Preliminary Studies On Malignant Transformation Of Murine Macrophages Cells Induced By Glioma Stem Progenitor Cells

Part I: Malignant transformation of fusion cells derived from theco-cultured glioma stem progenitor cells and macrophagesObjective We have hypothesized that the malignant transformation of macrophages(MPs) in glioma was induced by fusion of glioma stem progenitor cells(GSPCs) with MPs.The purpose of this study was to verify this hypothesis by co-culturing GSPCs and MPs.Methods Human GSPCs cell line SU3 transfected with red fluorescent protein(RFP)gene was co-cultured with peritoneal lavage MPs from EGFP mice(whole body expression of enhanced green fluorescent protein) in vitro.RFP+/GFP+dual-color fluorescent cell lineages were established with monoclonal pipetting techniques, then were analyzed on their biological phenotypes, including tumorigenic assay, surface marker expression.Results(1)A few dual-color fluorescent cells were observed several days later in the co-culture model later. Three GFP and RFP double positive monoclonal cell lineages were cloned and named after C3,C4 and C12.(2)Three types of cell subpopulation arose during continuous passage of C12 cells namely,RFP+cells, EGFP+cells and RFP+/EGFP+cells.But the proportion of EGFP+ cells increased gradually and they became the main population of C12 cells,meanwhile the proportion of RFP+/EGFP+cells decreased and maintained at a low level,the RFP+ cells almost disappeared.(3)EGFP+ cells in C12 had the following characteristics: ① malignant biological characteristics,such as lost of contact inhibition,rapid proliferation and chromosome aneuploidy;②high tumorigenic rate in nude mice(5/5);③expression of macrophage specific marker CD68,and most of chromosomes were telocentric chromosomes.Conclusion In vitro studies support our previous finding that human glioma stem progenitor cells could induce malignant transformation of host macrophages in xenografttumors via cell fusion.Along with the malignant transformed macrophages(ih CTC) in vivo obtained from solid tumor tissue of the tumor-bearing mice,our results further confirmed the possibility of malignant transformation of host macrophages in vitro in tumor microenvironment.The malignant transformation of host cells induced by fusion with tumor stem progenitor cells not only provided new finding to understand the progression of tumor and cancer heterogeneity,but also offered new targets for therapy against glioma.Part II: Social activities of co-cultured glioma stem progenitorcells and macrophages under live cell imagingsystem:cell conjunction,cell fusion,and entosisObjective :The roles of real-time monitoring of cytokine secretion at single immunocyte level,following the concept of immune cells sociology has been reported recently.However, the mutual interactions between glioma stem/progenitors cells and host macrophages in the immune cells society has never been reported previously, which deserve further investigations discover the important events which broke the immune balance of the cancer cells society.Methods: The dual fluorescence tracing method was applied to observe the co-cultured glioma stem/progenitor cells and host macrophages,interactions between the two kinds of cells were studied under live cell imaging system,fusion cells(yellow) in the co-culture system were mono-cloned,and cultured in vitro.Then their social activities were observed and recorded under the live cell imaging system,too.Results: Via real-time dynamic observation of the target cells,we noticed that①there were 6 types of conjunction pipes can be found, which functioned as transfering intercellular information; ② two activating cells formed hybrid cell after mutual interactions,and the hybrid cell can fuse with another cell again;③the fates of hybrid cell were to generate offspring cells by symmetrical division, or to undergo apoptosis;④the phenomenon of cell in cell,which can release out another cell, namely entosis,can be found,too.Conclusion:The research revealed the patterns of cell conjunction between glioma stem/progenitor cells and host macrophages,the processes of cell fusion,division and entosis.These were the cell sociology evidences from direct visualization of mutual interaction between glioma stem/progenitor cells and host macrophages,which offered basic knowledges for exploring the complex relationships among the residents in the tumor cell society.