Figure 6.
TRAIL involvement in NK activation
(A) Caspase 3/7 activity of A375, MEL-BRO and SK-MEL-28 was measured every hour after addition of rhTRAIL in increasing concentrations. Caspase 3/7 activity is shown as total green fluorescence area of the mean ± SEM of technical quadruplicates of a representative experiment.
(B) Caspase 3/7 and caspase 8 activity was measured after 3h of co-culture with A375 using various blocking combinations. Data is shown as mean ± SEM of the caspase activity relative to isotype ctrl, n = 2 donors.
(C–E) NLR-A375 cells were seeded at 1∗103 cells/well and were allowed to form spheroids for 2 days. NK cells were pre-incubated with the various blocking mAbs combinations and added to the spheroid in a final E:T ratio of approximately 25:1.
(C) Representative graph of total integrated intensity of the red calibrated unit (RCU) shown as the mean ± SEM of technical quadruplicates for donor #5.
(D) Percentage of cytotoxicity relative to isotype ctrl. Calculated by first determining % cytotoxicity of each condition using the formula 100-(AUC condition/AUC A375 only∗100) followed by calculating % of cytotoxicity relative to isotype ctrl (=100%). Data is shown as mean ± SEM of n = 3 donors, divided among 3 independent experiments. Statistical analysis was performed using paired t-test between de indicated condition.
(E) Representative images of A375 spheroid after 1h and 10h of co-culture with NK cells at 4× magnification. A375 cells are visible in red and NK cells are surrounding the A375 spheroid. Significance is shown as p < 0.05 ∗, <0.01 ∗∗, 0.001 ∗∗∗, 0.0001 ∗∗∗∗.