Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis

Abstract

Cell migration on 2D surfaces is governed by a balance between counteracting tractile and adhesion forces. Although biochemical factors such as adhesion receptor and ligand concentration and binding, signaling through cell adhesion complexes, and cytoskeletal structure assembly/disassembly have been studied in detail in a 2D context, the critical biochemical and biophysical parameters that affect cell migration in 3D matrices have not been quantitatively investigated. We demonstrate that, in addition to adhesion and tractile forces, matrix stiffness is a key factor that influences cell movement in 3D. Cell migration assays in which Matrigel density, fibronectin concentration, and beta1 integrin binding are systematically varied show that at a specific Matrigel density the migration speed of DU-145 human prostate carcinoma cells is a balance between tractile and adhesion forces. However, when biochemical parameters such as matrix ligand and cell integrin receptor levels are held constant, maximal cell movement shifts to matrices exhibiting lesser stiffness. This behavior contradicts current 2D models but is predicted by a recent force-based computational model of cell movement in a 3D matrix. As expected, this 3D motility through an extracellular environment of pore size much smaller than cellular dimensions does depend on proteolytic activity as broad-spectrum matrix metalloproteinase (MMP) inhibitors limit the migration of DU-145 cells and also HT-1080 fibrosarcoma cells. Our experimental findings here represent, to our knowledge, discovery of a previously undescribed set of balances of cell and matrix properties that govern the ability of tumor cells to migration in 3D environments.

Fig. 1.

Maximum cell speed is an optimum between oppositely acting adhesive forces. The adhesivity of DU-145 parental cells to the matrix was modulated by either adding fibronectin to the matrix or inhibiting binding to integrin with 4B4 antibody. (a) Box-and-whiskers plot with all of the raw data overlaid for DU-145 cell migration under specified conditions. The boxes represent 25th and 75th percentile with the median shown by the line bisecting the box. The mean is shown by a black dot inside the box. The whiskers represent 10th and 90th percentiles of the data. Cell speed values of <3 μm/h come from “oscillating” cells or simply active shape changes rather than actively motile cells. These cells are ignored from the cell speed analysis shown in b. (b) In 67% Matrigel concentration, cell speed decreases from 12 μm/h as fibronectin is included in the matrix. As integrin binding is blocked with 4B4 antibody, cell speed displays a biphasic character in which the maximum in cell speed shifts to higher fibronectin concentrations. (c) Cell speed of “motile cells” (see Materials and Methods) over the range of fibronectin concentrations displays a biphasic relationship with an adhesiveness factor, L × R. As the number of available receptors decreases, the loss in integrin activity is compensated by increase in ligand concentration, resulting in a constant relationship between speed and substrate adhesivity. The error bars represent SEM in results from five independent experiments where ≈15–20 cells were tracked per experiment.

Fig. 2.

Integrin inhibition shifts the maximum cell speed to lower Matrigel concentrations. DU-145 parental (a and b) and EGFR-overexpressing (c and d) cells were mixed with different concentrations of Matrigel and 4B4 antibody, and the cell speed was measured for 75–100 cells at each Matrigel and antibody data point. a and c show box-and-whiskers plot of cell speed distribution with raw data overlaid as described in Fig. 1. The number of motile cells varies with both integrin inhibition and matrix concentration. The average number of motile cells decreases with increase in concentration of anti-integrin blocking antibody, while average speed shows somewhat of a bimodal behavior with variations in gel density, reaching a maximum with intermediate gel concentration. In the absence of 4B4 antibody, the optimal cell speed of the parental cells was 12 μm/h at 67% Matrigel and for the EGFR-overexpressing cells was 24 μm/h at 60% Matrigel. The presence of 4B4 antibody slowed cell speed and shifted the maximum to lower Matrigel concentrations. In excess 4B4 antibody, cell speed reached a negligible value (3 μm/h) in both DU-145 parental and EGFR-overexpressing cells. The error bars represent SEM in results from five experiments where ≈15–20 cells were tracked per experiment.

Fig. 3.

Matrigel stiffness and steric properties influence migration. (a and b) The stiffness, G′, of Matrigel was measured for different concentrations of matrix (a) and for different amounts of exogenously added fibronectin at 67% Matrigel (b). Between 50% and 100% Matrigel, the storage modulus increases 5-fold. (c) The mean void area decreases as the Matrigel concentration is increased. (d) Aspect ratio (length of major axis/minor axis) of DU-145 parental cells as a function of mAb 4B4 concentration. The error bars show SEM for 10 different cells. The results for EGFR-overexpressed DU-145 are similar (data not shown). (e) Scanning electron microscopy images of Matrigel and collagen show starkly different matrix structures at the same resolution. Matrigel stock concentration is ≈10–12 mg/ml, and collagen I is typically used at a concentration of 1–2 mg/ml. (f) Collagen I structure at a concentration of 2.8 mg/ml. (g) Although HT-1080 cells show plasticity in migration in collagen matrices (10), they are unable to migrate when they are unable to either adhere or proteolyse the matrix (see Movies 1–6). (h) Similar to HT-1080 cells, DU-145 parental cells (EGFR cells show qualitatively similar behavior) show negligible motility in the presence of anti-integrin antibody or matrix metalloproteinase (MMP)-inhibiting mixture (see Movies 1–6).

Fig. 4.

The 3D migration landscape. (a and b) A contour plot of experimentally measured DU-145 parental cell speed as a function of fibronectin and Matrigel concentrations in the absence (a) and presence (b) of 2.5 μg/ml 4B4 antibody. The region of highest speed (red zone) lies at intermediate stiffness and low adhesion (bottom center) and shifts to the region of high adhesion and low stiffness (top left) when integrin binding is blocked. The rest of the landscape shifts and accommodates the changes due to this decrease in the effective number of available β1 integrins. (c) Experimentally measured speed of DU-145 parental cells as a function of receptor number and Matrigel stiffness. (d) Model-predicted dependence of cell speed on matrix stiffness and adhesiveness from the computational model of Zaman et al. (14). The quantitative differences between computation and experiment are due to assumptions of the model regarding the approximate number of receptors, the order of magnitude estimate of protrusion and drag forces, and limitations of the model in capturing the change in cell shape as a function of integrin inhibition.