The Role Of Dendritic Cells Pulsed With Tumor Stem-like Cells Associated Antigens Against Malignant Gliomas In Vitro

Aims:To isolate tumor stem-like cells from glioma first,and then characterize tumor stem-like cells including morphology,ratio of CD133+ cells,cell division and cell cycle,and expression of HLA-A,and HLA-DR;To compare the different effects between DCs implused by total tumor antigens extracted from stem-like cells(TSCs) and from non-TSCs on cytotoxicity in vitro;to improve the immunogenecity by irradiation or sorting CD133+ cells.Materials and Methods:Six primary glioma obtained from surgical patients and three cell lines (U87,U251,D341) were cultured in serum free medium(SFM,DMEM/F12 supplemented with EGF/bFGF).Proliferations and multilineage differentiation of these cells were investigated by limiting dilution analysis and immunocytochemistry. Cells were micro-injected into the brain of nude mice to determine tumorigenicity.To characterize the secondary tumor,cells from mice brain were re-cultured again,and histochemistry was performed with GFAP,MGMT.SHG62,SHG66(WHO gradeⅢ,andⅣ),as well as U87 cells were cultured in serum contained medium(SCM,DMEM with 10%FBS) or in SFM respectively,and switched in the two mediums.The ratio of CD133+ cells were analyzed with flowcytometry on the day 0,3,7,28,60,90 and 120.Clone formation was observed by limiting dilution analysis,and cell division was aslo observed according to the morphology under a microscope.Expression of CD133,Nestin,GFAP,CNPase was identified by immunocytochemistry,while expression of HLA-A,and HLA-DR was analyzed by flowcytometry.CD133+ cells were sorted by fluorescence activated cell sorting.The cell cycles of CD133+ and CD133- cells were analysed by flow cytometry using propidium iodide staining.The cells were sorted by magnetic cell sorting with CD 133.Total tumor antigens were extracted from cells cultured in the two different mediums after three freeze-thaw cycles or radiation by 6 Gray X ray.Corresponsive DCs derived from PBMCs were cultured in RPMI1640 with GM-CSF and IL-4,and then matured by antigens described afore.Autologous non-adherent PBMCs were co-cultured with DCs to get effect cells.HLA-A,HLA-DR,CD80 and CD86 of DCs before and after maturation were checked by flowcytometry,and effect cells were checked with CD3, CD16 and CD56.The proliferation of effect cells was checked with XTT assay.Then effect cells and targeted cells were cocultured with different ratio and cytotoxicity was investigated on scintillater after impregnation with thymidine labeled with tritium. Targeted cells before and after irradiated by 2 Gray X ray were explored here to compare the cytotoxicity.Results:Cells could propagate,differentiate to multiple lineages,and undergo tumorigenesis.Secondary tumors exhibited similar biological features with primary tumors.Cells cultured in the two mediums displayed different morphology.The ratios of CD133+ cells in primary gliomas decreased evidently with time(P＜0.05) while cell strains remained stationary levels.The ratio of CD 133+ cells was higher in SFM than in SCM(P＜0.01).The cells could be switched in the two mediums.The cells in SFM had a better ability of clone formation(P＜0.01),and they expressed more proteins such as CD133,Nestin,and HLA-A,but less HLA-DR,CD80,CD86,and GFAP. Asymmetric divisions were the main way of TSCs accretion.CD133+ TSCs were successfully separated from SHG62,SHG66,and U87 cell lines.The G0/G1 ratio in the CD133+ cell group was significantly higher than that in the CD133- cell group, while G2/M ratio was higher in the CD133-group.Cells contained 2.32±0.64%,5.37±0.82%CD133+ cells were obtained after magnetic sorting.Different tumor associated antigens were derived from different glioma cells.DCs were successfully matured by tumor stem-like associated antigens, as flowcytometric analysis determined that HLA-A,HLA-DR,CD80,CD86 of DCs were up regulated.Non adherent cells were multiplied significantly and CD3+ T cell ratio was obviously improved after co-cultured with corresponsive DCs(P＜0.01). Effect cells loaded with tumor stem-like cells associated antigens killed 70.2%±5.13% of non stem-like cells and 56.7%±7.81%of stem-like cells(P＜0.001),however effect cells loaded with non stem-like cells associated antigens eliminated only 36.6%±6.45%of non stem-like cells and 9.05%±3.49%of stem-like cells(P＜0.001). Furthermore,SAAs have better immunogenicity especially extracted from magnetically sorted stem-like cells.Antigens obtained from irradiated SCs(6 Gray) raised the cytotoxicity significantly to 75%(to GCs) and 59%(to SCs)(P＜0.05).No difference of CTL killing ability was detected between targeted glioma cells before and after irradiation(2 Gray)(P＞0.05).Conclusions:The cells in SFM showed the characteristics of tumor stem-like cells,The ratios of CD133+ cells dropped with time in primary gliomas,and stem cell mediums were favored for CD133+ cells.It indicates that supplementary factors or microenviroment is important to stem cell thriving.Hierarchy exists in malignant glioma cell lines.Asymmetric division is the primary mode of proliferation.CD133+ cells tend to stay in G0/G1 phases,indicating their quiescent nature.Lack of HLA-DR could be one of the way for TSCs to escape immune attack.Stem-like cells have their unique character of lots of proteins,such as CD133,Nestin,GFAP.All these remind us that TSCs have distinctive biological features.DCs implused with tumor stem-like cells associated antigens augment the protection against malignant glioma cells in vitro.X ray and antigens extracted from sorted cells with CD133 could improve the immunogenecity,and low-dose radiotherapy have no effect on immunotherapy.These data provide robust implications for clinical trials based on DC vaccination.