Analysis of cells in collagen gels

Imaris 9.1.2 software (Bitplane AG, Zurich, Switzerland) was used to segment cells by creating surfaces with a filter of 200 μm³ to discard cell debris. For organotypic spheroid assay analysis, cell surfaces were filtered by centre of image mass versus z depth, where the z value was determined by orientating the 3D image stacks to the plane of view showing the cells on the glass and cells attached to the glass were excluded from analysis. Cells from the 3D dissociated cell migration assay were tracked using autoregressive motion, applying a threshold of 1500s to filter track duration. Intensity, morphological and tracking data were then exported and further analysed using GraphPad Prism software (La Jolla, CA, USA).