Mitomycin C modulates tumor microenvironment and enhances radiosensitivity in rectal cancer

Fred Yi-Shueh Chen, Chih-Wen Chi, Hui-Ru Shieh, Chin-Ping Lin, Chun-Chuan Ko, Yi-Chun Chung, Jerry Chen-Yen Lai, Hung-Chi Tai, Yu-Jen Chen


Background: Neoadjuvant chemoradiation is the standard of care for locally advanced rectal cancer, however, pathological complete response (pCR) rate remains unsatisfactory. Tumor hypoxia in rectal cancer might be one factor of radioresistance. Mitomycin C (MMC), a hypoxia-activated prodrug (HAP), enhances radiosensitivity and is the standard adjunct to radiotherapy (RT) in anal cancer. Our study evaluated the effect of MMC and RT on rectal cancer and its tumor microenvironment (TME).
Methods: In vitro hypoxia was induced by 1% O2 culture for CT26 rectal adenocarcinoma cells and HIF-1α expression was validated with Western blotting. Clonogenicity and MTT assays were used for radiation and drug survival. In syngeneic CT26 model, tumors were treated as follows: control, MMC (2 mg/kg/day), RT (2 Gy × 2 fractions), and combination with MMC 2 h prior to RT. Tumor volume, body weight, and white blood cell count were monitored for 1 month. Immune cells in TME were analyzed using flow cytometry.
Results: MMC inhibited cell viability and enhanced radioresponse in CT 26 cells with delayed repair of DNA double strand break (P<0.05). Expression of Ifnb1 was augmented by RT and upregulated with addition of MMC. In vivo treatment using MMC plus RT suppressed tumor growth with durable effect in comparison to MMC or RT alone (P<0.05). In TME analysis, combination of MMC and RT recruited cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells into tumor and reduced infiltration of regulatory T cells.
Conclusions: The combination of MMC and RT may have synergistic therapeutic effect accompanied by modulation of TME towards favorable anti-tumor immunity.