FIGURE 2.

Tumor cell delivery of miR-126-3p increases their sensitivity to NK cell-mediated cytolysis. (A, B) HepG2 and SNU449 cells pretreated with control MNV or tMNV, or untreated controls, were cocultured with NK cells at 5:1 or 10:1 effector-to-target (NK: tumor cell) ratio. NK cell-mediated cytolysis was evaluated after 6 hours by LDH release. (C) Granzyme B and (D) perforin levels were evaluated in control MNV or tMNV pretreated HepG2 cells cocultured with NK cells. (E) Still images from live cell imaging of NK cells cocultured with HepG2 cells or SNU449 cells at 10:1 effector-to-target ratio identify NK cell-mediated cytotoxicity. (F) HepG2 cells were cocultured with NK cells. After 48 hours, adherent cells were harvested, and the expression of pro- and cleaved casp3 was evaluated by western blot. Representative immunoblot and quantitative data from 3 separate experiments are shown. (G) HepG2 cells were transfected with 2 siRNA to MICA, and an immunoblot for MICA was performed on lysates from transfected cells, showing robust silencing. (H) NK cell-mediated cytotoxicity was evaluated by LDH release in HepG2 cells transfected with NC or one of 2 siRNA to MICA and cocultured with or without NK cells at 5:1 and 10:1 effector-to-target ratios. *p < 0.05, **p <0.01, ***p<0.001, ****p<0.0001. Abbreviations: casp3, caspase-3; LDH, lactate dehydrogenase; MICA, major histocompatibility complex I-related chain A; MNVs, milk-derived nanovesicles; NC, nontargeting control; NK cells, natural killer cells; tMNVs, therapeutic MNVs.