Experimental Research On Malignant Transformation Of Tumor Stromal Cells Induced By Glioma Stem Cells Heterotopically Inoculated In Host Liver

Part I: Establishment and biological characteristics analysis of dual-color fluorescent tracing model of human glioma stem cells heterotopic transplanted in host liverObjective: To establish of red and green fluorescent tracing human glioma stem cells heterotopic transplantation model, for the purpose of further studies on heterotopic xenograft tumor microenvironment.Methods: The lentivirus carrying the red fluorescence protein(RFP) gene was transfected into human glioma stem cells SU3. Then SU3-RFP cells with stable expression of RFP were implanted into the liver of the nude mice with whole-body expressing green fluorescence protein(GFP). When symptoms of tumor-bearing mice appeared, the livers of part of tumor-bearing mice were harvested and observed under fluorescence imaging system. Meanwhile, the livers of other tumor-bearing mice were obtained after cardiac perfusion of 4% paraformaldehyde, then fixed in 4% paraformaldehyde, dehydrated in high concentrations of sucrose, packaged by OCT and cut in 5 μm thick serial frozen slices. After HE and DAPI staining, slices were observed under light microscope, fluorescence microscopic and confocal laser scanning microscope to analyze pathological features of the tumors.Results: SU3 cells retained their tumor stem cell properties after transfected with RFP gene. When transplanted into liver of GFP mice, tumor formation rate was 83%(15/18). Pathological studies and in vivo fluorescence imaging showed SU3-RFP cells can invade the liver and other abdominal organs. Frozen slices of xenograft tumors showed tumor cells with stable expression of RFP and host cells expressing GFP was intertwined, jointly participated in the composition of the tumor microenvironment inside tumor parenchyma. Besides, tumor cells were found to invade host blood vessels, migrate distantly, fuse with host cells, and phagocytosis of host cells which could be observed clearly with dual-color fluorescent tracing technigues.Conclusions: The establishment of dual-color fluorescent tracing glioma stem cells xenograft tumor model can provide great convenience for further exploring heterotopic transplanted glioma microenvironment. Furthermore, the application of dual-color fluorescent tracing glioma model in studying tissue remodeling of ectopic xenograft tumor possesses high practical value in investigating the invasion and metastasis of tumor cells, and mutual interactions with host cells.Part II: Malignant transformation of glioma stromal cells induced by glioma stem cells heterotopically inoculated in host liverObjective: The host gliocytes have the potential of undergoing malignant transformation induced by glioma stem cells(GSCs) in orthotopic glioma model. The purpose of this study is to explore whether malignant transformation of tumor stromal cells induced by GSCs is relevant to specific local microenvironment.Methods: Human glioma stem/progenitor cell line SU3 transfected with red fluorescent protein(SU3-RFP) gene were implanted into the liver of the nude mice with whole-body expressing green fluorescence protein(GFP). Then the xenograft tumors were harvested to prepare single cell suspension and analyzed with routine pathological examinations. GFP cells with high proliferative abilities were obtained from the cultivation of single cell suspension. Immortalized glioma stromal cells only expressing GFP and double expressing GFP/RFP were further monocloned with micro-pipetting techniques and under continuous passages. Cell phenotype analysis and tumorigenicity tests were also performed.Results: Immortalized glioma stromal cells were obtained from re-cultured xenograft tumor tissue. Three monoclonal cell lines B4, B9, B10 were established and proved to be host-derived cells. B4 was found to be a fusion cell, co-expressing GFP/RFP and dendritic cell markers CD1 a, CD83 and CD86. Both B9 and B10 were GFP+ cells, B9 expressed macrophages markers CD68 and F4/80 while B10 was positive in fibroblast marker proteins FAP-α, α-SMA and S100. Three cells were all aneuploid with a tumorigenicity rate of 100% in nude mice.Conclusions: Tumor stromal cells in tissue remodeling microenvironment from glioma stem cells also have the potential of malignant transformation in heterotopic xenograft glioma model. Malignant transformation may also occur outside the central nervous system and contribute to tumor heterogeneity. Further studies are warranted for elucidating the relationship between tumorigenesis, evolution and tumor microenvironment.