Soft matrix is a natural stimulator for cellular invasiveness

Abstract

Directional mesenchymal cell invasion in vivo is understood to be a stimulated event and to be regulated by cytokines, chemokines, and types of extracellular matrix (ECM). Instead, by focusing on the cellular response to ECM stiffness, we found that soft ECM (low stiffness) itself is sufficient to prevent stable cell-to-cell adherens junction formation, up-regulate matrix metalloproteinase (MMP) secretion, promote MMP activity, and induce invadosome-like protrusion (ILP) formation. Consistently, similar ILP formation was also detected in a three-dimensional directional invasion assay in soft matrix. Primary human fibroblasts spontaneously form ILPs in a very narrow range of ECM stiffness (0.1-0.4 kPa), and such ILP formation is Src family kinase dependent. In contrast, spontaneous ILP formation in malignant cancer cells and fibrosarcoma cells occurs across a much wider range of ECM stiffness, and these tumor cell ILPs are also more prominent at lower stiffness. These findings suggest that ECM softness is a natural stimulator for cellular invasiveness.

FIGURE 1:

ECM softness prevents AJ formation. (A) Primary human fibroblasts (3 × 10⁴ cells per well) were seeded onto gels of varying stiffness in 12-well plates and cultured for 24 h. Cell surface cadherin-11 was live stained by monoclonal anti–cadherin-11 (3H10) antibody for 1 h at room temperature, followed by fixation and secondary antibody staining. Plasma membranes were then permeabilized, and intracellular F-actins were stained by phalloidin. Cell nuclei were stained by DRAQ5. Arrows point to cadherin-11 adherens junctions. Scale bar, 100 μm. (B) Primary human fibroblasts (3 × 10⁴ cells per well) were seeded onto gels of varying stiffness in 12-well plates and cultured for 24 h. Cells were then fixed, and cell membranes were permeabilized. p120 catenin was stained by monoclonal anti–p120 catenin antibody. Arrows point to p120 catenin–positive adherens junctions. Scale bar, 50 μm. (C) Primary human fibroblasts were cultured as in A, and total cell lysates were subject to SDS–PAGE and Western blot for cadherin-11, p120-catenin, α-catenin, and β-catenin. β-Actin as protein loading control.

FIGURE 2:

ECM softness upregulates MMP secretion and promotes MMP activity. (A) Primary human fibroblasts (2 × 10⁴ cells per well) were seeded onto gels of varying stiffness in 12-well plates and cultured for 48 h. Cell culture medium from each condition was collected and subject to the RayBio Human MMP Array to determine the total secreted MMP protein quantity in each medium. (B) Three independent experiments (n = 3, ±SD) as in A were performed and quantified. Note that proliferation rates vary on gels of different stiffness. Total MMP quantities were normalized to total cell numbers on gels of varying stiffness at 48 h. Student's t test was used for the comparison of each mean with the mean of MMP protein quantity of the same MMP type on 0.2-kPa gels. *p < 0.05 considered significant. (C) Cell culture conditioned media were collected as in A, and the total MMP activity in each medium was measured and quantified (n = 3, ±SD) and normalized to cell number. Student's t test was used for the comparison of each mean with the mean of “0.1 kPa.” *p < 0.05 considered significant.

FIGURE 3:

ECM softness stimulates matrix degradation. (A) DQ collagens were cross-linked onto gels of varying stiffness. Primary human fibroblasts (2 × 10⁴ cells per well) were seeded onto gels of varying stiffness in 12-well plates and cultured for 4 h with or without MMP inhibitors. Cell nuclei were stained by DRAQ5. Green fluorescence indicated cleaved DQ collagen. Scale bar, 100 μm. (B) Five independent experiments (n = 5, ±SD) as in A were performed, and the fluorescence intensities of DQ collagens in these images were measured, quantified, and normalized to number of cells. Student's t test was used for the comparison of each mean with the mean of “No inhibitor 0.2 kPa.” *p < 0.05 considered significant.

FIGURE 4:

ECM softness induces spontaneous ILP formation. (A–C) Primary human fibroblasts were seeded onto gels of varying stiffness and cultured for 4 h. Cells were then fixed, and cell membranes were permeabilized for immunofluorescence (IF) staining. MMP-14 and integrin β1 were stained by primary antibodies, followed by fluorescent secondary antibodies. F-actin was stained by phalloidin–Alexa 647. All 2D images were projected from relevant confocal 3D stacks by the maximum projection method. Scale bar, 100 μm; in zoom panels, 10 μm. Arrows point to ILPs. (A) F-actin was stained by phalloidin–Alexa 647 and was defined as green color. (C) Top, zoom views of ILPs. Bottom, z-axis orthogonal views of ILPs showing their classic columnar shapes. The z-axis distances are 5× exaggerated. The vertical line defines the orthogonal view position. (D) Primary human fibroblasts were seeded onto gels of varying stiffness and cultured for 4 h. Cells were then fixed, but with no cell membrane permeabilization before IF staining. MMP-14 was stained by a primary antibody that specifically recognizes the MMP-14 extracellular hinge region domain. Cell nuclei were stained by DRAQ5. All 2D images were projected from relevant confocal 3D stacks by the maximum projection method. Scale bar, 100 μm.

FIGURE 5:

ECM softness induces spontaneous ILP formation. (A) Cells were prepared as in Figure 4B. MMP-14 and cortactin were stained by primary antibodies and followed by fluorescent secondary antibodies. F-actin was stained by phalloidin–Alexa 647. All 2D images were projected from relevant confocal 3D stacks by maximum projection method. Scale bar, 100 μm; in zoom panels, 10 μm. Arrows point to ILPs. (B) Gelatin–Oregon 488 was cross-linked to stiffness gels before cell seeding. Black areas represent degraded gelatins. Arrows point to ILPs.

FIGURE 6:

ECM softness–induced ILP formation is SFK dependent. (A) Primary human fibroblasts were seeded onto gels of varying stiffness and cultured for the hours indicated. Total cell lysates were subject to SDS–PAGE and Western blot for phospho-SFK (Y419 on c-Src). The anti–phospho-Src (pY419) antibody recognizes all activated SFKs. Total c-Src as protein loading control. (B) Three independent experiments (n = 3, ±SD) as in A were performed, and phospho-SFK Western blot band intensities were quantified. Student's t test was used for the comparison of each mean with the mean of SFK phosphorylation at 1 h on 0.2-kPa gels. *p < 0.05 considered significant. (C) Primary human fibroblasts were seeded onto gels of varying stiffness and cultured for 4 h with or without SFK inhibitors. Top, arrowheads point to cells unable to form ILPs upon SFK inhibition on gels of 0.2-kPa stiffness. Bottom, arrows point to cells unable to form stress fibers upon SFK inhibition on gels of 6.4-kPa stiffness. Scale bar, 100 μm. (D) Three independent experiments (n = 3, ±SD) as in C and Supplemental Figure S2C were performed, and the percentage of cells forming ILPs on gels of 0.2-kPa stiffness was quantified. In each experiment, 200 cells in total were counted. Student's t test was used for the comparison of each mean with the mean of “No src inhibitor.” *p < 0.05 considered significant.

FIGURE 7:

ECM softness differentially induces spontaneous ILP formation in primary cells, cancer cells, fibrosarcoma cells, and so on. Primary human fibroblasts (A) and MDA-MB-231 human breast cancer cells (B) were seeded onto gels of varying stiffness and cultured for 4 h. Invadosome marker proteins MMP-14 and cortactin were stained by primary antibodies and followed by fluorescent secondary antibodies. Arrowheads point to cells forming ILPs. Scale bar, 100 μm (A), 50 μm (B). Continues

FIGURE 7:

ECM softness differentially induces spontaneous ILP formation in primary cells, cancer cells, fibrosarcoma cells, and so on. Primary human fibroblasts (A) and MDA-MB-231 human breast cancer cells (B) were seeded onto gels of varying stiffness and cultured for 4 h. Invadosome marker proteins MMP-14 and cortactin were stained by primary antibodies and followed by fluorescent secondary antibodies. Arrowheads point to cells forming ILPs. Scale bar, 100 μm (A), 50 μm (B). Continues

FIGURE 8:

ECM softness differentially induces spontaneous ILP formation in primary cells, cancer cells, fibrosarcoma cells, and so on. (A) HS 93.T human fibrosarcoma cells were prepared as in Figure 7. Arrowheads point to cells forming ILPs. Scale bar, 100 μm. (B) Cells as indicated were seeded onto gels of varying stiffness and cultured for 4 h. Percentage of cells forming ILPs was quantified (n = 3, ±SD). In each experiment, 200 cells in total were counted.

FIGURE 9:

Similar ILP formation is detected in cells cultured in soft 3D matrices. (A) Primary human fibroblasts were live stained by DiI and then seeded into the 3D cell invasion assay as in Supplemental Figure S5. All 2D images were projected from relevant confocal 3D stacks by maximum projection method. Scale bar, 100 μm. Images were taken by a 20× objective with large pinhole. (B) Cells invading as in A were imaged by a 63× objective with small pinhole. All 2D images were projected from relevant confocal 3D stacks by maximum projection method. Scale bar, 100 μm. Arrows point at ILPs. (C) Schematics of ECM softness–induced ILP formation. (D) Schematics of stiffness cell-matrix adhesion curve and stiffness cell-matrix proteolysis curve.