Study On Temozolomide Resistance Of Glioma Stem-like Cell Promoted By Hypoxia-induced And Transdifferentiated Endothelial Cell Based On Microfluidic Chip

PartⅠ:Induce glioma stem-like cell to transdifferentiate into endothelial-like cell in hypoxic conditionObjective:To induce glioma stem-like cells(GSLC) to transdifferentiate intoendothelial-like cells(GDEC) in vitro hypoxic condition and further detect thevariation of character and function of these cells cultured in vitroMethods:Screened the RFP+SU3 glioma cell line and cultivated these cells inserum-free stem cell medium to obtain the GSLC, then put GSLC in specifictransdifferentiation medium and incubated in hypoxic chamber(1.5%O2) for 48 h.Set human brain microvascular endothelilal cell(HBMEC) as control group, thenobserved changes of distribution and shape of these cell from SU3 to GDEC.Select the vascular endothelial cell surface marks CD31 and v WF to verify theGDEC. Incubated the GDEC onto the matrigel to check the endothelial function tocheck whether the GDEC would form typical endothelial tube onmatrigel.Compared the the difference of GDEC organelles with HBMEC by theultrastructure under the electron microscopy. Finally, cultivated the GDEC inserum-free stem cell medium to verify the reversal conversion of GDEC intoGSLC and confirm the stability of GDEC endothelial cell mark when incubated inco-culture medium.Results: 1) After the GSLCs were cultured in specific transdifferentiationmedium in hypoxic condition for 48, the original haphazard distribution of cellchanged into regular distribution and the shape of cells transformed from irregularstructure into spindle endothelial-like cell. And all the induced cells presentedCD31 and v WF expression. 2) The GDECs possessed the character of normalendothelial cells that could form the endothelial tube on matrigel and wereequipped with more abundant organelles compared with normal endothelial cells3) The GDECs still have the properties of tumor cell that they could be inducedinto GSLC in serum-free medium. While incubated in endothelial co-culturemedium, the GDEC could maintained endothelial cell markers for a long time.Conclusion: In vitro, GSLCs in the hypoxic transdifferentiation mediumcould be induced into vascular endothelial-like cells that have endothelial characterand function. In addition, the GDECs have kept part character of tumor cell andcould change into various types of cells when cultured in different conditions.PartⅡ: Construction of real-time analysis model for the interaction between glioma stem-like cells and endothelial cells based on the concept of hypoxic nicheObjective: Based on the concept of tumor hypoxia niche, to build a effectivehypoxic model for the interactions between GSLCs and endothelial cells in vitro.Methods: Combine HE staining and immunofluorescence double stainingtechnique,the hypoxic perivascular niche in the glioma tissue were confirmed.Based on pathology parameter, utilized PDMS by "photolithography" to fabricatemicrofluidic chip, and then assembled two microfluidic chips to make adouble-layer device that was equipped with three "L" shaped channels in each layer.Added these devices into the hypoxic chamber to build a hypoxic co-culturesystem. Oxygen concentration and the distance between endothelial cell andGSLCs was real-time respectively detected by oxygen electrode and fluroscentprotein report system. The survival of GSLC、HBMEC and GDEC that respectivelyincubated in these hypoxic co-culture system for 24 h,48h and 72 h were severallydetected by Lived-Dead kit.The properties stability of GSLC、HBMEC and GDECthat incubated in these system for 48 h were respectively evaluated by cellfluorescent protein labeling technique and immunofluorescence labeling technique.With the cell fluorescent protein labeling technique and fluorescence trackingtechnology, we respectively inoculated endothelial cells and GSLC into top andbottom layer channel and built three culture groups of GSLC cultured alone, GSLCco-cultured with HBMEC and GSLC co-cultured with GDEC. The former twogroups were set as control, the GSLC proliferation and division under the GDECeffect in hypoxic condition were real-time analyzed.Results: 1) The hypoxic co-culture system assembled by hypoxic chamberand microfluidic devices provides a more real platform for the interaction betweenendothelial cell and GSLCs. 2)GSLC, GDEC and HBMEC respectively presentedgood growth after incubated in hypoxic co-culture system for24 h,48h and 72 h.Select the above cells incubated in hypoxic co-culture system for 48 h to real-timetrack the original cell surface markers by fluorescent protein labeling technique andimmunofluorescence labeling technique, the results presented that all the abovecells could effectively maintained stable original marks 3) Hypoxic co-culturesystem combined with fluorescent protein labeling technique and fluorescencetracking technology displayed that GDEC presented a more stronger ability topromote propagation of GSLC.Conclusion: Double-layer microfluidic chips coupled with a hypoxic chambercan be effectively applied to the study on hypoxic cell-cell interaction andsuccessfully mimic the crosstalk between GDEC and GSLC; Combinied with thecell fluorescent protein labeling technique and fluorescence tracking technology,thehypoxic co-culture system provides a real-time observation and detection fuction.PartⅢ:The effect of GDEC on the resistance to TMZ of GSLCs in hypoxic conditionObjective: To explore effects of GDEC on the resistance to thetemozolomide(TMZ) of GSLC in hypoxic condition.Methods: Based on the real-time analysis model of hypoxic co-culture systemin the Part Ⅱ,three chemotherapy co-culture groups of GSLC cultured alone,GSLC co-cultured with HBMEC and GSLC co-cultured with GDEC were built byadding 2000μmol/L TMZ to each group for 48 h, the former two groups were set ascontrol, 3 repeated assasys were perfomed for each group, the survival andapoptosis of GSLC under the effect of GDEC were real-time analyzed by laserscanning confocal technology and flow cytometry. From another three co-culturegroups,the Jagged1 and DLL4 expresison on the GDEC that respectively beforeand after co-cultured with GSLC were detected by Wester-blot andimmunofluorescent staining. The Notch ligands Jagged1 and DLL4 from the eachco-culture group medium were analyzed by Elisa kit. Finally, the activation of Hes1 in GSLC of each co-culture group,the main Notch pathway downstreameffector,were detected by quantitative PCR.Results: 1)From three chemotherapy co-culture groups applying TMZintervention for 48 h in the hypoxic condition, GSLC that co-cultured withendothelial cells emerged a higher cell survival proportion and less cell apoptosispercentage, among which GSLC that co-cultured with GDEC presented mostsurvival and least apoptosis. 2)After hypoxic co-culture with GSLC for 48 h,Jagged1 and DLL4 in GDEC displayed a higher expression than HBMEC bylateral comparison of Wester-blot and immunofluorescent staining results, whileJagged1 and DLL4 in GDEC emerge an increased expression than that beforeco-culture by longitudinal comparison of Wester-blot and immunofluorescentstaining results 2) The Jagged1 and DLL4 in GDEC and GSLC co-culture mediumexhibit highest concentration.; Hes1 in GSLC was upregulated most significantlyafter co-culture with GDEC when compared with control.Conclusion: Within hypoxic niche, GDEC could promote GSLC self-renewland resistance to TMZ through providing more Notch ligands(Jagged1 and DLL4)that activate GSLC Notch pathway.