Response of Mycn-nGEMM tumors to checkpoint inhibitors. (A) Kaplan–Meier curves for C57BL/6J mice injected with Mycn-nGEMM-high cell line in the renal capsule and treated with five doses of IgG or anti-PD-1+anti-CTLA-4 antibodies (n = 11 mice per group). (B) Kaplan–Meier curves for C57BL/6J mice injected with Mycn-nGEMM-low cell line in the renal capsule and treated with 5 doses of IgG or anti-PD-1+anti-CTLA-4 antibodies (n = 13 mice per group). (C) Tumor volume of Mycn-nGEMM-high tumors implanted in the renal capsule of C57BL/6J mice and treated with five doses of anti–PD-L1 antibody or corresponding IgG antibody (n = 6 mice per group). (D) Kaplan–Meier curves for C57BL/6J mice injected with Mycn-nGEMM-high cell line in the renal capsule and treated with five doses of IgG or anti–PD-L1 antibody (n = 9–10 mice per group). (E) Heatmap indicating levels of expression for each marker for all CD45⁺ PhenoSOM meta clusters identified with the mouse mass cytometry panel for Mycn-nGEMM tumors treated with IgG or anti-PD-L1. (F) CD206⁺/CD206⁻ macrophage ratio from mass cytometry analysis of renal capsule tumors from Mycn-nGEMM-low mice treated with three doses of anti–PD-L1 antibody or corresponding IgG. (G) Flow cytometry data from Mycn-nGEMM, BMDM-derived M1 or M2-like macrophages, and MC38 cells for isotype control and PD-L1. (H) Cell proliferation of Mycn-nGEMM, BMDM-derived M1 or M2-like macrophages and MC38 cells after treatment with IgG control and anti–PD-L1 for 72 h. Data represent mean ± SD from three independent experiments. (I)Mycn-nGEMM and MC38 cells (T; target) and mFCγRIV cells (E; effector) were cultured at E:T ratio is 5:1 under IgG control or anti–PD-L1 conditions. After 6 h, ADCC activity was determined. Data represent mean ± SD from three independent experiments. (J) Flow cytometry data from Mycn-nGEMM-high (top) and low (bottom) tumors after treatment with IgG, anti–PD-L1, and anti-CD4 and anti-CD8 were filtered for CD45⁺ and CD3e cells and showed cell populations for CD4 and CD8a (n = 5 mice per group). (K) Tumor volume was measured by ultrasound on day 21 after injection (n = 5 mice per group). Statistical significance was determined by unpaired t test (C and F), one-way ANOVA multiple comparisons test (K), and Kaplan–Meier survival analyses were performed by log-rank test (A, B, and D). *P < 0.05, **P < 0.01, and ns = no significance.