Fig. 1: ZD55-IL-24 inhibits tumor growth in B16-bearing immunocompetent mouse model not through the classic direct killing pathway, but through unknown indirect pathway.

A–D The antitumor efficacy of ZD55-IL-24 in B16-bearing immune-competent mouse model. A Timeline of tumor engraftment and treatments. B Tumor growth curves and C survival over time for mice inoculated with 10⁶ B16 melanoma cells s.c. in the right flank and treated 7 days later (the average tumor volume was about 80 mm³) with the indicated PBS or ZD55-IL-24. D Body weight changes of the B16 melanoma-bearing mice monitored during the therapy period. s.c. subcutaneous injection, i.t. intratumoral injection. Data are presented as the mean ± SEM. n = 10 mice per group per experiment. E–H The B16 cell killing effect of ZD55-IL-24 in vitro. E B16 cells were infected with ZD55-IL-24 at a series of MOI (PFU/cell) from 0 to 150, the appearance of cytopathic effect was monitored under microscope, and representative photographs were taken at day 2 post-infection F and cell viability was measured by CCK-8 assay. G The B16 cells at a density of 10⁴ cells/well cultured in 96-well plates were infected with 1500 MOI (PFU/cell) ZD55-IL-24, the appearance of cytopathic effect was monitored under microscope, and representative photographs were taken at 4 days later, and H cell viability was examined by CCK-8 assay. Results represent mean ± SEM of triplicate experiments and are expressed as a percentage of control cells. I–O The B16 cell killing effect of ZD55-IL-24 in vivo. I Tumors resected from B16-bearing C57BL/6 mice receiving PBS or ZD55-IL-24 treatment as indicated in A were analyzed 2 days after the last injection by immunohistochemical staining. Shown are representative micrographs of tumor sections immunostained for the proliferation marker Ki-67 (green), and J quantification of the number of Ki-67⁺ cells in I (Ki-67⁺ nuclei as % of total nuclei; n = 3). K Shown are representative images of tumor sections immunostained for the apoptosis marker TUNEL (red), and L quantification of the TUNEL⁺ cells in K (TUNEL⁺ cells as % of total cells; n = 9). Nuclei is counterstained with DAPI (blue). Data are mean ± SD. Scale bars, 300 µm. Shown is one of three independent experiments. M C57BL/6 mice with B16 tumors were treated as indicated in Fig. 4A, and then the tumor cells were isolated for flow cytometry analysis. Shown are representative flow cytometry plots for Ki-67, and N percentages or O average median fluorescent intensities (MFI) of Ki-67⁺ cells, mean ± SEM is shown. Data represent cumulative results from eight independent experiments.