An Inductive Role Of Glia Maturation Factor-beta In The Formation Of Glioma Cell-derived Neovessels And Its Preliminary Mechanism

Glioma is the most common type of primary brain tumor in adults. The vigorously proliferating microvessels are associated with high invasiveness and poor prognosis of malignant glioma, especially of glioblastoma multiforme(GBM). Despite recent improvements in antiangiogenic therapy for glioma patients, some vascular-targeting agents bring about therapy resistance, owing to complicate mechanisms of glioma vascularization. It is needed to identify specific regulators of the formation of glioma-derived neovessels.Sprouting angiogenesis was once regarded as an exclusive means for tumor neovessel formation. Recently, it has been found that tumor stem cells(CSCs) and malignant tumor cells could generate vascular cells to form new blood channels, which has provided a novel insight into the mechanism underlying tumor neovascularization.Tumor neovascularization is often associated with the reactivation of molecular pathways that regulate embryonic vasculogenesis. It is noted that neural stem cells(NSCs) and endothelial cells(ECs) co-locate in a neurovascular unit during embryogenesis. Moreover, NSCs and ECs are involved in the reciprocal coordination of neurogenesis and vasculogenesis, sharing some common regulators. In addition, NSCs has been reported to convert into ECs. It has been proved that such a dynamic interplay between glioma stem cells(GSCs) and tumor endothelial cells(TECs) is implicated in gliomagenesis. Moreover, transdifferentiation of GSCs or GBM tumor cells into TECs is respectively reported by several studies.Glia maturation factor-beta(GMF-β) was first identified as a glial differentiation factor. The protein factor can induce the differentiation of glial cells and the growth of some neurons, and promote neural regeneration. Previous studies suggested that GMF-β could exert a negative feedback control on cell proliferation, promote differentiation and induce chemosensitivity of glioma cells. However, GMF-β overexpression was reported to be related with poor prognosis in breast cancer and ovarian cancer.Based on the background of common regulators of neurogenesis and vasculogenesis, as well as manifold functions of GMF-β in glioma and other tumors, we hypothesis that GMF-β, a glial differentiation factor, could induce the formation of glioma cell-derived neovessels and thus promote glioma progression. Firstly, we examined the expression pattern of GMF-β in gliomas of all grades, and assessed its correlation with neovascularization as well as its clinical significance. Next, we further explore if GMF-β can induce invitro tube formation and endothelial differentiation of malignant glioma cells. Lastly, we verified the role of GMF-β in inducing TECs derived from malignant glioma cells in an orthotopic xenograft model.Main methods, results and conclusion as follow:1. By combined application of GMF-β/CD31 double immunohistochemical staining and histochemical periodic acid-schiff(PAS) staining methods, we examined the expression pattern of GMF-β in 146 human gliomas of all grades, and analysed its correlation with microvessel density(MVD), clinical prognosis and other clinicopathological parameters.(1)GMF-β was expressed by both glioma cells and TECs. Moreover, the levels of GMF-β expression were found being positively correlated with tumor grade and MVD in glioma cells(P<0.001; r=0.367,P<0.001), as well as in TECs(P<0.0001; r=0.557, P<0.0001).(2)In GBM, GMF-β expression levels in perivascular glioma cells(p GCs) were significantly higher than in glioma cells distant from microvessels(P<0.0001). Furthermore, GMF-β expressions in p GCs were positively correlated with those in TECs(r=0.770, P <0.0001) and MVD(r=0.620, P <0.0001).(3) High expression of GMF-β in TECs was an independent predictor of poor prognosis in glioma patients. Kaplan–Meier analysis and univariate Cox regression analysis showed that, GMF-β expressions in both tumor cells and TECs were unfavorable prognostic factors for glioma patients(both(P<0.001). However, multivariate survival analysis confirmed that it was GMF-β expression in TECs, not in tumor cells, being an independent predictor of both PFS(P<0.0001, HR=1.244, 95%CI=1.136-1.363) and OS(P<0.0001, HR=1.236, 95% CI=1.126-1.358) in glioma patients.(4) Correlation existed between GMF-β expression status and other clinicopathological parameters. It showed that GMF-β expression was associated with other indicators of poor prognosis and radio/chemotherapy. Pearson χ2 test indicated that higher GMF-β expression in tumor cells was correlated with higher tumor grade and elevated Ki67 index(both P<0.05), and dominated in patients receiving radiotherapy(P<0.05). On the other hand, higher GMF-β expression in TECs was closely related to higher tumor grade, older age of patients(both P<0.001), and dominated in patients receiving chemotherapy or radiotherapy(both P<0.05). In addition, tumors located in temporal lobe tend to have higher GMF-β expression in TECs(P<0.05).2. GMF-β gene(GMFB) expression was upregulated by lentiviral vector-mediated overexpression and was downregulated by lentiviral vector-mediated RNA interference. The effect of GMF-β on tubulogenesis and endothelial differentiation was observed in U87 glioblastoma cells.(1)A two-dimensional matrigel tube formation assay was performed to assess vasculogenic activity of U87 cells in vitro. Tubulogenesis was initiated in U87 cells, extremely relying on endothelial cell growth medium. By "pattern recognition" and "branch point counting" system, tubulogenic capacity of U87 cells was quantified and showed to be lower than human umbilical vein endothelial cells(both P<0.05).(2)Deferoxamine was added into the endothelial cell growth medium to mimic hypoxic stimulation. Under such conditions, parallel upregulation of GMF-β and CD31 m RNA expression was induced in U87 cells.(3) GMF-β knockdown impaired tubulogenic capacity of U87 cells. Tubulogenesis of U87-sh GMFB cells was showed to have significantly decreased when compared with that of U87 mock cells(P<0.001; P<0.0001).(4) GMF-β knockdown inhibited U87 cell proliferation. In contrast to U87 cells, the proliferation of U87-sh GMFB cells was more strongly inhibited(P<0.05).(5)GMF-β overexpression upregulated CD31 m RNA expression in U87-OE-GMFB cells, while GMF-β knockdown downregulated CD31 m RNA expression in U87-sh GMFB cells.3. We further verifed the role of GMF-β in inducing TECs derived from glioma cells in murine orthotopic glioma model. All xenografted glioma samples were subjected to immunohistochemistry(IHC)-double staining of h GMF-β and h CD31.(1) GMF-β overexpression resulted in increased quantities of glioma cell-derived neovessels in U87 glioblastoma xenograft tumors and shorter survivals of tumor-bearing mice. The statistical analysis showed that, a significant trend toward shorter survival was revealed in mice receiving U87-OE-GMFB cells as compared with mice in control group(P<0.01). Furthermore, human CD31-positive microvessel densities(h CD31-MVDs) in U87-OE-GMFB xenograft tumors were significantly decreased in contrast to U87-mock tumors(P<0.001).(2)GMF-β knockdown in U87 cells repressed tumor growth and the formation of glioma cell-derived microvessels. Statistical analysis demonstrated that gross tumor volumes and h CD31-MVDs in U87-sh-GMFB group were significantly decreased than those in U87-mock group(P<0.0001; P<0.001).In summary, GMF-β is specifically expressed by both glioma cells and TECs, which is associated with tumor grade, MVD and clinical prognosis. Especially, high expression of GMF-β in TECs is an independent poor prognostic indicator for glioma patients. GMF-β can induce tubulogenesis and CD31 expression of U87 glioblastoma cells in vitro, but also can promote tumor growth and the formation of glioma cell-derived neovessels in an orthotopic U87 xenograft model. Our results suggested that GMF-β should be a novel diagnostic marker and prognostic indicator, as well as a cell type-specific target for anti-vasculogenic therapy in glioma.