Enhanced tumor-specific immunogenicity and delayed tumor progression by TNP-470–treated DC vaccine in therapeutic B16 melanoma vaccination model. (A) Schematic diagram of therapeutic setting of vaccination model. Solid tumor was developed on mice by subcutaneous injection of B16 melanoma. When size of solid tumor reached around 150 mm³ (day 7), mice were immunized with PBS (solvent control), DC without tumor lysate pulsing (DC control), tumor lysate-pulsed TNP-470/vehicle-treated DC vaccine on day 7 and day 11, respectively. (B) Lymphocytes from immunized mice in different groups were collected and stimulated with tumor lysate (50 µg/mL) for 24, 48, and 72 hours ex vivo. Culture supernatant was detected for secretion of IL-2 (top) and IFN-γ (bottom). **P < .01 and *** P < .001 TNP-DC vaccine vs PBS solvent control; ^##P < .01 TNP-DC vaccine vs vehicle DC vaccine. (C) To quantify amount of tumor-specific CTLs from mice in different groups, lymphocytes were stimulated with tumor lysate (50 µg/mL) for 72 hours ex vivo, followed by re-stimulation of PMA/Ionomycin for 6 hours. Cells were then stained with CD3, CD8 and IFN-γ. Tumor-specific CTLs were determined by flow cytometric analysis of CD3⁺CD8⁺IFN-γ⁺ cells. ***P < .05 TNP-/vehicle DC vaccine vs PBS solvent control; ^##P < .01 TNP-DC vaccine vs DC control/ vehicle DC vaccine. Data presented as mean ± SD (n = 5). (D) Tumor-specific cytolytic activity. Lymphocytes from mice in different groups were collected and stimulated with tumor lysate (50µg/mL) for 72 hours ex vivo, then harvested as effector cells (E), and B16-F10 cells were used as target cells (T). Specific lysis was detected by LDH assay at E:T ratio = 100:1. **P < .01 vehicle DC vaccine vs PBS solvent control; ***P < .001 TNP-DC vaccine vs PBS solvent control. Data presented as mean ± SD (n = 5). (E) Solid tumor development in therapeutic setting of B16 melanoma vaccination model. Mice with solid tumor (around 150 mm³ on day 7) were vaccinated with vehicle PBS, DC without tumor lysate pulsing, tumor lysate-pulsed vehicle DC vaccine, TNP-470–treated DC vaccine. Tumor volume of solid B16 melanoma developed on mice from day 7 to day 15. Data are presented as mean ±SD (n = 5) *P < .05, TNP-DC vaccine vs vehicle DC vaccine; ^#P < .05, ^##P < .01 TNP-DC vaccine vs PBS solvent control.