Additional file 1: Supplementary material. of A 3D in vitro model of the human breast duct: a method to unravel myoepithelial-luminal interactions in the progression of breast cancer

Figure S1. Promoter efficacy in primary cultures of myoepithelial and luminal cells. a Representative images of GFP expression in myoepithelial and luminal cells 48 h following infection with neuraminidase-treated lentiviral particles driving GFP expression under either human/mouse CMV, human/mouse EF1α, CAG, PGK and UBC promoters. Scale bar = 20 μm. b Mean fluorescence intensity (MFI) values of myoepithelial and luminal cells 48 h post-infection with lentiviral particles as in (a). Images and values are representative of cells derived from two donors. Figure S2. Spheroids formed in Matrigel cultures express markers of both luminal and myoepithelial cells. Expression of cytokeratin (CK) 8 and P-cadherin in spheroids formed in Matrigel from co-culture of isolated myoepithelial and luminal cells over 21 days. Images are representative of cells derived from at least three donors. Scale bar = 20 μm. Figure S3. Objective and systematic calculation of cell and spheroid volumes. Representative workflow of spheroid analysis. Raw DAPI z-sections (a) are converted into greyscale images and a greyscale distribution profile (b). Greyscale images are then converted to binary images using a calculated threshold to indicate cell presence (c). The pixels that indicate cells are then translated into a geometrically accurate point cloud using the known image resolutions (d). Further post-processing using density-based spatial clustering of applications with noise (DBSCAN) is performed to identify the main body of cells (e). The point cloud representing the main spheroid is then extracted (f). The alpha-shape algorithm is applied using thresholds set as a function of the image resolutions to form triangulated bodies that represent the cells and body (g). The volumes of these bodies are then calculated alongside the resultant cell/body ratio. (PDF 1342 kb)