Fig 1: Immune evasive NPE-IE tumors possess a highly immunosuppressive TME, related to Figure 3(A) & (B) Gating strategy to define myeloid (A) and lymphoid (B) populations in NPE and NPE-IE tumors.(C) Representative histograms of PD-L1 expression on microglia and macrophages in NPE-IE tumors (linked to Figure 3E). Control represents fully stained sample minus anti-PD-L1 antibody.(D) Representative histograms of CD206, CD86 and CD11c expression on macrophages from NPE and NPE-IE tumors. Data derived from 3 tumors randomly down sampled for 50,000 live cells each. Control represents fully stained sample minus either anti-CD206, anti-CD86 or anti-CD11c antibodies.(E) Flow cytometry quantification of the frequency of macrophages, M-MDSC and microglia positive for expression of PD-L1(F) Flow cytometry quantification of the frequency of NK cells as a percentage of live cells.(G) Flow cytometry quantification of the frequency of CD11b+ DCs as a percentage of live cells.(H) Flow cytometry quantification of CD11b+ DCs positive for expression of PD-L1.(I) Survival analysis of NSG mice orthotopically injected with NPE-IE cells subjected to IP injection of aCSF-1R or PBS (n = 5 mice + ?CSF-1R; n = 3 mice +PBS; p = 0.2367).
Fig 2: Transcriptional and epigenetic reconfiguration occurs across mouse samples, with DNA hypomethylation occurring at key immune-associated genes, related to Figures 4 and 5(A) WGS copy number heatmap of log2 ratios of coverage for NPE and NPE-IE lines demonstrates genetic stability of mouse lines (gain of chrX evident as CNVs were called against male reference).(B) Principal Component Analysis (PCA) of RNA-seq data (top 500 most variable genes) for all cell line samples.(C) Heatmap of Z-scaled normalized counts for genes specific to all cell lines engineered with Nf1 loss ordered by log2 fold change (NF1 KO samples Vs. all others) (top). Z-scaled normalized counts of specific genes shown in heatmap across all mutants (Red trend line indicates mean Z-scaled expression) (bottom).(D) Pairwise comparisons of CpG methylation changes between lines (excluding NP double mutant) as density scatterplots, and DMR bar plots – highlighting the predominant hypomethylation within tumor derived samples.(E) Rank plots of promoter DMRs (+/- 2kb TSS) displaying hypomethylation in NPE-NSG-TD lines versus NPE samples (DMRs with > 50% methylation loss, overlapping genes within immune and interferon GO terms are highlighted in blue).(F) Promoter DMR methylation (%) across lines for Irf8, Nt5e and Cd274, genes (top), and correlation with RNA-seq normalized counts (bottom) (SCC, and p value reported).(G) RNA-seq normalized counts of Nt5e across analyzed lines.
Fig 3: Immune evasive NPE-IE tumors possess a highly immunosuppressive microenvironment(A) Fast interpolation-based t-distributed stochastic neighborhood embedding (FIt-SNE) maps of cell populations in NPE and NPE-IE tumors.(B) Quantification of cell population frequencies in (A) as proportion of total live cell population (n = 4 per condition).(C) Flt-SNE maps of myeloid (CD11b+) cells in NPE and NPE-IE tumors.(D) Quantification of macrophage, M-MDSCs, and microglia frequency in (C) as proportion of total live cell population (n = 4 per condition).(E) PD-L1 median fluorescent intensity (MFI) quantification on microglia and macrophages in NPE and NPE-IE tumors (n = 4 per condition).(F) Bioluminescent imaging of NPE-IE tumor progression in BL6 in vivo following intraperitoneal (i.p.) injection of aCSF-1R or PBS.(G) Survival analysis of BL6 mice orthotopically injected with NPE-IE cells and subjected to i.p. injection of aCSF-1R (n = 17) or PBS (n = 11).(H) Quantification of CD8+/CD4+ T cell population frequencies in NPE and NPE-IE tumors as a proportion of total live cell population in (A) (n = 6 per condition).(I) Phenotypic marker expression of CD8+/CD4+ T cell subsets from (A) (n = 6 per condition).For Flt-SNE plots, data were generated from 150,000 live cells randomly sampled from 3 tumors per condition (50,000 live events shown per tumor).See also Figure S3.
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