Collagen pore size determines physical migration limits of HT/MT1 cells in 3D collagen lattices. (A, C, and E) Single-cell migration rates in bovine dermal or rat tail collagen lattices of varying concentration or polymerization temperature (1.7 mg/ml) using anchored 3D matrices in migration chambers (shown in Fig. S1 A), monitored by time-lapse microscopy and analyzed by single-cell cell tracking. Migration paths (A and C, left) and averaged single-cell speeds (A and C, right; E) after 24 h of observation in the absence or presence of MMP inhibitor GM6001. Horizontal lines and boxes and whiskers show the medians, 25th/75th, and 5th/95th percentile (50–180 cells, 19–24 h of migration analyzed, 3–6 independent experiments). Dashed lines indicate references for 100% MMP-independent movement and 90% inhibition. ***, P < 0.0001; n.s., not significant. (B, D, and F) Correlation between median pore cross section (from Fig. 1, D–F) and proteolytic and MMP-independent cell speed (A, C, and E) for each collagen condition. Symbols show medians, and whiskers the 5th/95th percentiles. R² (describing how well the regression line approximates real data points), slopes, and significance between slopes from untreated and GM6001-treated populations were as follows: (B) bovine dermal collagen, untreated control 0.998/0.044; GM6001-treated population 0.991/0.040/n.s.; (D) rat tail collagen, untreated control 0.851/0.010, GM6001-treated cells 0.999/0.017/*, P < 0.05 (P = 0.045); (F) rat tail collagen with varied polymerization temperature, untreated control 0.960/0.007, GM6001-treated cells 0.972/0.017/*, P < 0.05 (P = 0.01). Dashed lines, 90% inhibition of migration. Bars: (A and C) 100 µm.