A Novel Thepapeutic Vaccine Of GM-CSF Surface-modified MB49 Cells Against The Metastatic Bladder Cancer

BackgroundUrinary bladder cancer is one of the most common malignant tumors threatening human health. Based on the estimates of National Cancer Society Institute, in 2008, bladder cancer is the fourth most common type of cancer in men and the ninth most common cancer in women in USA. In China, the incidence of bladder cancer ranks first during the genitourinary cancers, and ranks eighth the total cancer cases. Of all cases,75% were the superfical and 25% were muscle-invasive cancer. Currently, the most effective treatment with muscle-iinvasive bladder cancer is radical cystectomy with postoperative radiotherapy and chemotherapy. But about 50% of patients would suffer from recurrence 2 years after radical resection, and eventually die. It is mainly because micro metastases appeared in more than half of the patients with muscle-invasive bladder cancers, the efficacy of treatment could be poor. Therefore, new methods which can effectively control and even kill the micro-metastatics will greatly improve the treatment with invasive bladder cancer.Today, immunotherapy has become the most common effective treatment with bladder tumor after surgery. Some scholars used inactivated tumor cells as tumor vaccines to enhance the ability against tumors, but because of weak immunogenicity of tumor cells, the simple use of inactivated tumor cells is difficult to significantly improve the body's immune system against tumor cells. Then various cytokines (GM-CSF, IL-2, TNF-α) were found to enhance the body's antigen-presenting ability and increase activation and proliferation of T cell, which was confirmed by animal experiments. However, due to rapid metabolism of cytokines in vivo, while larger doses lead to a series of serious side effects, its effect were greatly limited and failed to achieve satisfactory treatment.Thus, some reseachers began to study how to combine cytokines and inactivated tumor firmly to get both anti-tumor effect of the two. The most common methods are the gene transfection (or transduction) method. However, the gene transfection (or transduction) have inherent disadvantages:the efficiency is so low that the effective concentration of the protein translated by therapeutic genes in vivo is difficult to reach and maintain long time,and also brings a potential safety problem of viral vector. So based on both the unique property of streptavidin (SA) to bind rapidly and almost irreversibly to any biotin-linked molecule and the outstanding ability of biotin to be incorporated easily into the proteins on the cell surface, we developed a novel protein-anchor platform to immobilize SA-tagged bioactive molecule on the surface of biotinylated MB49 bladder cancer cells so as to effectively induce antitumor immunity.There are no relevant reports about methods that Streptavidin (SA) labeled GM-CSF anchore MB49 bladder tumor cells in the treatment of bladder cancer metastasis currently. In this study, we first produce the SA-GM-CSF anchored membrane bladder tumor cell vaccine, and establish dual metastasis models in mice with MB49 bladder tumor cells, then through a variety of detective methods, confirme that SA-GM-CSF anchored bladder tumor cell vaccine inhibit the metastasis of bladder cancer occurrence and development, which provides reliable experimental basis and foundation for the treatments in future of human invasive bladder cancers.PurposeTo investigate SA-GM-CSF in the treatment of metastatic bladder cancer and its mechanism. To study the immune protection about treatment of SA-GM-CSF strategy.MethodsOur study was divided into two parts Vaccine preparationMB49 bladder cells were fixed in 30% ethanol (v/v) for 30min at room temperature and tested for viability by trypan blue (0.4%) staining. Ethanol-fixed MB49 cells (2×107 cells/ml) were incubated with lOmM fresh EZ-Link Sulfo-NHS-LC-Biotin (Pierce) for lh at room temperature, and washed three times with PBS including 100mM glycine. The biotinylated cells were incubated with SA-mGM-CSF at 100ng/106 cells for 1h, and washed three times with PBS. The presence of SA-mGM-CSF on cell surface was assayed by flow cytometry using FITC-conjugated anti-mGM-CSF monoclonal antibody.Experiments with MB49 cell vaccines in mouse models of MB49 bladder cancer1. Metastatic mouse models MB49 bladder cancerMouse subcutaneous model of MB49 bladder cancer:8 mice (6-8w) were prepared for the construction of mouse subcutaneous model of MB49 bladder cancer, which were injected with 1×106MB49 cells into the hind leg. Since the 5th day, we measured the tumor size with calipers every five days until the 30th day.Mouse lung model of MB49 bladder cancer:Mice were randomly divided into five groups and every mouse was inoculated intravenously in the tail vein with a total of 1×105,2×105,3×105,4×105,5×105MB49 cells respectively. After two weeks, pulmonary cancer tissues were collected from each experiment group, which were prepared for pathological examination.2. Protective immunotherapy with SA-GM-CSF surface-modified MB49 cells vaccineMice were randomly divided into five groups:(a)SA-GM-CSF modified group---experiment group; (b)soluble SA-GM-CSF group; (c) SA-GFP modified group; (d) Ethanol-fixed group; (e) PBS group and were given SA-GM-CSF surface-modified MB49 cells vaccine(1×106), soluble SA-GM-CSF, SA-GFP surface-modified MB49 cells vaccine (1×106) and PBS respectively, once every 4 days for four times. After the protective vaccination was performed, we challenge the immunized mice with a total of 1×106 MB49 bladder cancer cells injected subcutaneously in the hind leg or a total of 2×105 MB49 bladder cancer cells inoculated intravenously in the tail vein on day 23, then measured the tumor size every five days or recorded the survival time.3. Therapeutic efficacy of SA-GM-CSF surface-modified MB49 cells vaccines in the subcutaneous and lung metastatic mouse model of bladder cancerFemale C57BL/6 mice (6-8w) were injected with 1×106MB49 cells into the hind leg with to get subcutaneous model or 2×105 MB49 cells intravenously in the tail vein to obtain lung metastatic model. All mice were divided into five groups and each group had 12 mice. The treated group was SA-GM-CSF-conjugated ethanol-fixed MB49 cell vaccine.,and the other four groups are soluble SA-GM-CSF, SA-GFP-conjugated ethanol-fixed MB49 cells, ethanol-fixed MB49 cells and PBS. The mice were injected subcutaneous with 1×10 cell vaccines on the same day (day 0), and the procedure was repeated on days 4,8 and 12. On day 19 after tumor injection, 100ul blood was collected from each experimental group for measuring the proportion of CD4+ and CD8+T cells; splenocytes were pooled from each group for CTL; subcutaneous and pulmonary cancer tissues were collected from each group for histological and immunohistochemical analyses. In the course of the experiment, we measured the volumes of subcutaneous tumors and recorded the survival time.4. Tumor-specific immune response:In vivo:in order to further confirm the antitumor immunity, on one hand, we performed the CTL assays with MB49 bladder cancer cells. On the other hand, the mice in subcutaneous or lung metastatic experiment group survived were injected with 1×106 MB49 cells or RM-1 cells in the left or right hind leg respectively. Then we measured the volume of subcutaneous tumors every five days until the 30th day.In vitro:to investigate the effect of SA-GM-CSF-anchored MB49 cell vaccines to CTL function, the splenocytes were collected from each group and to prepare effector cells by stimulated with inactivated MB49 bladder cells in the presence of hIL-2 for 5 days. Then a varying number of effector cells was added to the target cells at the effector as target radios of 12.5:1,25:1,50:1 and 100:1, respectively. The percentage of cytotoxicity of each group was detemined at the LDH of each well.ResultFlow cytometric analysis showed that SA-mGM-CSF could be efficiently anchored on the surface of MB49 cells. About 94.8% cells were anchored with SA-mGM-CSF.According the result of bioassay experiments, SA-GM-CSF anchored on the biotinylated surface of MB49 cells still retained its GM-CSF bioactivity.Metastatic mouse models MB49 bladder cancer:Pathological examination histological and immunohistochemical analyses confirmed the success of the construction of metastatic mouse models MB49 bladder cancer.The results of experiments with MB49 cell vaccines in mouse models of MB49 bladder cancer show that:SA-GM-CSF surface-modified MB49 cells vaccine has the strongest immunogenicity(P<0.05). Compared with the control group, the mice of experiment group have slow tumor growth, small tumor volume, better mental state and longer survival time. The results of flow cytometric analysis and histological and immunohistochemical analyses show that and more CD4+ and CD8+T cells appeared in mice of experiment group(P<0.05). The results of tumor-specific lymphocyte cytotoxicity assays show that there were significant differences between SA-GM-CSF-anchored group and other groups (P<0.05) and the mice of SA-GM-CSF-vaccine-treated group were mostly tumor-free at MB49 injecting side (left leg) and obviously tumor at RM-1 injecting side (right leg), which were significant different from control group.ConclusionWe successfully prepared SA-GM-CSF surface-modified MB49 cells vaccine, and flow cytometric analysis showed that SA-mGM-CSF has a high anchor efficiency, and SA-GM-CSF anchored on the biotinylated surface of MB49 cells still retained its GM-CSF bioactivity. The results of experiments with MB49 cell vaccines in mouse models of MB49 bladder cancer show that the mGM-CSF surface-modified MB49 bladder cancer cell vaccine induced specific anti-tumor immunity and was efficient for metastatic bladder cancer. Taken together, we developed a novel way of generating the whole cell tumor vaccine for immunotherapy of muscle invasive bladder cancer, and it may provide some experimental basis for the application of SA-mGM-CSF in human muscle invasive bladder cancer treatment.