Role And Mechanism Of M2-like Tumor-associated Macrophages In Vasculogenic Mimicry Of Glioma Cells

BackgroundGlioma is the most common intracranial primary malignancy in the central nervous system,accounting for about 50%of the primary tumors in the brain.The most common subtype of glioma is highly malignant glioblastoma(GBM,WHO grade ?).In recent years,although a combination of surgery,radiotherapy and chemotherapy has made some progress in the treatment of glioma,the median survival time of glioblastoma patients is still only 14.6 months,with poor prognosis and high recurrence rate.Because gliomas are highly vascularized brain tumors,the prospect of anti-angiogenic drugs targeting vascular endothelial cells to treat gliomas has attracted much attention.However,the recent data has showed that this potential anti-angiogenic therapy appears short-term efficacy and poor efficacy after clinical application.Until vasculogenic mimicry(VM)proposed in 1999,it is gradually realized that there is another form of blood supply without endothelial cells involved in tumor tissues,which is characterized by tumor cells forming tubular structures that transport erythrocytes and plasma in order to nourish tumors.These structures have been found in a variety of malignant tumors,including gliomas,and the positive rate of VM is closely related to tumor grade and poor prognosis.Thus,tumor VM may explain the limitations of anti-angiogenic therapy.And inhibition of VM combined with anti-angiogenic therapy may have a better therapeutic effect for glioma patients.At present,the specific mechanisms of VM are still unclear.Recent studies suggest that VM is associated with tumor microenvironment.Further-study of the glioma microenvironment will be helpful to understand the mechanisms of VM formation.Immune cell infiltration is a significant feature of tumor microenvironment,which plays a key role in malignant biologic behavior and further affects tumor progression.Macrophages are the most abundant immune cell population in solid tumor.In glioblastoma tumor-associated macrophages(TAMs)constitute up to 30%of tumor mass.Moreover,TAMs infiltrated in glioblastoma tissues have been reported to express the elevated M2-like macrophage markers,CD 14,CD68,CD 163,and CD204.The number and types of macrophages infiltrated in glioma tissue are associated with the glioma grade and prognosis of glioma patients.In addition,it has been reported that macrophage infiltration contributed to proangiogenic effects and was associated with resistance to anti-angiogenic therapy in several solid tumors,including glioblastoma.These findings suggested that targeting macrophage-promoting angiogenesis in tumor progression can be a universal method to overcome antiangiogenic drug resistance.Another important hallmark of cancer is considerable cytokines and chemokines in tumor microenvironment.Increasing data described that these immune mediators could create a favorable environment to support tumorigenesis by driving the functions of immune cells and tumor cells.One such factor is the pleiotropic cytokine,interleukin-6(IL-6);a potent mediator that is omnipresent in the inflammatory microenvironment of most solid tumors,including glioma.By participating in immune regulation,promoting tumor cell proliferation,migration,survival and drug resistance,IL-6 could lead to poor prognosis of cancer patients.In addition,recent work indicated that IL-6 was associated with poor efficacy of anti-vascular endothelial growth factor(VEGF)antibody treatment to tumors.In gliomas,IL-6 could promote tumor invasion and angiogenesis by enhancing the migratory ability of vascular endothelial cells.Inhibition of IL-6 expression combined with bevacizumab therapy may be more effective to prevent glioma cell invasion and growth.These results suggest that IL-6 has important effects on vascular regulation in the tumor microenvironment,but whether IL-6 plays a significant role in the formation of glioma VM is largely unknown.Based on previous work,this study preliminarily adopted immunohistochemical methods to analyze a correlation of VM with tumor associated macrophages in human glioma tissues.The in vitro studies were employed to investigate whether M2-like macrophages and monocyte-derived M2 macrophages could affect VM formation and explore the potential regulatory mechanisms.Part?Expression of vasculogenic mimicry and CD163+ macrophagesin gliomaObjectiveTo detect the expression and clinical significance of VM and CJ163+ macrophages in glioma tissues,and to analyze the correlation between VM level and CD163+macrophage infiltration.Methods1.Tissue samples from 87 patients diagnosed with glioma were obtained during the surgical tumor resection at the Department of Neurosurgery,Qilu Hospital of Shandong University.The expression of VM and CD163+ macrophages in glioma was examined by immunohistochemistry,and the correlation between them was analyzed.2.The basic information of glioma patients was collected.The correlation of VM and CD163+ macrophages with different clinicopatUological factors and prognosis were further analyzed.Results1.We detected the expression of CD 163(a marker of human M2-like macrophages),CD31 and PAS(markers for vessel)in 87 human glioma specimens by immunochemical staining.The results showed that the VM(CD31-PAS+)level and CD 163 density in high grade(WHO?-? grade)was significantly higher compared with low grade(WHO ?-? grade)(P<0.01,P<0.01).More importantly,a correlation of CD31-PAS+ vessels and CD163+ cells was observed in glioma tissues(r2=0.416,P<0.001).2.The number of VM and CD 163+ macrophages increased with aggressive tumor biology defined by advanced WHO grade(P<0.001,P<0.001).VM channels increased with higher tumor burden as defined by tumor size(P<0.05).No correlation was observed in gender,age,tumor location,KPS and growth pattern of tumor.3.To determine whether there were prognostically significant association between VM,or CD 163 and patient survival,Kaplan-Meier survival curves were then plotted.The results showed that CD 163 low group had a significant survival advantage compared with CD 163 high group(P=0.006),and this survival advantage was also shown in VM low group(P<0.001).We further analyzed patient survival with the combination of CD163 with VM.More significant value was observed in overall patient survival than one single factor(P<0.001).Conclusion1.The levels of VM and infiltrated CD163+ macrophages were positively correlated with glioma grade.There was a significantly positive correlation between VM level and CD 163+ macrophage infiltration in human glioma tissues.2.High VM level and high density of CD163+ macrophages were both associated with poor prognosis of glioma patients.3.The levels of VM and infiltrated CD163+ macrophages were negatively correlated with clinicopathological parameters,such as gender,age,tumor location,KPS and growth pattern of tumor.Part?The function and mechanism of THP-1-derived M2 macrophages in glioma vasculogenic mimicryObjectiveTo explore the effects of THP-1-derived M2 macrophages on glioma VM formation and potential mechanisms.Methods1.The phenotype of THP-1 cells-derived macrophages was detected by qRT-PCR.2.To determine the effects of THP-1-derived M2 macrophages on VM formation of glioma cells,tubule formation assays were performed.3.The effects of THP-1-derived M2 macrophages on the expression of VM related markers in glioma cells were examined by qRT-PCR and Western blotting.4.Cytokine array and ELISA were performed to analyze the effects of THP-1-derived M2 macrophages on the secretion of IL-6 in glioma cells.5.To determine the relative timing of IL-6 and VM marker expression following stimulation,mRNA levels of IL-6 and VM markers in glioma cells were evaluated by qRT-PCR at indicated time points.6.The effects of IL-6 on the VM formation of glioma cells were detected by tubule formation assays and Western blotting.7.Tubule formation assays,qRT-PCR,ELISA and Western blotting were employed to detecte the effects of THP-1-derived M2 macrophages on the signal pathways of glioma cells.Results1?THP-1-derived M2-like macrophages expressed high levels of M2 type markers(CD163,CD206,IL-10,IL-1RA and CCL17)and low levels of M1 type markers(TNF-?;IL-1?and IL-12).2.Treatment by conditioned medium from THP-1-derived M2 macrophages(M2-CM)increased approximately 2 fold changes of the tubule structures in glioma cells,compared to THP-1-CM-treated groups or control groups(U251,P<0.001;A172,P<0.001).3.Compared to THP-1-CM-treated groups,transcription and secretion of IL-6 in glioma cells were markedly increased by M2-CM in time-dependent and dose-dependent manners.IL-6 transcript levels rose first,reaching near-peak expression at 6 hours,whereas VM marker expression lagged behind,rising steadily over 24 hours.4.The number of tubes was significantly decreased after adding IL-6 neutralizing antibody(anti-IL-6,1 ?g/ml)into the coculture system(U251,P<0.01;A172,P<0.01),and the protein levels of VM markers were abated as well.In addition,the number of tubes was increased by using recombinant human IL-6(rhIL-6,100 ng/ml)alone compared with their corresponding controls(U251,P<0.01;A172,P<0.05),and VM marker expression was augmented correspondingly.5.Glioma cells were stimulated with M2-CM at different time,and a transient upregulated phosphorylation of PKC pathway was measured by Western blotting.PKC inhibitor Bisindolylmaleimide I significantly inhibited IL-6 transcription(U251,P<0.01;A172,P<0.01)and secretion(U251,P<0.001;A172,P<0.001)induced by M2-CM,and further inhibited VM marker upregulation and VM formation promotion(U251,P<0.05;A172,P<0.05).Conclusion1.THP-1-derived M2 macrophages promoted VM formation of glioma cells in vitro.2.THP-1-derived M2 macrophages upregulated the production of IL-6 by activating the PKC signaling pathway of glioma cells,thus promoting the glioma VM formation.Part?The function and mechanism of monocyte-derived M2 macrophages in glioma vasculogenic mimicryObjectiveTo explore the effects of human peripheral blood monocyte-derived M2 macrophages on glioma VM formation and potential mechanisms.Methods1.To generalize our findings,we isolated monocytes from human peripheral blood mononuclear cells by magnetic cell sorting using CD 14 microbeads,which were induced to macrophages.2.The phenotype of human peripheral blood monocyte-derived M2 macrophages was detected by ELISA and qRT-PCR.3.qRT-PCR and ELISA were performed to analyze the effects of human peripheral blood monocyte-derived M2 macrophages on the secretion of IL-6 in glioma cells.4.To determine the effects of human peripheral blood monocyte-derived M2 macrophages on VM formation of glioma cells,tubule formation assays were adopted.5.Tubule formation assays were performed to analyze the effects of IL-6 secreted by glioma cells on VM formation.Results1.Monocyte-derived M2 macrophages expressed high transcriptional levels of M2 type markers(CD163,CD206,CCL18 and CCL17)and low transcriptional levels of M1 type markers(TNF-?,IL-?,IL-6 and IL-12).Monocyte-derived M2 macrophages secreted high levels of M2 type cytokines/chemokines(CCL22,CCL18,CCL17 and IL-10)and low levels of Ml type cytokines(IL1-?,IL-6,IL-12 and IL-8).2.Compared to other groups,transcription(U251,P<0.01;A172,P<0.001)and secretion(U251,P<0.001;A172,P<0.001)of IL-6 in glioma cells were dramatically increased by monocyte-derived M2-CM.3.The number of tubes in glioma cells was significantly decreased after adding IL-6 neutralizing antibody(anti-IL-6?1?g/ml)into the coculture system(U251,P<0.01;A172,P<0.01).These results indicated that anti-IL-6 could block monocyte-derived M2 macrophages-mediated promotion of glioma VM.Conclusion1.Human peripheral blood monocyte-derived M2 macrophages promoted the production of IL-6 in glioma cells in vitro.2.Human peripheral blood monocyte-derived M2 macrophages amplified the production of IL-6 in glioma cells and further promoted the glioma VM formation.