5-fluorouracil (FU)-treated CT-26 cells induced increased proliferation and interferon (IFN)-γ production by spleen cells. (A) Proliferation curve of FU-treated and non-FU-treated CT-26 cells after treatment with mitomycin C (MMC). No proliferation was seen with either of the tumor cells, which confirmed the efficacy of MMC. (B) Spleen cells from tumor-bearing mice had significantly higher proliferation after co-culture with FU-CT-26 cells compared with non-FU-CT-26 cells, at a responder:stimulator (R:S) ratio of 10:1. (*P < 0.05, t-test) (C) IFN-γ production by spleen cells after co-culture with 5FU-treated CT-26 cells compared with CT-26 cells. (*P < 0.05, t-test). Error bars represent the standard deviation. CON: control spleen cells cultured without tumor cells. Mixed lymphocyte tumor cell culture (MLTC) was performed to investigate the proliferation of and IFN-γ production by spleen cells. Spleen cells were harvested from tumor-bearing Balb/C mice. The same numbers of FU-CT-26 and non-FU-CT-26 cells were seeded into 96-microwell plates. Spleen cells were added at an R:S ratio of 10:1 on day 1, and proliferation of spleen cells was analyzed by MTT assay on day 4. As for IFN-γ production, the same numbers of FU-CT-26 and non-FU-CT-26 cells were seeded into 24-well plates. Spleen cells were added at an R:S ratio of 10:1. The supernatant was collected on day 4, and IFN-γ concentration was analyzed by ELISA.