FIGURE 3.

Restoration of miR-126-3p sensitizes MTS to NK-mediated cytotoxicity. 3D MTS were prepared with HepG2 cells and LX2 cells in a 4:1 tumor cell to stellate cell ratio, using a methylcellulose-based protocol. The MTS were pretreated with control MNVs or tMNVs, stained with Calcein AM (green), and subsequently cocultured with NK cells labeled with CT Deep Red. (A) Representative confocal microscopy images of treated MCS were captured at 24 and 48 hours post-coculture. Scale bars represent 100 µm. NK cell-mediated cytotoxicity within MTS was evaluated using (B) lactate dehydrogenase activity assay, (C) granzyme B ELISA, and (D) perforin ELISA. The values are expressed as the mean ± SD from 3 replicates. Statistically significant data were represented as follows: *p < 0.05, ***p<0.001. Abbreviations: CT, CellTracker; MNVs, milk-derived nanovesicles; MTS, multicellular tumor spheroids; tMNVs, therapeutic MNVs.