Local Radiotherapy for Murine Breast Cancer Increases Risk of Metastasis by Promoting the Recruitment of M-MDSCs in Lung

Abstract

Abstract Background Explore how exosomes released by X-ray irradiated tumor cells promote the formation of pulmonary metastatic nodules by inducing the recruitment of MDSCs to the lung. Methods BALB/c mice were injected with 4T1 tumor cells into the mammary adipose pad and allowed to grow for 28 days. On the 10th day of tumor inoculating, the primary tumor was irradiated by X-ray with a single dose of 20Gy. The mice were monitored for tumor growth, the number of pulmonary metastasis nodules and frequency of MDSCs. Antibody microarray and ELISA methods were used to analyze the altered cytokines (G-CSF, GM-CSF and CXCL1) in exosomes released by 4T1 cells after X-ray irradiation. The effects of the exosomes on the recruitment of MDSCs in the lung and facilitating the colonization of 4T1 in the lung were observed in normal BALB/c mice. Transwell method was used to observe the promotion of MDSCs migration by exosomes, lung tissue extracts from 4T1 tumor-bearing mice and GM-CSF via CCR2/CXCR2 signals, meanwhile, the effect of MDSCs on T lymphocytes function and migration of 4T1 cells were observed by the co-culture system. Results Even though radiotherapy reduced the burden of primary tumors and larger lung metastatic nodules (≥ 0.5 mm2), the number of smaller metastases (< 0.5 mm2) significantly increased. In addition, radiotherapy markedly potentiated M-MDSCs and reduced PMN-MDSCs recruitment in lung of 4T1 tumor-bearing mice. Moreover, the frequency of M-MDSCs in lung was positively correlated with the number of smaller metastatic nodules. Using co-culture experiments, we provided evidence that M-MDSCs of the lung in 4T1 tumor-bearing mice markedly inhibited T cell proliferation and killing activity, while M-MDSCs and PMN-MDSCs had a similar role in facilitating tumor cell dissemination. The G-CSF, GM-CSF and CXCL1 rich exosomes derived from irradiated 4T1 (ir/4T1-exo) could facilitate both M-MDSCs and PMN-MDSCs migration by CXCL1/CXCR2 signal. While the lung tissue extracts of irradiated mice or culture medium of macrophage treated with ir/4T1-exo showed an obvious tendency to chemotaxis on M-MDSCs. Mechanistically, ir/4T1-exo could induce macrophage to produce GM-CSF, which in turn further promoted CCL2 release in an autocrine manner to recruit M-MDSCs via CCL2/CCR2 axis. Conclusions Our work has identified an undesired effect of radiotherapy promoting immunosuppressive premetastatic niches by recruiting M-MDSCs to lung. Further studies on RT combined inhibition of CXCR2 or CCR2 signals were necessary.