Preliminary Study On The Role Of MDSCs In Guanylate Binding Protein 1-promoted Glioma Proliferation

[Objective]Guanylate binding protein 1(GBP1)is a member of GBPs family.Many cytokines,such as interferon,interleukin and TNF-alpha,can induce endothelial cells,fibroblasts and immune cells to produce GBP1.GBP1 is involved in many biological processes such as resistance to pathogenic microbial infection and immune regulation.GBP1 plays an important role in the growth,invasion and metastasis of digestive system tumors,such as head and neck tumors,breast cancer,myeloma and glioma,but the mechanisms are not clear.Our previous studies found that GBP1 could promote the proliferation of human glioma cells in nude mice,but there was no significant change in the proliferation of human glioma cells in vitro,which may be related to the tumor microenvironment.The aim of this study was to investigate the role of MDSCs in GBP 1-promoted glioma proliferation.[Methods]1.The mRNA and protein expression of GBP1 were analyzed by qRT-PCR and Western blot methods in U87-GBP1 overexpressed GBP1 and control cells U87-LacZ which were construced previously.The growth of glioma cells in vitro or in vivo were verificated by CCK-8 analysis or orthotopic implantation into nude mice.2.The peripheral blood mononuclear cells(PBMCs)were isolated from human peripheral blood using Ficoll-Paque regent.The CD 14 positive(CD14+)monocytes and CD3 positive(CD3+)T cells were isolated from PBMCs by immunomagnetic beads.3.The separated CD3+ T cells were activated by anti-human CD3 antibody and CD28 antibody,and then the activated T cells were stained by fluorescence staining carboxyfluorescein diacetate succinimidyl ester(CFSE).4.The culture media of U87-GBP1 and U87-LacZ cells were harvested.The CD14+monocytes were cultured in the media for 48 hr.The ratio of CD33+HLA-DR-cells(myeloid-derived suppressor cells,MDSCs)was analyzed by flow cytometry.The activated T cells stained with CFSE were co-cultured with CD14+monocytes treated with culture media of glioma cells.The inhibition rate of T cell proliferation was calculated by analyzing the attenuation of CFSE in T cells.5.The expression of chemokine(C-C motif)ligand 2(CCL2)in U87-GBP1 and U87-LacZ cells was detected by qRT-PCR and ELISA,respectively.U87-GBP1 and U87-LacZ cells were cultured in medium containing 125 mg/L of anti-human CCL2 antibody for 48 hours,and then the cell cultures were collected to culture the CD14+monocytes for 48 hours.The proportion of MDSCs was analyzed by flow cytometry.6.5*106 of U87-GBP1 and U87-LacZ cells were orthotopic implanted into BalB/C nude mice,respectively.After 7 days,the mice were divided into two groups,one group was injected with 2 mg/kg mass of RS 504393,the inhibitor of CCL2 receptor CC chemokine receptor 2(CCR2),intraperitoneally,the other group received the equal volume of DMSO.After 30 days of implantion,the xenografts and spleen tissues were separated and detected the proportion of CD11b+Gr-1+ cells(MDSCs)by flow cytometry.7.2 mL of human peripheral blood was collected from 21 patients with glioma and 19 controls(normal physical examinees in laboratory),respectively.Hemolysin was used to lyse red blood cells.Anti-human CD33-APC and HLA-DR-PE antibodies were added to label the cells.The proportion of MDSCs in the cells was detected by flow cytometry.[Results]1.qRT-PCR and Western blot were used to analyze the mRNA and protein expression of GBP1 in U87-GBP1 and U87-LacZ cells.Compared with the control cells U87-LacZ,the expression of GBP1 in U87-GBP1 cells increased significantly.U87-GBP1 cells proliferated faster in nude mice than U87-LacZ cells,and the weight of xenografts in U87-GBP1 bearing mice was larger than that of U87-LacZ bearing mice.However,there was no significant change in the proliferation between U87-GBP1 and U87-LacZ cells in vitro.2.CD14+ monocytes were cultured in U87-GBP1 and U87-LacZ cell culture media for 48 hours.The proportion of MDSCs in monocytes was significantly higher than that in control group,and the proportion of MDSCs in U87-GBP1 cell culture media treatment group was higher than that in U87-LacZ group.The MDSCs induced inhibited the proliferation of activated T cells,and the inhibition rate of U87-GBP1 group was higher.3.The mRNA and protein level of CCL2 in U87-GBP1 cells and cell culture media were significantly higher than those in U87-LacZ cells.The CD 14+monocytes were cultured in U87 cells culture media with anti-human CCL2 antibody.The proportion of MDSCs in the monocytes decreased significantly.4.U87-GBP1 and U87-LacZ cells can induce intracranial tumors in nude mice.The volume of xenografts in U87-GBP1 bearing mice was larger than that of U87-LacZ cells.After intraperitoneal injection of RS 504393,the growth of U87-GBP1 and U87-LacZ were significantly slowed down,and the volume of xenografts was smaller than that of untreated ones.The proportion of MDSCs in xenografts and spleen tissues of U87-GBP1 bearing mice were significantly higher than that of U87-LacZ bearing mice,but decreased significantly after treatment with RS 504393.There was no significant differenc in U87-LacZ group before and after treatment.5.The proportion of MDSCs in peripheral blood of glioma patients was higher thanthat of control group.[Conclusions]1.Glioma cells could induce normal CD14+monocytes to transform into MDSCs in vitro,which can inhibit the proliferation of activated T cells.GBP1 could enhance the transformation of CD14+monocytes into MDSCs.2.GBP1 could promote the overexpression of CCL2 in glioma cells.Anti-human CCL2 antibody used to neutralize CCL2 in cell culture could inhibit the transformation of monocytes into MDSCs.3.Blocking the binding of CCL2 to its receptor CCR2 by CCR2 inhibitor(RS 504393)could reduce the aggregation of MDSCs into tumor microenvironment and slow the proliferation of glioma cells in mice.It is suggested that GBP1 may promote the proliferation of tumors by promoting the expression of CCL2 and recruiting MDSCs to aggregate in tumor microenvironment to form immunosuppressive microenvironment.