Study On The Correlation Between Perfusion-weighted MR Imaging And The Characteristics Of Neovascularization Related Genes And Patterns In High Grade Gliomas

Background and purpose:Glioma is the most common primary intracranial tumor in adults,accounting for more than 70%of adult primary malignant tumors in brain.According to the World Health Organization(WHO)classification of tumors in the central nervous system,glioma is divided into grade I to grade IV based on cytological atypia,mitotic activity,microvascular proliferation and necrosis,grade I and grade II glioma are low grade glioma(LGG),grade III and grade IV glioma are high grade glioma(HGG).HGG has a high degree of malignancy and rapid tumor progression,even with the development of treatment methods,the prognosis is still poor.Especially for WHO IV glioblastoma,the median survival time is only 14.6months,and the 5-year survival rate is less than 5%.High-grade glioma has actived microvascular proliferation.Those neoplastic microvessels are characterized with incomplete endothelium and basement membrane,loss of pericytes,and disordered communication,forming a special tumor microenvironment with hypoxia,acidity and high intercellular pressure,futher stimulating the secretion of angiogenic factors such as hypoxia inducible factor(HIF)and vascular endothelial growth factor(VEGF),etc.to promote tumor angiogenesis,and is closely related to the growth,progress and metastasis of tumor.Glioma microvessels also participate in the formation of tumor vascular niche like structure,which can not only provide the necessary place and nutritional support for the survival of glioma stem cells,but also promote the self-renewal of stem cells through the cross-talking of vascular endothelial cells and tumor stem cells,thus play a key role in the treatment resistance and recurrence of glioma.Microvascular proliferation of high-grade glioma plays an important role in various biological behaviors of tumor,anti-angiogenic therapy provides a new idea and direction for the treatment of glioma.Neovascularization of glioma is a complex process regulated by multiple genes and multiple molecules.A variety of cytokines are involved,such as VEGF,Angiopoietin-1(ANG-2),Matrix metalloproteinase(MMPs),stromal cell-derived Factor 1?(SDF-1?),Platelet derived growth factor(PDGF)and HIF,etc.,play important role in promoting neovascularization in different stages of tumor development.Meanwhile,it has been confirmed that there are many patterns of neovascularization in high-grade gliomas,such as vascular co-option,angiogenesis,vasculogenesis,vascular mimicry and glioma-endothelial cell transdifferentiation.Different neovascularization patterns have different pathological processes and regulatory mechanisms,and their main regulatory genes are cross and independent.Among them,the high expression of VEGF gene is a key event in promoting tumor neovascularization,and participates in various ways of neovascularization.However,the anti-angiogenic therapy strategy of radiotherapy combined with temozolomide(TMZ)chemotherapy followed with bevacizumab(BEV)to inhibit the biological activity of tumor VEGF in glioma has not prolonged the overall survival of glioma patients.Even tumor vascular co-option and tumor invasion increased after Bev treatment,and others anti-angiogenic strategies failed to achieve the purpose of anti-tumor neovascularization and inhibit tumor growth in clinical practice.These results indicate that there are still unknown neovascularization related genes and the changes of neovascularization patterns after anti-angiogenic therapy,which lead to the failure of the treatment strategy to achieve the expected goals.Because of the obvious heterogeneity of high-grade gliomas,some tumor tissues obtained by surgery or biopsy cannot fully reflect the biological characteristics of the whole tumor.Therefore,it is necessary to find an effective means to comprehensively evaluate the expression of neovascularization related genes in tumor and non-invasive monitoring the therapeutic effect of anti-angiogenic therapy.Perfusion weighted MR imaging(PWI-MRI)is widely used in noninvasively monitoring of blood flow and blood vessels in tumors,and to evaluate the molecular characteristics and gene expression of tumors.According to the change of intravascular contrast agent signal intensity with time,the parameters CBV and CBF which reflect tumor blood perfusion can be obtained by DSC-MRI technology.DCE-MRI can not only obtain the information of tumor blood flow,such as plasma volume VP,extravascular extracellular space volume fraction Ve,but also obtain parameters which can reflect tumor vascular function,such as vascular permeability Indicators K_trans,and contrast agent reflux rate index kep.These parameters are not only related to the structure and function of tumor blood vessels,but also can reflect the molecular characteristics and gene expression of tumor.For example,K_trans is associated with the promoter methylation status of O6-methylguanine-DNA methyltransferase(MGMT)in glioblastoma.rCBV can be used to evaluate the status of isocitrate dehydrogenase(IDH)mutation and MGMT promoter methylation in glioma patients,The expression of tumor specific messenger RNA(m RNA)was also correlated with MRI features.Therefore,PWI-MRI can be used as a potential tool for noninvasively evaluation of the expression of neovascularization related genes in tumor and the changes of tumor neovascularization patterns after anti-angiogenic therapy.In summary,this study firstly collected surgical specimens of HGG,extracted primary tumor cells to establish orthotopic glioma mice models and performed transcriptome sequencing.According to the microvascular status of the tumor specimens and whether the orthotopic glioma model can be successfully established,novel neovascularization related genes that closely related to the early growth and progress of high-grade glioma were screened,and so did PWI-MRI biomarkers to predict the expression of those genes,so as to provide new targets and detection methods for anti-angiogenic therapy of HGG.Then,the differences of conventional and PWI-MRI characteristics between primary orthotopic xenograft glioma models and original patient tumors were compared,and the reasons of these differences were explored from the perspective of histopathology and gene expression,which provided experimental basis for the clinical application of animal derived MR biomarkers.Finally,BEV and TMZ were used to intervene the growth of orthotopic xenograft glioma models in mice alone or in combination,to explore the dynamic changes of neovascularization patterns during the treatment process and the MRI biomarkers that can reflect these changes.Exploring the reasons for the failure of anti-angiogenic treatment from the perspective of neovascularization patterns.So as to provide scientific and reliable experimental basis for the individualized and accurate diagnosis of glioma and the monitoring of anti-angiogenic therapy.Materials and Methods:Part ?:To explore the expression of neovascularization related genes and PWI-MRI biomakers in the early growth stage of high-grade glioma.1.30 cases of high-grade glioma were collected.DSC and DCE-MRI were performed preoperative,and tumor specimens were collected intraoperative.2.The tumor tissues were divided into three portions under aseptic condition.The first portion was used to extract primary tumor cells,which were used to establish orthotopic xenograft models in non-obese diabetic-severe-combined immunodeficiency(NOD-SCID)mice.The second portion was used to pathologically analyze the tumor vascular,including microvessel density(MVD),microvascular area(MVD)and Diameter.The third portion was used for transcriptome sequencing.3.CBV,CBF,K_trans,V_p,V_e and K_ep maps were obtained from the original images of DSC and DCE-MRI after post-processing.The mean r CBV,r CBF,K_trans,V_p,V_e and K_ep values of all tumor voxels were calculated as r CBV,r CBF,K_trans,V_p,V_e and K_ep values of the tumor.4.The cases were divided into xenograft forming group and nonxenograft forming group based on whether the xenograft was established.The perfusion MRI parameters r CBV,r CBF,K_trans,V_p,V_e and K_ep,MVD,MVA and microvessel diameter of tumor tissue,the expression of neovascularization related genes were compared between the two groups.5.siRNA was used to inhibit the expression of the above-screened genes in two primary tumor cells respectively,and to detect the changes in their vascularization ability.6.The expression of these genes and their relationship with tumor microvessels were dynamically monitored in orthotopic xenograft models.7.Receiver operating characteristic curve(ROC)was used to evaluate the PWI-MRI derived parameters with significant difference between the two groups,and the diagnostic threshold(cut-off value),specificity and sensitivity for identifying whether the above-screened genes are highly expressed in tumor tissues were obtained.Part ?:Study on MRI features and gene expression differences between the primary orthotopic glioma model and the corresponding original patient tumor.1.7 cases of high-grade glioma were collected.Conventional MRI,DWI-MRI and DCE-MRI were performed preoperative,and tumor specimens were collected intraoperative.2.The ADC and K_trans map of patients were obtained from the original images of DWI and DCE-MRI after post-processing.Five regions of interest(ROI)were selected at the largest slice of the tumor area using the hot spot method,then r ADC and K_trans values were measured respectively.The average values of r ADC and K_trans were used as the r ADC and K_trans values of the tumor.3.Primary glioma stem cell spheres were extracted and the corresponding orthotopic glioma models were established in NOD-SCID mice,5 glioma mice models were established in each case.4.In the late stage of tumor growth in NOD-SCID mice,the Bruker 7.0T MRI scanner for small animals with head surface coil was used to monitor the growth of xenografts.The MRI scanning included coronal T2WI,axial T1WI,T1WI contrast enhanced,DWI and DCE-MRI5.The ADC and K_trans map of xenografts were obtained from the original images of DWI and DCE-MRI after post-processing.Five regions of interest(ROI)were selected at the largest slice of the tumor area using the hot spot method,then r ADC and K_trans values were measured respectively.The average values of r ADC and K_trans were used as the r ADC and K_trans values of the xenograft.6.The differences of conventional MRI,DWI and DCE-MRI features between the original patient tumors and the corresponding xenografts were compared.7.The glioma tissues from patients and the corresponding xenografts of mice were embedded in paraffin,and then hematoxylin eosin staining(H&E staining)and CD34immunohistochemical staining were performed to explore the pathological basis of the difference in MRI features between the original patient tumors and the corresponding xenografts.8.The original patient tumors(patient tumors 1,2 and 3)and the corresponding xenografts(xenografts 1,2 and 3)were collected for transcriptome sequencing(Wuhan Seqhealth).The differences in m RNA expression levels between the original patient tumors and the xenografts were compared.Part ?:DCE-MRI evaluation of changes in neovascularization patterns of glioblastoma after anti-angiogenic therapy.1.Establishment of orthotopic U87 glioblastoma models in BALB/c mice.2.Drug administration was performed on day 21 after cell inoculation.The experimental groups were orally treated with 50 mg/kg TMZ for 5 consecutive days,intravenously administered 15 mg/kg BEV,or administered a combination of TMZ and BEV(TMZ treatment occurred 24h after the administration of BEV).The control groups were administered saline in the same way with corresponding experimental groups.3.Xenografts were scanned at 1,3 and 6 days after treatment.The MRI scanning included coronal T2WI,axial T1WI,T1WI contrast enhanced,and DCE-MRI4.The K_trans map of xenografts were obtained from the original images of DCE-MRI after post-processing.Five regions of interest(ROI)were selected at the largest slice of the tumor area using the hot spot method,then K_trans values were measured respectively.The average values of K_trans were used as the K_trans values of the xenograft.5.After MRI scanning,the brain tissues of tumor bearing mice were completely taken out and fixed.H&E staining,immunohistochemical staining(GFAP,CD34,TNC,and CD34-PAS)and transmission electron microscopy analysis were performed.The microvessel density(MVD),vascular co-option,sprouting angiogenesis,intussusceptive microvascular growth(IMG),and vascular mimicry were quantitatively analyzed.6.Spearman correlation analysis was used to compare the correlation between K_trans and neovascularization patterns.ResultsPart ?:To explore the expression of neovascularization related genes and PWI-MRI biomakers in the early growth stage of high-grade glioma.1.Orthotopic xenograft glioma models were successfully established from 9 cases of HGG surgical specimens.The ability of glioma surgical specimens to form xenografts was associated with microvascular density and diameter in the tumor tissue.Tumor tissues that could form xenografts had a higher microvascular density(P=0.003)and a smaller mean microvascular diameter(P=0.019),however,no association was found between the ability of glioma surgical specimens to form xenografts and microvascular area(P>0.05).2.The expression levels of BMPER(Bone morphogenetic protein endothelial cell precursor derived regulator),CXCL10(Chemokine ligand-10)and HOXA9(Homeobox A9)in the surgical specimens that could form xenografts were significantly higher than those in the surgical specimens that could not form xenografts(P=0.043,P=0.002,P=0.002).3.After inhibiting the expression of BMPER,CXCL10 or HOXA9 in two cases of primary glioma cells by siRNA,the tube formation capacities of these cells were significantly reduced(P<0.05,P<0.05,P<0.05,P<0.05,P=0.008,P<0.05).4.Dynamic monitoring of the orthotopic xenografts model showed that,on day 20 of tumor growth,BMPER was highly expressed,while CXCL10,HOXA9 and the tumor blood vessel marker CD34 were lowly expressed.On day 30,BMPER was highly expressed,CXCL10 and HOXA9 were lowly expressed,and CD34 was elevated.On day 40,BMPER expression was reduced,while CXCL10 and HOXA9 expression was elevated,which was spatially correlated with CD34 expression.In the late-tumor growth stage,BMPER,CXCL10and HOXA9 were all lowly expressed,while CD34 was highly expressed.5.The DSC-MRI derived parameters r CBV,r CBF and the DCE-MRI derived parameters K_trans and V_p of cases with high expression of BMPER,CXCL10 and HOXA9 were significantly higher than those in patients with low expression of these genes.ROC curves were used to further analyze the diagnostic efficacy of tumor r CBV,r CBF,K_trans and V_p in determining whether the above genes were overexpressed.The optimal diagnostic threshold of r CBV was 1.481,the area under the curve was 0.861,and the sensitivity and specificity were 100.00%and 75.00%,respectively.The optimal diagnostic threshold of r CBF was 1.289,the area under the curve was 0.847,and the sensitivity and specificity were 100.00%and75.00%,respectively.The optimal diagnostic threshold of K_trans was 0.209,the area under the curve was 0.957,and the sensitivity and specificity were 100.00%and 92.86%,respectively.The optimal diagnostic threshold of V_p was 0.139,the area under the curve was 0.871,and the sensitivity and specificity were 80.00%and 85.71%,respectively.Part ?:Study on MRI features and gene expression differences between the primary orthotopic glioma model and the corresponding original patient tumor.1.The growth pattern of the xenografts were divided into two categories.The first category included 6 cases of tumor with diffuse growth(xenografts 1,2,3,4,5 and 6);the second category was for the one tumor case with nodular growth(xenograft 7).The most significant difference noted in the MRI features between the 6 cases of diffusely grown xenografts and the original patient tumors was the mild enhancement occurring in the local area of the xenografts.K_trans map showed that there were no obvious abnormal high signals in xenografts.In addition,the internal signal of the xenografts was homogeneous in the absence of edema.A clear demarcation between the xenografts and the normal brain tissue was noted in the one case of tumor nodular growth between the two groups(xenografts and human tumor).In addition,the enhancement degree of the xenografts was significantly lower compared with the original patient tumor.The K_trans values of all the xenografts were lower compared with the corresponding values of the original patient tumors,and the r ADC values of all the xenograft samples were higher compared with the corresponding values of the original patient tumors(P=0.016,P=0.001).2.CD34 staining showed that the microvessel area and diameter of the 6 xenograft cases that exhibited diffuse growth were significantly lower compared with the corresponding patient tumors(P=0.009,P=0.007).There were no significant differences in the microvessel density between the xenografts and the patient tumors.H&E staining of the xenografts and human tumor tissues indicated that the boundary of the xenografts tissues was clear,whereas that of the patient tumor was unclear.3.There were significant differences in gene expression between xenografts and corresponding original patient tumor.There were 3590 differentially expressed genes between patient-1 and xenograft-1,5408 differentially expressed genes between patient-2 and xenograft-2,and 3590 differentially expressed genes between patient-3 and xenograft-3.GO analysis of the differentially expressed genes revealed that tumor cell characteristics and extracellular matrix-associated genes(cell activation,cell adhesion,cell migration,cell motility and extracellular matrix associated genes),angiogenesis-associated genes(angiogenesis and vasculature development)and immune-associated genes(immune response,immune system process and immune effector process)were highly expressed in the original patient tumors.The expression levels of the genes that were involved in cell cycle and nuclear division were increased in the xenografts.Part ?:DCE-MRI evaluation of changes in neovascularization patterns of glioblastoma after anti-angiogenic therapy.1.Four types of neovascularization patterns were identified by pathological staining of the tumor region,including vascular co-option,sprouting angiogenesis,IMG and vascular mimicry.2.In the BEV group,The amount of sprouting angiogenesis and IMG began to decrease3 days after treatment compared with control groups(P<0.05,P=0.002).The MVD and the amount of IMG were both decreased compared with control group(P<0.05,P=0.001)and an actual decrease was observed in the number of co-option and sprouting angiogenesis 6 days after treatment(P<0.05,P<0.05),whereas the amount of vascular mimicry was signifcantly increased(P<0.05).3.In the TMZ group,the amounts of sprouting angiogenesis,IMG and MVD were all significantly decreased compared with control groups(P=0.001,P<0.05,P<0.05)and an actual decrease was observed in the number of vascular co-option(P=0.001)3 days after treatment.However,6 days after treatment,the MVD was significantly increased compared to the control group(P=0.003),and no significant differences were found in the amounts of vascular co-option,sprouting angiogenesis,IMG and vascular mimicry between the experimental group and the control group.IMG was the most sensitive neovascularization pattern to TMZ and decreased 1 day after treatment compared with control groups(P=0.001).4.In the BEV and TMZ combined group,the combination of BEV and TMZ significantly reduced the MVD and the amount of vascular co-option,sprouting angiogenesis and IMG 1 day(P<0.05,P<0.05,P<0.05,P=0.01)and 3 days(P=0.003,P<0.05,P<0.05,P=0.025)after treatment compared with control groups,but the amount of vascular mimicry(VM)was not different between the experimental and control groups.However,6 days after treatment,the only decrease was observed in sprouting angiogenesis(P<0.05).5.In the BEV treatment group,the K_trans significantly decreased from day 1 through day6 after treatment compared with control groups(P<0.05,P<0.05,P<0.05).In the TMZ treatment group,K_trans significantly decreased 3 days after treatment but increased on the 6th day after treatment compared with control groups(P<0.001,P=0.007).In the BEV and TMZ combination treatment group,the variation in Ktrans was similar to that in the BEV treatment group.K_trans was significantly reduced from day 1 to day 6 after treatment compared to the control groups(P=0.022,P<0.05,P<0.05).6.Spearman correlation analysis showed that K_trans was positively correlated with sprouting angiogenesis in all experimental groups after drugs administration,r=0.9068?0.9806?0.8641(P<0.05)in BEV and TMZ alone or in combination group respectively.(All the P values<0.05).Conclusion1.BMPER,CXCL10 and HOXA9 promote early tumor growth and progression by stimulating neovascularization of primary HGG,which can be used as a new target for anti-angiogenic therapy of glioma;DSC-MRI and DCE-MRI derived parameters r CBV,r CBF,K_trans and V_p can be used as imaging biomarkers to predict the expression of BMPER,CXCL10 and HOXA9.2.Patient-derived orthotopic xenograft glioma models in mice could not replicate the MRI features of the corresponding original patient tumors by comparing conventional MRI,DWI-MRI and DCE-MRI characteristics of these two distinct groups.The differential expression of certain genes may underlie the differences observed in the MRI features between original patient tumors and the corresponding xenografts.Together,the results of the present study showed that MRI biomarkers obtained from PDXs should be interpreted with caution.3.The increase in the number of vascular mimicry after BEV intervention and the increase in the MVD after TMZ treatment in the tumor area may be important reasons for the failure of anti-angiogenic therapy for glioblastoma;After the administration of BEV and TMZ alone or in combination,the change in the number of sprouting angiogenesis was positively correlated with K_trans value,indicating that K_trans can be used as a potential effective imaging biomaker to monitor the changes of sprouting angiogenesis after drug administration.