Establishment Of The Balb/c Nude Mice Expressing EGFP And Malignant Transformation Of The Tumor Stroma Cells In Glioma Stem Cells Orthotopic Transplantation Model

Part I: Establishment of transgenic EGFP Balb/c nude mice andEGFP/RFP dual-color fluorescent tracer glioma orthotopictransplantation model.Objective: To incubate transgenic enhanced green fluorescent protein (EGFP) nudemice used for orthotopic xenograft of glioma cells labeling with red fluorescence protein.To establish the dual-color fluorescence tracing (GFP/RFP) tumor model for the furtherinvestigation of the interaction between tumor and host in the microenviroment.Methods:（1）Transgenic female C57BL/6mice expressing EGFP were crossed withBALB/C nude male mouse. Male F1mice were crossed with brood female F1mice andcontinuous inbreeding to the tenth generation according to the criterion, and then followthe closed group management（.2）U87-MG and SU3glioma cell lines was transfected withthe DsRFP gene carried by lentivirus, and C6glioma cell line was traced with CM-DiI, toestablish three lines of glioma cells with red fluorescence expressing(SU3-RFP，U87-RFPand C6-CM-DiI);（3）1X105red fluorescence cells were injected slowly to the caudatenucleus of the EGFP nu/nu mice with the help of the stereotactic instrμment andmicro-injector. Tumor-bearing mice were killed when clinical symptoms appears，and toobserve and detect the dual-color tracing with whole-body in-vivo fluorescence imagingand frost slice techniques under the fluorescence microscope and laser scanning confocalmicroscope.Results:（1）Establishment of the transgenic EGFP nude mice, C.B6-EGFP, withapproximately50%of the offspring of these mice were nu/nu EGFP, and the other halfnu/+EGFP. Almost all of the essential tissues, except the hair and erythrocyte, expressing the green fluoresce at470nm excitation light;（2）SU3-RFP,U87-RFP and C6-CM-DiIalmost100%express red fluorescence under the fluorescence microscope;（3）In theRFP/GFP transplant tumor tissues, RFP+cells were observed growing in wherever themwent including brain parenchyma、ventricle、cerebral vascular under the fluorescencemicroscope and/or laser scanning confocal microscope, and interweaving or fusing withthe GFP+cells.Conclusions:(1) Established EGFP+BALB/C nu/nu inbred mice are appropriate fortransplantation of human tumor cells labeled with RFP.Established RFP/GFP solid tumorscount for a lot in the study of tumor tissue remodeling between tumor cells and host cells. Part II: Malignant transformation of the host stroma cells in the gliomastem/progenitor xenograft tumorObjective: Malignant transformation of the tumor stroma cells in the xenograft tumorshas been reported already, but the mechanism is unclear. Glioma stem/progenitor cellstransfected with red fluorescent protein (RFP) gene are transplanted to the transgenicenhanced green fluorescent protein (EGFP) gene nude mice, establishing dual-colorfluorescent protein tracing tumor model, to study the malignant transformation of thetumor stroma and its possible mechanism in the process of tissue remodeling.Method: Established glioma stem/progenitor cell line, SU3was tranfected with redfluorescent protein gene, named SU3RFP. In vivo, cells were transplanted into EGFP nudemice subcutaneous, celiac and intracranial, respectively; in vitro, peritoneal lavage cells(PLC) and bone marrow derived cells (BMDC) were directly co-cultrue with the SU3RFP.Subcultruing cells from the solid tumor were separated for EGFP positive cells andEGFP/RFP double positive cells by fluorescence activated cell sorting (FACS), thenimmortalized EGFP positive and EGFP/RFP double positive cells were cloned, furtheranalysis of these cells’ growth characteristics in vitro were performed, such as cloningefficiency, motility and invasion ability, and tumorigenic rate, mRNA level was detectedby RT-PCR, and origin of the cells were identified by RT-PCR and immunohistochemistry.Real-time dynamic observation on the interaction between tumor and host by co-cultrue of SU3RFP and PLC under the time-lapse living cell station was performed.Result: Tumorigenic rate of SU3RFP in EGFP mice (subcutaneous, celiac andintracranial) was100%(15/15). RFP, EGFP, and EGFP/RFP positive cells were allobserved by the laser scanning confocal microscope (LSCM) and flow cytometry.EGFP/RFP double-positive cells were cloned, and they not only express biomark of theglioma stem/progenitor cells, nestin, but also express marks of bone marrow mesenchymalcell, CD105, and their tumorigenic rate was also100%, but indirected cells is notumorigenic, which suggested that malignant transformation of the stroma cells maybecaused by cell fusion. Possible ways of communication between tumor and host wereobserved time-lapse living cell station: cell fusion, axon transmit, entosis and vesiculartransport.Conclusion: Tumor initiating cells, SU3RFP fused with the host stroma cellsspontaneously and induced them undergo malignant transformation, showing thatdual-color fluorescent tracing tumor model was of great importance in the research oftumor tissue remodeling, especially the malignant transformation of the tumor stromal cellsas well as the targeted therapy.