Fig. 8: ZD55-IL-24 eradicates established melanoma in A375-bearing immunocompromised mouse model mainly through the classic direct killing pathway, but not through the antitumor immunity pathway and anti-angiogenesis pathway.

A, B The antitumor efficacy of ZD55-IL-24 in A375-bearing immunocompromised mouse model. A Tumor growth curves over time for BALB/c nude mice inoculated with 2 × 10⁶ A375 cells s.c. in the right flank and treated with PBS or ZD55-IL-24 as indicated in Fig. 1A. B Body weight changes of the treated mice monitored during the therapy period. Data are presented as the mean ± SEM. n = 8 mice per group per experiment. C, D Fluorescence microscopic analysis of viral infection and exogenous gene expression in A375 cells. C The human melanoma A375 cells were infected with ZD55-EGFP at a MOI (PFU/cell) of 0 and 1000, and the viral infection and exogenous gene expression were monitored under the fluorescence microscope on Day 0, Day 1, Day 2, and Day 4 after infection. D quantification of the EGFP-positive A375 cells in C (n = 9). Error bars indicate mean ± SD. Shown is one of three independent experiments. Scale bars, 300 µm. E Representative transmission electron microscopy images of A375 cells treated with ZD55-IL-24 at a MOI (PFU/cell) of 0 and 250. Shown is one of three independent experiments. Nuclei and viral particles are indicated by the black and red arrow, respectively. Scale bar: 4 μm. Inset: high-power view, Scale bar: 100 nm. F Western blot analysis of viral infection and exogenous IL-24 expression in A375 cells infected with ZD55-IL-24 at a series of MOI (PFU/cell) as indicated. Shown is one of three independent experiments. G, H The cytotoxicity of ZD55-IL-24 in A375 cells in vitro. G The A375 cells at a density of 10⁴ cells/well cultured in 96-well plates 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 phase-contrast images were taken at 2 days later, and H cell viability was examined by CCK-8 assay. Scale bars, 300 µm. Results represent mean ± SEM of triplicate experiments and are expressed as a percentage of control cells. I–P Flow-cytometric analysis of immune cells infiltration in tumors, and recruitment and activation in spleens. Tumors and spleens resected from A375-bearing BALB/c nude mice receiving PBS or ZD55-IL-24 treatment indicated in Fig. 4A were analyzed by flow cytometry. I Shown are representative flow cytometry plots of tumor-infiltrating total myeloid cells and neutrophils in right tumors. J Representative flow cytometry plots of tumor-infiltrating NK cells and NKT cells in right tumors. K Percentages of innate immune cells in right tumors. L Percentages of adaptive immune cells in right tumors. M Percentages of innate immune cells in spleens. N Percentages of adaptive immune cells in spleens. O Percentages of innate immune cells in left tumors. P Percentages of adaptive immune cells in left tumors, mean ± SEM is shown. Data represent cumulative results from seven to eleven (I–L), eight to nine (M, N) or nine (O, P) independent experiments. Q, R The anti-angiogenic effect of ZD55-IL-24 in A375-bearing immunodeficient mouse model. Tumors resected from A375-bearing BALB/c nude mice receiving PBS or ZD55-IL-24 treatment indicated in Fig. 4A were analyzed by immunohistochemical staining. Q Representative images of tumor sections immunostained for the endothelial marker CD31 (green) to label the blood vessels in tumors. R Quantification of the CD31⁺ cells in Q (n = 10). Nuclei is counterstained with DAPI (blue). P PBS, Z ZD55-IL-24. Scale bars, 300 µm. Data are mean ± SD. Shown is one of two independent experiments.