Mechanisms Of Glioblastoma Neovascularization And Their Targeted Theranostics With MRI

Background and ObjectivesGlioblastoma(GBM),which is the most frequent primary malignant brain tumor in central nervous system in adults,is one of the most vascular-rich tumors and has short median survival and poor prognosis regardless of optimal treatment and evolving standard of care.Anti-vascular endothelial growth factor(VEGF)therapy has had significant efficacy in GBM with more than half of responders,but this effect is transient in most patients,acquired antiangiogenic resistance may occur.Treatment with an inhibitor of VEGF results in reduced tumor growth rate with decreased tumor vascularization followed by a massive necrosis and hypoxia leading to post-therapy relapse with a plexus composed of enlarged glomeruloid vascular proliferations(GVPs).Emerging evidence has detailed several mechanisms,including enhanced invasion and alternative angiogenesis,by which glioma adapts to and circumvents antiangiogenic therapy.Recent studies demonstrate that a population of glioma stem-like cells(GSCs)transdifferentiate into vascular endothelial cells within glioma,possibly via an intermediate endothelial progenitor cells(EPCs),which may provide new perspectives on the mechanisms of the resistance to anti-VEGF therapy currently in use.On the other hand,emerging evidence indicates that tumors with intensive GVPs showed more resistant to anti-VEGF therapy than these with predominant classic angiogenesis.Although treatment with bevacizumab,an inhibitor of VEGFR tyrosine kinase,could reduced tumor growth rate by decreased tumor vascularization,the massive necrosis and hypoxia in GBM are usually followed by the formation of a plexus composed of enlarged GVPs that is believed as a major cause for relapse.Therefore,GVPs is one of the bevacizumab-resistant neovascularization.Our previous study showed that GVPs were mainly distributed close to area of necrosis,where locates TF.These findings provide new ideas on the mechanisms of resistance to anti-angiogenesis therapy.Therefore,it is prior to find new methods on anti-angiogenesis therapy and their corresponding MR imaging.To test the hypothesis,our study aims to investigate from the endothelial composition at the cellular and molecular level to GVPs at structural level.In part one,rat C6 brain glioma model and C6 glioma cells were used to explore the composition of tumor vascular endothelium.In part two,56 GBM patients were included,mice U87 brain glioblastoma model and U87 glioblastoma cells were used to investigate the molecular mechanism of GVP formation mediated by TF and correlation with MRI perfusion indexes.In part three,based on the mechanism of glioma neovasculature above,to investigate the targeted theranostic carrier for cancer cells transdifferetiation into the endothelial cells and targeted therapy for GVP formation.Our findings may provide a rational base for target screening for anti-angiogenesis,targeting therapy,monitoring course of the disease,and evaluate the therapeutic efficacy.Materials and MethodsThis research was composed of in vitro,in vivo,and clinical sample.In vitro experiments were conducted on U87 glioblastoma cell line,C6 glioma cell line,and rat spleen-derived endothelial progenitor cells.In vivo experiments,nude mouse U87 brain glioblastoma model and rat C6 brain glioma model was established.In clinical study,human diagnosed with glioblastoma without treatment were chosen.The composition of vascular endothelium and their MRI in vivo tracking in glioblastoma1.CD 34 and GFAP immunohistochemical staining were performed to observe the co-localization of the CD34 and GFAP in GBM clinical sample and the co-localization of the endothelial cell marker CD34,CD31,v WF and GFP in frozen section of C6 brain glioma model.2.C6 glioma cells were transfected with lentivirus containing GFP,and the transfection efficiency was detected.3.C6 glioma cells were cultured in hypoxia.The co-localization of CD34,CD31,v WF and GFP were detected.4.Transdifferentiation efficiency of C6 glioma cells was detected by flow cytometry,endothelial function of C6 glioma cells was evaluated by tube formation assay after DAPT,sunitinib and normoxia treatment.5.HIF-1α、Notch 1、Flk1,and p-Flk1 expression were detected by western blot analysis.6.SD rat spleen-derived mononuclear cells were isolated by density gradient centrifugation.The medium was changed every 3 days.EPCs were characterized as morphology,double positive for Di I-ac LDL uptake and lectin binding by immunofluorescence assay,and tube formation in vitro.7.EPCs were labeled with USPIO,injected into the rat with glioma via tail veil.MRI T2-weight imaging,T2 map,SWI were performed to evaluate the number,distribution of EPCs,and their relationship with microvessels in tumor.8.Prussian blue staining was used to detect the distribution of EPCs in tumor.The homing exogenous EPCs were quantified by flow cytometry.The mechanism of GVPs formation and their correlation with MRI characteristics in glioblastoma1.Fifty-six GBM patients were involved in the clinical study.Microvessel density,microvascular area,and diameter were assessed by CD34 immunohistochemical staining,TF,VEGF,HB-EGF expression were detected by immunohistochemistry in GBM clinical samples.2.TF expression in U87 glioblastoma cell line was detected by western blot and immunofluorescence analysis.3.Sh RNA was used to knock down the TF expression in U87 glioblastoma cell line,and knock-down efficiency was detected.4.Glioblastoma mouse model was established,TF expression and GVPs were detected by immunohistochemistry.The correlation of TF expression and GVPs distribution was analyzed.5.Four groups were divided in tumor cells in vitro experiments,including Parental group,Anti-TF abs group,NS group,and TFsh group.The effect of TF on U87 glioblastoma cell proliferation,migration,and apoptosis were evaluated by MTT,scratch wound and transwell assay,and flow cytometry,respectively.6.Conditioned medium(CM)was collected,including Parental-CM,NS-CM,TFsh-CM.The effect of the different CMs on the proliferation,migration,tube formation,and PAR2/HB-EGF expression were assessed.7.Some of the GBM patients involved were performed conventional MRI and perfusion MRI(DCE-MRI and VSI-MRI).8.The correlation between TF expression in tumor and indexes of DCE-MRI and VSI-MRI were analyzed to obtain the MRI biomarkers of TF and bevacizumab-resistant neovascularization.Targeting labeling and therapy for tumor microvessels and their MRI evaluation1.Immunofluorescence staining and transmission electron microscope were performed to investigate whether exogenous EPCs integrated into the vessels containing TDECs or not.2.HIF-1α,Notch 1,Flk1,and p-Flk1 expression were detected by western blot analysis.3.Two groups were divided including control group and BBG group.MRI was performed to detect the tumor volume,perfusion,the number and distribution of USPIO-EPCs in tumor with or without P2X7 receptor suppression.Immunofluorescence staining was performed to investigate the effect of P2X7 receptor on exogenous EPCs integrated into the vessels containing TDECs.4.Four groups of animal model were established,including Parental group,NS group,TFsh group,Bevacizumab group.5.Survival analysis was evaluated,and the statistics were done to analyzed the significance.6.The volume and invasion of GBM were detected by conventional MRI,the angiogenesis and flow perfusion of GBM were detected by perfusion MRI at different time points.7.Tumor invasion was analyzed by H.E.staining.Microvessel density,microvascular area,and diameter were assessed by CD34 immunohistochemical staining.8.TF,VEGF,HB-EGF expression were detected by immunohistochemistry and western blot analysis.ResultsThe composition of vascular endothelium and their MRI in vivo tracking in glioblastoma1.Tumor-derived endothelial cells integrating into tumor vessels and its mechanismThe immunohistochemistry revealed that some endothelial cells,lining the vessel lumen of tumor vessels,not only expressed endothelial cell markers(CD34,CD31,v WF),but also expressed GFAP/GFP in clinical GBM specimens and C6 glioma mouse model.Moreover,the C6 glioma cells were found to transdifferentiate into endothelial cells and had the endothelial function by immunofluorescence,flow cytometry,tube formation in vitro.Therefore,tumor-derived endothelial cells(the cancer cells transdifferentiation into endothelial cells)could be a target for anti-angiogenesis therapy.The protein expression of HIF-1α,Notch1,Flk1,and p-Flk1 in glioma tissue and C6 glioma cells with higher transdifferentiation frequency were significantly higher than those with lower transdifferentiation frequency by western blot analysis.Moreover,transdifferentiation frequency of C6 glioma cells decreased by DAPT,sunitinib and normoxia treatment.2.In vivo MRI tracking and quantifying of EPCs homing and migration into the tumor vessels.T2-weighted imaging and T2 map revealed that hypointensity was detected at the rim of the tumor on day 1 after EPCs transplantation,the hypointense area nearly covered the entire tumor foci on day 7 after EPCs transplantation,which was confirmed by Prussian blue staining.SWI could track the integration of EPCs into glioma vessels,which was confirmed by immunofluorescence.The mechanism of GVPs formation and their correlation with MRI characteristics in glioblastoma1.TF contributed to GVPs in GBM clinical samples and their clinical significanceIn total 56 GBM patients,TF expression in tumor positively correlated with malignant progression of GBM.GVPs were mainly distributed in the hypoxia of GBM,close to area of necrosis,which was consistent with TF expression area.While,another distinct vascular type “classic” angiogenesis,evident by evenly distributed capillary-like microvascular sprouting,located similar to the region of VEGF expression.Further,we found that TF expression correlates well with GVPs,while VEGF expression correlates well with classic sprout angiogenesis.2.The molecular characteristics of TF mediated GVPs formation in GBMTF suppression decreased PAR2 and HB-EGF expression in tumor tissue.Further,TF expression contributes to the proliferation,migration,and angiogenesis of endothelial cells after endothelial cells treated with different CMs,via PAR2/HB-EGF signal pathway.On the other hand,TF inhibition deceased migration of the tumor cells,leading to decrease of GVPs formation.Therefore,TF suppression decreased GVPs by down-regulating PAR2/HB-EGF pathway in ECs and reducing cancer cells invasion.3.In vivo MRI evaluation of TF expression to reflect GVPs in patients with GBMAnatomic and advanced MRI were performed to assess TF expression to reflect GVPs in clinical GBM in vivo.In TF-high expression GBM,Ktrans map derived from DCE-MRI showed high permeability of contrast across the BBB into EES in tumor region,VSI map showed large microvessel size in tumor,which indicated that the vessels in TF-high expression tumor could be high in permeability,large in area,consistent with the characteristics of GVPs.While,in TF-low expression GBM,Ktrans map and VSI map revealed relative low permeability and small microvessel size.Further,Ktrans hot-spot value and VSI hot-spot value positively correlated well with TF expression in GBM.These results showed that Ktrans hot-spot value based on Extended Tofts Linear model and VSI hot-spot value,which can be used to assess functional and structural changes of tumor vascularization,could be surrogate imaging biomarkers for TF expression in GBM patients.Targeting labeling and therapy for tumor microvessels and their MRI evaluation1.The targeting theranostic carrier of the cancer cells transdifferentiation in glioblastoma and their modulationExogenous EPCs homed and integrated into the vessels containing TDECs by immunofluorescence and transmission electron microscope analysis.There was no significant difference between EPC group/EPC-CM group and control group about the transdifferentiation frequency and the protein expression of HIF-1α,Notch1,Flk1,and p-Flk1 in glioma tissue and C6 glioma cells.Therefore,EPCs may be a best vehicle to deliver the therapeutic genes,targeting glioma cells transdifferentiation more s ufficiently and effectively.T2 weighted imaging and T2 map showed that BBG inhibited exogenous EPCs homing and migration into the glioma in vivo.SWI,immunofluorescence and transmission electron microscope analysis showed BBG inhibited exogenous EPCs integrating into the vessels containing the tumor derived endothelial cells,which indicated that P2X7 receptors could modulate exogenous EPCs integrating into the vessels containing the TDECs.T2-weighted imaging and DCE-MRI showed P2X7 receptors exerted no significant promoting effect on C6 glioma cells proliferation,gliomas growth and angiogenesis.2.Targeted TF suppression decreased GVPs formation in mice brain GBM model and prolonged the survival time of mice with GBMMRI T2-weighted imaging showed that the volume and invasive area of tumors in TFsh group was significantly decreased.DCE-MRI showed the perfusion of tumors in TFsh group was also significantly reduced.SWI showed the hypointensity lessen in TFsh group,especially the peri-tumor.TF targeted therapy showed TF suppression decreased MVD,MVA,diameter,resulting in the decrease of the GVPs.While,bevacizumab therapy showed bevacizumab decreased MVD,but not MVA and diameter,resulting in the decrease of the classic angiogenesis.And the mice treated with TF suppression survived longer than those in control groups.Conclusion1.This study provides insight into the underlying mechanism of tumor neovascularization in GBM: 1)Cancer cells transdifferentiation into endothelial cells integrated into tumor vascular endothelium;2)TF modulated GVPs by inducing PAR2/HB-EGF pathway in endothelial cells and increasing cancer cells invasion.2.USPIO labeled exogenous EPCs could be a targeting theranostic carrier to the cancer cells transdifferentiation into endothelial cells,which could be modulated by P2X7 receptor to improve the tracking and therapeutic efficacy.3.Targeted TF suppression decreased GVPs formation in mice brain GBM model and prolonged the survival time of mice with GBM.MRI could evaluate the the rapeutic efficacy non-invasively and dynamically.Moreover,Ktrans hot-spot value and VSI hot-spot value could be the imaging biomarkers to evaluate TF expression and GVPs in GBM.Our findings may provide a rational base for target screening for anti-angiogenesis,targeting therapy,monitoring course of the disease,and evaluate the therapeutic efficacy.