Fibroblast growth factor receptor (FGFR) and fibroblast growth factor (FGF) overexpression in human primary uveal melanoma (UM). Analysis of The Cancer Genome Atlas (TCGA) dataset was performed on a cohort of 80 UM patients. (A) Pie chart showing the percentage of samples with mRNA overexpression of the different FGFRs. (B) Overall survival of patients with or without FGFR alterations. (C) Pie chart showing the percentage of samples with mRNA overexpression of different members of the FGF family. Some samples showed the overexpression of more than one FGF family member. (D) Overall survival of patients with or without FGF alterations.

Correlation between FGF/FGFR expression and chromosome 3 /BAP1 status in UM. Analysis of the expression of all members of the FGFR and FGF families was performed on the cohort of 80 UM patients present in the UM TCGA dataset. FGF/FGFR genes that showed a significant differential expression between chromosome 3 (monosomic, red symbols; disomic, open symbols) and BAP1 (mutated, blue symbols; wild-type, open symbols) status.

Effect of long-pentraxin 3 (PTX3) overexpression on B16-LS9 cells. (A) RT-PCR analysis of Fgf2 and Fgfr expression in B16-LS9 cells. (B) Western blot analysis of the phosphorylation of FGFR1 and FGFR3 and of the downstream signaling proteins ERK_1/2 and AKT in B16-LS9 cells following 30 min treatment with 30 ng/mL FGF2. (C) RT-PCR analysis of human PTX3 (hPTX3) expression in WT_LS9, mock_LS9, and hPTX3_LS9 cells. Inset: Western blot analysis of PTX3 protein levels in the extracts of the same cells. (D) Western blot analysis of the phosphorylation of FGFR1, FGFR3, FRS2, and ERK_1/2 proteins in WT_LS9, mock_LS9, and hPTX3_LS9 cell extracts. (E) WT_LS9, mock_LS9, and hPTX3_LS9 cells were seeded in 48-well plates at 10⁴ cells/well in medium containing 0.4% FBS. After 24 h (T₀), medium was changed, and cell were counted 24 and 48 h thereafter. Data are the mean ± SEM of three independent experiments in triplicate. (F) Cells were seeded as in (E). At T₀, cells were treated with 30 ng/mL FGF2 and counted 24 h thereafter. Data are the mean ± SEM of three independent experiments in triplicate (G) WT_L69, mock_LS9, and hPTX3_LS9 cells were seeded at 50 cells/cm². After 10 days, cell colonies were stained with crystal violet and quantified by computerized image analysis. Representative images of mock_LS9 and hPTX3_LS9 cell colonies are shown on the right. Data are the mean ± SEM of 15 fields for each triplicate sample. (H) A mechanical wound was performed in WT_LS9, mock_LS9, and hPTX3_LS9 cell monolayers. After 18 h, cell migration at the leading edge of the wound was quantified by computerized image analysis. Representative images of wounded mock_LS9 and hPTX3_LS9 cell monolayers are shown on the right. Data are the mean ± SEM of six microscopic fields. (I) Mock_LS9 and hPTX3_LS9 cells were injected subcutaneously (s.c.) in syngeneic mice at 50,000 cells/graft and tumor growth was measured with calipers. Data are the mean ± SEM (n = 16). (J) Red fluorescent WT_LS9, mock_LS9, and hPTX3_LS9 cells were injected into the bloodstream of 48 hours post fertilization (hpf) zebrafish embryos (80–100 cells/embryo). During the next 3 days, the growth of fluorescent metastases in the tail vascular plexus was quantified by fluorescence microscopy followed by computerized image analysis. Data are the mean ± SEM of three independent experiments (n = 20) and were normalized to metastasis areas at day 1. (K) WT_LS9 cells were injected s.c. in wild-type and transgenic TgN (Tie2-hPTX3) mice (50,000 cells/graft) and tumor growth was measured with calipers. Data are the mean ± SEM (n = 18). (L) WT_LS9 cells were injected into the spleen of wild-type and transgenic TgN (Tie2-hPTX3) mice (20,000 cells/graft). After 14 days, livers were harvested, and metastases were counted. Representative images of harvested livers are shown on the right. Data are the mean ± SEM (n = 5). In (B) and (D), the right panel shows the densitometric analysis of immunoreactive bands normalized to α-tubulin protein levels. *p < 0.05; **p < 0.01, Student’s t-test (F,L), one-way (E,H,J) and two-way (I,K) analysis of variance.

Effect of the pan FGF-trap NSC12 on B16-LS9 cells. (A) Western blot analysis of FGFR1 and FGFR3 phosphorylation in B16-LS9 cells treated for 12 h with increasing concentrations of NSC12. The right panel shows the densitometric analysis of immunoreactive bands normalized to GAPDH protein levels. (B) Effect of NSC12 treatment on the proliferation of B16-LS9 cells. Viable cells were counted after 24 h of incubation with increasing concentrations of NSC12. Data are the mean ± SEM (n = 3). (C) B16-LS9 cells were seeded at 50 cells/cm² and treated with 2.5 µM NSC12. After 10 days, cell colonies were stained with crystal violet and quantified by computerized image analysis. Data are the mean ± SEM of 15 fields for each triplicate sample. (D) A mechanical wound was performed in a B16-LS9 cell monolayer followed by incubation with 3.0 µM NSC12. After 18 h, cell migration at the leading edge of the wound was quantified by computerized image analysis. Data are the mean ± SEM of six microscopic fields. (E) B16-LS9-luc cells were injected into the eye of 48 hpf zebrafish embryos (100 cells/embryo). Then, embryos were incubated with increasing concentrations of NSC12 at T₀. Tumor growth was evaluated 3 days after grafting by measuring the cell luminescence signal. Data are the mean ± SEM (n = 20). (F) B16-LS9-luc cells were grafted in the liver of syngeneic mice (50,000 cells/graft). Next, vehicle or NSC12 (7.5 mg/kg) were injected i.p. every other day and tumor growth was imaged with IVIS Lumina III for the following 14 days. Data are the mean ± SEM (n = 9). Representative images of control and NSC-12 treated mice imaged 14 days after grafting are shown on the right. *p < 0.05; **p < 0.01, Student’s t-test (C,D), one-way (E) and two-way (F) analysis of variance.

Effect of the pan FGF-trap NSC12 on human UM cells. (A) Western blot analysis of the phosphorylation of FGFR1 and FGFR3 and of the downstream signaling proteins FRS2 and ERK_1/2 in Mel285, 92.1, Mel270, and OMM2.3 cells after 3 h treatment with 15 µM NSC12. (B) Effect of NSC12 treatment on the proliferation of UM cells. Viable cells were counted after 24 h of incubation with increasing concentrations of NSC12. Data are the mean ± SEM (n = 3). (C) Kinetics of PARP and caspase-3 cleavage following incubation of MEL285 cells with 15 µM NSC12. (D) Cytofluorimetric analysis of apoptosis induced in Mel285 cells (upper panels) and 92.1 cells (lower panels) after 12 h treatment with 15 µM NSC12. (E) Western blot analysis of the levels of β-catenin in Mel285, 92.1, Mel270, and OMM2.3 cells after 3 h treatment with 15 µM NSC12. (F) Boyden chamber chemotaxis assay performed on Mel285 cells treated for 4 h with 6.0 µM NSC12. Data are the mean ± SEM of five fields for each triplicate sample. (G) A mechanical wound was performed in a Mel285 cell monolayer followed by 18 h incubation with 6.0 µM NSC12. After 18 h, cell migration at the leading edge of the wound was quantified by computerized image analysis. Representative images of untreated and NSC12-treated cells are shown on the right (black lines highlight the front of cell migration). Data are the mean ± SEM of 6 microscopic fields. In (A,C,D) the right panel shows the densitometric analysis of immunoreactive bands normalized to GAPDH protein levels. ** p < 0.01, Student’s t test.