P30 15

Eight chamber glass slides (1.5 Lab-Tek II; Nunc) were coated with 0.01% poly-l-lysine (PLL) and dried at 60 °C for 1 h. Slides were coated with His-ICAM-1 (2.5 µg/mL; produced in house) alone or with His-MICA (2.5 µg/mL; Sino Biological), B7-H6-Fc (2.5 µg/mL; R&D Systems), αNKG2A (5 µg/mL; R&D Systems), or αNKp30 (10 µg/mL; P30-15; Biolegend or 210845; R&D Systems) in phosphate-buffered saline (PBS) overnight (4 °C). Bilayers were prepared as previously described (8 (link)) and functionalized with His-ICAM-1 (2.5 µg/mL) alone or with His-MICA (2.5 µg/mL), biotinylated-αNKp30 (10 µg/mL; P30-15), or biotinylated-αCD3 (5 µg/mL; OKT3; a gift from Andy Shepherd, GSK).

Spheroids were generated by seeding 1–3000 tumor cells into a Nunclon Sphera round-bottom 96-well plate (ThermoFisher Scientific) in 200 µl cRPMI. On day 3, medium was renewed, and 20 µg of EVs were added to appropriate wells in duplicates, together with 12 µM CellEvent Caspase-3/7 Green Detection Reagent (ThermoFisher Scientific). Spheroids were monitored every hr in an IncuCyte S3 instrument (Sartorius) for up to 5 days. Spheroid apoptosis was also monitored by fluorescence microscopy (FLoid Cell Imaging Station), and further analyzed by ImageJ. To assess involvement of receptor-ligand interactions, EVs and spheroids were co-cultured in presence of 10 µg/ml of antibodies toward DNAM-1 (DX11, Invitrogen), NKp46 (9E2, BioLegend), NKp30 (P-30–15, BioLegend), FasL (NOK-1, Invitrogen), NKG2D (5C6, eBioScience), or MICA/B.