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. 2017 Sep 19;8:607. doi: 10.1038/s41467-017-00452-4

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a–g Prevalence of CD20⁺ B cells in metastatic melanoma tissues and increased IGF-1 expression in TAB cells. a, b Presence of CD20⁺B cells. Representative immunostaining (TMA, 79 cores from 48 patients) for CD20 (red) and a three melanoma marker combination (CSPG/β3 integrin/HMB45 (green)) plus DAPI nuclear stain (blue). a Left and middle panels: sample with predominant distribution of CD20⁺, triple melanoma marker^-ve B cells in the tumor stroma (red; left panel: isotype control). Right panel: close-up with infiltration of single CD20⁺ B cells (red) among melanoma cells (green). Scale bars: 50 μm. Far right 2 panels: CD20⁺ B cells are DAPI⁺ and triple melanoma marker^-ve. Scale bars: 10–20 μm. b Tissue FAXS analysis, the MFI was displayed in scattergrams for FITC-labeled CSPG/β3 integrin/HMB45+ and Texas red (TXR)-labeled CD20+ cells. Cutoff values were set based on isotype control staining (left panel). CD20+ B cells gated as CD20+, triple melanoma marker^-vecells (right panel, red frame). c Representative immunostainings from additional metastases (six patients’ biopsies) stained for IGF-1 (red) in CD20⁺ (grayish white) CSPG^-ve (green) B cells. Scale bars: 10 μm. d, e Immortalized B cells (48 h, B-cells-only cultures) from melanoma tissues (n = 5; TAB cells; red bars) show increased mRNA/protein expression of IGF-1 when compared with immortalized B cells from peripheral blood (n = 3; NB cells; blue bars) and pooled total PBL of healthy volunteers (black bars). d mRNA transcripts were determined by qPCR with levels indicated as RQ values normalized to GAPDH and relative to control normal B cells from healthy volunteers. Bars represent mean + SE of duplicate samples. Results are representative of three independent experiments for each sample. e B-cell supernatants (48 h, B-cells-only cultures) were used for protein expression of IGF-1 (ELISA). Bars represent mean + SE of duplicate samples. f, g TCGA-SKCM patients’ data set analyses (n = 473). f Kaplan–Meier survival curves of 158 melanoma patients with tumors containing a high lymphocyte infiltrate (above median, see Methods) who were divided into high and low IGF-1 expression (by median). Log-rank test shows poor OS in patients with high IGF-1 expression. g Box plots showing significantly (p < 0.0001, t-test) increased B-cell (MS4A1, CD20) expression levels in melanomas with high tumor IGF-1 expression