Preliminary Studies On 3-bromopyruvate Targeting Glucose Metabolism Pathway In Malignant Transformation Of Host Micro-environment Stromal Cells Induced By Glioma Stem Cells

Part I:Experimental studies on malignant transformation of micro-environment stromal cells induced by glioma stem cellsObjective:It was well known that several kinds of cells in the tumor micro-environment（TME）establishment by glioma stem cells can promote tumor progression.However,whether these cells underwent malignant transformation,and relevant molecular changes kept largely unknown.Methods:Immortal EGFP⁺cells were monocloned either from in vitro co-culture of glioma stem cells labeled with red fluorescence protein（SU3-RFP）and murine MSCs of mice that expressing EGFP protein,or from their xenografts in vivo.These cells were further studied with chromosome analysis and tumorigenicity assay.Cell surface markers of these cells were evaluated through RT-PCR,flow cytometry and immunochemistry（IHC）techniques.Results:（1）Six EGFP+cell lines have been cloned,four of them were from xenografts in vivo,the other two were from co-culture models in vitro,all of which bearing characteristics of self-renewal,heteroploid of chromosomes and 100%tumorigenicity rate,suggest malignant transformation of these cells.（2）Cell surface marker analysis showed origin of the six cell lines were macrophage(t M?_1,2),dentritic cell(tDC_1,2),fibroblast（tFB）,oligodendrocyte（tOG）,respectively.（3）All of these six cell lines expressed Sca-1 and c-myc,part of them had Sox-2 and Nanog expression,which suggest that they bear partial characteristics of mesenchymal stem cells or pluripotent stem cells.Conclusions:（1）Stromal cells in tumor microenvironment can promote growth of primary tumors.Besides,these cells can also undergo malignant transformation,which was relevant to remodeling of TME initiated by tumor stem cells.（2）Solid tumors are consist of millions of heterogeneous cells,however,it is not appropriate to regard them simply as tumor heterogeneity,however,there may be two kinds of malignant cells with different origins in solid tumors.Part II: Effects of 3-Br PA on glucose metabolism of malignant transformed stromal cells in glioma micro-environmentObjective: To pursue the effect and relevant mechanisms of 3-Br PA on glucose metabolism of malignant transformed stromal cells in glioma microenvironment.Methods: t DC2 and t M?2 cells were cultured at 105 cells/ml at different concentration（75¹25umol/L）of 3-bromopyruvate in 96-well dish for 24 hours.Then suppression ratio of cell proliferation was measured by CCK-8 and lactate concentration were measured from culture medium.c-myc and MCT1 expression were evaluated through q PCR and western blot.The effect of 3-bromopyruvate on invasive ability was evaluated by transwell assay.Results: ⑴Proliferation of t DC2 and t M?2 cells were inhibited by 3-Br PA,and the suppression ratio was positive relavant with the concentration of at 3-Br PA.⑵ Lactate concentration of t DC2 and t M?2 cells decreased gradually with increasing of 3-Br PA.⑶3-Br PA could downregulate both transcriptional expression and protein level of c-myc and MCT1 of t DC2 cells.⑷Transwell assay showed numbers of t DC2 and t M?2 cells that pass through membrane decreased obviously at 100 umol/L concentration of 3-Br PA（p<0.05）.Conclusions: Pharmacological inhibition of glycolysis metabolism synergistically potentiated the synthetic lethal targeting of myc by 3-bromopyruvate due in part to the metabolic disturbance caused by c-myc/MCT1 signaling pathway in malignant transformed stromal cells,which represents an attractive therapeutic option for future glioma treatment.