Fig 1: 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
Fig 2: TAB cells modulate melanoma cells to express FGFR-3 and its ligand FGF-2 for tumor stroma-tumor cell cross-talk. a WM3749 co-cultured (72 h) with TAB cells (red bar) show increased FGFR-3 mRNA expression when compared with tumors only (open bar) or co-cultured with NB cells (blue bar). b Lysates obtained from pools of melanomas co-cultured (72–120 h) with NB- or TAB cells were probed in western blot with anti-FGFR-3 antibody (left panel), results expressed as relative intensity after β-actin normalization (right panel). c Melanoma cells co-cultured with TAB cells (72 h) show increased phospho-FGFR-3 expression (right panel; immunofluorescence assays) when compared with melanoma cells alone (left panel) or melanoma cells co-cultured with NB cells (middle panel), scale bars: 40 μm, images captured by Nikon inverted microscope. d Melanoma cells co-cultured with TAB cells (red bar) show increased FGF-2 mRNA expression when compared with melanoma cells alone (open bar) or melanoma cells co-cultured with NB cells (blue bar). e 451Lu and WM989treated with IGF-1 (25 ng/ml/daily for 5 days; red bar) show increased FGFR-3 expression when compared with untreated controls (blue bar), flow cytometry results expressed as net % expression of control antibody. IGF-1 treated melanoma cells (red bars) show higher expression of FGFR-3 compared with untreated cells (blue bars). Bar represents mean + SD of replicate samples. f NB cells treated with FGF-2 (10 ng/ml/daily for 4 days; red bar) show high IGF-1 mRNA expression relative to untreated NB cells (blue bar). g 451Lu and WM989 co-cultured (72 h) with TAB cells in the presence of an anti-IGF-1 neutralizing antibody (10 μg/ml) show decreased FGFR-3 mRNA expression in tumor cells (blue bar) when compared with controls (red bar). h TAB cells co-cultured (72 h) with 451Lu and WM989 in the presence of an anti-FGF-2 neutralizing antibody (1 μg/ml) show decreased IGF-1 mRNA expression in B cells (blue bar) when compared with controls (red bar).Experiments in a, d and f–h were performed using qPCR. In Figures a, d–h, bars represent mean + SE of duplicate samples and are representative of at least two independent experiments. i Summary of cross-talk between melanoma and B cells: FGF-2 is constitutively expressed by tumor cells, released into the microenvironment to bind FGFR-3 on the B cells, activated B cells express increased levels of pro-inflammatory cytokines. IGF-1 released by TAB cells modulates tumor cells to increase their growth, heterogeneity and therapy resistance
Fig 3: Anlotinib targets FGFR3 and inhibits FGFR3 phosphorylation in OSCC. A and B The tyrosine kinase inhibitor (TKI) targets of anlotinib were assessed by quantitative real-time PCR (qRT-PCR) and western blotting. C The expression and phosphorylation levels of FGFR3 were detected in concentration gradient anlotinib treated in SCC9 and SCC25 cells using the western blotting
Fig 4: Shisa3 interacts with FGFR to impact EGFR-TKI sensitivity. a. Cell lysates from PC9/ER cells transfected with Flag-Shisa3 were immunoprecipitated with Flag or IgG and blotted with FGFR1, FGFR3, and Flag antibodies. b. Western blot analysis in cellular extracts of indicated cells. c. Representative images (× 200 magnification) of FGFR1 staining by immunohistochemical analysis in tumor tissues. Scale bars, 50 μm. d. Histogram of the negative relationship between shisa3 and FGFR1 expression in lung adenocarcinoma tissues with EGFR mutations (n = 102). e, f. Kaplan-Meier analysis of the DFS (e) and OS (f) of lung adenocarcinoma patients (n = 102) in connection with FGFR1 expression. G. PC9/ER-control and PC9/ER-shisa3 cells were incubated with the following agents: gefitinib (0.5 μM), osimertinib (0.1 μM), BGJ398 (1 μM), or a combination of gefitinib/osimertinib and BGJ398. g: n.s. p > 0.05; *p < 0.05; **p < 0.001; ***p < 0.0001
Fig 5: FGFR3 expression level affects antitumor activity of anlotinib in OSCC. A FGFR3 silenced effects were examined by western blotting. B Cell proliferation inhibition assay showed that the cytotoxic ability of anlotinib in OSCC cells (24 h) after transfected with siFGFR3 or treated with rhFGF. C Cell apoptosis assay showed the ratio of apoptosis cells of anlotinib-treated (24 h) OSCC cells after transfected with siFGFR3 or treated with rhFGF. D Western blotting was used to detect the expression of protein and phosphorylated protein of FGFR3, AKT and mTOR and apoptosis-related proteins in indicated treatment OSCC cells. *P < 0.05; **P < 0.01; ***P < 0.001
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