Fig. 5 |. CBD-IL-12 decreases metastatic tumour burden by triggering activation of innate and adaptive compartments of the immune system in the pulmonary metastatic model of B16F10 melanoma.

a, 5 × 10⁵ B16F10 cells were injected i.v. on day 0. Mice were treated with either 25 μg IL-12 (n = 3) or with equimolar CBD-IL-12 (n = 4) i.v. once on day 8 and sacrificed on day 17. Metastatic burden was quantified using ImageJ software and normalized by total area of the lung. b-i, 2.5 × 10⁵ B16F10 cells were injected i.v. on day 0. Mice were treated with either PBS (n = 6), 25 μg IL-12 (n = 12) or with equimolar CBD-IL-12 (n = 12) i.v. once on day 9 and lungs were collected on day 18. 2 × 10⁶ live cells/well were plated for flow cytometric analysis. b, Percentages of CD3⁺CD8⁺ T cells within live cells. c, Frequency of CD3⁺CD4⁺CD25⁺Foxp3⁺ Tregs within lung-infiltrating immune cells (% of CD45⁺). d, Frequency of CD3⁺CD8⁺CD44⁺CD62L⁻ effector CD8⁺ T cells within total CD8⁺ T cells. e, Ratio of effector CD8⁺ T cells to Tregs. f-h, Percentages of DCs (CD11c⁺MHCII⁺F4/80⁻) (f), CD103⁺ DCs (g), and CD11b⁺ DCs (h) within live cells. i, Frequency of MHCII⁺CD80⁺ macrophages within total macrophages (defined as CD11b⁺F4/80⁺). Lines represent mean ± SEM. Antitumor efficacy experiment (a) was performed twice, with similar results. Flow analysis was performed once on independent biological samples. Statistical analyses were done using unpaired, two-tailed t-test with Welch’s correction (a), ordinary one-way ANOVA with Tukey’s test for parametric data (b,c,d,f,g,h,i) and Kruskal-Wallis test followed by Dunn’s multiple comparison for nonparametric data (e).