Figure 2

CD271 expression characterizes melanoma metastases. (A) Left panel: Visualization of CD271/NGFR expression levels (log2) at different melanoma progression stages (GSE46517): MET (n = 54), PM (n = 31), melanocytic nevus (Nevus; n = 9), NS (n = 7) and melanocytes (Mel.; n = 1). Right panel: CD271/NGFR expression levels (log2) of brain metastases (Brain; n = 29, GSE50493) as well as extracranial metastases, lymph node (LN; n = 12), soft tissue (ST; n = 10), small intestine (S. int.; n = 4), lung (Lung; n = 6), Bowel (n = 1), Spleen (n = 4), Adrenal (n = 1), Bone (n = 3), Liver (n = 2). (B) Supervised clustering of brain metastases with high expression of CD271/NGFR (red, patients 15, 37, 42, 51 and 55) and very low expression (green, n = 13) of 99 genes, among them 62 genes with increased (≥1.5fold, log2; p < 0.05) and 37 genes with decreased (≤1.5fold, log2; p < 0.05) expression. (C) Left and right panel: Gene-set enrichment analysis (GSEA) of CD271^high (BM-CD271^high) and CD271^low (BM-CD271^low) brain metastases (study GSE50493) shows enrichment of CD271-responsive genes (n = 156; NES = normalized enrichment score; FDR = false discovery rate). Venn-diagram depicts the number of CD271-responsible genes, distinctly or commonly up-regulated in CD271^high brain metastases in two independent studies (GSE50493, GSE44660; FC_log2 ≥ 1.1, p ≤ 0.05). (D) Left panel: Representation of the migratory capacity of A375^GFP and A375^CD271/NGFR cells indicated by the wound width [µm], 24 hours after wounding. Right panel: Enrichment of genes involved in focal adhesion formation (Broad institute’s MSigDB) in A375^CD271/NGFR cells as determined by GSEA (E) Confocal microscopy-based serial imaging of mouse brain slices, 48 h after injection of 10 000 A375^GFP or A375^CD271/NGFR cells. Left panels: GFP expression of image sections, Z0/0, Z7/14, Z14/14 (inset) as well as CD271 expression (red) for Z14/14 of slices injected with A375^GFP are shown. Right panels: image sections Z0/7and Z7/7 of slices injected with A375^CD271/NGFR are shown. Areas depicting migrating CD271⁺ cells are magnified. DAPI served as nuclear stain, scale bars indicate 50 µm. White dotted lines indicate initial tumor borders/non-migrated cells.