Transwell analysis

Assays were performed as described previously. HUVECs (6 × 10⁴ at seeding in 200 μl) were cultured for 2 days in a 12-well Transwell chamber with polyester membrane of 8 μm pore size (Corning). To construct an endothelial monolayer model, the medium was changed daily until the cells were completely confluent. HUVECs were stimulated with exosomes (isolated from 10-cm Petri dishes) or phosphate-buffered saline (PBS) as a control for 24 h. For overexpression and interference experiments, 3 × 10⁴ HUVECs were transfected with circ-IARS and miR-122 overexpression or interference plasmid (Sangon Biotech, China) using Lipofectamine 3000 (Invitrogen, USA). Cells cultured until confluence. For transmigration experiments, the medium from the upper chamber was removed, and 3 × 10⁴ GFP-labeled AsPC-1 or Hs 766 T cells were added to 200 μl of serum-free endothelial cell culture medium. In all experiments, tumor cells that passed through the endothelial monolayer and attached to the lower side of the filter were imaged (Olympus) and counted to assess changes in endothelial monolayer permeability. Each experiment was repeated at least three times.