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

Table1_The deubiquitinase USP8 regulates ovarian cancer cell response to cisplatin by suppressing apoptosis.DOCX

The identification of therapeutic approaches to improve response to platinum-based therapies is an urgent need for ovarian carcinoma. Deubiquitinases are a large family of ubiquitin proteases implicated in a variety of cellular functions and may contribute to tumor aggressive features through regulation of processes such as proliferation and cell death. Among the subfamily of ubiquitin-specific peptidases, USP8 appears to be involved in modulation of cancer cell survival by still poorly understood mechanisms. Thus, we used ovarian carcinoma cells of different histotypes, including cisplatin-resistant variants with increased survival features to evaluate the efficacy of molecular targeting of USP8 as a strategy to overcome drug resistance/modulate cisplatin response. We performed biochemical analysis of USP8 activity in pairs of cisplatin-sensitive and -resistant cells and found increased USP8 activity in resistant cells. Silencing of USP8 resulted in decreased activation of receptor tyrosine kinases and increased sensitivity to cisplatin in IGROV-1/Pt1 resistant cells as shown by colony forming assay. Increased cisplatin sensitivity was associated with enhanced cisplatin-induced caspase 3/7 activation and apoptosis, a phenotype also observed in cisplatin sensitive cells. Increased apoptosis was linked to FLIPL decrease and cisplatin induction of caspase 3 in IGROV-1/Pt1 cells, cisplatin-induced claspin and survivin down-regulation in IGROV-1 cells, thereby showing a decrease of anti-apoptotic proteins. Immunohistochemical staining on 65 clinical specimens from advanced stage ovarian carcinoma indicated that 40% of tumors were USP8 positive suggesting that USP8 is an independent prognostic factor for adverse outcome when considering progression free survival as a clinical end-point. Taken together, our results support that USP8 may be of diagnostic value and may provide a therapeutic target to improve the efficacy of platinum-based therapy in ovarian carcinoma.</p

Additional file 2: of Simultaneous E-cadherin and PLEKHA7 expression negatively affects E-cadherin/EGFR mediated ovarian cancer cell growth

Figure S1. IHC with anti-E-cadherin on: upper panel, FFPE sections from fallopian tubal epithelium; and lower panel, eight FFPE samples of solid masses from HG-SOC patients. Control, a section only processed with the secondary antibody. Bar, 50 μm. Figure S2a. Representative phase contrast images of OAW42 MCAs and evaluation of live/dead cells; bar, 50 μm. The empty box highlights the image reported in Fig. 1f. b. Upper panel: representative phase contrast images of MCAs of control (CO) and E-cadh siRNA-treated OAW42 cells grown in Matrigel® for 6 days. Lower panel: measurement of OAW42 MCA area using ImageJ software. c. Control (CO) or E-cadherin siRNA-treated OVCAR5 cells. Upper panel: cell viability assay performed on silenced OVCAR5 cells; the number of cells was evaluated. Lower panel: E-cadherin levels in OVCAR5 cells after 5 days of culture. d. E-cadherin levels in treated cells of Fig. 2c. Control, (CO) or pooled E-cadherin siRNA. e. Western blotting on lysates from OAW42 starved (−) or EGF treated cells. Figure S3. Representative phase contrast images or fluorescent marked OAW42 and OVCAR5 live/dead cells; bar, 100 μm. Figure S4a. Western blotting on total cell lysates from six EOC cell lines. b. IF on fixed Caco2, OAW42, and OVCAR5 cells. c. Upper panel: representative western blotting on lysates from Caco2 cells infected with a control (NT) or with PLEKHA7 shRNA (shPLEKHA7). Starved cells (−). Lower left panel: western blotting with anti-PLEKHA7 Ab. Lower right panel: quantitative P-EGFR/EGFR ratio on PLEKHA7 silenced cells as above. Figure S5a. Confocal IF performed on LZRS or LZRS-PLEKHA7 infected OAW42 cells. Bar, 20 μm. The panel reports the stacks with single Ab of the merge images of Fig. 5d. b. Left panel: representative phase contrast images of LZRS or PLEKHA7 OAW42 MCAs grown in Algimatrix™. Right panel: cell viability assay of cells extracted from the sponge. (PDF 791 kb

Additional file 1: of Simultaneous E-cadherin and PLEKHA7 expression negatively affects E-cadherin/EGFR mediated ovarian cancer cell growth

Table S1. List of antibodies used in this study. Table S2. Quantitative evaluation of P-MAPK on E-cadherin silenced cells stimulated with EGF 20 ng/ml. The table reports the ratio between the target protein and β-actin, as the percentage of the control, from three different experiments performed on both OAW42 and OVCAR5 cells of Fig. 2a and from Fig. 2b. Table S3. Selected EOC samples from the publicly available datasets analyzed in the present study. (PDF 83 kb