Fig. 6: Treatment with an ALKi and anti-PD-1 antibody results in protection from a secondary tumor challenge in an HCC mouse model.

a Scheme of the experimental procedure for the HCA-1/R1 tumor regression study. C3H mice were challenged with stable HCA-1/R1 cells and given crizotinib and an anti-PD-1 antibody as indicated in Fig. 5a. Mice with complete tumor regression were rechallenged 45 days after tumor-cell injection (n = 12). Similarly aged C3H mice (n = 12) were injected with HCA-1/R1 cells and used as tumor-bearing controls. TdLNs and splenocytes were harvested from mice for flow cytometric analysis 7 days after tumor rechallenge (n = 6 per group). Figure was created with Adobe Illustrator. b The sizes of the tumors over time in the rechallenge and tumor-bearing control groups (n = 6 per group). c The overall survival rates for the rechallenged and tumor-bearing control mice (n = 6 per group). d, e The numbers of CD8⁺IFN-γ⁺ T cells and Th1 cells in the TdLNs (d) and spleens (e) of the indicated groups of mice (n = 6 per group). The error bars represent mean ± SD values. f, g The percentages (f) and absolute numbers (g) of CD8⁺ and CD4⁺ T_CM, T_EM, and naïve T-cell subsets in TdLNs. h, i The percentages (h) and absolute numbers (i) of CD69⁺CD103⁺ and CD69⁺CD103⁻ subsets of CD8⁺ Trm cells in TdLNs. j, k Percentages (j) and absolute numbers (k) of Klrg1⁺CD127⁻CD8⁺ Teff cells and Klrg1⁻CD127⁺CD8⁺ Tmem cells in TdLNs. For g, i and k: n = 6 for non-tumor and tumor-bearing control groups; n = 5 for rechallenge group. The error bars represent (mean ± SEM) values. a–c Representative results from 2 independent experiments. The results were analyzed using two-way analysis of variance (ANOVA; b), a log-rank test (c), a two-sided unpaired Student t-test (d, e), or one-way ANOVA (g, i, and k).