Fig. 9.

Effect of ECM proteins and collagen density on α5β1-integrin-facilitated cell invasion. (A) Concentration of fibronectin in the medium secreted by T24, MDA-MB-231 and 786-O cancer cells with low, medium and high α5β1 integrin expression after 3 days of invasion into 3D collagen matrices. (B) Concentration of secreted fibronectin in the medium after 3 days of siRNA-mediated knockdown of α5 integrin. Unspecific siRNA served as control and five different siRNAs were used for specific α5 knockdown (α5 si1–si5). (C) The percentage of invasive cells strongly increased in 3D matrices containing 100 μg/ml fibronectin (embedded) or RGD peptide (embedded), and slightly increased in 3D matrices containing 100 μg/ml vitronectin (embedded). (D) Five different collagen concentrations of 0.6, 1.2, 2.4, 3.7, and 5.8 mg/ml were tested for cell invasion. After 3 days, α5β1^high cells were able to invade into 1.2–5.8 mg/ml collagen gels, whereas α5β1^low cells were able to migrate into 0.6–3.7 mg/ml collagen gels. (E–H) The invasion profiles of α5β1^high (E) and α5β1^low (G) cells were not altered by the addition of fibronectin, RGD peptide or vitronectin. Invasion profiles of α5β1^high (F) and α5β1^low (H) cells in 3D ECMs with increasing collagen concentrations, as indicated. (I) Confocal images of 3D fibronectin–collagen matrices. Left: reflection mode shows the collagen fibers. Right: fluorescent image of an anti-fibronectin staining of the same matrix. (J) Confocal images of a 3D collagen matrix with an invasive α5β1^high cell. Left: reflection mode shows the matrix fibers and the cell. Middle: fibronectin staining of the same matrix and the cell. Right: bright field image of the same field of view. (K) Left: the percentage of invasive α5β1^high and α5β1^low cells strongly decreased in 3D matrices containing 100 μg/ml soluble fibronectin or 100 μg/ml soluble RGD peptide. The invasion profiles of α5β1^high (middle) and α5β1^low cells (right) show reduced invasiveness after addition of soluble (s) fibronectin and RGD peptide. The bar graphs show means + s.e.m. **P<0.01, ***P<0.001.