15353-j-veluchamy

4 NK cell ADCC enhances treatment efficacy in colorectal cancer | 107 Materials and Methods Cell lines Cell lines A431, COLO320, SW480, Caco-2, SW620, HT-29 were obtained from ATCC and cultured in Dulbecco's modified medium (DMEM; Invitrogen, Carlsbad CA, USA) containing 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal calf serum (FCS; Integro, Zaandam, The Netherlands) Cell cultures were passaged every 5 days. Cultures were maintained in a 37°C, 95% humidity, 5% CO 2 incubator. Peripheral blood NK cell isolation and activation from whole blood specimens Mononuclear cells (MNCs) were isolated from peripheral blood using Lymphoprep™ (STEMCELL Technologies, The Netherlands) density gradient centrifugation from buffy coats obtained from anonymous healthy blood donors (Sanquin Blood Supply, Amsterdam) with written informed consent for research use, in accordance with the ‘‘Code for Proper Use of Human Tissues’’ as formulated by the Dutch Federation of Medical Scientific Organizations (www.fmwv.nl ) 44 . CD56 + NK cells were isolated from MNCs using a MACS Human NK cell isolation kit (Miltenyi Biotech, BergischGladbach, Germany) according to themanufacturer’s instructions. The cell number and purity of the isolated NK cell fraction were analyzed by flow cytometry. Isolated NK cells were activated overnight with 1000U/ml IL-2 (Proleukin ® ; Chiron, München, Germany) and 10ng/ml IL-15 (CellGenix) for use in cytotoxicity assays. NK cell purity and viability were checked using CD3 PE, 7AAD (BD Biosciences), CD56 APC Vio 770, and CD16 APC (Miltenyi Biotech). The parameters compared before and after activation were NK purity (CD56 + %, 83 ± 9 % vs. 82 ± 9%), NK CD16% 88 ± 10 % vs 85 ± 11%) and NK viability (91 ± 3 % vs 86 ± 2%) respectively. Anti- EGFR monoclonal antibodies used for the cytotoxicity assay The anti-EGFR mAbs cetuximab (Erbitux ® ) and panitumumab (Vectibix ® ) were purchased through the VU University medical center pharmacy. Biotinylation of anti-EGFR mAbs To assess the binding of cetuximab and panitumumab to EGFR expressing target cells, anti-EGFR mAbs were concentrated using Amicon ultra centrifuge 0.5ml 30K tubes (EMD Millipore, Netherlands). Concentrations of the mAbs were adjusted to 20mg/ml and then biotinylated using Biotin-N-hydroxysuccinimide ester (Sigma Aldrich, St Louis, USA) according to the manufacturer’s instructions. The biotinylated anti-EGFR mAbs were incubated with A431 (1x10 6 ) cells for 1hr, washed twice in ice cold PBS and stained with