WEE1 Inhibition Promotes Radiotherapy-Induced Myeloid Suppressor Cells through cGAS/STING-STAT3-IRF3 Pathway

Abstract

This study is the first to propose the WEE1 inhibitor combined with radiotherapy affect tumor microenvironment through cGAS/STING-STAT3-IRF3 pathway, and the effects of DNA damage caused by WEE1 inhibitor combined with radiotherapy on cGAS/STING-STAT3-IRF3 pathway and MDSC (myeloid-derived inhibitory cells) in tumor microenvironment were systematically expounded. MDA-MB-231 cells, A549 cells and HT29 cells were irradiated with 2GY, 12GY and 20GY X-ray respectively, and cell cycle tests were conducted. WEE1 inhibitor combined with radiotherapy was used to cells. EDU kit was used to detect the changes of DNA damage repair and proliferation of tumor cells after irradiation. Western-blot experiment was used to detect the expression changes of cGAS/STING-STAT3-IRF3 pathway related proteins TBK1, P-TBKI, Sting, P-Sting, IRF3, P-IRF3 and cGAS. Immunofluorescence double-label experiment was used to detect the expression changes of target molecules in tumor cell control group and experimental group. The tumor-bearing model of mice was established, and the survival time and tumor volume of mice were detected. The differential expression of CD11B, LY6C, CD45, IFN-γ, CD8a and other immune-related molecules was detected by flow cytometry. The results of cell cycle experiment show that WEE1 inhibitor combined with radiotherapy can effectively inhibit G2/M phase of cell cycle compared with the control group. Compared with the control group, the tumor volume of mice in WEE1 inhibitor combined with radiotherapy was significantly reduced and the survival time of mice was significantly prolonged. The infiltration of CD8T cells and the secretion level of IFN-γ in tumor tissues of mice were detected by flow cytometry. The WEE1 inhibitor combined with radiotherapy group was significantly higher than that of the control group. The results of immunofluorescence double-label experiment showed that WEE1 inhibitor combined with radiotherapy reduced the infiltration of MDSC in tumor microenvironment after radiotherapy compared with the control group. Western-blot results showed that inhibition of WEE1 could significantly down-regulate the expression of TBK1, P-TBKI, Sting, P-Sting, IRF3, P-IRF3, P-STAT3 and cGAS through STATE3, which indicated that the radiation resistance induced by WEE1 inhibitor combined with radiotherapy was related to cGAS/STING-STAT3-IRF3 pathway. WEE1 targeted therapy combined with radiotherapy can improve the microenvironment of inhibitory tumor and effectively alleviate radiotherapy resistance. The combination of WEE1 inhibitor and radiotherapy significantly inhibited the growth of tumor compared with single treatment, and the combined treatment achieved obvious inhibitory effect on tumor.