In vitro permeability assay

Transwell filter inserts (pore size 1.0 mm, 12 mm diameter, polyester membrane, Corning, New York, USA) were coated with fibronectin (10 mg/ml; Sigma). Then, HUVECs were seeded (0.3 x 10⁶ cells/ well) and grown on transwell filters for 48 hr until reaching confluency. Confluent monolayers of HUVEC cells were treated with similar amounts (10–100 mg) of 4T1-EVs, PBS (as a negative control) or with 100 ng/ml TNF-a (as a positive control) overnight. FITC-dextran (MW ~70,000; Sigma) was added to the top well at 25 mg/ml for 20 min at 37 ̊C, and fluorescence was measured in the bottom well using a fluorescence plate reader (Berthold Tris Star 2; 485 nm excitation and 520 nm emission).  Cells were washed for three times and were fixed for immunofluorescence (described below). At the end the polyester membrane of each transwell was cut carefully using a carbon steel surgical blade. Then using a high precision tweezer, the polyester membrane was transferred to a slide and mounted with DAPI-containing Vectashield.