Anti–PD-L1 treatment of Mycn-nGEMM tumors depletes CD206⁺ TAMs and remodels the intra-tumoral immune landscape. (A) Mycn-nGEMM-low cells were transplanted in the renal capsule of C57BL/6J hosts on day 0. After BLI imaging and randomization, treatments started on day 7 with 200 µg of anti–PD-L1 (or corresponding IgG) injected i.p. every 3 days for a total of 5 doses. (B) Growth curves of Mycn-nGEMM-low tumors treated with IgG or anti–PD-L1 antibodies, as assessed by BLI. Grey area indicates treatment window. Data represent mean values ± SEM (n = 5 mice per group). (C) Kaplan–Meier curves for C57BL/6J mice injected with Mycn-low cell line in the renal capsule and treated with 5 doses of IgG or anti–PD-L1 antibodies (n = 10 mice per group). (D) Volcano plot comparing abundance of tumor-infiltrating leukocyte (TIL) subpopulations in anti–PD-L1–treated tumors (red) and IgG-treated tumors (blue) using the mouse mass cytometry immune cell panel (n = 3 tumors per group). Statistically significant clusters in volcano plots are highlighted in opaque color and indicated with a cell type label. (E) Mass cytometry data from Mycn-nGEMM tumors were filtered on CD45⁺CD11b⁺F4/80⁺ TAMs, then gated manually to identify cells positive or negative for CD206 or MHC-II. Frequencies of TAMs expressing CD206 and MHCII was quantified for IgG-treated and anti–PD-L1–treated tumors (n = 3 tumors per group). (F) Percentage of CD11b⁺F4/80⁺ TAMs expressing MHC-II, CD206, or PD-L1 markers in Mycn-nGEMM tumors after treatment with IgG or anti–PD-L1 antibodies (n = 3 tumors per group). (G) Mass cytometry data from Mycn-nGEMM tumors were filtered on CD45⁺ cells, then gated manually to identify cells positive or negative for CD3e, CD4, or CD8. Frequencies of T cells expressing CD4 and CD8 was quantified for IgG-treated and anti–PD-L1–treated tumors (n = 3 tumors per group). (H) Percentage of CD4⁺ T cells and CD8⁺ T cells in Mycn-nGEMM tumors after treatment with IgG or anti–PD-L1 antibodies (n = 3 tumors per group). (I) After CD4⁺ T and CD8⁺ T cell depletion by neutralizing antibodies and treatment of IgG and anti–PD-L1, tumor volume was measured by ultrasound on day 21 after injection (n = 5 mice per group). (J) Kaplan–Meier curves representing percentage of tumor-free mice for naïve (n = 10 mice) and anti–PD-L1–cured mice (n = 7 mice) rechallenged with Mycn-nGEMM-low cells in the right flank. Statistical significance was determined by two-way ANOVA (B), unpaired t test (F and H), and one-way ANOVA multiple comparisons test (I). Kaplan–Meier survival analyses were performed by log-rank test (C and J). *P < 0.05, **P < 0.01 and ns = no significance.