Study Of Interactions And Their Mechanisms Between Glioma Stem Cells And Endothelial Cells

Malignant glioma is the most common tumor in central nervous system. The besttreatment so far is combination of surgery, radiation therapy, and temozolomide. It hasn’t stillachieved the improvement we respected. Cancer stem cell (CSC) was proved existing in mostof malignant tumors, including glioma, and responsible of tumor initiation, propagation,resistance to radiotherapy and recurrence. CSC niche is one of the theories about regulatingCSC self-renewal. To investigate the interaction between glioma stem cell (GSC) and itsniche may be a new way to understand glioma development.The techniques of transwell co-culture system, PCR, Western-blot, RNAi assay, ELISA,proliferation assay, migration assay, xenograft experiment were used to explore theinteraction between GSC and endothelial cells and its mechanism. The study was divided intothree parts. First, we tend to observe the changes of self-renewal and stemness related geneexpression in GSC and GL261cells after cultured with endothelial cells. Hedgehog pathwaywill be checked in order to test its role. Second, proliferation and migration abilities ofendothelial cells will be detected after cultured with GSC. Hedgehog pathway will be checkedin order to investigate its mechanism. Third, Shh and Hhip will be detected in supernatant ofco-culture system to determine the main factor mediate the phenomenon. The main resultsand conclusions are as the followings:Results:1. Endothelial cells enhanced self-renewal of GSC and stem-like features of glioma cells.1.1Limit dilution assay showed that tumor sphere generated by GSC cultured withendothelial cells are more and larger than that of control, especially at5cells per well (24.3%vs11.3%) and10cells per well (39.4%vs25.8%). Tumor sphere generated by GL261cellsco-cultured with endothelial cells were larger than that by GL261cells alone.1.2The sizes of xenografts generated by GSC injected with endothelial cells were larger than that by GSCs alone (0.87±0.25(cm3) VS0.23±0.046(cm3)). Xenografts generated byGL261cells injected with endothelial cells were larger than that by GL261cellsalone(0.093±0.068(cm3) VS0.039±0.043(cm3)).1.3PCR and Western-blot results showed that expression of Olig2and Bmi1wereup-regulated4.5times and2.7times in GSCs co-cultured with endothelial cells than that ofcontrol, respectively.2. Hedgehog pathway was important in regulating of GSC self-renewal.2.1PCR and Western-blot showed that expression of Gli1was up-regulated in GSC andGL261cells co-cultured with endothelial cells than that of control.2.2After knocking down of Smo expression in GSC, Gli1expression of GSC wasdown-regulated. Sphere formed by shSmo-GSC was smaller than that of control.2.3Western-blot assay showed expression of Oligo2and Bmi1were down-regulated inshSmo-GL261cells.2.4Xenografts generated by shSmo-GL261cells were smaller than that of control, and itwas diminished that endothelial cells enhanced tumorigenity of GL261cells in mouse.3. Bmi1was an important factor in endothelial cells promoting stem-like features ofglioma cells.3.1The expression of Bmi1increased in promoting stem-like features of GL261cells byendothelial cells.3.2CD133positive ratio of shBmi1-GL261cells was reduced than that of control(0.78%VS0.05%).3.3Sphere formation assay showed that spheres generated by shBmi1-GL261cells weresmaller than that of control.4. Proliferation and migration abilities of endothelial cells were enhanced when culturedwith GSCs4.1CCK-8proliferation assay revealed that the wells of endothelial cells cultured withGSC had higher absorbance at24h,48h,72h than that of control.(0.044,0.072,0.10VS0.033,0.046,0.064).4.2Compared to control, wound-healing assay showed endothelial cells migrated fasterwhen cultured with GSC at24h (270μm VS160μm). Transwell migration assayshowed endothelial cells migrated faster when cultured with GSC (260/field VS150/field). 5. Hedgehog pathway was important in GSC regulating proliferation and migrationabilities of endothelial cells.5.1PCR and Western-blot experiments showed that Gli1expression was up-regulated9.5times in endothelial cells cultured with GSC than that of control.5.2After knocking down of Smo expression of endothelial cells, CCK-8proliferationassay showed lower absorbance at72h than that of control (0.027VS0.020).5.3Compared to control, wound-healing assay revealed that migration ability ofendothelial cells reduced at8h (84μm VS53μm) and24h (173μm VS120μm) afterknocking down of Smo expression of endothelial cells. Transwell migration assay showedmigration ability of endothelial cells reduced at8h(65/field VS22/field) and24h (244/field VS150/field).6. Shh from endothelial cells was the important mediator of the activation of hedgehogpathway.6.1PCR assay showed the expression of Shh was up-regulated both in GSC andendothelial cells in co-culture system than that of control(3.8times,2.0times). ELISAexperiments showed that Shh and Hhip concentrations both increased in co-culturesupernatant.6.2PCR assay showed Gli1expression in GSC was up-regulated when endothelial cellco-cultured with GSC that Shh expression was knocked down, while Gli1expression in GSCwasn’t up-regulated when GSC co-cultured with endothelial cell that Shh expression wasknocked down. It indicated that Shh from endothelial cells was more important in activatinghedgehog pathway of GSCs than that from GSCs.Conclusions:1. Endothelial cells enhance stem-like features of GSCs and glioma cells by activatinghedgehog pathway and up-regulating Bmi1.2. GSCs enhance endothelial cells proliferation and migration by activating hedgehogpathway.3. Shh secreted from endothelial cells may be an important factor in activating hedgehogpathway of GSCs.