Matrigel-Coated Tumor Spheroid Assay

MCTS were prepared according to the hanging-drop method as before [44 (link)]. Formed MCTS were then transferred to wells of Lab-Tek™ Chambered Coverglass which had been coated with 70 μl of Matrigel (5 mg/ml) in SFM. Spheroids were covered with an additional 70 μl of Matrigel (5 mg/ml) in SFM and, after 20 min at 37°C, covered with 400 μl of complete medium. Nitroxoline (2.5 or 5 μM), CA-074 (5 μM), CA-074Me (5 μM) or DMSO (0.05 %) were added to the Matrigel and the medium and the growth of spheroids was monitored for up to three days by measuring the spheroid dimensions under a light microscope, using an ocular micrometer. Spheroid volume was calculated according to the equation: V = (π × (spheroid length) × (spheroid width)²)/6. In a separate experiment, CatB activity was assayed using a Magic Red^TM Cathepsin B Assay Kit according to manufacturer's instructions (Immunochemistry Technologies, Bloomington, MN). Images of tumor spheroids were obtained using an Olympus IX 81 motorized inverted microscope and Cell^R software (Olympus, Tokyo, Japan).