Clonogenic Metastasis Assay

Lung metastasis was assessed using the clonogenic metastasis assay as described by Pulaski and Ostrand-Rosenberg (43). Briefly, the lungs were harvested from tumor-bearing mice and digested with collagenase IV solution (1.5 mg/ml, Sigma) at 4°C for 1 h. The slurry was filtered using 40 μm cell strainers and cultured in complete DMEM containing 6-thioguanine (final concentration 60 μM) at 37°C for 10–14 days when the metastatic 4T1 colonies became visible. The colonies formed in each culture dish were fixed with methanol, stained with 0.3% methylene blue and counted.