15353-j-veluchamy

4 NK cell ADCC enhances treatment efficacy in colorectal cancer | 109 SW80, SW620 and HT-29) were washed and co cultured with activated NK cells. To measure degranulation by NK cells, anti-CD107a PE (Miltenyi Biotech, Germany) was added in 1:20 dilution to the wells. After incubation for 4h at 37°C, 75 µl supernatant was collected and stored at -20°C for analysis of cytokine production. Cells in the remaining volume were harvested and stained with 7AAD (1:20). Degranulation of NK cells was measured by detecting cell surface expression of CD107a. After 4 hrs of incubation at 37°C, CD56 APC Vio 770 (1:25) and CD16 APC (1:25) (Miltenyi Biotech, Germany) were added to the co-cultures and NK CD107a degranulation was measured for CD56 + NK, CD56 + CD16 + NK and CD56 + CD16 - NK cells. IFNγ production assay Production of IFNγ by target cell-stimulated NK cells was measured in the supernatant of the co-cultures by ELISA (Sanquin, Amsterdam, The Netherlands). Absorbance was measured at 450 nm with a Multiscan MCC/340 ELISA reader (Titertek, Huntsville, Alabama, USA). Primary colon tissue dissociation and storage Colon cancer tissues from patients participating in trials conducted at the VU University medical center in Amsterdam, The Netherlands were collected and processed as described 45 after written informed consent and used for this study according to protocols approved by the VUmc IRB (IRB00002991; IORG number 0002436) 46 . Tumor material was further confirmed histologically and were screened for HLA class I (Clone W6/32), and EGFR expression. Tumor tissues were washed thrice, scraped, cut into small fragments and digested mechanically using collagenase A (Roche Diagnostics, The Netherlands) based growth medium. Digestion was carried out in a sterile glass flask with continuous stirring for 45 mins. This step was repeated twice, and the single cell suspension was collected through a 45µm sterile filter. Cells were counted and then frozen under controlled conditions in liquid nitrogen. Primary tumor cells were thawed, ficoll based density grade separation was done to remove dead cells and finally resuspended in DMEM +10% FCS medium for the cytotoxicity assays. RAS typing The mutational status of KRAS exon 2/3/4, NRAS exon 2/3/4 and BRAF exon 15 was assessed by high resolution melting (HRM) assay followed by Sanger sequencing of HRM- PCR products with an aberrant melt curve, essentially as described previously 47,48 . Statistical analysis Statistical analysis was performed using Graph Pad Prism software. Differences between conditions were determined using two-way anova with multiple comparisons between