| Osteosarcoma is one of the most common primary malignant bone tumors mostly in youth and adolescents. The main treatment for osteosarcoma was surgical excision including amputation and joint amputation before the1970's. The average time is 8 months from surgical treatment to lung metastasis. Less than 25% of patients had a 5-year survival rate. With the development of surgery and screenage technology and achievement of alliance of neoadjuvant chemotherapy, radiotherapy and the immunotherapy, the osteosarcoma therapy has achieved great achievement, with 5-year disease-free survival exceeds 80%. However, the metastasis, recurrent and chemotherapy-resistant cases has not been solved. Therefore, novel strategies to prevent proliferation of malignant cells are urgently needed.A promising approach may be the prophylactic vaccination directed against a tumor-associated antigens(TAA)or tumor-special antigens(TSA)from the whole tumor cells. The major objective of active specific immunotherapy is to generate tumor- specific cytotoxic T lymphocyte (CTL) responds, but to induced CTL responds depend on the use of professional antigen presenting cells loaded with tumor antigen. In the antigen-presenting cells, dendritic cell (DC)has been considered to be the most potent professional antigen-presenting cell (APC)that can initiates primary immune responses. Its ability to stimulate and regulate T- and B-cell responses makes DC ideally suited to serve as an adjuvant for the purpose of cancer immunotherapy. DC also can be involved in the pathogenesis of graft versus host disease or host versus graft disease after transplantation as well as immunization against viral infections and immunosuppression in autoimmune diseases.Considering the expected emergence of antigen-loss variants and the lack of known tumor-specific antigens in most cancers, in this study, the DC-based vaccine is generated using whole osteosarcoma cell body through electrofusion process. One important advantage of immunization with the whole tumor cell is the potential to induce an immune response against all possible tumor antigens, known or unknown. Electrical cell fusion is an essential step in some of the most innovative methods in modern biology, such as the production of monoclonal antibodies, the cloning of mammals, and vaccination against cancer. Compared with the chemically induced cell fusion via polyethylene glycol (PEG), electrical cell fusion is a method with higher efficiency. More importantly, this technique makes it practical to combine two allogeneic APC or tumor cell together.The allogeneic fusion hybrids are particularly attractive because they would enable the use of allogeneic DC or tumor cells, both providing the costimulatory fusion or MHC molecules required to induce CTL in naive T-cell precursors. Both of the options seem to project a practical advantage. For in a clinical setting, it is not easy to cultivate original tumor cell from osteosarcoma patients, who have suffered from the complications of expectant treatment, thermotherapy by microwave or radio-frequency, et al. Allogeneic tumor cells can save the necessary process of original tumor cell collection and make cell cultivation easier. While allogeneic DC can be generated conveniently from stored peripheral mononuclear cells from normal healthy volunteers from the general population, which avoids the strike to draw additional peripheral blood from cancer patients, who have been very weak.Part I Bolting of UMR-106 Rat Osteosarcoma Cell Line Susceptible Population and Establishment of Animal Model of Rat Osteosarcoma and Its Lung MetastasisObjective To introduce a method of bolting of UMR-106 rat osteosarcoma cell line susceptible population and breeding its inbred strain, and establish animal model of rat osteosarcoma and its lung metastasis.Methods 10 couples 4-week-old SD rats from closed population were prepared and the UMR-106 osteosarcoma cell line was injected subcutaneously into the SD rat thigh lateral at the amount of 1×10~7. The survival couples who were susceptible to the osteosarcoma cell line were selected, and mated six weeks later. After pregnancy and delivery, the offspring emerged, who would also be challenged with UMR-106 cell line 4 weeks later……The whole process were be repeated for 4 times and as a matter of course, the the 4th generation rats from inbred strain was obtained, who were more susceptible to the osteosarcoma cell line. 30 SD rats from inbred strain of 4 weeks old were divided into 3 groups. The UMR-106 cell line was injected subcutaneously into the SD rat thigh lateral at the amount of 1×10~6,5×10~6 and 1×10~7. Neoplastic diameters were measured every week after inoculation to build the tumor growth curve. 6 weeks later, the rat'double lungs and tumors were prepared. Pathological study was made under microscope by HE stains.Results The 4-week-old rats from closed population seemed unsusceptible to the UMR-106 cell line and only 30% of them were infected. After bolting and propagation, the 4th generation rats from inbred strain became so susceptible to the osteosarcoma cell line that in the1×10~7 group all tumors developed and 70% of them metastasized to lungs, while the tumors of 1×10~6 and 5×10~6 groups didn't develop well,nor lung metastasis were observed.Conclusions The SD rats from closed population showed up certain genetic diversity, while their offspring from inbred strain became identical and demonstrated no diversity in oncogenicity. The UMR-106 cell line has strong carcinogenic capability and high lung metastasis frequency. The animal models depending on it offer a method for the animal experiments of comprehensive therapy to osteosarcoma.PartⅡInduction, Proliferation and Identification of Dendritic Cells from Rat Bone Marrow Mononuclear Cells in vitroObjective To establish a method to induce dendritic cells from the bone marrow mononuclear cell of rats in vitro,and to identify the process of phenotypes'variation on DC.Methods BMMCs were isolated by Ficoll-Hypaque density gradient separation and then cultured in RPMI1640-10% FCS medium in polystyrene flask under the condition of rGM-CSF and rIL-4 at 37℃and 5% CO2. On d6 of culture, immature DC were washed and suspended in medium containing nrhTNF-αto generate mature DC. On d9 after purified by monoclonal antibody OX62 and magnetic beads, DC was identified by morphological features under both inversion microscope and electron microscope. While DC'surface antigens expression was analyzed by FACS every three days.Results After culture and induction,DC displayed typical morphology with elongated dendritic processes viewed by inversion microscope as well as electron microscope. DC expressed increasing level function associated surface antigens,including OX62 1.78%,MHCⅡ13.68%,CD80 4.77%,CD86 5.77% on d0; OX62 32.31%,MHCⅡ32.14%,CD80 23.68%,CD86 15.44% on d3; OX62 63.13%,MHCⅡ68.36%,CD80 43.06%,CD86 54.78%; and OX62 72.41%,MHCⅡ84.71%,CD80 79.06%,CD86 74.80%on d9. While the rat osteosarcoma associated surface antigen CD44 was always negatively expressed on DC.Conclusions Mature DC with typical morphology and surface antigens expression could be generated from rat bone marrow mononuclear cells under the condition of rGM-CSF, rIL-4 and nrhTNF-α,and DC's surface antigens expression was increased progressively with culture days added, which presents the feasibility for further clinical application of DC in the immunotherapy of cancer.PartIII Preparation and Identification of Allogeneic DC-osteosarcoma Electrofusion VaccineObjective To establish a method to prepare allogeneic DC-osteosarcoma electrofusion vaccine with the Wistar rats'bone marrow derived dendritic cells and SD rats derived osteosarcoma cell line UMR-106,and to identify the phenotypes'variation on the fusion hybrids.Methods Allogeneic DC and tumor cells were mixed at a 5:1 ratio and suspended in 0.3 M glucose solution containing 0.1mM Ca (CH3COO)2, 0.5mM Mg (CH3COO)2, and 10% bovine serum albumin. After centrifugation, the cells were resuspended in the same fusion medium without bovine serum albumin. Routinely, 0.5ml of cell suspension containing 6×10~6 cells were processed using a specially designed electroporation cuvette. For electrofusion, a pulse generator (model ECM 2001) was used. Electrofusion involves two independent but consecutive steps. The first reaction is to bring cells in close contact by dielectrophoresis, which can be accomplished by exposing cells to an alternating (ac) electric field of relatively low strength. Cell fusion can then be triggered by applying a single square wave pulse of 250V to induce reversible cell membrane breakdown in the zone of membrane contact. The entire process was repeated a second time to maximize fusion efficiency. The fusion mixture was allowed to stand for 5 min before suspending in complete medium and then incubated at 37℃overnight. The electrofusion products were purified by monoclonal antibody OX62 (a DC marker not expressed on tumor cells) sticking to the magnetic beads (Miltenyi Biotec) and then was identified by morphological features under both inversion microscope and electron microscope. The allogeneic DC- osteosarcoma products'surface antigens expression was analyzed by FACS and co-staining against both kinds of markers allows for the detection of double-positive hybrids.Results The allogeneic DC-osteosarcoma vaccine were achieved through electrofusion pulse, and the electrofusion products displayed typical morphology of both component cells viewed by scan electron microscope as well as multi-nucleus ultrastructure viewed by transmission electron microscope. FACS analysis of a purified fusion products by monoclonal antibody OX62 and magnetic beads showed both tumor cell marker and DC marker were highly expressed, for CD44 in the case of osteosarcoma was 50.02%, OX62 in the case of DC was 83.55%, while the percentage of double-positive cell was 49.43%.Conclusions This study demonstrated it was feasible to generate a large number of allogeneic DC-osteosarcoma hybrid cells by the electrofusion technique. The electrofusion products displayed typical morphology and phaenotype of both component cells. The allogeneic tumor vaccine affords a promising new approach for the immunotherapy of osteosarcoma.Part IV In vitro Induction of Anti-tumor CTL Effect against Osteosarcoma Cell Line by Allogeneic DC-Osteosarcoma Fusion VaccineObjective This study was designed to investigate the potentiality to induce T cell proliferation and osteosarcoma-specific cytotoxic T lymphocytes of tumor vaccine produced by electrofusion between rat osteosarcoma cells and allogeneic DC.Methods The allogeneic DC-osteosarcoma fusion cells were purified by monoclonal antibody OX62 and magnetic beads and then irradiated with 30 Gy with radioactive ray of 60Co to ensure inactivation of the tumor cells and DC. Coculture of SD bone marrow derived T lymphocytes with the tumor vaccine was to induce T cell proliferation. Then cytotoxic T lymphocytes assay was assessed according to results of MTT assay. 10 athymic mice were challenged subcutaneously with 1×10~6 UMR-106 cells and then divided into two groups randomly 3 days later. The tumor-bearing athymic mice were immunized with 1×107 activated T lymphocytes for 3 times. The condition of tumor development was observed for 4 weeks since tumor challenge.Results After T cells were cultured with allogeneic DC-osteosarcoma fusion cells, effective activation of T cells was observed. The immunization using allogeneic DC-osteosarcoma vaccine induced UMR106-spcific CTL responses which were statistically significant (P<0.05) compared with corresponding control groups using saline or DC. 28 days later, the experiment of athymic mice showed that the induced CTL inhibited the growth of implanted tumor in athymic mice, and the volum of athymic mice's implanted tumor was (945±125)mm3 in treatment group, and (4867±375)mm3 in control group respectively(p<0.05).Conclusions The present study provided valid evidence of the potentiality of allogeneic DC-osteosarcoma fusion cells to induce effective T cell proliferation and osteosarcoma-specific cytotoxic T lymphocytes. The fusion cells may thus represent a promising strategy for DC-based immunotherapy of osteosarcoma and play certain role in the clinical treatment and prevention.Part V Specific Active Antitumor Effects of Tumor Vaccine Produced by Electrofusion between Osteosarcoma Cell Line and Allogeneic Dendritic Cell in Rats in vivo.Objective This study was designed to investigate the potency and antitumor effects of allogeneic tumor vaccine produced by electrofusion between SD rats derived osteosarcoma cell line UMR-106 and Wistar rats'bone marrow derived DC.Methods In this study of immunization against tumor challenge, groups of 10 rats were immunized intradermally with 1×10~6 electrofused cells after irradiated with 30Gy 60Co-ray on days 0 and 14. One week after the second immunization (day 21), the rats were challenged double-blind with a lethal dose of tumor cells and were then monitored for tumor growth and survival over time. For the tumor challenge, vaccinated SD rats were prepared and the UMR-106 osteosarcoma cell line was injected subcutaneously into the SD rat thigh lateral at the amount of 1×10~7. In contrast to the results obtained with the fusion products, immunization with physiological saline, tumor cells that underwent the electrofusion process alone, DC that underwent electrofusion alone, or a mixture of these two populations was also made to induce antitumor protection. To determine whether the immunization could result in a long-term immunological memory, rats injected with UMR106-allogeneic DC fusion products that survived an UMR-106 tumor challenge were rechallenged with a second lethal dose of tumor cells 10 weeks later and then monitored for tumor growth and survival again for 7 weeks. In the active immunization therapeutic model, tumor cells were injected on day 0 and vaccination with electrofusion products was done on days 3, 7, and 14. Then the animals'survival curve was carried out.Results In the foregoing study of immunization against tumor challenge, 70% of the rats immunized with 1×10~6 electrofused cells were typically able to reject tumor challenge and remained tumor-free. And then all of the survival rats were able to reject this secondary tumor challenge and remain to live on 7 weeks later. In the active treatment study, little or no antitumor protection was observed in the rats treated with1×10~6 electrofused cells, a dose sufficient to achieve substantial tumor protection in pretreatment studies. However, substantial antitumor protection (60% long-term survivors) was obtained with a higher dose of the vaccine (2×10~6 electrofused cells).Conclusions Preimmunization with irradiated electrofusion products were found to provide partial to complete protection from tumor challenge in the UMR-106 tumor model. Vaccinated survivors developed long immunological memory. The therapeutic potential of this type of approach was suggested by the ability of UMR106-DC electrofusion products to induce tumor rejection in a substantial percentage of hosts bearing pre-established tumor cells. These results tended to indicate that treatment with electrofused tumor cells and allogeneic DC might be capable of inducing a potent antitumor response and could conceivably be applied to a wide range of cancer indications for which tumor-associated antigens have not been identified.
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