Microfluidic Based Vasculogenic Mimicry Models Establishment In Glioma Microenvironment And Mechanism Investigation

Objective: Vasculogenic mimicry is crucial to tumor infiltration and metastasis in tumor genesis.Mesenchymal stem cells homing tumor tissues could promote tumor vessels sprouting and growth which contributes to the development of tumor.So far,the majority of current approaches have focused on the effect of single factor upon tumor angiogenesis.This article established a microfluidic system that integrate multiple factors,including 3D co-culture tumor cells,extracellular matrix,hypoxia and human umbilical vein endothelial cells(mesenchymal stem cells)to investigate the angiogenesis of tumor tissue by recapitulating the microenvironment in vivo.Method: The chip is designed and fabricated with polydimethylsiloxane(PDMS)by soft-lithography in Dalian Institute of Chemical Physics.The molded PDMS was irreversible sealed to a plate PDMS membrane by procreating with plasma.Human glioma cells(U87)were cultured on one microchannel and mouse bone marrow mesenchymal stem cells(MSC)or Human umbilical vein endothelial were embedded in the vertical Matrigel perfused microchannel.After cultured in hypoxia or normoxia,the cell morphology,cell migration were evaluated by Image-Pro Plus 6.0.The device was compatible with fluorescent microscopy to exam the apoptosis,MSC-derived endothelial specific antibody expression and the mesenchymal transition of U87.Result:(1)The tumor microenvironment-mimicking chipThe tumor microenvironment-mimicking chip is consisting of five interconnected micro-channels.including three main channels(500μm in width,150-160μm in depth)and two vertical gel purfused micro-channels(200μm in width,70-80μm in depth).The interval between main micro-channel and gel micro-channel has two rows collateral micro-channel(80μm in width).After gel be injected into the gel chambers,it can stay in gel channel by liquid surface tension.Cytokine exchanged through gels,so,with this design,the cells inoculated into two contiguous main micro-channels co-cultured in a non-contact type.(2)Cells assayIn hypoxia,the migration distance and network area of U87 were both significantly increased(P<0.01).Under hypoxia condition co-culture with EC compared with normoxia condition co-culture with EC,The migration distant and networks area of U87 were no statistical significance in the expression level.Under normoxia and hypoxia condition,The EC cells did not migrate neither under normoxia nor hypoxia condition.And the apoptosis rate of EC cells significantly increase(P<0.05).Compared the MSC migration distant and networks area when MSC co-cultured with U87 and U87hif-2αrespectively.The migration distant and networks area of MSC were significantly increase(P<0.01).Under normoxia and hypoxia condition,both in the two groups MSC could express CD31,hypoxia condition group was higher.But there was no express in normoxia condition.Conclusion: This study used the microfluidic chip techniques as platform to set up the brain tumor microenvironment in vitro.We investigated the interactions between tumor,endothelium and MSC in the process of tumorygenesis.The MSC played a role in early stage of tumor vasculogenesis in hypoxia,which could promote the development of tumor.