The Affect Of Endothelial Cells On Invasive Growth Of Gliomas And The Relation Of Notch Signaling Pathway

Background and objective:Glioma is the most common and the most difficult to cure primary malignanttumor in central nervous system, accounting for50%of the adult brain tumor.Although surgery methods are continuously improved, and radition therapy,chemotherapy, immunotherapy, gene therapy, and other auxiliary therapy arecomprehensive applicated, and the clinical efficacy certain some progress, its overallprognosis poorly improved. Regardless of the level of differentiation, glioma has thebiological characteristics of invasive growth, rich vascularization, no envelope.Generally, it is assumed that high invasiveness, rich vascularization, and theinteraction with vascular cells of tumor cells is the root cause of glioma is difficult tocure, easy to relapse. Research has shown that invasive growth of gliomas is closelyrelated to the micro environment. The interaction of vascular endothelical cells andglioma cells may promote the invasive growth of glioma,but the mechanism is stillunclear. Notch signaling pathway plays a key role in new angiogenesis and thedevelopment of central nervous system, the activation of Notch signaling closely withangiogenesis and glioma cell proliferation, thus we put forward assumption, in theinvasive migration process of vascular cells promote glioma, the Notch signaling mayplay an important regulatory role. We proposed to explore the mechanisms of effectof glioma cells invasivly grow on vascular cells by observing the influence ofendothelial cells invasive glioma cells and the corresponding changes of relatedfactors of Notch signaling pathway.Content and methods:1We attempted to set up a co-culture systems of U87glioma cells and HUMECsin vitro. U87cells and HUVECs were cultured in medium containing10%FBS at37°C temperature、5%CO2and95%humidity incubator，using the logarithmic growthphase cells for experiments.The cells were divided into three groups according to theexperiment requirements，the single culture group,normal U87co-culture with ECs group,U87co-culture with ECs which Notch signaling were blocked by DAPT group.2To observe the affect of ECs on the invasive growth of glioma cells. HUVECsand U87were co-cultured with Transwell co-culture systerm in vitro，to measure theinvasiveness of glioma cells that induced by Ecs in different status.3HUVECs and U87were co-cultured with Transwell co-culture systerm invitro，to contrast the change of the gene expression of Cathepsins B、MMP-9、MMP-2、Notch-1、Notch-2、Hes-1and Dll4by RT-PCR on the U87cells in co-culturewith the single culture U87cells.4To observe the vitro vascular mimicry in glioma cells under different culturedconditionResults:1Co-cultured with ECs, the invasiveness of glioma U87cells can be inhanced,but blocking Notch signaling of ECs can reduce the increasing amplitude of theinvasiveness in glioma cells. The number of the glioma cells that pass through thematrigel is: single cultere group40.0±3.92, normal co-culture group81.5±2.89，DAPT group61.8±4.72（P<0.05）.2RT-PCR results shows that glioma U87cells showed the expression ofCathepsins B、MMP-9、Notch-1、Notch-2、Hes-1、Dll4mRNA were markedlyincreased after co-culturd with Ecs. In contrast, the up-regulated expression ofCathepsins B、MMP-9、Notch-1、Notch-2、Hes-1mRNA were limited when the Notchsignaling pathway was blocked in Ecs. But the expression of DLL4mRNA had nosignificant change, and the expression of MMP-2mRNA was no obvious changed，which haven’t statistically significant. The Notch12-ΔΔCTof different group is1、6.87、1.82；The Notch22-ΔΔCTof different group is1、22.78、14.52；The Hes12-ΔΔCTof different group is1、3.89、3.20；The MMP-92-ΔΔCTof different group is1、16.45、2.33；The CB2-ΔΔCTof different group is1、29.24、13.00；The DLL42-ΔΔCTofdifferent group is1、9.38；3The glioma U87cells which cultured after co-cultured with Ecs group intoafunicular and mutual fusion that connected into an network structure in vitrovasculogenic mimicry. In the control group, the single culture U87cells cultured after 12h still growth disorders separately, haven’t obvious regularity. In contrast，the U87cells in DAPT group began to deformation. Some of these connected to each other,form a network structure, but less than the nomal co-culture group.Conclusions:1Ecs can induce the co-cultured U87cells to enhance the invasive, andup-regulated the gene expression of Notch signaling and invasive factors.2Blocking the Notch signaling pathway in ECs can limit the co-culture U87cells invasive, and reduced the up-regulating gene expression.3Tumor invasive metastasis may be the consequences of the change of theextrecellular matrix caused by interaction between tumour cells and vascularendothelical cells.