Phenotype And Xenograft Tumor Pathology Study Of A2B5~+/CD133~-and CD133~+/A2B5~-Glioma Stem Cell Spheres

Objective To explore the phenotype and xenograft tumor pathology of A2B5⁺/CD133^-and CD133⁺/A2B5^-glioma stem cell spheres（GSCs），reveal the biological characteristics ofthese two kinds of GSCs.Methods SHG-139glioma cells derived from grade II astrocytoma as well as SHG-44glioma cells derived from SHG-44glioma cell line were cultured under neural stem cellmedium（NSCM） and glioma stem cell spheres were collected, immunocytochemistry wasperformed to detect their expression of CD133, Nestin, A2B5, Vimentin, VEGFR-2, NG2and IDH1^R132H; secondary sphere forming assay was used to confirm their self-renewalcapacity; spheres were induced using serum-containing medium, the expression of themarker of differention GFAP，β-III Tubulin and GalC were detected; gene chip technologywas used to detect the differentially expressed genes between SHG-139s GSCs and itsdifferentiated progeny cells, pathway-net analysis was performed; then the xenograft tumormodels were established, we detected the expression of CD133, A2B5, Nestin, VEGFR-2,GFAP, S-100, CD31, CD34of these xenograft tumor tissue using immunohistochemistryselectively; comparison of intracranial xenograft tumor tissue of both SHG-139s gliomastem cell spheres and SHG-44s glioma stem cell spheres was performed.Results The SHG-139glioma stem cell spheres（SHG-139s） and SHG-44glioma stemcell spheres（SHG-44s） were collected successfully with the application of NSCM culturecondition. Immunocytochemistry confirmed SHG-139s express A2B5⁺/CD133^-whileSHG-44s express CD133⁺/A2B5^-.They both expressed Nestin positively, but theexpression of Vimentin、 VEGFR-2、 IDH1^R132H、NG2varied. After induced usingserum-containing medium, both SHG-139s and SHG-44s differentiated. However, the expression of GalC was higher in SHG-139s compared with SHG-44s, while theexpression of β-III Tubulin was higher in SHG-44s compared with SHG-139s afterdifferentiation. Gene chip study of SHG-139s GSCs and its differentiated progeny cellsrevealed the important differentially expressed genes related to stemness maintenance andinvasiveness; pathway-net analysis showed MAPK signaling pathway, TGF-beta signallingpathway and adhesion related pathway are key differentially expressed pathway. Thesubcutaneous xenograft tumor model and intracranial xenograft tumor model of both GSCswere established successfully. Oligodendroglioma component was found in the SHG-139sintracranial xenograft tumor while other models didn’t show pathology appearance like this.Immunohistochemistry showed A2B5+tumor cells of SHG-139s intracranial xenografttumor are invasive; compared with SHG-44glioma cells-formed xenograft tumor, SHG-44glioma stem cells-formed xenograft tumor were more invasive; some SHG-44s gliomacells surrounding like a vessel were postive for anti-human specific antibody CD34whileno similar phnomenon is shown in SHG-139s xenograft tumor.Conclusions Both A2B5⁺/CD133^-and CD133⁺/A2B5^-glioma stem cells possess theability of self-renewal, multi-differentiation and tumorigenicity, but they vary in molecularphenotype, differentiation inclination and xenograft tumor pathology, the study thus revealthe heterogenity of gliomas from the perspective of GSCs subpopulation.Gene chip techcombined with bioinformatics analysis has revealed the key differentially expressed genesand the related pathway which control the stemness maintenance and invasiveness ofA2B5⁺/CD133^-GSCs, thus lay the foundation for further study of the targeted therapy ofthis GSCs subpopulation.