OBJECTIVES: Many strategies are pursued to enhance tumor vaccine immune response, including the utilization of cytokines. We have developed a novel protein-anchor technology to immobilize cytokines on tumor cell surface. Here we reported the preparation of tumor cell vaccines by immobilizing GM-CSF or IL-2 on MB49 bladder cancer cells and evaluated their antitumor efficacy (administrated alone or sequentially) in a metastatic mouse model. MATERIALS AND METHODS: SA-mGM-CSF or SA-hIL-2 surface-modified MB49 cells were prepared as vaccine. Mice were treated with MB49 cell vaccines (administrated alone or sequentially). Survival time, tumor growth, flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and cytotoxic T lymphocytes (CTL) assay were used to evaluate the antitumor efficiency of the vaccines in the pulmonary metastatic model of bladder cancer. RESULTS: GM-CSF vaccine induced more mature dendritic cells in the mice spleen. Combination with subsequent IL-2 vaccine significantly increased CD4(+), CD8(+), and IFN-γ(+)CD8(+) T but not CD4(+)Foxp3(+) T cell population and induced the highest production of IFN-γ, IL-12, but not IL-10. Furthermore, the splenocytes from the sequentially combined vaccines group showed the most potent cytotoxicity on MB49 cells. Finally, the sequentially combined vaccines evidently extended the survival time of mice (the median survival time of PBS, ethanol-fixed, anchored GM-CSF, anchored IL-2, and anchored GM-CSF + anchored IL-2 groups were 34, 37, 45, 47, and 59 days, respectively) and effectively protected the mice against a second MB49 cells but not RM-1 cells challenge. CONCLUSIONS: This study demonstrated that sequential administration of GM-CSF and IL-2 surface-modified MB49 cells vaccines could effectively induce specific antitumor immune response.