The Research Of Relationship Between Stem Cells' Marker ABCG2 And Glioma Neovascularization As Well As Patients' Survial Prognose

PartⅠThe correlation of expressions of stem cell marker ABCG2 with angiopoiesis associated proteins in glioma tissues analyzed by tissue-microarray.Object:The ABCG2 is regarded as the marker of tumor stem cells,and reported to be associated with tumors' multidrug-resistance.However,it is still unknown whether it is related to the neovascularization.In this study,we aim to explore the correlations of ABCG2 and VEGF,VEGFR and CD34 during the process of angiopoiesis in glioma,and to prove the importance of ABCG2 to angiopoiesis.Methods:A tissue microarray containing 90 specimens that include all pathological grades of human brain glioma tissues was constructed.The expressing of ABCG2,VEGF and VEGFR(flt-1) in brain tumor was detected by EnVision immunohistochemical staining.CD34 monoclonal antibody was used to label vascular endothelial cells and tumor microvasculars density(MVD) was evaluated according to the numbers of CD34 positive labeled microvasculars.Six normal brain tissues were employed as control.Results:The positive expression rates of ABCG2,VEGF,VEGFR and CD34 increased with the pathological grades.The expression of ABCG2 were statistically different(P＜0.05) among each pathological grades of gliomas except the gradeⅠ—Ⅱ.Spearman rank-correlation analysis showed the positive correlation between the expression of ABCG2 and pathological grades;moreover,the expression level of ABCG2 was notably related with MVD in tumors,γ=0.540,P＜0.001.The MVD in gliomas of gradeⅢ—Ⅳwere higher than that in gradeⅠ—Ⅱ.The genes of VEGF, VEGFR(flt-1) and CD34 were also analysized,which showed that the average value of MVD in tumors expressing VEGF~+,VEGFR~+(flt-1) and ABCG2~+ was significantly higher than that in the ABCG2~- tumors,P＜0.001.Conclusions:VEGF,VEGFR and CD34 have been generally recognized as the representative genes inducing the angiogenesis in tumors,while ABCG2 is a novel gene which only expresses in stem cells.The results of tissue-array containing glioma specimens of different pathological grades indicated their intimate the correlations.The results also suggested that the ABCG2~+ cells had the multipotential to differentiate into tumor cells and tumor vessel cells,which explored a new direction for the research on vasculogenic mimicry in tumor.PartⅡThe co-expressions of stem cell marker ABCG2 with angiopoiesis associated proteins in glioma tissue microarray.Object:The study aimed to detecte the co-expression of ABCG2 and angiopoiesis associated proteins on base of direct correlation between them,and further to supply the research of glioma stem cells' transformation into angiocellulars in gliomas with morphological evidence.Methods:The co-expression of ABCG2,VEGF,CD34,vWF and GFAP in human glioma tissues by tissue-microarray was detected by laser scanning confocal microscope(LSCM).Results:There are co-expression cells of CD34/ABCG2,vWF/ABCG2, CD34/VEGF,VEGF/CD34 in glioma vessels,but no co-expression cells of GFAP/ABCG2.And co-expression cells of CD34/ABCG2,VEGF/CD34 were also observed in normal brain tissue,but no co-expression cells of ABCG2/GFAP,either.Conclusions:The co-expression of stem cells marker ABCG2 and vascular endothelia cells marker CD34 and vWF,but no co-expression ABCG2 and GFAP, indicated that glioma stem cells have ability to differentiate into glioma vascular endothelial cells for the co-expression of CD34/VEGF were also observed.The differentiation is also related with cell secretion of VEGF,but not with GFAP. PartⅢParticipation of cells coexpressing ABCG2 and nestin in glioma angiopoiesisObject:It is known that stem cells marker nestin and CD133 have close relationship in glioma stem cells.This article aims to investigate the relationship between nestin and another stem cells marker ABCG2,and to investigate the participation of cells coexpressing the two markers in glioma angiopoiesis.Methods:Glioma specimens with most plentiful vessels and highest pathological grades selected from 90 human glioma were immunostained against nestin and ABCG2, then observed with LSCM.Results:Some cells composing glioma vessels co-expressed nestin and ABCG2, and the heterogeneity among vessels were also observed.Nestin was expressed not only in the glioma vascular endothelial cells but also in tumor cells away from vessels.There were no co-expression of nestin and GFAP in glioma vessels.Conclusions:The glioma stem cells not noly have the potentiality to differentiate into their parent tumor cells but also into cells with phenotype of tumor vessel cells, which provide the tumor vessel formation theory:vasculogenic mimicry and mosaic vessels with new evidence.PartⅣAs a high risk factor,ABCG2 could effect survival of glioma patientsObject:While ABCG2 was a key gene for glioma angiogenesis,as discussed in first part and third part,it is worthy to make clear whether ABCG2 were associated with patient prognosis,which is the object of this part.Methods:A tissue microarray that include all pathological grades of human brain glioma tissues was constructed,and the expressions of PCNA,P53,VEGF,IGFBP-2, C-MYC and ABCG-2 were detected by EnVision immunohistochemical staining method in the tissue microarray.Cox proportional hazards models were usedunivariable analyses,and multivariabte analyses were conducted to characterize the association between survival and both clinical and biological markers.Results:Univariable analyses show that histopathology grade,ABCG2,P53 and VEGF was correlated with the prognosis(P＜0.05).ABCG2,P53 and VEGF immunopositivity enter the final multivariable model,and their high expression show poor prognosis.Conclusions:This study demonstrates ABCG2 being a high risk factor in glioma patients.This provides further evidence for target molecule theraty.