Figure 1. Human tumor cells express variable, but significant, levels of Siglec-7 and -9 ligands.
(A–C and E) Immunofluorescence binding studies by flow cytometry (A and C) or immunofluorescence confocal microscopy (B and E) using recombinant Siglec-Fc (human IgG1) fusion proteins coupled to secondary PE-conjugated (Fab′)2 goat anti-human Fc antibody. (A) Broad analysis of ligand expression to Siglec-7 and -9 on different tumor cell lines, as well as on primary melanocytes (n = 2) and PBMCs (n = 9) from healthy donors for comparison. Values are expressed as geometric mean fluorescence intensity (GMFI) ratio of specific staining compared with secondary antibody only. (B) Expression of Siglec-7 and -9 ligands on A375 melanoma cells was localized to the cell surface. Staining was lost upon neuraminidase treatment (sialic acid dependency). Original magnification, ×630. (C) Expression levels of Siglec-7 and -9 ligands on CLL and AML cells, as revealed by flow cytometric analysis. Histograms are representative of 3 CLL and 3 AML patients. (D) Lectin immunohistochemistry for Siglec-7 and -9 ligand expression in paraffin-embedded tissues representative of melanoma, BCC, SCC, and CTCL sections. Scale bars: 100 μm (melanoma, BCC, and SCC); 50 μm (CTCL). (E) Paraffin-embedded primary tissue biopsy sections of malignant melanoma lesions in epidermal skin layers, costained for the melanoma marker Melan-A and Siglec-7 or Siglec-9 ligands. Scale bars: 50 μm. Data are representative of at least 2 (B), 3 (D), or 5 (E) independent experiments.