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. 2020 Jul 10;12(7):1860. doi: 10.3390/cancers12071860

Figure 1.

Figure 1

Distinct transcriptional signatures of neutrophils isolated from cSCC lesions and surrounding skin of DMBA/PMA-treated mice highlight their protumor functions. (A) Percentage of neutrophils among CD45+ cells in the skin surrounding papillomas (papilloma skin), within papillomas, in the skin surrounding tumors (tumor skin), and within tumors, n = 6 mice per group, * p < 0.05, ** p < 0.01, Mann–Whitney U test. (B) Microarray analysis of cell-sorted neutrophils: two-dimensional PCA of the 15% most variable probe sets with a minimum log2 average expression of 6. Neutrophils were purified from pools of skin surrounding papillomas (n = 6 mice, 2 experiments), skin surrounding tumors (n = 9 mice, 2 experiments), papillomas (175 papillomas from n = 8 mice, 3 experiments), and tumors (24 tumors from n = 5 mice, 2 experiments). (C) DEGs in pairwise comparisons. The four contrasts were analyzed based on a minimum log2 average expression of 6, an absolute logFc of at least 1, and an adjusted p value p ≤ 0.05. (D) IPA upstream regulator enrichments from the analysis of DEGs in papilloma versus papilloma skin (outer circle), tumor versus tumor skin (middle circle), and tumor versus papilloma (inner circle). The most significant (−Log10 ≥ 1.39) upstream regulators are shown. A negative z-score (blue) denotes an inhibited pathway. A positive z-score (red) stands for an activated pathway. (E) Hierarchical clustering of genes differentially expressed between lesions and their respective skin control identified 10 clusters. The DEGs were selected based on an average log2 expression level across all conditions of at least 7, an absolute log2-fold change ≥ 1, and an adjusted p value ≤ 0.05, in at least one of the two contrasts, papilloma versus papilloma skin and tumor versus tumor skin. (F) Average intensity expression levels and adjusted p values of Cd274 (PD-L1), Vegfa, Olr1 (LOX1), Siglec 5 (Siglec F), Nos2, and Arg1 transcripts are shown, * p < 0.05, *** p < 0.001, limma differential expression analysis. cSCC: cutaneous squamous cell carcinoma; DMBA: 7,12-dimethylbenz[a]anthracene; PMA: phorbol 12-myristate 13-acetate; PCA: principal component analysis; DEGs: differentially expressed genes; IPA: Ingenuity Analysis Pathways; PD-L1: programmed death-ligand 1; LOX1: lectin-type oxidized LDL receptor 1; Siglec F: sialic acid-binding immunoglobulin-type lectins.