Patients and tissues
All experiments using human tissues were performed in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Fudan University. The clinical samples of lung cancer patients were obtained from Fudan University Shanghai Cancer Center (Shanghai, China). The tissues consisted of samples from 132 patients with NSCLC, including 100 cases of adenocarcinoma (ADC), 23 cases of squamous cell carcinoma (SCC), and 9 cases of other types of NSCLC at different stages. On each tissue, healthy lung tissues were also included.
Wound healing assay
Wound healing was analysed using IBIDI Culture-Inserts (IBIDI GmbH, Martinsried, Germany). Cells were dissociated from plates using 0.05% (w/v) trypsin and seeded into culture-insert plates at a concentration of 2 × 104 cells per culture well. After 24 hours of incubation, culture inserts were removed. Photographs of the movement of cells into the scratch area were taken after 24 h using a light microscope.
Cell migration
Cell migration was measured using transwell inserts (Corning Life Sciences, Tewksbury, MA) according to manufacturer’s instructions. Briefly, transwell chambers (8 mm pore size) were coated with 50 μL of diluted matrigel. Cells suspended in serum-free medium at a density of 1.5×105 cells/mL were seeded (0.1 ml) in the upper chambers and 0.5 mL medium containing 10% FBS was added to the lower chambers, and PBS, 10 nmol/L thrombin, 25 nmol/L r-hirudin, 50 nmol/L DTIP, 10 nmol/L thrombin + 25 nmol/L r-hirudin and 10 nmol/L thrombin + 50 nmol/L DTIP were added to both of the upper and lower chambers. After culturing for 24 hours, cells were fixed in methanol and stained with 0.1% crystal violet. The cells on the bottom of the filters were counted.
F-actin staining assay and confocal microscopy
Cells were grown on 35-mm glass-bottom dishes and fixed with 4% paraformaldehyde for 15 minutes and then permeabilized with 0.5% Triton X-100 in PBS for 10 minutes. The cells were then blocked with 1% BSA for 20 minutes. Cells were incubated with YF-488 phalloidin (US Everbright®Inc) for 30 minutes and then stained with DAPI for 5 minutes. The cells were observed under a confocal microscope.