Figure 2. Biochemical cues.

(a) Reflection confocal micrograph. Singular of 3D collagen matrices. Scale bar, 10 μm. (b) Correlation plot of fibre alignment versus cell density. (c) Correlation plot of cell speed versus fibre alignment. (d) Correlation plot of average inter-fibre spacing versus cell density. (e) Method to prepare condition medium: medium is incubated for 24 h with a collagen matrix containing a high density of cells, 50 cells mm⁻³ (HD), which is then filtered using a 0.45-μm filter, and added to a matrix containing a low density of cells, 10 cells mm⁻³ (LD). (f) The addition of conditioned medium (CM) from a matrix containing a high cell density (HD) increases the speed of cells in a matrix containing a low cell density (LD). The HD cell speed in the presence of fresh medium (FM) is recapitulated in LD when using CM. (g) Secretomic analysis of CM harvested from human fibrosarcoma cells indicates that levels of interleukin 6 (IL-6) and interleukin 8 (IL-8) increase as a function of HT1080 cell density in the matrix, while levels of other major cytokines do not significantly change. (h) Secretomic analysis of conditioned medium from human breast carcinoma cells (MDA-MB-231) confirms our observations with HT1080 cells. (i,j) Increasing density of human fibrosarcoma cells in the matrix increases the concentrations of secreted IL-6 (A) and IL-8 (B), as analysed by ELISA. (k,l) Increasing cell density of human carcinoma cells in the matrix increases the concentrations of secreted IL-6 (A) and IL-8 (B), as analysed by ELISA. In all panels, data is represented as mean±s.e.m. from three independent experiments. *P<0.05; **P<0.01; ***P<0.001 (ANOVA).