3D time-lapse microscopy reveals EGF-stimulation leads to increased 3D U87MG migration in collagen mediated by increase in cell speed and concentration-dependent increase in directional persistence. (A) Representative 3D image of eGFP-expressing U87MG cells seeded in varying concentrations of collagen matrix captured by 3D time-lapse confocal microscopy. Images were taken over 10 h with 15-min intervals and cells tracked using Imaris tracking software. Image dimensions: 870 μm x 660 μm x 80 μm. (B) Representative 3D Wind-Rose plots depicting migratory tracks over 10 h of 30 random cells determined as motile in 3.0 mg/ml collagen matrix. Cells that moved more than one cell length were determined as motile. (C) Ratio of number of motile cells to total number of cells tracked in presence (red) or absence (black) of 50 ng/ml EGF. Cells that moved more than one cell length were determined as motile. (D) Cell dispersion quantified as random motility coefficient. Random motility coefficient of each cell is calculated from cell speed and persistence time of the persistent random walk model. (E) Cell speeds of cells determined as motile. (F) Directional persistence time fitted from the persistent random walk model. High persistence times indicate directed movement, whereas low persistence times indicate erratic movement according to the model. Supplementary Figure S2 shows box-and-whisker and scatter plots of individual cells. Data shown as mean ± SEM. n = 3–5 biological experiments (C). n = 18–105 motile cells (D–F) out of ∼100–250 total cells tracked. p values indicate statistics obtained from two-way ANOVA across treatment conditions.

EGF-induced barrier-free, 2D migration exhibits increased cell speed but decreased directional persistence. (A) Representative 2D Wind-Rose plots depicting migratory tracks over 10 h of 40 random cells determined as motile on 10 μg/cm² collagen-coated 2D surfaces. (B–E) Migration parameters for 2D cell movement obtained from 2D time-lapse microscopy analysis of U87MG cells migrating on collagen-coated surfaces of varying densities in presence (red) or absence (black) of 50 ng/ml EGF. (B) Motile fraction, (C) random motility coefficients, (D) cell speeds, and (E) persistence times. Cells that moved more than one cell length were determined as motile. Parameters have been obtained analogously to Figure 1, C–E. Supplementary Figure S3 shows box-and-whisker and scatter plots of individual cells. Data shown as mean ± SEM. (B) n = 3 biological experiments. (C–E) n = 83–118 motile cells. p values indicate statistics obtained from two-way ANOVA across treatment conditions. (F) Time course of Rac1-GTP levels of U87MG cells on collagen-coated 2D surfaces (gray) or in 3D collagen matrix (black) after stimulation with 50 ng/ml EGF using commercially available Rac-GTP ELISA normalized to total Rac1 levels measured by semiquantitative immunoblot. Data shown as mean ± SEM. n = 2 biological replicates.

EGF stimulation increases MMP release and 3D matrix degradation. (A) MMP-1 release was measured using a quantitative immunoprecipitation bead-based MMP-1 detection assay. Cells were seeded on surfaces coated with 10 μg/cm² collagen and serum-starved. Media was collected at various times after changing to serum-free media (SF) or to media containing 50 ng/ml EGF. (B) Cell surface MT1-MMP expression was measured by semiquantitative Western blot for cell surface MT1-MMP. Cells were seeded on collagen I and serum-starved. Cell surface proteins were biotinylated, and cells were lysed at indicated times after changing media to serum-free or EGF-containing media. Cell surface proteins were precipitated with streptavidin beads and prepared for SDS-PAGE. (C) Bulk matrix degradation by U87MG cells in 3D collagen gels were measured using a FITC-collagen dequenching assay. Cells were embedded in a matrix consisting of 5% quenched FITC-labeled collagen and serum-starved. FITC-collagen released into media was quantified at indicated times after changing media to serum-free or EGF containing media. 1 μM GM6001 (GM), a broad matrix metalloproteinase inhibitor, was used as negative control.

Inhibition of MMPs does not affect 2D migration, but abrogates 3D migration. U87MG cells were seeded in 3.0 mg/ml collagen matrices (A) or on 2D surfaces (B) coated with 10 μg/cm² collagen were stimulated with 50 ng/ml EGF in presence or absence of 1 or 5 μM of a broad MMP inhibitor GM6001 for 3D and 2D experiments, respectively, for 8 h and then tracked for 10 h. (A) Cells that moved more than its cell length were determined as motile. n = 3–5 biological experiments, p = 0.002. (B) 2D cell speed (left) and persistence (right) were calculated for motile cells. Data shown as mean ± SEM.

Concentration-dependent matrix-confined movement governs low persistent movement in unstimulated cells. (A) Confocal reflection micrographs of collagen matrices of varying concentrations. (B) Representative time-lapse images of an U87MG cell migrating with low persistence time in serum-free cells. White sphere and yellow line indicate the centroid of the cell and the migratory track, respectively. Dimensions of the box: 122 × 112 × 80 μm. (C) Scatter plot for goodness of fit (R²) of cell migration tracks to the persistent random walk model. Only cells migrating in untreated, serum-free (top, black) or in 50 ng/ml EGF treatment (bottom, red) with persistence (P) <50 min were plotted. Lines indicate mean of the distribution and the numbers on right indicate the percentage of cells with R² <0.6 (threshold chosen as poor fit).

Modulation of MMP activity results in correlated modulation of 3D directional persistence. (A) Bulk matrix degradation of U87MG cells was quantified in presence of varying concentrations of GM6001 and 50 ng/ml EGF as described above. Horizontal dashed lines indicate mean values of EGF control and serum-free control from Figure 4C. R² = 0.96 for logarithmic fit. (B–D) Motile fraction, cell speed, and persistence times of cells migrating in 3.0 mg/ml collagen matrices in presence of various concentrations of GM6001 and 50 ng/ml EGF. Migration data were plotted against FITC-collagen release values determined in A. Data point in gray for B and C indicates migration data in serum-free condition at 3.0 mg/ml matrix concentration from Figure 2. Data shown as mean ± SEM. n = 3 biological samples for A, n = 3–5 biological experiments for B, and n = 7–64 motile cells for C and D. R² = 0.92 for linear fit in D. Supplementary Figure S3 shows box-and-whisker and scatter plots for single cell migration parameters.