Mohammadreza Dastouri, Fatima Elmusa
Anti-CD226 antibody-mediated stimulation of NK-92 cells (sNK-92) represents a potential immunotherapeutic approach; however, its cytotoxic and apoptosis-associated effects in hormone-independent solid tumors remain insufficiently characterized. This study investigated the activity of sNK-92 cells against PC3 castration-resistant prostate cancer and SH-SY5Y neuroblastoma cell lines, using PNT1A normal prostate epithelial and BJ normal dermal fibroblast cells as non-malignant controls. Cytotoxicity was assessed by CCK-8 assay at target-to-effector (T:E) ratios of 1:1, 1:5, and 1:10, and markers historically associated with the intrinsic (BAX, caspase-9), extrinsic (caspase-8), and executioner (caspase-3) apoptotic pathways were evaluated quantitatively by ImageJ-based corrected total cell fluorescence (CTCF) analysis. sNK-92 cells produced significant ratio-dependent cytotoxicity against PC3 and SH-SY5Y cells, reaching 23.76% and 26.29%, respectively, at the 1:10 T:E ratio, with sNK-92 producing significantly greater cytotoxicity than unstimulated NK-92 at this ratio in both cell lines and additionally at the 1:5 ratio in SH-SY5Y cells; no significant reduction in CCK-8 viability was detected in PNT1A or BJ cells at any ratio. Quantitative immunofluorescence analysis demonstrated substantially increased relative fluorescence intensity of all four apoptosis-associated markers in sNK-92-treated PC3 and SH-SY5Y cells compared with their corresponding controls and, in most comparisons, with NK-92-treated cells, whereas changes observed in the PNT1A and BJ non-malignant models examined were markedly smaller. These findings provide preliminary quantitative evidence that anti-CD226-stimulated NK-92 cells exert a preferential cytotoxic effect on the tumor cell models examined, relative to the non-malignant models tested, and induce changes in apoptosis-associated markers consistent with engagement of apoptotic signaling. Further orthogonal validation is warranted.