Identification And Characteristics Analysis Of Fusion Between Glioma Stem Cells With Bone Marrow Derived Stroma Cells

Part I:Identification of fusion between GSCs and bone marrow derivedstroma cells in vitroObjective: Through dual-color fluorescent tracing co-culture model in vitro, interactions between the tumor stem cells and stromal cells in microenvironment were observed, including whether fusion occurs,and existence of the fused cells were verified in multiple levels.Methods:Human glioma stem-cell lines SU4 was transfected with red fluorescent protein(RFP) gene. Bone marrow mesenchymal stem cells(BMSCs) or macrophages( Mφ) from Balb / c nude mice with whole body expression of green fluorescent protein(EGFP), and SU4-RFP cells were co-cultured, respectively, to observe the interactions between SU4-RFP and BMSCs or Mφ, a cell fusion was also observed. Monoclonal RFP / EGFP double positive cells with high proliferative ability, fusion of SU4-RFP cell with BMSCs or Mφ, and were named after F-BMSCs and F-Mφ. The RFP/EGFP gene and protein of the fusion cells were detected and analyzed by fluorescence in situ hybridization(FISH) and Western blotting, and compared with SU4-RFP,BMSCs and Mφ. Then expression of cell-specific markers were studied by immunocytochemistry staining in SU4-RFP, BMSCs, M φ and F-BMSCs, F-M φ. Finally Karyotype analysis of the chromosome features of SU4-RFP, BMSCs, F-BMSCs, F-Mφ were performed.Results: Active interactions can occur between SU4-RFP and BMSCs or Mφ cells, respectively. Fusion cells co-expressed RFP and EGFP two fluorescent proteins. FISH,Western blotting detection further confirmed F-BMSCs, F-Mφ co-expression of RFP&EGFP genes, and the corresponding fluorescent proteins. To detect the expression of cell surface markers, immunocytochemistry results showed positive expression of Nestin in SU4-RFP cells, BMSCs highly expressed its specific markers CD105 and CD44, Mφ cells expressed their high specificity marker CD68. Fusion cells F-BMSCs co-expressed glioma stem cell marker Nestin and BMSCs markers CD105, CD44; F-Mφ co-expressed glioma stem cell marker Nestin and Mφ marker CD68. Karyotype analysis showed that F-BMSCs, F-Mφ were telocentric chromosomes dominant, F-BMSCs karyotype contains two centromere chromosomes, F-Mφ contains only one centromere chromosome.Conclusions: Active interactions can occur in co-culture system of GSCs with BMSCs or Mφ. Spontaneous fusion could be observed between GSCs and BMSCs or Mφ.Part II: Preliminary analysis of biological characteristics of fusion cellsbetween glioma stem cells and bone marrow derived stroma cellsObjective: To compare the biological characteristics of the fused cells and glioma stem cells.Methods: Colony formation assay, CCK-8 test, transwell and apoptosis analysis were performed to detect and compare proliferation, invasion and apoptosis of F-BMSCs, F-Mφ and SU4-RFP cells.Results: Colony formation experiments showed that cloning efficiency of the fusion cells F-BMSCs, F-Mφ was higher than that of SU4-RFP cells(P <0.05). CCK-8 test showed that F-BMSCs, F-Mφ proliferation rate were statistically higher than that of SU4-RFP(P <0.05). Transwell invasion assay showedinvased cell number of the fusion cells was higher than SU4-RFP(P <0.05). The early apoptotic cells proportion of F-BMSCs and F-Mφ was less than SU4-RFP(P <0.05).Conclusions: Cloning efficiency, proliferation and invasion ability of the fusion cells significantly enhanced,with decreased apoptosis, suggesting that fusion between glioma stem cells and bone marrow-derived stroma cells would lead to higher degree of malignancy of the fusion cells. Thus, by blocking glioma stem cells fusion with stroma cells, may become a potential target for glioma therapy, which worth further study.