Cells and culture conditions
B16M cells (B16F10 subline) were cultured in DMEM supplemented with 10% FCS and penicillin-streptomycin (Sigma Chemicals Co., St. Louis, MO) [10]. B16M-conditioned medium (B16M-CM) was obtained from subconfluent cells cultured for 12 hours as previously described [13].
Hepatic metastasis model and treatment schedule
Syngeneic C57BL/6J mice (male, 6-8 weeks old) were obtained from IFFA Credo (L'Arbreole, France). Animal housing, care, and experimental conditions were conducted in conformity with institutional guidelines, in compliance with the relevant national and international laws and policies. Hepatic metastases were produced by intrasplenic injection of B16 cells as previously described [14]. Mice were killed on the twelfth day afterwards. Liver tissue was processed for histology [14]. Fifteen 4 μm-thick tissue sections of formaldehyde-fixed liver (five groups, separated 500 μm) were stained with H&E. An integrated image analysis system (Olympus Microimage 4.0 capture kit) connected to an Olympus BX51TF microscope was used to quantify the number, average diameter, and position coordinates of metastases. Percentage of liver volume occupied by metastases and metastasis density (foci number/100 mm3) were also determined [14]. In order to study the effect of RVL (Sigma-Aldrich Chemicals Co, St Louis, MO) on hepatic metastasis development, some mice (10 per group) received 1 mg/kg/day RVL dissolved in ethanol (5%), via intragastric tube, from day 1 to 12. Control mice received the same volume of vehicle. Each experiment was carried out three times.
Quantitative assay on hepatic retention of circulating melanoma cells
B16M cells were stably transfected with a construct containing the Photinus pyralis luciferase gene coding sequence under transcriptional control of the cytomegalovirus promoter and the neomycin resistance gene [16]. Three hundred thousand viable luciferase-transfected B16M cells were intrasplenically injected into C57BL/6J mice (n = 30). Some mice received 1 mg/kg/day RVL five days prior to B16M-Luc cell injection. All mice were killed 18 hours later, and livers were analyzed for luciferase activity (Promega Co., Madison, WI) as described previously [16].
Isolation of hepatic sinusoidal cells and enriched primary culture of endothelial cells
HSE cells were isolated from syngeneic C57BL/6J mice (male, 6-8 weeks old) identified, and cultured as described previously [16]. Briefly, isolated mouse liver cells were obtained by two-step collagenase perfusion. A non-parenchymal liver cell fraction was further purified by centrifugation in a Percoll® gradient (Amersham; Uppsala, Sweden). Kupffer cells were then removed by selective adherence to plastic substrate, HSE cells were phenotypically characterized by flow cytometry analysis with specific antibodies against CD31 (PECAM-1 Sigma, St Louis), HLA class II and CD40, (both from BD Biosciences); CD106 (VCAM-1) and CD14 (both fromBD Pharmingen, San Diego, CA), smooth muscle alpha actin (ASMA, Sigma-Aldrich, St Louis, MO). HSE cells were seeded at 2 × 105 cells/well in RPMI-1640 culture medium (Sigma Chemicals, St. Louis, MO) supplemented with 10% FCS (Life Technologies, Gaithersburg, MD) onto 24-well tissue culture plates pre-coated with type I collagen solution (0.03 mg/ml) (Collagen Biomaterials, Palo Alto, CA) and allowed to spread for 45 min at 37°C and 5% CO2.
Enzyme immunoassay of IL-18 concentration in hepatic blood and supernatants of cultured cells
Serum samples were obtained from hepatic (suprahepatic vein) blood of adult male C57/B1/6J mice 18 hours after intrasplenic injection of B16M cells. Some mice received (1 mg/kg/day) RVL from day 1 to 5 via intragastric tube prior to melanoma cells injection. Primary cultured HSE cells were incubated in the presence of absence of 2.5 μM RVL or recombinant VEGF antibody for 30 min, after which B16M-CM or 10 ng/ml of recombinant VEGF were added. Eight hours later, the supernatant from the treated HSE cells were collected. IL-18 concentration in serum from hepatic blood or in culture cell media was detected using a competitive enzyme immunoassay (R&D Systems, Minneapolis, MN).
Immunohistochemical analysis of p65 nuclear translocation
B16M cells (1 × 104 cells/well) were grown on 8 μm-chamber glass slides. Cells were serum-starved for 24 h and treated with IL-18 (100 ng/ml) for 30, 60 and 120 min. In some experiments, cells received 2.5 μM RVL 30 min prior to IL-18 treatment. Once treatment time had finished, cells were fixed in 4% formaldehyde (in PBS) for 30 min at room temperature and permeabilized in 1% SDS for 10 min. Non-specific binding was blocked for 1 hour with 10% bovine serum in PBS buffer. Cells were incubated with 1.5 μg/ml rabbit anti-p65 polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 1 h at room temperature. Further wash steps removed unbound antibody prior to the addition of the secondary Alexa 594 goat anti-rabbit antibody. Images were acquired on a BD Pathway™ Bioimager.
Western blot analysis of p65 in nuclear fractions
Subconfluent cultures of B16M cells were treated as above described for 60 min. Then, they were harvested and incubated in lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM DTT, 0.1% Nonidet P-40 and 0.5 mM PMSF) for 20 minutes on ice. The crude nuclei were collected by centrifugation, further incubated in 20 mM HEPES (pH 7,9), 0.4 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF) for 20 minutes on ice and clarified by micro-centrifugation and frozen. Fifty micrograms of nuclear extracts were resolved on 12% of SDS-PAGE and p65 protein was analyzed by Western blot using rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Lamin-B expression was used as loading control.
Western Blot analysis of VCAM-1
Freshly isolated HSE cells were seeded onto 24-well plates for 24 hours. Then, they were cultured for 12 hours in the presence of basal medium, B16M-CM or 1 ng/ml IL-18. In some experiments, HSE cells received 2.5 μM RVL 30 min prior to B16M-CM or IL-18 treatment. HSE cells were disrupted in 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, 2 mM EDTA, 10 mM NaF, 10 μg/ml leupeptin, 20 μg/ml aprotinin, and 1 mM PMSF. Then, 40 μg of protein from cell lysates were separated by 12% SDS-PAGE followed by Western analysis using rat anti-mouse VCAM-1 monoclonal antibody and β-tubulin (both from Santa Cruz Biotechnology, Santa Cruz, CA) and the appropriate secondary antibodies.
Blot was imaged using a Syngene G-Box gel imaging system (Synoptics Ltd., Cambridge) and band density analyzed using Gene Tools analysis software.
Measurement of H2O2 production from B16M cells in vitro
B16M cells were cultured in DMEM without phenol red with 20 μmol/L 2', 7'-dichlorofluorescein-diacetate (DCFH-DA) as described [12]. H2O2 produced by incubated cells oxidizes DCFH to the highly fluorescent DCF so that fluorescence intensity is directly proportional to the amount of H2O2 produced by the cells. DCF fluorescence was recorded at 485/22-nm excitation and 530/25-nm emission filters. Non-DCFH-DA-incubated cells were used to determine basal autofluorescence. B16M cells were treated with 1 ng/ml IL-18, for 2 hours. In some experiments B16M cells were incubated with 2.5 μM of RVL 30 minutes prior to IL-18 addition. H2O2 production per well was quantified in arbitrary fluorescence units after subtracting basal autofluorescence.
Proliferation assay
B16M cells were cultured overnight in DMEM plus 10% FCS. Then, they were incubated for 72 hours in the presence of 0.1 ng/ml IL-18 supplemented with 0.5% FCS. Control cells received the same culture medium without the cytokine. In some experiments, 2.5 μM RVL was added to control and IL-18-treated B16M cells. After 48 hours incubation, B16M cell proliferation was measured using sulforhodamine 101 protein assay, as described previously [15].
B16M cell adhesion assay to primary cultured hepatic endothelial cells
B16M cells were labeled with 2', 7'-bis-(2-carboxyethyl)-5,6-carboxyfluoresceinacetoxymethylester solution (Molecular Probes, Eugene, OR) and added to primary culture of HSE cells (2 × 105 cells/well). Eight minutes later, wells were washed three times with fresh medium. The number of adhering cells was determined using a quantitative method based on a previously described fluorescence measurement system [14]. HSE were treated with B16M-CM for 6 hours prior to the addition of B16M cells. In some experiments, HSE cells received 2.5 μM RVL or vehicle 30 minutes prior to B16M-CM. In other experiments, B16M cells were pretreated with IL-18 for 6 hours prior to the adhesion assay. In this case, B16M cells received 2.5 μM RVL or vehicle 30 minutes before IL-18.
Statistical analyses
Data were expressed as means ± SD. Statistical analysis was performed by SPPS statistical software for Microsoft Windows, release 6.0 (Professional Statistic, Chicago, IL). Homogeneity of the variance was tested using the Levene test. If the variances were homogeneous, data were analyzed by using one-way ANOVA test with Bonferroni's correction for multiple comparisons when more than two groups were analyzed. For data sets with non-homogeneous variances, ANOVA test with Tamhane's posthoc analysis was applied. Individual comparisons were made with Student's two-tailed, unpaired t test (program Statview 512; Abacus Concepts, Inc., for Macintosh). The criterion for significance was P < 0.01 for all comparisons.