Analysis of CYSLTR1/CYSLTR2 expression and UM patient survival from The Cancer Genome Atlas (TCGA). Kaplan–Meier survival curves demonstrate a statistically significant relationship between high (red) CYSLTR1 expression and disease-free survival (A) (n =80; Log-rank; p = 0.03; HR 1.05; 95% CI 1.03–1.07) or overall survival (B) (n = 80; Log-rank; p = 0.02; HR 1.06; 95% CI 1.04–1.08) in UM patients. Low CYSLTR1 expression is shown in blue. Similarly, Kaplan–Meier survival curves demonstrate a statistically significant relationship between high (red) CYSLTR2 expression and disease-free survival (D) (n =80; Log-rank; p = 0.002; HR 1.35; 95% CI 1.25–1.45), or overall survival (E) (n =80; Log-rank; p = 0.0001; HR 1.01; 95% CI 1–1.02) in UM patients. Low CYSLTR2 expression is shown in blue. The third quartile was used as the cut-off point for high versus low expression for all Kaplan–Meier survival curves. Both Log-rank p-values (categorical variable) and Cox p-values (continuous variable) were calculated and are displayed for (A,B,D). Log-rank and likelihood ratio (LHR) test p-values were calculated and are displayed for (E). Samples were scored using gene expression profiles and categorized into high and low CYSLTR1 (C) and CYSLTR2 (F) expression using the third quartile as cut-off. Expression profiles of interest were manually selected from the Molecular Signatures Database (MSigDB) which includes gene sets from Hallmarks and Biocarta curated pathways. Colour values correspond to the median values of the enrichment scores. Patient tumours with high expression of CYSLTR1 show a corresponding significantly altered expression for the terms corresponding to Inflammatory Response, INF-γ, TNF-α, Angiogenesis and GPCR signalling (C) (* p < 0.05). Samples with high expression of CYSLTR2 show an associated significantly altered expression for the terms Inflammatory Response, IFN-γ, Glycolysis, TNF-α, Angiogenesis and GPCR signalling (F) (* p < 0.05). Differences were assessed using a non-parametric Wilcoxon test. Differences were considered statistically significant when p-value < 0.05.

Examination of the prognostic value of CysLT₁ and CysLT₂ protein expression in primary UM samples by manual pathology. (A) Representative cores from the UM patient TMA designated with a score of 1, 2, or 3 for CysLT₁ staining intensity. (B) Kaplan–Meier survival curve stratified based by high (red) or low (blue) CysLT₁ expression and death by metastatic melanoma (n = 51; Log-rank; p = 0.122; HR 2.04; 95% CI 0.81–5.12). (C) High expression of CysLT₁ (red) is significantly associated with reduced overall survival in primary UM patients (n =51; Log- rank; p = 0.034; HR 2.34; 95% CI 1.04–5.25) (D) Representative cores from the TMA designated with a score of 1, 2 or 3 for CysLT₂ staining intensity. (E). Kaplan–Meier survival curve stratified based by high (red) or low (blue) CysLT₂ expression and death by metastatic melanoma (n = 50; Log-rank; p = 0.341; HR 1.15, 95% CI 0.58–2.28). (F) Kaplan–Meier survival curve stratified based by high (red) or low (blue) CysLT₂ expression and death by any cause (n = 50; Log-rank; p = 0.697; HR 1.15; 95% CI 0.58–2.28). The median was used as the cut-off point for high versus low expression for all Kaplan–Meier survival curves. Number of events indicates the number of deaths due to metastatic melanoma (B,E). Number of events indicates the number of deaths due to any cause (C,F).

Examination of the prognostic value of CysLT₁ and CysLT₂ protein expression in primary UM patient samples by digital pathology analysis. (A) Representative cores from the UM TMA designated with a score of Low, Intermediate, or High expression for CysLT₁ staining following digital analysis (Scale = 40 µm). (B) High expression of CysLT₁ (red) is associated with reduced survival from metastatic melanoma in primary UM patients (n = 49; Log-rank; p = 0.012; HR 2.755; 95% CI 1.205–6.3) (C) High expression of CysLT₁ (red) is associated with reduced overall survival in primary UM patients (n = 49; Log-rank; p = 0.011; HR 2.755; 95% CI 1.205–6.3) (D) Kaplan–Meier survival curve stratified based by high (red) or low (blue) CysLT₂ expression and death by metastatic melanoma (n = 49; Log-rank; p =0.165; HR 1.796; 95% CI 0.773–4.172). (E) Kaplan–Meier survival curve stratified based by high (red) or low (blue) CysLT₂ expression and death by any cause (n = 50; Log-rank; p =0.332; HR 1.461; 95% CI 0.674–3.167). The third quartile was used as the cut-off point for high versus low expression for all Kaplan–Meier survival curves. Number of events indicates the number of deaths due to metastatic melanoma (B,D). Number of events indicates the number of deaths due to any cause (C,E).

CysLT₁ and CysLT₂ are expressed in uveal melanoma cell lines. qPCR analysis confirmed the expression of CysLT₁ (A) and CysLT₂ (B) mRNA in UM cell lines, Mel285, Mel270, and OMM2.5 (n = 3). Western blot analysis confirmed the expression of CysLT₁ (C) and CysLT₂ (D) in Mel285 and OMM2.5 cells (n = 2). (E) Characteristics of UM cell lines used in this study. Statistical analysis was carried out using a paired t-test. Data are expressed as mean + SEM.

CysLT₁ antagonists reduce cell viability in uveal melanoma cell lines. Graphs represent the effects of treatment with varying concentrations of dacarbazine (DTIC), quininib (Q1), 1,4-dihydroxy quininib (Q7), montelukast (Mon), and HAMI 3379 (HAMI) for 24 and 96 h in Mel285 (A,E) (n = 3/n = 6), OMM2.5 (B,F), (n = 3/n = 4), Mel270 (C,G) (n = 3) and ARPE-19 (D,H) (n = 4/n = 6) cell lines. Treatment with 50 μM of quininib, 1,4-dihydroxy quininib, and montelukast for 96 h significantly reduced cell viability in Mel285 (E), OMM2.5 (F), and Mel270 (G) cells. (D) Quininib analogues had no effect on ARPE-19 cells at 10 or 50 μM following 24 treatment. (H) 50 μM of quininib reduced ARPE-19 viability at 96 h. Viability of cells was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 5000 cells were seeded and treated in triplicate for each individual experiment. Statistical analysis was performed by ANOVA with Dunnett’s post hoc multiple comparison test. Error bars are mean + S.E. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Quininib and 1,4-dihydroxy quininib reduce colony formation in Mel285 and OMM2.5 cells. Graphs show the percentage survival fraction of clones at 24 (A,C) and 96 h (B,D) post treatment with quininib (Q1), 1,4-dihydroxy quininib (Q7), montelukast (Mon), or HAMI 3379 (HAMI). Images of clones captured by GelCount™ system (Oxford Optronix) after 10 days of culture following treatment with DMSO control or 20 μM quininib (Q1) or 20 μM 1,4-dihydroxy quininib (Q7) for 24 or 96 h. Clones were stained with 0.5% crystal violet before counting. 1500 cells (Mel285) or 9000 cells (OMM2.5) were seeded and treated in duplicate in 6-well plates for each individual experiment and individual experiments were conducted three times (n = 3). Statistical analysis was performed by ANOVA with Dunnett’s post hoc multiple comparison test. Error bars are mean + S.E. *** p < 0.001; **** p < 0.0001.

ELISA of cell conditioned media demonstrates that 24 h treatment with 20 µM quininib analogues decreases inflammatory markers in Mel285 cells but increases inflammatory markers in OMM2.5 cells. In OMM2.5 cells, 24 h treatment with quininib (Q1) significantly increased the secretion of IL-10 (A), IL-13 (B), IL-1B (C), IL-8 (D), IL-12p70 (E), IL-2 (F), IL-6 (G), and TNF-α (H). In Mel285 cells, 24-h treatment with 20 μM quininib (Q1) and 1,4–dihydroxy quininib (Q7) significantly reduced the levels of IL-2 (F) and IL-6 (G). Treatment with 20 μM montelukast or HAMI 3379 had no effect on the secretion of inflammatory markers in either cell line. Conditioned media were collected from three separate experiments and analysed by ELISA (n = 3). All secretions were normalised to total protein content. Statistical analysis was performed by ANOVA with Dunnett’s post hoc multiple comparison test. Error bars are mean + S.E. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

ELISA of cell conditioned media demonstrates that 24 h treatment with 20 μM quininib analogues increases angiogenic markers in Mel285 cells and OMM2.5 cells. In Mel285 cells, 24 h treatment with 20 μM quininib (Q1) significantly increased the secretion of VEGF-C (A) and bFGF (B) Treatment with 20 μM 1,4-dihydroxy quininib (Q7) significantly increased the secretion of Flt-1 (C) In OMM2.5 cells, 24 h treatment with quininib significantly increased the secretion of Flt-1 (C) and VEGF-A (D) Treatment with 20 μM montelukast or HAMI 3379 had no effect on the secretion of inflammatory markers in either cell line. Conditioned media were collected from three separate experiments and analysed by ELISA (n = 3). All secretions were normalised to total protein content. Statistical analysis was performed by ANOVA with Dunnett’s post hoc multiple comparison test. Error bars are mean + S.E. * p < 0.05, ** p < 0.01, *** p < 0.001.

Quininib analogues inhibit oxidative phosphorylation, but not glycolysis, following 24 h treatment in Mel285 and OMM2.5 cell lines. Analysis of cellular metabolism in Mel285 (A–F) and OMM2.5 (G–L) UM cell lines. Oxygen consumption rate (OCR), a measure of oxidative phosphorylation, was evaluated in Mel285 (n = 5) (A) and OMM2.5 (n = 4) (G) cells using the Seahorse Biosciences XFe24 analyser following 24 h treatment with 20 μM of test compound or DMSO control. (A) 20 μM quininib (Q1) and 20 μM 1,4-dihydroxy quininib (Q7) significantly reduced OCR in Mel285 cells versus DMSO control. (G) 20 μM quininib, 1,4-dihydroxy quininib, and montelukast significantly reduced OCR in OMM2.5 cells versus DMSO control. Extracellular acidification rate (ECAR), a measure of glycolysis was evaluated in Mel285 (B) and OMM2.5 cells (H) following 24 h treatment. OCR:ECAR ratio was measured in Mel285 (C) and OMM2.5 (I) cells following 24 h treatment. Basal respiration was significantly reduced in Mel285 (D) and OMM2.5 (J) cells following 24 h treatment with quininib analogues at 20 μM. (E) ATP production was significantly reduced in Mel285 cells following 24 h treatment with quininib. (K) ATP production was significantly decreased in OMM2.5 cells following 24 h treatment with quininib and 1,4-dihydroxy quininib. (F) Maximal respiration was significantly reduced in Mel285 cells following 24 h treatment with quininib. (L) Maximal respiration was significantly reduced in OMM2.5 following 24 h treatment with quininib, 1,4-dihydroxy quininib, and montelukast. Data are expressed as mean + SEM. Statistical analysis was carried out using a paired t-test to compare within the same cell line. Data was normalised to cell number, as assessed by crystal violet assay. * p < 0.05; ** p < 0.01; *** p < 0.001.

CysLT₁ antagonists inhibit the growth of UM cell lines in in vivo zebrafish xenograft models. Zebrafish cell line-derived xenograft models were developed using Mel285 and OMM2.5 cell lines. Labelled UM cells were implanted into the perivitelline space (PVS) or vitreous of 48 hpf (hours post-fetilisation) Tg(fli1a:EGFP)^y1 zebrafish embryos and treated with the maximum tolerated dose of test compound, or DMSO control. (A) Treatment with 3 μM quininib (Q1), 10 μM 1,4-dihydroxy quininib (Q7) and 20 μM montelukast (Mon) significantly reduced the growth of Mel285 xenografts in the zebrafish eye (p < 0.0001). (B) Treatment with 10 μM 1,4-dihydroxy quininib and 20 μM montelukast significantly reduced the growth of OMM2.5 xenografts in the zebrafish eye (p = 0.0258 and p = 0.0439, respectively). Relative change in tumour volume was evaluated as the size of the tumours at three days post implantation (3 dpi/5 dpf) relative to the size immediately after implantation (at 0 dpi/2 dpf).