Figure 4.

Integrin-αvβ3 is necessary for FN assembly. (A) Quantification of cancer cell invasion alone or in the presence of control CAFs, α5-depleted CAFs, and β3-depleted CAFs from patient 3. (B) Quantification of cancer cell invasion in the presence of CAFs from patient 2, with or without cilengitide treatment. (A and B) Invasion index is defined as the ratio between the number of invading nuclei of GFP cancer cells and the area of the spheroid contour. All quantification results are expressed as box and whiskers (minimum to maximum) of at least n = 3 separate experiments. P-values are compared with cancer cells alone (in gray) and to cancer cells with CAFs (in black) using Newman-Keuls multiple comparisons test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (C) Maximum intensity projections of CAFs from patient 2 treated with siRNA scrambled control, siRNA against integrin-α5 or integrin-β3, or cilengitide. Bar, 100 µm. Magnified regions are represented by the white squares. F-actin is stained with phalloidin-rhodamine (green), FN is immunostained (magenta), and collagen is acquired using second harmonic generation (cyan). Bar, 20 µm. (D, left) Immunostaining of FN (green) in control CAFs, FN-depleted CAFs, α5-depleted CAFs, and β3-depleted CAFs from patient 2, 3 d after plating. F-actin was stained with phalloidin-rhodamine (red), and DNA was stained with DAPI (blue). Bar, 40 µm. (Right) Graph represents the percentage of assembled FN relative to control conditions defined as the amount of fluorescence in a monolayer (integrated density) normalized to the number of nuclei. Quantification results are expressed as column bars with mean ± SEM. Depleted CAFs were compared with control CAFs for n = 10 frames over n = 4 separate experiments. P-value is calculated using Newman Keuls multiple comparisons test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).