| Myeloid-derived suppressor cells(MDSCs)are a group of immature,suppressive cells.Studies have found that MDSCs play an important role in the tumor microenvironment.Tumor-derived cytokines and growth factors can stimulate the expansion or activation of MDSCs,which will promote tumors growth and generate immune tolerance environment through various mechanisms.MDSCs are one of the main reasons for the failure of tumor immunotherapy.It's believed that therapeutic strategies targeting MDSCs will reduce immunosuppressive effects and enhance responses to immunotherapy.Calcitriol is the active form of vitamin D3,which exerts biological functions by combining with vitamin D receptor(VDR).In addition to regulating calcium and phosphorus homeostasis,calcitriol also has effects on tumor growth and homeostasis of the immune system.However,the role of calcitriol on glioma and its microenvironment in MDSCs is still unknown.In this study,C57 BL / 6J mice were pretreated with calcitriol for two weeks and the same dose of normal saline was used in the control group.Then mouse glioma cells(GL261)were used to construct an orthotopic tumor model,the survival time of mice was recorded,and the survival rate of the two groups of tumor-bearing mice was calculated.Compared with control group,the survival time of tumor-bearing mice injected with calcitriol was significantly shortened(p <0.05),and the tumor area was also obviously larger(p <0.05).In addition,to explore the mechanism by which calcitriol aggravates glioma,we evaluated the direct effect of calcitriol on the viability of GL261 cells L929(mouse fibroblasts)using CellTiter-Blue.We found low concentration of calcitriol(<10 ?M)had no significant effect on the viability of GL261 and L929 cells,while high concentration(?10 ?M)of calcitriol showed cytotoxicity on both cells.In order to elucidate whether MDSCs is involved in the effect of calcitriol in glioma,mice bone marrow cells were isolated and cultured in vitro and the differentiation of MDSCs was detected by flow cytometry.Calcitriol induced bone marrow cells to differentiate into M-MDSC,a significant increase of M-MDSC was observed after calcitriol treatment(p <0.001).Also,real time PCR results showed that calcitriol promoted the expression of vitamin D receptor(VDR)and arginase 1(ARG1),a gene related to MDSC suppressive function.Furthermore,the infiltration of MDSCs,CD4 and CD8 a T cells in brain,spleen and bone marrow was detected using flow cytometry on day 18 after tumor implantation.Compared with control group,the proportion of mononuclear MDSC(monocytic-MDSC)in the brain tissue area was significantly increased(p <0.05)by calcitriol,but no significant difference was found in the spleen and bone marrow.The percentages of CD4 and CD8 a T cells in brain tissue,spleen,and bone marrow were not changed after calcitriol administration.Finally,in order to further confirm the biological significance of calcitriol in gliomas,immunohistochemical staining and western blot was performed to detect the expression of VDR in normal brain and glioma.The results showed that the VDR expression level in glioma was significantly higher than that in normal brain,and VDR was prominently increased by calcitriol.Kaplan-Meier survival analysis of GBM patients from TCGA database showed that VDR expression was inversely related to the survival time of GBM patients.In conclusion,VDR was high expressed in glioma and negative associated with the prognosis of patients.Calcitriol administration induced the expression of VDR in mouse glioma model and promoted the progression of glioma and shorten the survival time of tumorbearing mice via enhancing the infiltration of M-MDSC in tumor microenvironment. |
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