Sequential Administration Of GM-CSF And IL-2Surface-Modified MB49Cells Vaccines Against The Metastatic Bladder Cancer

BackgroundBladder cancer is one of the most common urologicmalignancy. Although radical cystectomy with pelvic lymphadenectomy and adjuvant chemotherapy were performed to treat this kind of patients, the recurrence rate was up to50%for the micro-metastases at the time of surgery. Therefore, some novel therapies are essential to address and prevent the recurrence of bladder cancer.At present, immunotherapy is considered to be an efficacious method for carcinoma treatment. In order to enhance the antitumor immunity, cytokines are used to improve tumor antigen presentation or enhance T cell response to cancer cells. But the traditional methods are difficult to induce the desired antitumor immunity for the high metabolic rate of the cytokine, low gene transfer efficiency or transgene expression level and bio-safety concerns. We have developed a novel technology which immobilized the streptavidin (SA)-tagged bioactive cytokines on the biotinylated tumor cell surface to prepare tumor cell vaccines. This kind of novel therapeutic vaccines not only retained the biological activity of cytokines but also maintained high local cytokine concentration, reduced metabolic rate and side effects.IL-2is widely recognized as T cell growth factor, it plays an important role in immunoregulation. However, recent studies have confirmed that the biological effect of IL-2had both positive and negative effect in the generation of T-cell responses, which depended on the stage of T cell differentiation. In the initial stage of immune response, IL-2therapy is detrimental to the survival of rapidly dividing effector T cells and may has a beneficial effect on the proliferation and function of Treg cells. While IL-2treatment increases the proliferation and survival of antigen-specific T cells after the initial stage, especially in the case of the presence of a certain number of mature dendritic cells (DCs). GM-CSF is a cytokine that promotes the growth and maturation of DCs and initiates the immune response. Therefore, theoretically, a sequential combination of GM-CSF and IL-2will be more potential for immune response. In this paper, on the basis of our protein-anchor technology, sequential administration of GM-CSF and IL-2surface-modified MB49cells vaccines were evaluate for their effects in the treatment of MB49metastatic mouse model. Our study was divided into two parts:Chapter1The preparation and biological activity of GM-CSF or IL-2-modified MB49cells vaccine1. ObjectiveTo prepare GM-CSF or IL-2-modified MB49cells vaccine and study their biological activity2. Methods(1) Vaccines preparationOur previous study has shown that30%ethanol (v/v) could completely inactivate tumor cells and keep the cell shape and size intact. Ethanol-fixed MB49cells (2×107cells/ml) were incubated with lOmM fresh EZ-Link Sulfo-NHS-LC-Biotin (Pierce) for1h at room temperature, and washed three times with PBS including100mM glycine. The biotinylated cells were incubated with SA-mGM-CSF or SA-hIL-2at100ng/106cells for1h, and washed three times with PBS. The presence of SA-mGM-CSF or SA-hIL-2on the surface of MB49cells was assayed by flow cytometry.(2) The biological activity of GM-CSF or IL-2-modified MB49cells vaccine2×105GM-CSF-modified MB49cells were pelleted and resuspended in1ml of PBS. Cells were lysed by three cycles of freezing and thawing. Membrane fractions were harvested by centrifugation at15,000×g at4℃for5min and resuspended in1ml of complete medium.SA-mGM-CSF bioactivity was assessed by bone marrow cells (BMCs) proliferation with SA-GFP as the negative control. Aliquots of cell membrane fractions were added to1×104bone marrow cells in each well, and then incubated for3days at37℃in5%CO2humidified incubator. A total of4h before the end of the assay,20ul of5mg/ml3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was added to each well. The reaction was stopped by adding200ul dimethyl sulfoxide to each well and the optical density (OD) at570nm was read on a microplate reader.Human peripheral blood lymphocytes (hPBLs) were used to assess the activity of SA-hIL-2immobilized on the surface of ethanol-fixed MB49cells according to the method mentioned above.3.Results(1) Flow cytometry results showed that SA-mGM-CSF or SA-hIL-2could be efficiently anchored on the surface of ethanol-fixed MB49cells. The modification efficiency were98.41%and97.95%, respectively.(2) SA-mGM-CSF or SA-hIL-2fusion protein anchored on the biotinvlated surface of MB49cells still retained their bioactivity in a dosage-dependent manner. 4. ConclusionsWe have the vaccines of GM-CSF or IL-2surface-modified MB49cell, SA-mGM-CSF or SA-hIL-2could be efficiently anchored on the surface of ethanol-fixed MB49cells and still retained their bioactivity.Chapter2Experiments with MB49cell vaccines(administrated alone or sequentially) in mouse models of MB49bladder cancer1.ObjectiveTo evaluate the GM-CSF or IL-2surface-modified MB49cell vaccines (administrated alone or sequentially) in the treatment of metastatic bladder cancer and its mechanism.2. Methods(1) Metastatic mouse model of MB49bladder cancer and the therapeutic or protective immunotherapy with MB49cells vaccinesA total of2×105MB49cells suspended in100ul of PBS were injected intravenously into the tail vein of C57BL/6/female mice (6-8w) to established lung metastatic model. Mice were then randomly divided into five groups e.g. PBS, Ethanol-fixed, Achored GM-CSF, Anchored IL-2and Anchored GM-CSF+Anchored IL-2(sequentially combined vaccines) group. For the sequentially combined vaccines group, the mice were injected subcutaneously with1×106GM-CSF-modified MB49cells vaccine on day0, day4and day8, and then injected subcutaneously with IL-2-modified MB49cells vaccine on day12, day16and day20. For the other four control groups, the mice were injected subcutaneously with PBS, Ethanol-fixed MB49cells, only GM-CSF surface-modified MB49cells vaccine or only IL-2surface-modified MB49cells vaccine, respectively.In addition, the protective vaccination was performed to immunize the mice according to the method mentioned above, and then challenged the immunized mice with MB49bladder cancer cells on day27(one week after vaccination).(2) Memory immune experimentOn day60after tumor challenge, the survived mice in the sequentially combined vaccines group were injected intravenously with2×105MB49cells again with the untreated mice as negative control, and the survival time were recorded.(3) Tumor-specific immune response:In vitro:On day27after tumor challenge, splenocytes were isolated from the survived mice of each group and stimulated by inactivated MB49cells puls20U/ml of hIL-2(R&D systems) for5days. These splenocytes served as effector cells and were adjusted at1×105cells/ml in the RPMI-1640medium. MB49cells served as target cells and were adjusted to the density of1×104/mL. Both were added to a96-well plate at the desired E:T ratios. After4h of incubation, the supernatant was collected and the LDH activity was measured by CytoTox96non-radioactive cytotoxicity assay to assess the cytotoxicity of tumor-specific cytotoxic T lymphocytes (CTL). In vivo:On day60after tumor challenge, the survived mice in the sequentially combined vaccines group were injected subcutaneously with1×106MB49cells (in the left hind leg) and RM-1cells (in the right hind leg) on day60after therapeutic vaccination.(4) Investigate the related immunological mechanisms1) Flow cytometry analysis of dendritic cells in the spleen:On day12after tumor injection, splenocytes were isolated from each experimental group, and the splenocytes were washed with PBS+1%BSA, and then stained with PE-labeled anti-mCD11c and FITC-labeled anti-mCD80antibodies for30min at room temperature. The double positive cells considered to be mature dendritic cells were measured by flow cytometer.2) Flow cytometry analysis of T-cell subsets in the blood after therapeutic immunotherapy with MB49cells vaccinesTo determine the effect of the therapeutic vaccines on different T-cell subsets in mice,100ul blood was collected from each experimental group at the indicated time points. The total number of lymphocytes was calculated; To enumerate the CD4+and CD8+T cells populations in the blood of each experimental group on day12and day27, cells were stained with PE-labeled anti-mCD4and FITC-labeled anti-mCD8; To enumerate the IFN-y+CD8+and CD4+Foxp3+T cells populations in the blood of each experimental group on day27, cells were first stained with FITC-labeled anti-mCD8 or FITC-labeled anti-mCD4(Biolegend), then fixed and permeabilized before they were stained with PE-labeled anti-IFN-γ or PE-labeled anti-FoxP3antibody. T-cell subsets were measured by flow cytometer.3) Histological and immunohistochemical analysesOn day27after tumor injection, tumor tissues were collected from each experiment group and embedded by freeze embedding medium. The frozen sections (6-7μm) were fixed in cold acetone for15min and incubated with anti-mCD4or anti-mCD8for1h at37℃. Then the anti-mCD4or anti-mCD8antibody was displayed through the anti-rat Ig SABC kit and counterstained with haematoxylin and eosin.4) Determination of IFN-γ, IL-4, IL-10or IL-12concentrations by ELISAOn day27after tumor injection,100ul blood was collected from each experiment group and congealed at room temperature for20min, then the supernatant was harvested by centrifugation at3000r.p.m for5min. The concentrations of IFN-γ, IL-4, IL-10or IL-12were measured by ELISA.3. Results(1) Therapeutic and protective efficacy of GM-CSF and IL-2surface-modified MB49cells vaccines in the metastatic mouse model of MB49bladder cancerAfter being treated with sequentially combined vaccines, GM-CSF vaccine induced more mature dendritic cells in the mice spleen. Combination with subsequent IL-2vaccine significantly increased CD4+, CD8+and IFN-y+CD8+T but not CD4+Foxp3+T cells population and induced the highest production of IFN-y, IL-12but not IL-10. Significant differences in survival time were observed between the sequentially combined vaccines group and the other four groups (P<0.05). On day60after therapeutic vaccination, the survived mice in the sequentially combined vaccines group were injected intravenously with2×105MB49cells again with the untreated mice as negative control. In addition, the study on the protective vaccination also showed that the mice of the sequentially combined vaccines group have the longest survival time (P<0.05).(2) Memory immune experiment:The results showed that significant survival differences existed between the two groups, which indicates that the mice treated with the sequentially combined vaccines produced a long lasting protection (P<0.05).(3) Specific immune responseThe results of CTL showed that there were significant differences between the sequentially combined vaccines group and other groups at the tumor-specific cytotoxic activities. In order to fully prove that the sequentially combined vaccines could induce tumor-specific immune response in vivo, the survived mice in the sequentially combined vaccines group were injected subcutaneously with1×106MB49cells (in the left hind leg) and RM-1cells (in the right hind leg) on day60after therapeutic vaccination, the tumor volume was recorded. The results showed that the sequentially combined vaccines could effectively protect the mice against a second MB49cells but not RM-1cells challenge, which demonstrated that the sequentially combined vaccines could effectively induce tumor-specific T cell immunity.4.ConclusionsWe have successfully generated a kind of novel therapeutic vaccine, this study further demonstrated that the sequentially combined vaccines could induce better antitumor immunity in the metastatic mouse model of MB49cells bladder cancer than a vaccine alone. These findings may provide some experimental basis for the application of this kind of vaccines in human bladder cancer treatment.