Materigel

In the migration experiment, Materigel gel was not paved. In the invasion experiment, Materigel® [Corning™, USA] and Opti-MEM® I Reduced-Serum Medium [Thermo™, USA] were diluted at a ratio of 1: 8, with 50 μL of each chamber spread at the base of the upper-chambers and incubated for 1 h to render it semi-solidified. CNE-2Z and HNE-1 cells were inoculated into the upper-chambers within serum-free RPMI-1640 medium, and 600 μL RPMI-1640 medium carrying 10% FBS were introduced into the lower wells. Regarding the migration experiment, cells were grown for 24 h. Regarding the invasion experiment, cells were grown for 48 h. The upper-chambers were consequently removed, fix-treated using 4% paraformaldehyde for 15 min, dyed using crystal violet for 10 min, subjected to PBS wash-step, followed by careful removal of excess dye using a cotton swab, and finally observed/counted. The cellular population density within five randomly selected fields-of-vision under light microscopy were determined, with all experimental runs performed on three separate occasions.

HUVECs (5 × 10⁴/well) were seeded onto materigel (Corning, #356230, USA) coated 48-well plates. Then, EVs (10 μg/ml) obtained from different sources were added to the plates seeded with HUVECs. HCQ (100 μM) or PBS was added to the cell culture with EV-depleted FBS. After incubation for 6 h at 37 ℃ in a humidified atmosphere with 5% CO2, the tube structure formation was imaged using microscope. The Image J was used to quantify the tube length.