Rat tail collagen 5

U87-NT cells were seeded at 3 × 10³/well in low adherence 96 well plates (ThermoFisher, Altrincham, UK) as previously described [14 (link)]. Three days after seeding, spheroids contained within the wells were embedded in rat tail collagen V (Corning Life Science, Glendale, AZ 85301, USA), polymerisation was achieved with 1 M NaOH. The inhibitors CCG-1423 and Rhosin-HCl (Tocris, Bristol, UK) were resuspended in DMSO and added at a predetermined anti-migratory concentration (CCG-1423: 500 nM, Rhosin: 1 µM). Control spheroids were mock-treated with DMSO-supplemented medium only. Spheroids were allowed to grow for 72 h in collagen, fixed with 4% PFA and labelled with Phalloidin-TRITC (ThermoFisher, Altrincham, UK) in their collagen plugs. Image z-stacks of spheroids were acquired on a Zeiss 880 LSM confocal microscope (Zeiss, Birmingham, UK) with an EC plan-neofluar 10× objective. For analysis maximum projections of the z-stacks were used. The resulting grey-scale images were inverted and using the free-hand selection tool in ImageJ, the outline of the spheroid as well as of the gaps between migration cells were drawn and the area measured (for illustration see Supplement Figure S2).