cy enhances tumoricidal effects of 5-day spacing high-dose RT by increasing antitumor immune responses.