Fig. 2

Surface MUC21 on cancer cells suppresses ADCC activity but has no impact on CDC. (A-B) Raji-tet-MUC21 cells were cultured with or without Dox (1 µg/mL) for 24 h. (A) Raji-tet-MUC21 cells were co-incubated with NK-92-CD16 cells at an E:T ratio of 0.5:1 for 4 h in the presence of varying concentrations of human IgG1 or rituximab. NK-92-CD16 cytotoxicity against Raji cells was measured by the luciferase activity of the surviving Raji cells. (B) Representative FACS analysis (above panel) of the surface CD107a expression of NK-92-CD16 cells cultured with Raji-tet-MUC21 cells in the presence of 10 µg/ml of hIgG1 or rituximab. A summary graph (below panel) showing the percentage of CD107a expressing NK-92 cells. (C) FACS analysis of surface MUC21 expression in wild-type NCI-H441 cultured in both 2D and 3D conditions, as well as MUC21 knockdown NCI-H441 cells (shMUC21) cultured in 2D. (D-E) The NCI-H441 cells, which were stably expressing scramble shRNA (shCTL) or shRNA targeting MUC21 (shMUC21), were cultured in 2D condition with NK-92-CD16 cells at an E:T ratio of 0.5:1 for 4 h. The co-culture was conducted in the presence of human IgG1 (0.1 µg/ml) or cetuximab (0.1 µg/ml). (D) NK-92-CD16 cytotoxicity against NCI-H441 cells was measured by luciferase activity in surviving NCI-H441 cells. (E) A summary graph of surface CD107a expression of NK-92-CD16 cells. (F) A549-tet-MUC21 cells were cultured with or without Dox (1 µg/ml) for 24 h and then co-incubated with NK-92-CD16 cells at an E:T ratio of 0.5:1 for 4 h in the presence of either human IgG1 (0.1 µg/ml) or cetuximab (0.1 µg/ml). (G) Raji-tet-MUC21 cells were cultured with or without Dox (1 µg/ml) for 24 h and incubated with indicated concentration of rituximab for 15 min followed by addition of 10% human complement. Cell viability was then measured by luciferase activity. Statistical significance was determined by a two-way ANOVA with Holm-Sidak comparisons in (A) or one-way ANOVA with Holm–Sidak multiple comparisons in (B), (D) and (E) or multiple t tests with correction for multiple comparisons using the Holm–Sidak method in (F). *P < 0.01, **P < 0.01, ***P < 0.001, ****P < 0.001; and ns, not significant