Fig. 7

The antitumor activity of the trametinib and anti-PD-1 combined treatment depends on trametinib-mediated Id1 inhibition. A Western blot analysis of Id1 protein in parental and LLC Id1-flag LLC cells. β-Actin was used as control. B Left panel: Subcutaneous growth of LLC tumors in mice treated with trametinib and anti-PD-1 combination (TRAM + anti-PD-1; TRAM; 5 days per week; anti-PD-1; twice weekly) (n = 6 mice per group) or vehicle (control) (n = 5 mice per group). The follow-up of tumor size and the tumor volumes at day 14 are shown. Right panel: Subcutaneous growth of LLC Id1-flag tumors in mice treated with trametinib and anti-PD-1 combination (TRAM + anti-PD-1; TRAM; 5 days per week; anti-PD-1; twice weekly) (n = 6 mice per group) or vehicle (control) (n = 6 mice per group). The follow-up of tumor size and the tumor volumes at day 17 are shown. C Immunohistochemical quantification and representative images of Id1 immunostaining in the tumors from the experiment shown in B. Scale bar: 200 μm. D Flow cytometric quantification of total tumor-infiltrating CD8⁺ T cells (CD45⁺, CD3⁺, CD8⁺, CD44⁺), Treg cells (CD45⁺, CD3⁺, CD4⁺, CD25⁺, Foxp3⁺), total MDSCs (CD45⁺, CD11b⁺, Ly6C⁺), PMN-MDSCs (CD45⁺, CD11b⁺, Ly6C⁺, Ly6G^High). CD8 T cells/Treg ratio is also shown. Data are expressed as the percentage of total leukocytes (CD45⁺), except for Treg cells, which are expressed as the percentage of total CD4⁺ T cells. Data are expressed as means ± SD. Comparisons between experimental groups were performed by two-sided t-test (parametric) or two-sided Mann–Whitney U-test (non-parametric)