OBJECTIVEThe aim of this study was to clarify whether the fusion of bone marrow mesenchymal stem cells (MSCs) with tumor cells can promote tumor angiogensis.

METHODSHuman glioma stem/progenitor cells (GSPCs) (SU3 cells) were transfected with red fluorescent protein (RFP) gene. Bone marrow mesenchymal stem cells (MSCs) were harvested from nude mice with whole-body green fluorescent protein (GFP) gene expression. Then the two kinds of cells were co-cultured in vitro. At the same time SU3-RFP was transplanted into the brain of GFP-expressing nude mice to establish xenograft tumors. The co-cultured cells, GFP/RFP double positive (yellow) cells and blood vessels obtained from the xenograft tumors were observed under fluorescent microscope and laser scanning confocal microscope.

RESULTSAfter five passages in vitro, MSCs maintained the proliferative activity and highly expressed CD105. CD105 was also expressed in the femurs of GFP-expressing nude mice, tumor cells, blood vessels of SU3 xenograft tumors, and clinical malignant gliomas. When MSCs were co-cultured with SU3-RFP, the ratio of yellow cells co-expressing RFP and GFP was significantly increased after extended time and continuous passages. According to the flow cytometry, yellow cells co-expressing RFP and GFP were 83.7% of the cultured cells. In tissue slices of the xenograft tumors, bundles of yellow vessel-like structure and cross-sectioned yellow vascular wall structures including vascular wall stroma cells were observed with RFP and GFP expression, and were identified as de novo formed vessels derived from fusion of MSCs with SU3-RFP cells.

CONCLUSIONCell fusion occurs between tumor cells and host MSCs and it promotes tumor angiogenesis.

Animals ; Bone Marrow Cells ; physiology ; Cell Communication ; Cell Fusion ; Cells, Cultured ; Glioma ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; Mesenchymal Stromal Cells ; Mice ; Mice, Nude ; Microscopy, Fluorescence ; Neoplasms ; Neovascularization, Pathologic ; Stem Cells ; Transfection ; Transplantation, Heterologous

Objective To explore the metabolite profiles of mesenchymal stem cells(MSCs)underwent death using 9.4 T high resolution MR spectroscopy.Methods MSCs were cultured and treated for 6,12 and 24 hours in a stimulated condition which included hypoxia,serum deprivation and changes of microenvironment.Cell death and the mortality was detected by light microscopy,Hocchst staining and flow cytometry analyses.The morality of stem cells was analyzed using one-way analysis of variance (ANOVA).Cell metabolite extraction was prepared by methanol-chloroform(M/C) method and analyzed on a 9.4 T MR device.1H-MR spectroscopy was obtained and the metabolite concentration of each time point was calculated and compared using one way ANOVA,the difference between two groups was analyzed by SNK test.Results Necrosis was the major form of cell death in the built model.The morality of every time sets was 16±4(0 h),658±61 (6 h),1 571 ± 154(12 h) and 2 816± 178(24 h) respectively,and the difference between each groups were statistically significant (F=298.96,P＜0.01).After induced stem cells death for 6,12 and 24 h,the metabolite concentrations at 0.89 ppm was (1.48±0.69),(2.32±0.63)and (2.15±0.45)nmol/mg respectively,and increased compared to thc control[(1.41 ±0.25)nmol/mg]with statistical significance (F=329.57,P＜0.01).The metabolite concentrations at 1.28 ppm was (6.42±0.31),(7.26±0.32)and (7.01 ±0.61)nmol/mg,respectively,and increased compared to the control[(5.76 ±0.74)nmol/mg]with statistical significance (F=19.56,P＜0.01).The metabolite concentrations at 1.60 ppm was (2.36±0.31),(2.29±0.16)and (2.31 ± 0.24) nmol/mg respectively,and increased compared to the control[(1.96 ± 0.27)nmol/mg]with statistical significance (F=4.35,P＜0.05).After induced stem cells death for 12 hours,the metabolite concentrations at 0.89 ppm was increased compared to 6 hours with statistical significance (P＜0.05).The metabolite concentrations at 1.28 ppm was increased compared to 6 hours with statistical significance (P＜0.05).After induced stem cells death for 24 hours,the metabolite concentrations at 0.89 ppm was decreased compared to 12 hours with statistical significance (P＜0.05).Conclusions There are some specific characteristics on MRS of MSCs underwent death,and the fatty acid peak may serve as a biomarker for cell death.

Objective To investigate whether the malignant transformation of macrophages ( Mφ) in glioma mesenchyme was induced by the fusion of glioma cells ( SU3 ) and Mφ.Methods SU3 cells transfected with red fluorescent protein genes were co-cultured in vitro with Mφexpressing enhanced green fluorescent protein.The cell lineages with RFP+/GFP+dual-color fluorescence were established by using monoclonal selection method.A series of tests for analyzing cancer-related phenotypes, tumorigenicity and specific markers for murine macrophage were performed.Results (1) A few of dual-color fluorescent cells were observed in the co-culture.Three monoclonal cell lineages (C3, C4 and C12) were obtained success-fully.(2) Three types of cells including RFP+, EGFP+and RFP+/EGFP+cells were formed during the cul-ture of monoclonal C12 cell lineage.The percentage of EGFP+cells was increased along the extended culture time and increased passages.Then, EGFP+cells gradually became the predominant cell population.Nota-bly, the percentage of RFP+/EGFP+cells were decreased and maintained at a low level, but the RFP+cells almost disappeared.(3) EGFP+cells from monoclonal C12 cell lineage showed the malignant characteristics such as loss of contact inhibition, rapid proliferation andchromosome aneuploidy, as well as high tumorigenic rate in nude mice (5/5).They also expressed macrophage specific marker CD68 and showed a large number of telocentric chromosomes.Conclusion The results of this study suggested that the malignant transforma-tion of host macrophages as previously observed in solid tumor might be induced by cell fusion occurred be-tween human glioma cells and macrophages.Along with the previous evidences showing the isolation of the malignantly transformed macrophages ( ihCTC) from solid tumor tissue of tumor-bearing mice, the results confirmed an objective existence of malignant transformation of host macrophages in tumor microenvironment. The malignant transformation of host cells induced by fusion with tumor cells revealed not only a new under-standing for the progression of tumor and cancer heterogeneity, but also new targets for cancer therapy.

Objective To investigate the effect of thymosin ?1 (T?1) on cellular immune function in gastric cancer patients through observing its treatment on the differentiation of T-lymphocyte subsets from screened peripheral blood mononuclear cells (PBMCs). Methods PBMCs were obtained by centrifugation of blood samples from 18 healthy subjects and 32 patients with gastric cancer,and then cultured in the presence of culture medium with addition of T?1 at 50,10 and 1 ?g/ml for 2 d. T lymphocyte subsets (such as CD4+,CD8+ and CD4+ CD25+ Foxp3+ T cells) and Th1/Th2 multiplex cytokines were detected by flow cytometry (FCM). Results After PBMCs isolated from healthy people and patients were incubated with or without T?1,there was no significant change in percentage of CD4+,CD8+ peripheral lymphocyte subsets and ratio of CD4+/CD8+. There was no obvious change in the percentage of CD4+ CD25+ Foxp3+ T lymphocyte subsets in the normal control,but a significant increase was observed in the cells from patients with gastric cancer after treatment (P