Extended Data Fig. 6: Effects of HO-1 antagonist (ZnPPIX) on immune infiltrate and tumor growth.

a, Mean ±- SEM tumor volume of B16 implants in WT mice treated with vehicle or ZnPPIX; n = 5 mice per group. b, FACS quantification of TNF and CD206 expression in F4/80^hi and F4/80^lo TAMs subsets; n = 6 mice per group. c, Quantification of IFNγ expression in B16 tumor-infiltrating CD8⁺ T lymphocytes and CD8 : T_reg ratio in vehicle- and ZnPPIX-treated mice; n = 5 mice per group. d, TAMs purified from MN/MCA1 tumors growing in WT mice treated or not with ZnPPIX were analyzed by RT-PCR for the expression of representative M1 and M2 cytokine genes; shown are pooled data from n = 2 technical replicates from n = 4 mice. e, FACS quantification of HO-1 expression in F4/80^hi and F4/80^lo TAMs subsets and in MN/MCA1 (left, n = 5 mice) or B16 tumor cells (right, n = 3 mice) in WT mice. f, (left) Mean ± SEM tumor volume of subcutaneous B16 implants in n = 5 Hmox1^fl/fl and n = 5 Hmox1^fl/flLyz2-Cre treated with vehicle or ZnPPIX; (right) proliferation rates of B16 and MN/MCA1 cells in primary tumors and lung metastasis of mice treated with vehicle or ZnPPIX; B16: n = 5 (vehicle), n = 4 (ZnPPIX); MN/MCA1: n = 4 (tumor) and n = 3 (metastasis). g, Representative images of K1735-M2 lung metastasis and quantification of K1735M2 melanoma cells in C3HN mice treated or not with ZnPPIX; n = 7 (Vehicle) and n = 6 (ZnPPIX). h TAMs purified from MN/MCA1 tumors growing in WT mice treated or not with CORM-2 were analyzed by FACS for pSTAT1, pSTAT3, pSTAT6 expression (top, n = 3 mice) and by RT-PCR for the expression of representative M1 and M2 cytokine genes (bottom, n = 4 mice), i, FACS quantification of CD31⁺ cells, E-cadherin⁺, N-cadherin⁺ and Vimentin⁺ mCherry⁺ MN/MCA1 cells in primary tumors of T lymphocyte-depleted MN-MCA1-bearing WT mice, treated or not with ZnPPIX, n = 5 (Vehicle and ZnPPIX) and n = 4 (anti-CD4/CD8+ZnPPIX). j, Mean ± SEM tumor volume and size of B16 tumors growing in WT mice treated with Vehicle, anti-PD-1 antibody, ZnPPIX, or anti-PD-1 antibody plus ZnPPIX are shown, n = 2 (Vehicle), n = 3 (anti-PD-1 and ZnPPIX) and n = 4 (anti-PD-1+ZnPPIX). Data are mean ± SEM. *P < 0.05 **P < 0.01 ***P < 0.001, between selected relevant comparisons. In a, b, f, j, two-way ANOVA with Sidak’s multiple comparisons test. In c, d, f, g, h, two-tailed unpaired Student’s t-test or Mann–Whitney U-test. In e, i one-way ANOVA and Kruskal-Wallis test with Dunn’s multiple comparisons test. The data in a–c, are representative of two independent experiment; in d–j one experiment was performed.