Supplementary Figure S3 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S3 - PDF file 9497K, A: Immunochemical analysis of IL-18BP expression in cells from EOC ascites. Double staining with anti-IL-18BP (red, rabbit mAb clone EP1088Y, Epitomics) and anti-macrophage (brown, mAb HAM-56, Ventana Medical Systems) antibodies is shown. Biotin-labeled goat anti-rabbit followed by alkaline phosphatase-conjugated streptavidin and peroxidase-conjugated anti-mouse (BioSpa) were used as secondary antibodies. Fast Red and DAB (Sigma) served as substrates. Bar=100�m. Some of the cells stained by the anti-macrophage Ab were also stained by anti-IL-18BP Ab (see enlarged inset). However, the brightest IL-18BP positive cells were negative for the anti-macrophage Ab. B: Two-color immunofluorescence analysis of IL-18BP protein versus leukocyte surface markers expression in cells from EOC ascites. Numbers indicate the % of cells in each quadrant. IL-18BP positive cells were CD13 positive and CD14 low or negative (as all the CD14 positive cells were within the CD13 positive population: lower right panel). FITC: fluorescein isothiocyanate; APC: allophycocyanin; PE: phycoerythrin. CD14APC and CD14PE were from Miltenyi Biotec, CD13PE from BD Pharmingen and NKp46PE from Beckman Coulter. Cells were analyzed on a FACSCalibur (Becton Dickinson) flow cytometer</p

Supplementary Figure S2 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S2 - PDF file 2513K, A and B: Analysis of the correlation between IL-18 and IL-18BP protein levels in EOC sera (n. 47) (A) and ascites (n. 17) (B). No significant correlation (P=ns) was found by Pearson's test. C: Analysis of the correlation between IL18 and IL-18BP protein levels and IFN-? in the ascites of 15 EOC patients. IFN-? levels are low to undetectable and show no correlation with IL-18 or IL-18BP levels (P=ns). Pearson's correlation coefficients are shown (r). Lines represent the best-fit linear regression analysis with the 95% Confidence Interval</p

Supplementary Figure S6 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S6 - PDF file 185K, A: Analysis of the correlation between IL18BP and EBI3 mRNA levels in high grade (Type II) tumors in two microarray datasets of EOC. A significant correlation was found between EBI3 and IL18BP in both datasets, suggesting a relationship between the expression of EBI3 and IL18BP mRNA in EOC cell primary tumors. Pearson's correlation coefficients are shown (r). Lines represent the best fit linear regression analysis with the 95% Confidence Interval. B: Association between different EBI3 mRNA expression levels and Progression Free Survival (PFS) in Type II EOC of the Tothill microarray dataset. High EBI3 mRNA levels are associated to a shorter PFS time. Median PFS was 13 months for EBI3 levels higher than third quartile versus 21 months for EBI3 levels lower than first quartile (P=0.016). Solid line: cases with EBI3 levels lower than first quartile (n.52). Dashed line: cases with EBI3 levels higher than third quartile (n.49). P values were determined using log-rank test.</p

Supplementary Figure S4 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S4 - PDF file 640K, Immunochemical analysis of IL-18BP expression in EOC cell lines and xenotransplants. Immunochemistry with an anti-IL-18BP Ab shows virtually no reactivity in EOC cell lines (A upper left panel, B left panel), whereas IL-27-cultured A2774 cells (B middle panel) and areas of orthotopic SKOV3 (A) and A2774 (B right panel) xenotransplants express IL-18BP. The sections were observed with a Nikon Eclipse 80i light microscope equipped with a color camera imaging head, using a 40x objective. Bar=100micron</p

Supplementary Figure S1 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S1 - PDF file 744K, Receiver Operating Characteristic (ROC) curve for IL-18BP serum levels at diagnosis in patients with all types and stages of ovarian tumors (malignant n. 48 versus normal controls n. 13): the area under the curve (AUC) value is significant with the 95% confidence interval indicated in parentheses. SE=standard error</p

Supplementary Figure S5 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S5 - PDF file 470K, Biological effects of IL-27 on cells isolated from the ascites and on EOC cell lines. A-C: Involvement of IL-27 in IL-18BP expression in EOC cells. IL-18BP secretion is detected by ELISA in culture supernatants following A2774 and SKOV3 EOC cell stimulation with IL-27 (10 to 100 ng/ml for 48 h). The effect of IFN-gamma is shown for comparison (A). IL-27 stimulation (48 hours, at the indicated doses) mediates IL-18BP secretion by A2780 and OVCAR5 EOC cell lines, as detected by ELISA (B). ** P<0.005 at all dose levels versus untreated control. IL-27 up-regulates IL18BP mRNA expression in A2774 and SKOV3 cell lines, as assessed by RT-qPCR (C). D: A 10 minutes treatment with IL-27 (10 ng/ml) induces STAT1 tyrosine phosphorylation in A2774 and SKOV3 cells, as detected by Western blot analysis on cell lysates. Tubulin or beta-actin was used as loading controls. Numbers indicate molecular weight markers in kDa. E: Immunofluorescence and confocal microscopy analysis of constitive and IL-27-induced STAT1 tyrosine phosphorylation in EpCAM-positive and EpCAM-negative cells isolated from EOC ascites (specimen A98). Immunofluorescence was performed incubating 5x105 EOC cells with FITC-labeled anti-EpCAM antibody (Miltenyi). Cells, fixed and permeabilzed with BD Cytofix/Cytoperm solution (BD Pharmingen) were then incubated with rabbit anti-IL-18BP antibody (Epitomics) followed by DyLight 549-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories). PermaFluor (Thermo Scientific)-mounted slides were analyzed by confocal fluorescence microscopy using an Olympus (Olympus Optical) laser-scanning microscope FV500 equipped with an Olympus IX81 inverted microscope. Digital images were acquired through PLAPO 60x objectives, with the Fluoview 4.3b software program. Images were acquired sequentially as single trans-cellular optical sections, archived in TIFF and mounted using Photoshop. Merged images are shown. Bar=50micron. Arrows indicate EpCAM-positive pSTAT1-positive ascites cells, arrowheads indicate EpCAM-negative pSTAT1-positive ascites cells</p

Supplementary Figure S7 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S7 - PDF file 603K, Immunochemical analysis of EBI3 expression in EOC. Immunochemistry with an anti-EBI3 Ab shows EBI3 expression predominantly by reactive cells in both ascites (A) and tumor tissues (B). Arrows indicate examples of tumor cell nests. The sections were observed with a Nikon Eclipse 80i light microscope equipped with a color camera imaging head, using a 40x objective. Bar=100�m</p