OPTIMIZATION OF THE FLOW CYTOMETRIC METHOD FOR ANALYZING NK CELL CYTOTOXIC ACTIVITY IN BREAST CANCER

Objectives: To establish a protocol for analyzing NK cell cytotoxic activity (NKAc) in breast cancer patients using the flow cytometric technique. Methods: We first optimized the protocol based on the previously published studies, focusing on choosing fluorescent dye concentration, incubation time length, and Effector:Target cell ratio (E:T ratio). Subsequently, we preliminarily applied the established protocol to characterize NKAc in some breast cancer patients and healthy controls. Lastly, we compared the NKAc versus NK cell secretory activity (NKAs) data obtained from the study. Results: The fluorescent dye concentration could be used according to the manufacturer's suggestion (CFSE: 2.5 - 10µM; Zombie NIR: 1:1000 - 1:100 dilution). The co-culture period of effector and target cells and the E:T ratio could be 4 hours and 5:1, respectively. NKAc was reproducible for 1 month. 2/7 breast cancer patients had NKAs < 200 pg/mL and NKAc < 10%. Conclusion: It is feasible to examine NKAc in breast cancer patients via the flow cytometric method established in this study. Further studies are needed to validate the diagnostic and prognostic value of NK cell function tests.