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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. 2 — Melanomas convert normal B cells to a tumor-associated phenotype with high growth factor production for tumor stroma-tumor cell cross-talk. a NB cells from healthy volunteer (NB cells) co-cultured with melanoma tumor lineWM3749 (tumor-conditioned; red bars) show increased expression of growth factors and inflammatory cytokines, including IGF-1 (see Supplementary Fig. 4 for fresh NB cells), IL-1α/β and PDGF-A/-B (Supplementary Fig. 3b) when compared with unconditioned NB cells from the same healthy volunteer (blue bars). The B cells were harvested after 14 days (viability > 90%) and were analyzed by qPCR as in Fig. 1d. Results are representative of 2 independent experiments. b–e Upregulated protein or mRNA expression and phospho-signaling of FGFR-3 in B cells and melanoma cells as determined by qPCR or immunostaining. b NB cells co-cultured with melanoma cells for 48 h (red bars) show increased phosphorylation of FGFR-1 and FGFR-3 when compared with unconditioned B cells (blue bars) as determined by phospho-RTK array analysis. c NB- (dark blue bar) and TAB cells (dark brown bar) co-cultured with melanoma cells (WM3749) for 48 h show increased FGFR-3 expression compared with unconditioned normal (blue bar) or TAB cells (red bar); qPCR as in Fig. 1d. Results are representative of two independent experiments. d Detection of FGFR-3 expression in TAB cells in additional 10 metastatic melanoma patients’ biopsies by laser scanning microscopy. The 6 larger images on the left show FGFR3 expression in a CD20⁺ (grayish white) CSPG^-ve (green) B-cell (white arrow) as well as in melanoma cells (CSPG⁺; green). The right panel is a close up into a CD20+ (grayish white) lymphocyte cluster of the tumor stroma in direct apposition to melanoma cells (part of a melanoma cell can be seen in the upper right corner by staining for CSPG; green). Nuclei were counterstained with DAPI (blue). Isotype-matched antibodies were used as a control. Representative images are shown. Scale bars: left 25 μm, right 10 μm. e Detection of FGFR-3 expression in NB or TAB cells after co-culture (48 h) with melanoma cells (WM3749) detected by immunostaining. Cytospin preparation of normal B or TAB cells after co-culture with melanoma cells show upregulation of FGFR-3 as determined by staining of B cells with rabbit anti-FGR-3 antibody followed by Alexa-Fluor 488 conjugated anti-rabbit antibody. Scale bars: 40 μm. Images were captured using Nikon fluorescent microscope