Glioma-associated Neovascularization Measured With Dynamic Contrast-enhanced MRI

BackgroundsGlioma is one of the most aggressive tumor in humanity, highly proliferated neovascular is one of its distinct hallmark. Neovascularization describes the process that tumor gain blood supply, previously reported patterns of neovascularization including vascular co-option, angiogenesis, vasculogenesis, mosaic vessel formation, vascular mimicry and endothelial cell transdifferentiation. Glioma-associated neovascularization changes rapidly during tumor progression, and different patterns of neovascularization show various sensitivity to antiangiogenic drugs, thus investigation of dynamic changes about these patterns will make a positive promotion in glioma therapy.Magnetic resonance imaging is a non-invasive imaging tool which can provide inside information of tumor pathology. Dynamic contrast-enhanced MRI has already gained great utility in tumor diagnose and treatment detection. Parameters of DCE-MRI can demostrate tumor vessel permeability and perfusion in vivo, however the utility in neovascularization remained unclear.Sprout angiogenesis, vascular co-option, IMG and vascular mimicry are associated with glioma progression. In this article, we investigated the patterns of neovascularization using dynamic contrast-enhanced MRI, and explored its utility in demonstrating glioma-associated neovascularization.Materials and MethodsWe used three different glioma cell lines, i.e. C6, U87, U251 to establish animal model, while DCE-MRI and T2-WI MRI were taken to measure dynamic changes of glioma –associated neovascularization.1. Establishment of C6, U87, U251 model: The needle of microsyringe advanced to the depth of 5 mm and 1×106 resuspended in 10 μL DPBS was injected.2. Survival analysis: Total survival time of each tumor bearing animal was obtained and the survival rate was also analyzed.3. Pathology investigation1). Using HE staining to investigate characteristic of glioma pathology;2). CD34, TNC and PAS-CD34 immunohistochemistry method was used to analyzed patterns of glioma-associated neovascularization.3). α-SMA, Desmin staining was used to compare the maturity of neovascularization.4).PCNA, MMP-2 analysis was made to observe whether different patterns of neovascularization could affect the proliferation and invasion of glioma.4. MRI scaning of glioma1).Characteristic of T2 WI and DCE-MRI was observed in 6 time points;2).Parameters of DCE-MRI in 6 time points such as Ktrans,Kep and Vp were calculated;3).Correlation of MRI parameters and neovascularization was made.5. Statistical analysisData are presented as mean±standard deviation(SD). Statistical significance was evaluated by SPSS software, version 18.0. Survival rate was analyzed using Kaplan-Meier method, and statistical significance was compared using Log-rank test. Comparation of DCE-MRI and neovascularization parameters was made by using one-way ANOVA analysis. Correlation between DCE-MRI and neovascularization parameters was made by Spearman correlation analysis, and Pearson Correlation Coefficient was also calculated. Significance was defined as P < 0.05.Results1. Neovascularization quantitative analysis in glioma anamial model1). C6, U87 and U251 glioma models were diagnosed as glioma WHOⅢ/Ⅳ according to the 2007 WHO criterion.2). Median Survival Time of C6 glioma was 30 days, which was much shorter than U87 and U251 model.3). Proliferation of glioma was very high for all three models the PNCA index in glioma cell showed no significient difference(P < 0.05).4). Two different ways of vascular maturity existed in glioma model. Recruitmet of α-SMA positive vascular pericytes was observed in U87 and U251 model, while C6 model showed a way of Desmin positive vascular pericytes recruitmet.5). Four patterns of neovascularization were identified in glioma tissues, which was sprout angiogenesis, vascular co-option, IMG and vascular mimicry. Unlike vascular mimicry, other three patterns showed significient difference between C6, U87 and U251 models(P < 0.05).2. Dynamic changes of neovascularization and MR imaging1). C6 glioma showed an invasive growth pattern under lightmicroscope, and vessel region was 437.8±153.6μm from the tumor edge.2). Vascular co-option was much less at PID 12 and 16 than any other time points(P<0.05).Sprout angiogenesis was the most important pattern of neovacualrization and numbers of stalk cells increased as tumor developed(P<0.05). IMG was only observed after PID 4, and numbers of folds across the vessel lumen gained rapidly with tumor growth(P<0.05). At all six time points, vascular mimicry existed less than any other three patterns of neovascularization.3). T2 WI MRI indicated that tumor volume increased as glioma developed(P<0.05) with parameters inside tumor of DCE-MRI changed in the mean time(P<0.05). However, Ktrans and Kep outside tumor decreased after a short ascending period at PID 12 and PID 16(P<0.05).4). Correlation analysis showed that Ktrans and Kep of DCE-MRI outside tumor correlated with vascular co-option negatively while Kep, Vp inside tumor correlated with sprout angiogenesis and IMG positively(P<0.05).Conclusion1. There exist at least four patterns of neovascularization in glioma, which are sprout angiogenesis, vascular co-option, IMG and vascular mimicry.2. These four patterns of neovascularization show a dynamic variance during tumor development.3. DCE-MRI parameters correlate with patterns of neovascularization, indicating that DCE-MRI could be a well-defined imaging-based biomarker for glioma neovascularization.