Spheroid invasion assays were performed as described previously (32 (link)). 1,000 231-GFP or 2,000 PyMT-GFP cells were seeded in round-bottom, low-attachment 96-well plates and centrifuged at 3,000 g for 3 minutes to promote spheroid formation. Spheroids were allowed to grow for three days before addition of an ECM mixture of 1 mg/mL collagen I (354236; Corning), 10 mmol/L NaOH, 7.5% 10X DMEM, and 50% DMEM with or without 20 µg/mL native human collagen IV (ab7536; Abcam). Once ECM had solidified, 50 μL of culture media was added to maintain moisture and humidity in the well. In inhibitor studies, this media included DMSO as a vehicle control or small molecule inhibitors. Spheroids were imaged in 3D on the day of ECM addition and after 4 days of growth using a Keyence BZ-X710 microscope (Keyence).
For 3D single-cell invasion assays, 20,000 231-GFP or PyMT-GFP cells were suspended in ECM mixture described above with or without 20 µg/mL collagen IV and seeded into 48-well tissue culture plates. Once ECM had solidified, 50 μL of culture media to maintain moisture and humidity in the well. In inhibitor studies, cells were treated with DMSO as a vehicle control or small molecule inhibitors 1 hour before imaging. Cells with imaged live in 3D over 16 hours using a Keyence BZ-X710 microscope and invasive speed was quantified as in reseeding experiments.
For 3D single-cell invasion assays, 20,000 231-GFP or PyMT-GFP cells were suspended in ECM mixture described above with or without 20 µg/mL collagen IV and seeded into 48-well tissue culture plates. Once ECM had solidified, 50 μL of culture media to maintain moisture and humidity in the well. In inhibitor studies, cells were treated with DMSO as a vehicle control or small molecule inhibitors 1 hour before imaging. Cells with imaged live in 3D over 16 hours using a Keyence BZ-X710 microscope and invasive speed was quantified as in reseeding experiments.
