PD-L1 is expressed on TAMs in both Mycn-nGEMM and human NB tumors. (A) t-SNE representation of GFP, CD11b, and PD-L1 signals in Mycn-nGEMM-high and Mycn-nGEMM-low tumors (n = 3 tumors per cell line) shows that PD-L1 is expressed mostly on myeloid cells in Mycn-nGEMM tumors. (B) Representative serial IHC of human MYCN-amplified NB tumor and pseudo-fluorescence visualization. Scanned images (10×) of serial IHC of Phox2b (left, nuclear staining, red), PD-L1 (middle, membrane staining, brown), and CD163 (right, membrane staining, red) of a representative formalin-fixed paraffin-embedded section of a human MYCN amplified NB obtained after chemotherapy. Scale bar = 200 µm. (C) Magnified region from B (dotted black lines) of deconvoluted images stacked to demonstrate IHC of Phox2b, Phox2b with PD-L1, Phox2b with CD163, and Phox2b with CD163 and PD-L1. Scale bar = 100 µm. (D) Quantitative image analysis of MYCN-amplified tumors for CD163, Phox2b, and PD-L1. Boxplot of the percent positivity of Phox2b, CD163, and PD-L1 in MYCN-amplified NB tumors. Percent positivity is also shown for Phox2b⁺PD-L1⁺ cells (mean 2.2%) and CD163⁺PD-L1⁺ cells (mean 34.1%). (E) PD-L1 expression assessed by flow cytometry on BMDM co-cultured with indicated cell lines (Mycn-high, Mycn-low, SB28, MC38, or B16F10) or treated with IFNγ for 72 h. Mean fluorescent intensity (MFI) for the PD-L1 signal is indicated for each condition. Data represent mean ± SD from two independent experiments. Statistical significance was determined by one-way ANOVA multiple comparisons test (D); ***P < 0.001.