The Amplification Effect Of Microvesicles Produced By CML Cells On Myeloid-derived Suppressor Cells

ObjectiveTyrosine kinase inhibitor(TKI)has become a classical therapy model for chronic myeloid leukemia(CML),but taking it for life time has always been a drawback of TKI.A new therapy goal has been proposed with the progress of TKI discontinuation researches.Recently,immune component in bone marrow microenvironment play a pivotal role in the success of TKI cessation.Myeloid derived suppressor cells(MDSC)are a group of heterogeneous cells that exert immunosuppressive function and provide an immunosuppressive microenvironment suitable for the survival of tumor cells.Our previous study had found CML patients who relapse after cessation have increased MDSC proportion.In addition,recent studies suggest that tumor-derived vesicles efficiently induce the proliferation of MDSC.The aim of this study is to explore the effect of microvesicle(MV)produced by K562 cells after different treatment on MDSC,to seek the important reasons for differential amplified MDSC after discontinuation,then construct a foundation for the later TKI discontinuation research.MethodsIn this work,the IC50 value of Imatinib and Dasatinib were detected by using CCK8 assay.K562 cells were treated with a concentration gradient lower than IC50 for different therapy and discontinuation times,then,the apoptosis rate of cells were analyzed for the aim of choosing appropriate treatment time.MV produced by K562 cells after different treatment were collected by density gradient centrifugation method.Peripheral blood mononuclear cells(PBMC)were isolated by Ficoll density gradient centrifugation method.After adding MV to PBMC,the proportion of MDSC was tested by flow cytometry.Mice experiments: Balb/c mice were divided into nine groups,and MVs were injected into mice through tail vein.Peripheral blood of 100~200ul were collected from angular vein of mice at 0,3,5 days after injection,and the MDSC ratio was detected by flow method.The mice were sacrificed on the day 5.Not only the spleens were taken to compare size but also the ratio of MDSC in bone marrow and spleen cells was tested.Results1.Compared with the control group,K562 cell-derived MV promoted the increase of MDSC and its subtypes G-MDSC and M-MDSC(P<0.05).2.Whether in the Imatinib group or the Dasatinib group,the changes of MDSC and its subtypes G-MDSC and M-MDSC induced by MV produced by K562 cells treated with TKI were not statistically significant compared with those in the no treatment group.3.After treatment with imatinib or dasatinib,the proportion of MDSC and its subtypes G-MDSC and M-MDSC caused by MV after TKI discontinuation was higher than that before TKI cessation(P<0.05).4.Animal experiments showed that MV from K562 cells promoted the increase of MDSC;Compared with the no treatment group,the proportion of MDSC induced by MV generated by K562 cells treated with TKI was significantly reduced(P<0.05).Compared with that before TKI discontinuation,the proportion of MDSC caused by MV after TKI cessation was significantly increased.ConclusionsBoth in vivo and in vitro experiments showed that MV produced by K562 cells after different treatment had the effect of inducing MDSC amplification,suggesting that the important reason for differential outcome after drug withdrawal in CML patients may be that the MV generated by leukemia stem cells had different amplification effects on MDSC,and MV could be an important indicator for TKI cessation and intervention target.