Experimental Study Of Dendritic Cells Pulsed With Brain Tumor Stem Cells For Immunotherapy Of Glioma

Glioma is the most common intracranial tumor.It present as diffuse toumors with invasion into normal brain tissue.After surgery,radiation or chemotherapy the glioma often recur or progress. The clinical therapeutic effect to glioma is disappointing. Despite the improvement of local therapies, such as surgery and radiation, the mean survival time for patients carrying a diagnosis of glioblastoma multiforme remains virtually unchanged. On average, patients survive 12 to 18 months, with few patients surviving beyond 2 years. Therefore it is urgent to find effective therapies of treating glioma.Recently,with the development of immunology and molecular biology, gene therapy and immunotherapy are rapidly gaining momentum. One of the most promising immunotherapeutic approaches for the treatment of glioma is the vaccination of cancer patients with dendritic cells (DC) pulsed with tumor antigens.As the most impotant functional antigen presenting cell, DC can capture,process and present antigen to lymphocyte.Recently, much research of different kinds of DC vaccine has got satisfactory antitumor effect. DC can present kinds of antigen such as the whole tumor antigen specific antigen,neoplasm DNA or RNA. The most impotant function of DC is processing and presenting antigen to lymphocyte,which can evoke anti-tomour effect.It is unknown about the specific and related antigen of glioma,so the anti-tumor effect is limited.BTSC were first reported by Ignatova.He observed small part of glioma cell posseses the abilities of self-regenerate, which caused the proliferation of glioma,namely BTSC. Similar to the NSCs, BTSC posses the abilities of self-regenerate and multipotential differentiation,which were connected to originate of glioma.BTSC expressed CD133+ and Nestin.Singh discovered only 100 CD133+cells could generate glioma in rat brain,but 105 CD133-cells could not do it. The result showed BTSC had strong antigenicity.In our study,C6 cell line was cultured in serum-free medium with EGF, bFGF to obtain tumor cell spheres. The morphous of BTSC was observed under microscope before and after differentiation.Specific markers such as CD 133 and nestin, were employed to recognize BTSC by immunofluorescence cytochemistry techniques. The monocytes from rat bone were cultured in RPMI-1640 containing GM-CSF and IL-4 to obtain DC. Dendritic cells pulsed with brain tumor stem cells can provide good protect for tumor-bearing rat. Pathological section of the tumor have the most inflammation and CD8+T lymphocytes.ObjectiveGlioma is the malignant intracranial tumor. Bescause it is very hard to be cured with the conventional therapy including surgery,radiation and chemotherapy,the recurrence rate of glioma is much high.The immunotherapy of glioma has become hot spot. Dendritic cells are the professional antigen presenting cells in vivo.DC vaccine has been applied in clinical trials,which has been safely and effectively. BTSC are the reasons of glioma occuring and recurring.They have good antigenicity of glioma. The aim of our study is to investigate the effect of dendritic cells pulsed with brain tumor stem cells. It will provide the experimental foundation for clinical immunotherapy of glioma.Methods 1. The culture and identification of BTSCC6 glioma cells were firstly cultured in DMEM containing 10% FCS. C6 cells which were in exponential phase of growth were transplanted to serum-free medium containing B27 supplement, basic fibroblast growth factor(20ng/ml) and recombinant epidermal growth factor(20ng/ml). The expression of special markers CD 133 and Nestin was detected by immunofluorescence. Method of flow cytometry were used to determine cell surface molecule of BTSC. Divided 10 nude mouse into two groups randomly, each group had 5 rats.One group accept BTSC by subcutaneous injections,the other accept C6 cells by the same way. we applied immunofluorescence to detect the expression of special markers of mature nerve cells such as GFAP.2. The isolation and culture of DC.Rat bone marrow monocytes isolated from rat femurs and tibias were cultured in RPMI1640 containing 10% FCS, granulocyte macrophage-colony stimulating factor(GM-CSF,5ng/ml)and interleukin-4(IL-4,5ng/ml). The mature DC were harvested after 12 days.The mature DC were analyzed by morphology with phase microscope; The expressions of specific marker OX62 was analyzed by flow cytometry. C6 glioma cells of logarithm growth period were collected through digestion and calculated.We prepare BTSC and C6 cells antigen by freezing-melt method. The antigen and DC were cocultured by the ratio 3:1.The function of two kinds of vaccines were examined by mixed lymphocyte rection (MLR).3. The treatment of glioma by DC vaccineTo established rat C6 brain-glioma model. C6 tumor cells were cultured in vitro and then the C6 cells at logarithmic growth phase were injected intracranially under the stereotactic monitor into the right caudate nucleus.40 Wistar rats were divided into group A,B,C and D randomly, each group had 10 rats. Group A was injected with DC-BTSC vaccines of 1 X 107 cells through tail veins once a week for 3 weeks. Group B was treated with DC-C6 vaccines. The same number of DC and the same volume PBS were applied to group C and group D. Tomor samples were examined with HE staining and immunohistochemistry. The survival time of rats was analyzed by Log-rank survival analysis.Results1. C6 glioma cells were cultured in serum-free medium. After five to seven days,they presented as typical tumor spheres,which appeared globular and floating in medium. The expression of special markers CD 133 and Nestin marked by immunofluorescence was positive. When cultured in serum-supplemented medium,BTSC began to differentiate and express mature neurocyte antigens such as GFAP. Flow cytometry results showed:the average positive rate of MHC-Ⅱ,CD80 and CD86 was 60%,51% and 62% respectively,which was significantly higher than that of C6 cells(9%,6%,4%). Tumor spheres were transplanted to rats in group A and tumor nodules were seen after several weeks.But no tumors were formed in group B injected with attached cells in serum-free medium.The size of formed tumor in nude mouse was related to cell density injected.The pathological section of transplanted tumor with HE staining showed typical characterization of C6 cells.When the cultural condition was changed into serum-supplemented medium,BTSC attached and appeared to reticulate.The characterization of differentiated cell was the same as C6 cells.2. When cultured with cytokines,after 10 days, PBMCs attached developed to free-floating DC, which had typical morphological dendrites. The positive rate of special surface marker OX62 on DC was 86% by flow cytometry analysis,which was low on PBMCs uninduced with cytokine.MLR showed:the stimulation index of DC-BTSC was significantly higher than that of DC-C6 at the same ratio (P< 0.01),which was the highest at stimulator to responder ratio of 1:10.3. After 3 weeks implanted C6 tumor cells by stereotaxis, the cerebral hemisphere of rats was observed the cauliflower-like growth of tumor. But no tumors were formed in the group protected by DC-BTSC vaccine. The pathological sections stained with HE showed the growth of tumor cells and intratumoral necrosis in group B,C and D.In contrast,they were not shown in group A. Immunohistology staining demonstrated the infiltration with CD8+T lymphocyte in group A,B and C.The MST of rats were as fllowing:(39±3.12)days of group A,(29±2.01)days of group B,(23±1.42)days of group C and (19±1.02)days of group D. The survival analysis showed group A was statistically significant in constrast to the other groups(P<0.05).Conclusion1. We could get enough BTSC by culturing C6 glioma cells in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. The characterization of differentiated cell was the same as C6 cells.2. The expression of MHC-Ⅱ,CD80 and CD86 on BTSC was higher than that of C6 glioma cells.3. The growth of tumor in nude mouse injected with BTSC was faster than that of nude mouse injected with C6 glioma cells.4. We got enough DC by culturing rat bone marrow cells in RPMI1640 containing GM-CSF and IL-4.5. MLR showed:the stimulation index of DC-BTSC was significantly higher than that of DC-C6 at the same ratio,which was the highest at stimulator to responder ratio of 1:10.6. DC loading with BTSC lysates can provide immunity protect for the rats loaded with C6 glioma cells,which could prolong those live time.7. DC loading with BTSC lysates can provide a higher level of immunity protect against gliomas than those loading with C6 cells,which provide a new method in the immuntherapy of brain glioma.