Fig. 2. TNF- induces 3D and 2D scattering of 2A4 mammary epithelial cells. (A) 2A4 cells grown for 9 days in a collagen gel in defined medium have formed compact ball-like structures. (B) 2A4 cells were grown for 3 days in a collagen gel to allow the formation of spheroidal cell clusters, at which time 10 ng ml^-1 TNF- was added for an additional 6 days. TNF- treatment has resulted in the dissociation of preformed colonies and the dispersal of individual cells within the collagen matrix. (C) 2A4 cells seeded in plastic wells (2D culture) in DMEM-F12 with ITS+ Premix and grown for 4 days have formed discrete islands of closely apposed cells. (D) 2A4 cells were grown in DMEM-F12 with ITS+ Premix for 2 days to allow formation of small compact colonies before addition of TNF- (10 ng ml^-1) for an additional 48 hours. TNF- has induced a pronounced 2D scattering of 2A4 cells. (E) 2A4 cells maintained for 5 days in the presence of TNF- are fully scattered. (F) Parallel cultures were treated 48 hours with TNF- to induce cell dispersion, at which time the medium was changed and the cells were incubated for an additional 72 hours without TNF-. The cells have re-established close intercellular contacts and formed compact epithelial-like colonies. (A-F) Scale bars, 100 µm. (G) Time course and dose dependence of TNF--induced 3D scattering. 2A4 cells were grown in collagen gels for 3 days and subsequently incubated for an additional 6 days with or without increasing concentrations of TNF-. Quantification was carried out by counting the number of apparently single cells per photographic field after 3 days and 6 days of treatment. Data represent the mean number of single cells ± s.e.m. P<0.05 for values of 1 ng ml^-1 TNF- compared with controls (3 days); P<0.01 for values of 1 ng ml^-1 TNF- compared with controls (6 days); P<0.005 for values of 3 ng ml^-1 TNF- compared with controls (3 days). (H) Time course and dose dependence of TNF--induced 2D scattering. 2A4 cells were grown for 2 days in gelatine-coated wells and subsequently incubated with or without increasing concentrations of TNF-. Scattering was quantified by counting cells that were completely isolated or showed only a minimal area of contact with their neighbours. Data represent the mean number of isolated cells ± s.e.m. in nine randomly selected photographic fields. P<0.05 for values of 3 ng ml^-1 TNF- compared with controls (24 hours); P<0.0025 for values of 10 ng ml^-1 TNF- compared with controls (24 hours); P<0.025 for values of 1 ng ml^-1 TNF- compared with controls (48 hours); P<0.0005 for values of 3 ng ml^-1 TNF- compared with controls (48 hours). Scale bars, 100 µm.