Figure 2.
CAFs deposit FN to induce cancer cell invasion. (A) Quantification of cancer cell invasion alone or in the presence of CAFs from patient 2, depleted or not for FN. 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 < 0.001). (B) Time-lapse sequence of CT26 cancer cells and CAFs from patient 1 in collagen. CT26 cancer cells express LifeAct-GFP (green), CAFs are stained with a lyophilic carbocyanine dye (red), and collagen is acquired by reflection (blue). Time is in hours and minutes (HH:mm). Bar, 200 µm. The magnified region is represented by the white square. Bar, 100 µm. (C, top) Overlaid images of collagen I matrices containing cancer cell spheroids alone or together with control or FN-depleted CAFs from patient 1 generated using CurveAlign. The yellow line indicates the edge of the spheroid, and the green lines indicate fiber orientation with respect to the closest point on the spheroid edge. Bar, 100 µm. (Bottom) Rose plots representing the frequency of distribution of the absolute angles of collagen fibers within the range of 0–90° with respect to the closest point on the spheroid edge. (D, left) Maximum intensity projections of CAFs from patient 1 treated with siRNA scrambled control (CAFsiCtrl) or with siRNA targeting FN (CAFsiFN). F-actin is stained with phalloidin-rhodamine (green), FN is immunostained (magenta), and collagen is acquired using second harmonic generation (cyan). Bar, 20 µm. (Right) Quantification of collagen fiber width and length using CT-FIRE. P-value is calculated using Mann–Whitney test for at least n = 3 stacks over n = 2 separate experiments. (E, left) Control and FN-depleted CAFs from patient 1 cultured in collagen I gels 1 d after embedding. (Right) Percentage of gel contraction of control and FN-depleted CAFs from patient 1 calculated using the formula P-value is calculated using Mann–Whitney test for n = 3 over n = 6 separate experiments. (F, left) Traction force map of control and FN-depleted CAFs from patient 1 on collagen-coated polyacrylamide gels with Young’s modulus of 5 kPa. Color code gives the magnitude of traction stress in Pa, which corresponds to forces of piconewton/squared micrometers. (Right) Corresponding mean force (strain energy) exerted by CAFs over a 30-min time lapse. P-value is calculated using Mann–Whitney test for n = 10 cells over n = 2 separate experiments. (G) Maximum intensity projections of cancer cell spheroids in collagen I gels with CAFs from patient 1 treated with siRNA scrambled control, siRNA against FN, or blebbistatin at day 3. Bar, 100 µm. Magnified regions are represented by the white squares. CT26 cancer cells express LifeAct-GFP (green), F-actin is stained with phalloidin-rhodamine (red), FN is immunostained (cyan), and collagen is acquired using reflection (white). Bar, 50 µm. (H) Quantification of cancer cell invasion alone or in the presence of control or FN-depleted CAFs treated with BB94 from patient 3. 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).