Activin promotes skin carcinogenesis by attraction and re-programming of macrophages (2016)
Figure 4

Figure 4. Activin re-programs macrophages towards a TAM-like phenotype

A. Scheme of the experimental setup. Pretumorigenic ear skin from 3-6 mice per genotype was pooled, and macrophages were enriched by FACS-sorting of F4/80+CD11b+ cells and subjected to RNA sequencing. Each replicate represents 1 of 3 independent FACS experiments (n=3).

B. Analysis of FACS data of sorted macrophages shows the relative percentage of CD45+CD11b+F4/80+ cells in each genotype and the absolute number of sorted cells used for RNA sequencing. Representative flow cytometry gating strategy for sorting is shown in Fig EV3B. N=5-9. Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons Test.

C. Differential expression of genes was analyzed using the edgeR software package. IPA was performed with filtered lists of up- and downregulated genes that exhibited an FDR-value of <0.1 in different genotype comparisons (wt/Act vs wt/wt; HPV8/Act vs HPV8/wt; HPV8/Act vs wt/wt). The number of differentially expressed genes used for analysis is indicated. Data are presented as pseudo-heatmap with magnitudes of Activation Z-scores color-coded as indicated in the legend; all non-grey comparisons are –log10(p-value)>2.5 unless otherwise indicated.

D. Gene Set Enrichment Analysis (GSEA) was performed to compare transcriptomes of genotype comparisons (wt/Act vs wt/wt; HPV8/Act vs HPV8/wt; HPV8/Act vs wt/wt, [all]/Act vs [all]/wt) to 3 published datasets of Tumor-Associated Macrophages (TAM) (Franklin et al. 2014; Ojalvo et al, 2009; Galletti et al, 2016). Gene sets are composed of top genes upregulated (TAM UP) or downregulated (TAM DOWN) in TAM vs normal tissue macrophages. Positive Normalized Enrichment Score (NES) corresponds to enrichment of gene set in activin-upregulated genes; negative NES corresponds to enrichment in activin-downregulated genes. Data are presented as pseudo-heatmap with magnitudes of NES color-coded as indicated in the legend; all non-grey comparisons are FDR<0.10.

E. Differential GSEA was performed on combined genotype comparison [all]/Act vs [all]/wt and two datasets of human AK vs normal skin (GSE2503; Nindl et al, 2006) and GSE63107 to compare these transcriptomes to gene sets derived from top upregulated genes in these datasets (top); and gene sets derived from top upregulated genes in datasets of differential human macrophage activation (IFN-, TNF-α, LPS, IL-4 vs non-treated) and selected human myeloid/lymphoid cell type-specific gene expression profiles (macrophage vs monocyte, dendritic cell, and T-cell) (Xue et al, 2014; bottom). Activin target genes enriched in the AK transcriptomes are shown (right). Data are presented as pseudo-heatmaps with magnitudes of Normalized Enrichment Scores (NES) and Log2(Fold Change) color-coded as indicated in the respective legends; all non-grey comparisons are FDR<0.05 for NES and p<0.10 for Fold Change.

F. Venn diagram showing overlapping differentially expressed genes (FDR≤0.05, |log2Ratio|≥1) in different genotype comparisons.

G. Heatmap of 28 differentially expressed genes (FDR≤0.05, |log2Ratio|≥1) overlapping in all 3 comparisons (wt/Act vs wt/wt mice, HPV8/Act vs HPV8/wt and HPV8/Act vs wt/wt). Genes with a published pro-tumorigenic function are highlighted in red.

Complete lists of differentially regulated genes from different genotype comparisons, including corresponding fold change and exact FDR values, are presented in dataset EV2. Complete list of diseases and functions identified by IPA for each genotype comparison, including exact p-values, are shown in dataset EV3. Complete original and filtered gene sets, ranked gene lists used for the GSEA, and original GSEA reports, including exact FDR and p-values, can be found in dataset EV4.