The Experimental Study Of Fusion Vaccine Of DC/C6 Glioma Cells To Prevent And Treat The Intracranial Glioma In Rats

Objective: Glioma is one of the most common intracranial malignant tumors of human beings. Although many traditional procedures such as surgical resection , radiotherapy and chemotherapy were applied to the treatment of glioma, a radical cure effect is still impossible. Tumor recurrence occurs to almost all the patients. Some lesions locating in functional area are not indications for surgical resection. Medium survival time (MST) of these patients is only 1 year. Current treatments have not changed the natural history of such incurable neoplasm. Therefore, the development of new therapies of treating malignant glioma is urgent. As the most impotant functional APCs, its isolation, proliferation and culture of DC are possible in vitro. Research of different kinds of DC vaccine became a hotspot as a new anti —tumor method. An approach of DC/C6 fusion cell as vaccine toward the prevention and treatment of C6 glioma was developed. In our experiment. The aim of is to provide more detailed theoretical basis for clinical application of DC vaccine.Meaterial and Methods:1. Isolation culture^ proliferation and identification of DCs: Rat bone marrow cells were collected and bone marrow monocytes were harvested by centrifugation. Non-adherent bone marrow monocytes were cultured in RPMI-1640 with additive of rGM-CSF and rIL-4 . Morphology^ expression of special surface marker and functional ability were observed in rat bone marrow—derived DC .2. Fusing DCs with C6 glioma cells and observation of biological properties of DC/C6 vaccine in vitro: PEG was used to fuse DCs with C6 glioma cells. The mixture of different cells was marked with 0X62 —RPE and selected by flow cytometry. We selected and purified DC/C6 fusion cells accoding to its stronger adherent ability than those of DCs. Immunofluorescence of GFAP-FITC was used to identify the DC/C6 fusion cells. Functional ability of fusion cells was examined by MLR and CTL in vitro. Tumor challenge of DC/C6 fusion cells were conducted in-situ and subcutaneously.3. Prevention and treatment of C6 glioma with fusion vaccine in vivo: Firstly, 30 rats were divided into group A> B and C randomly(10 rats per group). Group A were treated with a vaccination of lxlO7 fusion cells 1 time each week. 1 week after 3 times of immunization, tumor challenge with lxlO6C6 glioma cells were implanted in-situ .The survival conditions and longevity of tumor challenged rats were observed . As contrast groups, the rats in group B and C were immunized with DCs or PBS respectively. 40 days after the tumor implantation, all survival rats were sacrificed and observededintracranial tumor growth. Secondly, another 30 rats were used in the immunotherapeutic experiment. Intracranial tumor implantation of C6 glioma cells was performed with the aid of stereotactic guidance. 5 days after tumor implantation, 10 rats in group A were given a vaccination of lxlO7 fusion cells through the tail vein 1 time each week. After 3 times of immunotherapy, the survival conditions and longevity of vaccinated rats were observed . As two contrast groups, DCs and PBS were used in group B and C respectively. The same procedure as group A was conducted in group B and C. Analysis of survival time was conducted to evaluate the results we got. Pathological examinations were applied to rat brain tumor samples. Results:1. DC yield from bone monocytes cultures of 10 days was (4.9 + 0.12)xl07 per rat. Morphological property was observed in some cells at day 5 cultures of DC progenitors. Special surface marker positive expression of 0X62 was observed. Flow cytometric analysis of DCs demonstrated about 86% OX62+ cells. The results of MLR induced by cultured DC of different period to syngeneic spleen lymphocytes showed that the strongest proliferation of MLR occurred to DC at day 10 cultures. MLR of syngeneic spleen lymphocytes is stronger than that of heterogeneous spleen lymphocytes (PO.05).2. PEG as fusion additive was applied to fuse DC with C6 glioma cell and a fusion rate of 16% was gotten. Immunofluorescence of adherent OX62+ cells expressed positive GFAP-FITC. MLR induced by the same numbers of fusion vaccine and DCs showed that a more powerful proliferation of syngeneicspleen T cell in the groups of fusion vaccine as stimulator (P<0.05). Special CTL induced by fusion vaccine show antitumor response in vitro. There is a positive correlation of anti-tumor effect between the effector cells (DC/C6 fusion cells) and the target cells (C6 glioma cells) in vitro. No positive result of tumor challenge was found weather fusion cells inoculation in-situ or subcutaneously.3. In immunological prevention experiment, resistance against intracranial tumor growth was acquired in rats pretreated with DC/C6 fusion cells (group A) after 3 times vaccination. Rats in group A were free of tumor at 40 days after inoculation of C6 glioma. By contrast, a shorter survival time was observed both in group B and group C. Intracranial tumors stained with HE showed active proliferation of tumor cells -, newly formed blood vessels and hemorrhage. In immunotherapy experiment , the MST of rats were as following: 38.5 days of group A^ (23±1.05) days of group B and (21±1.05 )days of group C. There were 5 rats still alive after 40 days of tumor implantation in group A. At necropsy, no tumor was found in 2 of these 5 survival rats. In contrast to that of group B and group C, a significant longer longevity of rats in group A was found statistically (PO.01). A considerable number of dead cells was found in many areas of tumor from DC/C6 vaccinated rats which died in the later period. Moreover, histological examination showed larger number of lymphocytes infiltrated in tumor and immunohistology staining with anti-CD8Mcab revealed that most of the infiltrating lymphocytes were positive CD8+ T lymphocytes. But the same phenomena did not appear in rats brain tumors of DC group or PBS group.Conclusions:1.According to our improved Chen-Woan cells culture method, we got enough DCs for andvanced experiment by centrifugating bone marrow cells ? harvesting non — adherent bone marrow monocytes and using cytokines (GM-CSF^ IL-4) to promote the DCs yield. These DCs have a strong immunological activity to syngeneic MLR. As stimulator cells, DCs at day 10 cultures have the most powerful ability of MLR. A peak response of MLR will be achieved at stimulator to responder ratio of 1:10.2. We selected and purified DC/C6 fusion vaccine by procedures as followed: cell fusion with PEG -. selecting DCs and DC/C6 fusion cells with OX62-RPE marker by flow cytometry .. collecting adherent 0X62+cells according to different adherent ability between DCs and DC/C6 fusion cells. Fusion vaccine appeared double properties of DC and C6 cell. Besides expression of OX62+ as special surface marker of DCs, fusion cells expressed positive GFAP as antibody specific to C6 line by immunofluorescence. Moreover, fusion cells got stronger adhesive ability as that of C6 cells than DCs. Tumor challenge experiment with fusion vaccine showed that it was safe in vivo.3. As stimulator cells, fusion cells appeared more powerful ability of MLR than that of DCs. Cytotoxicity assays showed that fusion cells effectively presented tumor antigen to CTL and induced tumor-specific CTL immune response to C6 glioma cells in vitro.4. Fusion vaccine can protect vaccinated rats against C6 glioma challenge and immunotherapy of fusion vaccine is sensitive to C6 glioma model. Doublemechanisms of cellular and humoral immunity contribute to anti-tumor activity. The cellular immunity of anti-tumor responses mainly depends on CD8+ T lymphocytes.