Figure 1.
Figure 1. CAFs induce invasion of cancer cells through matrix remodeling. (A) Maximum intensity projections of cancer cell spheroids in collagen I, with or without fibroblasts, at day 3. CT26 cancer cells express LifeAct-GFP (green); F-actin (red) and DNA (cyan) were stained with phalloidin-rhodamine and DAPI, respectively. Bar, 100 µm. (B) 3D rendering of spheroids at day 3. Invasion is quantified using the invasion counter software. Red dots represent nuclei of invading GFP cancer cells (migrated out of the spheroid). (C) Quantification of cancer cell invasion alone (blue box) and in the presence of NAFs (green boxes) or CAFs (red boxes) for three different patients. 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). (D, left) Quantification of the density of NAFs and CAFs from all three patients around the spheroid. Fibroblast density is defined as the number of nuclei of non-GFP cells normalized to the surface area of the spheroid contour in 3D. P-value is calculated using Mann–Whitney test. (Right) Quantification of the mean distance of NAFs and CAFs from the spheroid. The mean distance from the spheroid was defined as the distance from the nuclei of non-GFP cells to the closest point along the cancer cell spheroid contour. P-value is calculated using Mann–Whitney test. (E, left) Schematic representation of the experiment. Cancer cells were embedded in collagen gels (a). CAFs were either mixed with cancer cells in the collagen droplet (b), their conditioned media was added to cancer cells (c), or they were plated around the collagen droplet (d). (Right) Quantification of cancer cell invasion alone, in the presence of CAFcm or CAFdm, or in the presence of CAFs for three different patients. Invasion index is defined as the ratio between the number of invading nuclei of GFP cancer cells and the area of the spheroid contour. 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). CC, cancer cells.

CAFs induce invasion of cancer cells through matrix remodeling. (A) Maximum intensity projections of cancer cell spheroids in collagen I, with or without fibroblasts, at day 3. CT26 cancer cells express LifeAct-GFP (green); F-actin (red) and DNA (cyan) were stained with phalloidin-rhodamine and DAPI, respectively. Bar, 100 µm. (B) 3D rendering of spheroids at day 3. Invasion is quantified using the invasion counter software. Red dots represent nuclei of invading GFP cancer cells (migrated out of the spheroid). (C) Quantification of cancer cell invasion alone (blue box) and in the presence of NAFs (green boxes) or CAFs (red boxes) for three different patients. 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). (D, left) Quantification of the density of NAFs and CAFs from all three patients around the spheroid. Fibroblast density is defined as the number of nuclei of non-GFP cells normalized to the surface area of the spheroid contour in 3D. P-value is calculated using Mann–Whitney test. (Right) Quantification of the mean distance of NAFs and CAFs from the spheroid. The mean distance from the spheroid was defined as the distance from the nuclei of non-GFP cells to the closest point along the cancer cell spheroid contour. P-value is calculated using Mann–Whitney test. (E, left) Schematic representation of the experiment. Cancer cells were embedded in collagen gels (a). CAFs were either mixed with cancer cells in the collagen droplet (b), their conditioned media was added to cancer cells (c), or they were plated around the collagen droplet (d). (Right) Quantification of cancer cell invasion alone, in the presence of CAFcm or CAFdm, or in the presence of CAFs for three different patients. Invasion index is defined as the ratio between the number of invading nuclei of GFP cancer cells and the area of the spheroid contour. 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). CC, cancer cells.

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