The Relationship And Mechanism Between Abnormal Differentiation Of MDSCs And Tumor Cells Drug Response To Oxaliplatin In Tumor Microenvironment

BACKGROUD AND OBJECTIVE:Colorectal cancer(CRC)is one of the most common malignant tumors characterized by high morbidity and mortality.The CRC prognosis is closely related to tumor staging.In early stage,CRC can be resected by surgical removal of the lesion,and the 5 year survival rate of early CRC patients was as high as 90%.Chemotherapy is the most important treatment for those patients with advanced CRC,but the drug resistance may make chemotherapy failure,which reduced the quality of life and shortened the survival time of the CRC patients.The mechanism of chemotherapeutic drug resistance is still unclear.Recent studies have shown that drug resistance have close relation to tumor microenvironment.As one of the most crucial immunoregulators in tumor microenvironment,myeloid-derived suppuressor cells(MDSCs),their number and differentiation may be one of the mechanisms of chemoresistance.The present studies showed that the recruitment and differentiation of MDSCs in different chemotherapeutic drugs is still controversial.Our previous study found that the number of MDSCs increased with the increasing dose of oxaliplatin,and inhibited the differentiation of MDSCs to M1-liked MDSCs.The objective of this study is to clarify the crosstalk among oxaliplatin,tumor cells and MDSCs in the tumor microenvironment of oxaliplatin chemotherapy,so as to explain the change of MDSCs' number and differentiation in our previous studies and provide new ideas for solving oxaliplatin resistance.Methods1.The effect of oxaliplatin and CT26 cell supernatant on the number of MDSCs in the tumor microenvironment of oxaliplatin chemotherapyThe primary bone marrow cells were induced into MDSCs in vitro,and treated with different doses of oxaliplatin and CT26 cell supernatant respectively.Flow cytometry was used to detect the proportion of MDSCs apoptotic cells in each group.2.The effect of oxaliplatin and CT26 cell supernatant on MDSCs differentiation in tumor microenvironment of oxaliplatin chemotherapyThe primary bone marrow cells were induced into MDSCs in vitro,and treated with different doses of oxaliplatin and CT26 cell supernatant respectively.Flow cytometry was used to detect the proportion of M1-liked MDSCs and M2-liked MDSCs in each group.3.The effect of MDSCs' abnormal differentiation on CT26 cells' drug response to oxaliplatinCT26 cells were respectively treated with M1 and M2 macrophage supernatants collected in vitro.Flow cytometry was used to detect the proportion of CT26 apoptotic cells in each group.The effects of M1 and M2 macrophage supernatants on invasion and metastasis of CT26 cells were observed by cytotoxicity test,wound heeling test and transwell migration assays.4.statistical analysisAll statistical analysis were performed on SPSS 22.0 statistical software.The measurement data were expressed by mean±standard deviation.when conformed to normal distribution and variance homogeneity,statistical significance of two groups were determined by using Independent-Samples T Test,or using Wilcoxon rank sum test.The two independent sample rates were compared between two groups of count data with the method of Chi-Square Test.The difference was statistically significant inP<0.05.Result1.With the increase of oxaliplatin dose,the proportion of MDSCs apoptotic cells increased in a manner of dose-dependence.In the presence of CT26 cell supernatant,the proportion of MDSCs apoptotic cells induced by oxaliplatin decreased significantly,while the proportion of living cells increased significantly.2.The proportion of M1-liked MDSCs decreased significantly,with the increase of oxaliplatin concentration,while the proportion of M2-liked MDSCs increased,when treated with low dose oxaliplatin.The high dose of oxaliplatin had no significant effect on the proportion of M2-liked MDSCs.With the treatment of CT26 cell supernatant,the proportion of M2-liked MDSCs increased,and the proportion of M1-liked MDSCs had no obvious change.3.The proportion of CT26 apoptotic cells induced by oxaliplatin decreased significantly,When added with M2 macrophage supernatant.While the proportion of CT26 apoptotic cells induced by oxaliplatin increased under the condition of M1 macrophage supernatant.With the treatment of M2 macrophage supernatant,oxaliplatin IC50 and migration ability of CT26 cells were significantly increased,while it has the opposite effect treated with Ml macrophage supernatant.Conclusion1.Oxaliplatin can promote MDSCs apoptosis and reduce MDSCs infiltration in tumor microenvironment.While CT26 cells accelerate MDSCs infiltration in tumor microenvironment through a variety of ways,including inhibition of MDSCs cytotoxicity induced by oxaliplatin,promoting recruitment of MDSCs to turnor microenvironment and bone marrow mobilization.The effect of CT26 cells on the infiltration of MDSCs in tumor microenvironment is significantly stronger than that of oxaliplatin.2.Oxaliplatin and CT26 cells promoted MDSCs differentiation to M2 macrophages,and oxaliplatin inhibited MDSCs differentiation to M1 macrophages,while CT26 cells had no significant effect on MDSCs differentiation to M1 macrophages.3.M2 macrophage supernatant attenuated CT26 cell apoptosis induced by oxaliplatin and increased IC50 and oxaliplatin resistance of CT26 cells,whereas M1 macrophage supernatant was on the opposite.In addition,M2 type macrophage supernatant increases the migration ability of CT26 cells.