Protease-indipendent angiogenic properties and invasion were evaluated by in vitro capillary morphogenesis and 3D-Boyden chamber assays, as described above, with Matrigel coating in the presence of a physiological protease inhibitor cocktail, consisting of α2-antiplasmin (plasmin inhibitor; 5 μg/ml), Cystatin (cysteine protease inhibitor; 5 μM), PAI 1 (plasminogen activator inhibitor; 10 ng/ml), TIMP1, TIMP2 and TIMP3 (metallo-protease inhibitors; 0,5 μg/ml each one) purchased by Abcam. A completely artificial protease inhibitor cocktail (composed by Ilomastat, leupeptin, pepstatin A, E-64 and aprotinin; Sigma Aldrich) used in a previous study [13 (link)] was also used. Concentrations used were selected according to the manufacturer’s instructions and literature [16 (link), 17 (link)]. Protease inhibitor cocktails were added to un-polymerized Matrigel solution on the upper surface of the porous filter. To induce the amoeboid phenotype, cells were treated overnight with the protease inhibitor cocktails at the same concentrations used in the invasion assay. Cell viability upon protease inhibitors treatment was evaluated by Trypan blue dye (Sigma) exclusion assay.
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