Cell culture and cell lines
The human ovarian cancer cell lines IGROV-1, SKOV-3 and OVCAR-3 were obtained from Dr. M. Mallmann, MD (Life and Medical Sciences Institute Bonn, University of Bonn, Germany), the ovarian cancer cell lines A2780 and OVCAR-4 were provided by Westdeutsches Tumorzentrum, University of Duisburg-Essen, Germany. IGROV-1, SKOV-3 and OVCAR-3 were cultured in standard medium RPMI-1640 (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS, Biochrom, Berlin, Germany), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Karlsruhe, Germany). A2780 and OVCAR-4 were cultured in modified medium consisting of RPMI-1640 and DMEM (high-glucose Dulbecco’s Modified Eagle Medium, Invitrogen) (3:1 vol/vol) supplemented with 10% FCS (Biochrom), 1% penicillin/streptomycin (Invitrogen) and 1% sodium pyruvate (Invitrogen). Tumour cells were incubated in plastic culture flasks (Greiner, Solingen, Germany) at 37°C and 5% CO2 and continuously passaged by treatment with Accutase (PAA, Pasching, Austria) for 5 minutes at 37°C.
Isolation of NK cells and monocytes from PBMC’s of healthy donors
Blood samples (50–150 ml) of healthy donors were obtained in citrate monovettes (Sarstedt AG & Co., Nümbrecht, Germany) and diluted (1:1, vol/vol) with phosphate-buffered saline (PBS) and separated by density centrifugation (Biocoll Separating Solution, Biochrom AG, Berlin, Germany) at 25°C, 300g for 30 minutes. The mononuclear cell (MNC) fraction was collected, washed repeatedly with PBS and counted (CasyCounter; Innovatis-Roche, Bielefeld, Germany). For further isolation of NK cells and monocytes the magnetic cell separator NK isolation kit II and CD14-beads (Miltenyi Biotec, Mönchengladbach, Germany) were used according to manufacturer’s protocol. The separated immune cells were used for experiments immediately after isolation. Freezing of NK cells at −20°C resulted in a significant loss of activity. Therefore, all experiments in this study were performed with freshly isolated and purified NK cells. Purity of cell subsets was routinely tested and ranged from 90% to 97%.
Stimulation of purified NK cells and monocytes
Purified NK cells and monocytes, single or in co-culture (0.5 x 106 cells/well, cell-cell-ratio 1:1) were stimulated with 10 μg/ml of PstS-1 (obtained from M. Singh, PhD, Lionex GmbH, Braunschweig, Germany) in a 24 well plate (Greiner Bio-One, Frickenhausen, Germany). In some experiments 10mm Tissue Culture Inserts with 0,4 μm Anapore® Membrane (NUNC, Roskilde, Denmark) were inserted during stimulation to inhibit cell-cell contact between monocytes and NK cells. CD69- and NKG2D-expression on NK cells and the expression of CD80, CD86 and CD11c on monocytes were determined on day one and three of stimulation. NK subsets were differentiated by the addition of anti-CD16. Supernatants were collected for cytokine-analysis by ELISA and BioPlex assay.
Human IFN-γ- and IL-18-ELISA
The supernatants of stimulated NK cells and unstimulated controls were recovered over stimulation time (one and three days) and examined for the presence of IFN-γ, IL-12, -15 and −18. Detection of IFN-γ was performed with an anti-human IFN-γ-ELISA-kit (R&D Systems, Wiesbaden, Germany), IL-18 was detected by an IL-18-ELISA kit (Benders MedSystems (eBioscience, San Diego, USA). Both kits were used according to manufacturer’s protocol.
BioPlex assay for IL-12 and IL-15
For the detection of IL-12 and IL-15 a multiplex immunoassay (BioPlex assay) was used. Magnetic microspheres dyed with two fluorochromes and conjugated with specific monoclonal antibodies against the target protein were used according to the manufacturer’s instructions (Millipore Corporation, Billerica, USA). Each experiment was performed in duplicate using a 96-well plate. The analytes concentration was determined from the standard curve by analysis of mean fluorescent intensity values using a Bio-Plex array reader (Bio-Rad, Laboratories, Hercules, CA) with software (Bio-Plex Manager™ 6.0 Software).
Flow cytometric analysis (FACS)
For FACS-analysis the following antibodies were used: anti-CD107a-FITC (clone H4A3), anti-CD86-RPE (clone 2331 FUN-1), anti-CD11c-APC (clone B-ly6), anti-CD16-PE-Cy7 (clone 3G8) and all from BD Bioscience, Heidelberg, Germany. Anti-CD80-PE (clone MAB104) and anti-CD69-FITC (clone FN50) from Invitrogen, anti-CD14-PerCP/Cy5.5 (clone HCD14) from BioLegend GmbH, Fell, Germany, anti-NKG2D-RPE from R&D Systems, anti-MICA (clone AMO1) from Immatics, Tübingen, Germany, anti-HLA-classI-RPE (clone W6/32) from Dako. Corresponding isotypes were used as controls. Cells were analysed on a FACS Canto II using Diva software 6.0 (Becton Dickinson, Heidelberg, Germany).
CD107a degranulation assay
Since the lysosomal-associated membrane protein-1 (LAMP-1 or CD107a) in NK cells is expressed during degranulation and correlates with NK cell-mediated tumour cell lysis
 the expression of CD107a on NK cells was used to evaluate natural and antibody-mediated NK cell cytotoxicity. NK cells stimulated with monocytes with or without PstS-1 for one and three days were seeded on a flat-bottom 96-microtiter well plate (Greiner Bio-One). Tumour cells of the ovarian cancer cell lines A2780, IGROV-1, OVCAR-3, OVCAR-4 and SKOV-3 were coincubated in 1:1 cell ratio. The monoclonal anti-EGFR-antibody Cetuximab (Erbitux®, ImClone Systems, Bristol-Myers Squibb, New York, USA und Merck KGaA, Darmstadt, Germany) concentrated 1 μg/ml was directly added in experiments evaluating the ADCC of NK cells. Samples without antibody and unstimulated NK cells served as controls. NK cells were labelled with anti-CD107a-FITC or isotype mIgG1-FITC 1:20, the golgi-stop monensin (BD Golgi-stop, BD Bioscience) was added 1:600 after one hour incubation at 37°C in 5% CO2. After 5 hour incubation at 37°C in 5% CO2 cells were resuspended in 200 μl azide-PBS and immediately analysed in the flow cytometer.
RNA isolation and quantitative polymerase chain reaction
For quantitative polymerase chain reaction (qPCR) of IFN-γ, IL-12, IL-15 and IL-18 mRNA from co-cultured NK cells and monocytes stimulated with or without PstS-1 for one and three days were isolated using RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA concentrations and purity were determined at 260 nm/280 nm photometrically. For cDNA-synthesis the RNA of three donors and independent experiments was pooled and reverse-transcribed using Super Script II RNase H- Reverse Transcriptase kit (Invitrogen) and hexamer-random primer (Invitrogen) according to manufacturer’s protocol. The cDNA was used as template for the quantitative real-time PCR (Light cycler 2.0, Roche Diagnostics GmbH, Mannheim, Germany) using specific primer for IFN-γ, IL-12, IL-15 and IL-18 (Invitrogen). The following sets of primers were used: IFN-γ sense 5′-GAGTGTGGAGACCATCAAGGAAG-3′ and antisense 5′-TGCTTTGCGTTGGACATTCAAGTC-3′, IL-12A(p35) sense 5′-TGCCTTCACCACTCCCAAAACC-3′ and antisense 5′-CAATCTCTTCAGAAGTGCAAGGG-3′, IL-15 sense 5′-AACAGAAGCCAACTGGGTGAATG-3′ and antisense 5′-CTCCAAGAGAAAGCACTTCATTGC-3′, IL-18 sense 5′-GATAGCCAGCCTAGAGGTATGG-3′ and antisense 5′-CCTTGATGTTATCAGGAGGATTCA-3′. The conditions for amplification were as follows: initial denaturation at 95°C for 10 minutes, followed by 50 cycles of 10 s at 95°C, 20 s at the primer-specific temperature of 60°C, 20s at 72°C for extension. The amplified PCR products were separated by 2% agarose gel electrophoresis and visualised after ethidium bromide staining.
Western blot analysis for the determination of the EGFR-expression of various ovarian cancer cell lines was performed. Protein of total lysates (20 μl of each cell lines) were separated by SDS-7.5%-polyacrylamide gel and immunoblotted according to semi-dry-blot-method onto polyvinylidene difluoride membrane (PVDM, Roche Diagnostics). The membrane was incubated with the monoclonal primary antibody anti-EGFR (BioLegend) followed by AP-conjugated secondary goat-anti-rabbit IgG. Bands were visualised after application of the substrate CDP-star (Roche Diagnostics) and chemiluminescent transformation. The chemiluminescent signal was recorded with ChemiDoc-It Imaging system (UVP, Upland, California, USA) after exposure time of 20 minutes.
Data are presented as mean and standard error of several independent experiments. For statistical evaluation unpaired t-test was performed assuming statistical significance level of p ≤ 0.05. The statistical calculations and illustrations were performed using SigmaPlot for Windows Version 11 (Systat Software GmbH, Erkrath, Germany).