Experimental Research On Brain Tumor Stem Cells And Their Application For Immunotherapy

1.Aim and MeaningBrain tumors damaged human beings health seriously.It was reported that 2%of cancer patients belong to the malignant brain tumors ones,and 20～25%of them were showed in child tumors.Among malignant brain tumors,gliomas are detected most frequently and account for 40.49%of malignant brain tumors.Therefore, glioblastoma,is one of the commonest brain tumors.Middle life span of patients who were diagnosed as the glioblastoma,is usually for 15 months.Bescause it is very hard to be eradication with conventional antitumor treasurement including surgery, radiation treatment and chemicotherapy,the recurrance rate of glioma is much high. It is showed that there are cells among the glioma tissues behaving like stem cells, whose number is much rare in the glioma.These cells are the reasons of glioma occuring and recurring,which are called brain tumor stem cells(BTSCs).Ignatova(2002) and colleagues firstly reported that there were BTSCs in the gliomas.They used special medium which was used to culture neural stem cells (NSCs) to incubate the glioma cells.Cloning of cells were then observed.It were observed that genes of nestin(the marker of NSCs),neurone specific enolase(NSE), and glial fibrillary acidic protein(GFAP) expressed in the cloning cells and the differentiating cells respectivily.The results showed cells had the characteristics of NSCs.They named the cells as the tumor stem-like cells,and belived the cells were connected to originate of glioma.Then,Singh and colleagues(2004) found that a small part of cells in the glioma caused the proliferation of glioma,which expressed CD133,namely BTSCs.Similar to the normal NSCs,the BTSCs can proliferate, differentiated into neuron-like and glial-like cells,and had same rate and phenotype with primary glioma.They were proved as the BTSCs but not the cells pollutted from the normal NSCs.Hemmati and colleagues(2003) discovered that there were cells in the glioma tissure to be similiar with the NSCs,and suggested them to be possibly involved in the formation of glioma,to be able to form spheres in vivo, posess the abilities of self-regenerate and multipotential differentiation.The difference between the BTSCs and the NSCs was that the former showed an exceptional ability of proliferation.BTSCs had the same feature as follow:(1)Express specific markers of NSCs like Nestin and CD133;(2) Possess the abilities of self-regeneration,proliferation,differentiation,and becoming everlasting cells; (3)Differentiated tumors from the BTSCs have the same phenotype and karyotype with primary tumor tissues.Dendritic cells(DCs) are professional antigen presenting cells in vivo,which strongly ingest and process antigen,and highly express major histocompatibility complex(MHC-â… ,â…¡) and costimulating molecule.They can effectively present antigen to T cells and stimulate first immune response.For the function of DCs, tumor vaccine basing on DCs becomes as one of the important methods of tumor immunotherapy.Combining the characteristic of BTSCs,the fusion between BTSCs and DCs were used to form tumor vaccine to observe the function of immunotherapy in vitro.There are several ways to construct tumor vaccine basing on DCs,such as DCs pulsed by tumor cell mRNA,DCs pulsed by polypeptide of tumor cell,DCs pulsed by decomposer of tumor cell,DCs modified by antigen gene,et al.Nowadays, fusion of DCs and tumor cells had become an effective way of tumor immunotherapy. Meanwhile,the discovery of BTSCs also provides a new train of thought on tumor immunotherapy.The fusion of DCs and BTSCs would be more effective on tumor immunotherapy.In this experivent,we isolated both CD133⁺ and CD133^- glioma cells through immune magnetic cell sorting assay from the patients' tumor samples,and drew patients' blood autologusly to get DCs and T cells.After the generation of fusions between DCs and two object cells(CD133⁺ and CD133^- glioma cells) respectively, we studied the immune responses caused by two kinds of fused cells.2.MethodsThere are three parts of expreiment2.1 Getting and sorting CD133⁺ and CD133^- cells from primary glioma cells.To get the glioma tissues from patients during surgery.After digested and dissociated,primary glioma cells were cultured in the DMEM/F12 medium containing 10%FBS for 3～5 days.Then,parts of primary cells were examined with Flow Cytometry to detect the rate of CD133⁺ cells in the primary cells.Other parts of primary cells were used for Magnetic cells separation to get the CD133⁺ cells. CD133⁺ and CD133^- sorted cells were then resuspended in the DMEM-F12 with cytokines including B27,EGF,bFGF and LIF.After the spheres were observed with light microscope,the immunocytochemistry method was used to test the expression of CD133 antibody on the CD133⁺ cells spheres.We compared the number of spheres of CD133⁺ cells and CD133^- cells with light microscope under 20 random fields of view.Suspended spheres cultured within serum-free medium were transferred to the medium contain 10%FBS.At the 5^th day after transfer culture,the spheres were adherent,and then tested on phenotypic analysis with immunocytochemistry.2.2 Induction and identification of DCs.Adherent peripheral blood mononeuclear cells(PBMCs) were separated from human peripheral blood and the DCs were contained by wall-adherent method from the PBMCs.The DCs were cultured with RPMI-1640 containing GM-CSF and IL-4. On the 5^th day.TNF-αwas added to the culture system to induce their maturation, and then were observed with light microscope.The matured DCs were harvested on the 8^th day.Their phenotypic were assayed with flow Cytometry.2.3 Preparation of the fusion samples about DCs-CD133⁺cells or DCs-133^- cells, Preparation of autologus T cells,and Immune responses of fused cells.2.3.1 Autologous DCs were mixed with separated autologous CD133⁺ cells or CD133^- cells respectively at 5:1 ratio,and then were suspended and washed two times in the Cytofusion Medium.Electrofusion was performed by PA-4000 and PA-101 (Cyto Pulse Sciences,Ltd.).After the cellular fusion was finished,the fusion cells were incubated in the chamber at 37℃with 5%CO₂ for 30mins,then co-cultured with the separated T cells.In some cases,fusion hybrids were detected with fluorescence microscope and flow Cytometry.2.3.2 T cells were isolated from patients through the use of the RosetteSep Lymphoid enrichment kit(StemCell Technologies,Ltd.) in accordance with the manufacturer's protocol.More than 90%of T cells were harvested through this separation according to FACS analysis.T cells were cultured in the RPMI 1640 containing 10%FBS,20ng/ml recombinant human interleukin(IL-2)2.3.3 T cells were grouped into five categories to be cocultured with DC-CD133⁺ glioma electrofused cells,DC-CD133^- glioma electrofused cells, DC-glioma mixed cells,glioma cells only and DCs only,respectively.All the groups were culture in RPMI 1640 medium containing 10%FBS,20ng/ml IL-2.After one week of incubation,the cytotoxic T lymphocy(CTL) responses were performed by the use of CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit(Promega Ldt.) according to the manufacturer's instruction and protocol.IFN-γrelease was tested via ELISA.After incubating for 48h,supernatants were removed and stored at -20℃. Samples were thawed and analyzed for human IFN-γby ELISA kit.2.4 Statistical analysisResults are expressed as mean±SEM.The t test was used for comparisons of two groups,one way ANOVA was used for comparisons of multiple groups. Difference is considered significant at P＜0.05.3.Result 3.1 CD133⁺ cells were isolated from primary glioma cells sucessfully,and tested to have the characters of BTSCs.Number of BTSCs was much less in the tissues of glioma,most of which contained only aproximate 1%.However,high grade of glioma tissues(such as Gradeâ…£) had more than 10%of BTSCs3.2 After 8 days culture with cytokines,PBMCs developed into DCs,which revealed typical morphological dendrites,high level of costimulatory molecules and significant stimulating function on T cells proliferation.It indicated the matured DCs with bioactivity were successfully induced from the PBMCs.3.3 fusion hybrids of DCs-CD133⁺ cells and DCs-CD133^- cells were acquired through electrofusion method.FACS tests proved that purity of T cells can be more than 90%,which were isolated through RosetteSep Lymphoid enrichment kit.ELISA method showed that T cells were stimulated by fusion hybrids.The amount of IFN-γsecretion of T cells previously stimulated by the two fused cells(DCs-CD133⁺cells and DCs-CD133^-cells) was observed much higher than the other groups,while there was not statistically significant difference in the amount of IFN-γsecreted by stimulated T cells between the two kinds of fused cells.Cytotoxicity experiments were detected via Lactate dehydrogenase(LDH) release assay.The results showed that T cells stimulated by the two types of fused cells were effective in inducing cytotoxicity of autologous tumor cells,however,the differences between DC-CD133⁺glioma cells and DC-CD133^-glioma cells were not obvious.The T cells stimulated by autologous glioma cells alone,the mixtures of DCs and glioma cells,or autologous DCs only,showed much less capacity of responding to the target cells (glioma cells) comparing to the the ones stimulated by groups of both DC-CD133⁺ and DC-CD133^-glioma cells.3.ConclusionCD133⁺ cells can be cultured within the DMEM/F12 medium containing B27, EGF,bFGF and LIF,after isolated through Immune Magnetic separation.CD133⁺ cells suspending in DMEM-F12 medium with cytokines can proliferate some spheres. After transferred into medium containing serum(10%FBS),the spheres showed the adherent and differentiation.However,CD133^-cells did not growth permanently within such condition.Mature DCs with typical dendritic cells morphology could be generated from enriched PBMCs of glioma patients with GM-CSF,IL-4 and TNF-α.Fusion cells of DCs and glioma cells maintained the important characteristics of both DCs and tumor cells,and could induce effective CTL activity against tumors in vitro.Fusion cells can effectively stimulate T cells to secrete IFN-γ.CTL responses showed that the fusions of both DCs-CD133⁺ cells and DCs-CD133^- cells can effectively stimulate T cells to kill glioma cells.However,DCs, glioma cells,mixture of DCs and glioma ceils did not generate strongly CTL responses.There were not statistically significant difference between two types of fusions(DCs-CD133⁺ cells and DCs-CD133^- cells).The results of release of IFN-γamong groups indicated that the fusion cells of both DCs-CD133⁺ and DCs-CD133^-can stimulate T cells to secrete IFN-γsignificantly.However,there are not significant differences between two types of fusions(DCs-CD133⁺ and DCs-CD133^-) statistically.