A flexible network of vimentin intermediate filaments promotes migration of amoeboid cancer cells through confined environments

Abstract

Tumor cells can spread to distant sites through their ability to switch between mesenchymal and amoeboid (bleb-based) migration. Because of this difference, inhibitors of metastasis must account for each migration mode. However, the role of vimentin in amoeboid migration has not been determined. Because amoeboid leader bleb-based migration (LBBM) occurs in confined spaces and vimentin is known to strongly influence cell-mechanical properties, we hypothesized that a flexible vimentin network is required for fast amoeboid migration. To this end, here we determined the precise role of the vimentin intermediate filament system in regulating the migration of amoeboid human cancer cells. Vimentin is a classic marker of epithelial-to-mesenchymal transition and is therefore an ideal target for a metastasis inhibitor. Using a previously developed polydimethylsiloxane slab-based approach to confine cells, RNAi-based vimentin silencing, vimentin overexpression, pharmacological treatments, and measurements of cell stiffness, we found that RNAi-mediated depletion of vimentin increases LBBM by ∼50% compared with control cells and that vimentin overexpression and simvastatin-induced vimentin bundling inhibit fast amoeboid migration and proliferation. Importantly, these effects were independent of changes in actomyosin contractility. Our results indicate that a flexible vimentin intermediate filament network promotes LBBM of amoeboid cancer cells in confined environments and that vimentin bundling perturbs cell-mechanical properties and inhibits the invasive properties of cancer cells.

Keywords: amoeboid; bleb; bundling; cancer; cell migration; cytoskeleton; epithelial-to-mesenchymal transition (EMT); metastasis; simvastatin; vimentin.

Figure 1.

Vimentin localizes to the cell body of leader bleb–forming cells. A and A', localization in cells adhered to fibronectin (A', left panel) and uncoated glass (blebbing; A', center panel) and confined under PDMS (forming a leader bleb; A', right panel) of either endogenous (A, adhered) or transiently expressed vimentin (A', vimentin-FusionRed). B, percent of confined cells with vimentin localized in the cell body or leader bleb. C and C', lysates from A375-M2 and A549 cells probed for endogenous vimentin and pan-keratin. Densitometry (C', n = 3) was used to determine the ratio of vimentin in A375-M2 versus A549 cells. Statistical significance was determined by a one-sample (hypothetical value = 1) Student's t test. Error is S.E. All data are representative of at least three independent experiments. *, p ≤ 0.05.

Figure 2.

RNAi of vimentin promotes rapid leader bleb–based migration. A, montage of an A375-M2 cell under PDMS depleted of vimentin by RNAi expressing F-tractin–mEmerald and H2B-FusionRed. B, Western blot confirming depletion of vimentin by RNAi in A375-M2 cells. B', densitometry (n = 3) was used to determine the -fold change in vimentin after RNAi. Error is S.E. C and D, quantitative evaluation of leader bleb (C) and cell body (D) area in control (nontargeting), vimentin RNAi, and rescue (RNAi + Vimentin-FusionRed) cells. Statistical significance was determined by an ordinary one-way ANOVA followed by a post hoc multiple comparisons test. E, migration tracks for nontargeting (left panel, n = 40) and vimentin RNAi (right panel, n = 43) cells under PDMS. F, amalgamated instantaneous speeds for nontargeting, Vimentin RNAi, and rescue cells. Data were normalized around the average for nontargeting collected at the time of vimentin RNAi or rescue. Statistical significance was determined by an F test. Error is S.E. G, cartoon of the gel sandwich approach for measuring cell stiffness. H, cell stiffness for nontargeting (n = 77), vimentin RNAi (n = 30), and rescue (n = 23) cells. Statistical significance was determined by an ordinary one-way ANOVA followed by a post hoc multiple comparisons test. h, height; d, diameter. I, Western blots of endogenous p-RLC (S19) and RLC in nontargeting and vimentin RNAi cells. I', densitometry (n = 3) was used to determine the -fold change in p-RLC after RNAi. Statistical significance was determined by a one-sample (hypothetical value = 1) Student's t test. Error is S.E. In Tukey box plots, + and lines denote the mean and median, respectively. All data are representative of at least three independent experiments. **, p ≤ 0.01; ***, p ≤ 0.001; NS, not significant.

Figure 3.

Overexpressing vimentin in confined cells. A, montage of an A375-M2 cell overexpressing vimentin and the marker of F-actin, F-tractin, under PDMS. B and C, quantitative evaluation of leader bleb (B) and cell body (C) area for cells overexpressing EGFP alone and vimentin-FusionRed. Statistical significance was determined by two-tailed (unpaired) Student's t tests. D, instantaneous speeds for cells overexpressing EGFP alone (n = 41) and vimentin-FusionRed (n = 41). Statistical significance was determined by an F test. Error is S.E. E, cell stiffness for EGFP alone (n = 34) and vimentin-FusionRed–overexpressing (n = 31) cells. Statistical significance was determined by a two-tailed (unpaired) Student's t test. In Tukey box plots, + and lines denote the mean and median, respectively. All data are representative of at least three independent experiments. **, p ≤ 0.01; ***, p ≤ 0.001; NS, not significant.

Figure 4.

Vimentin bundling inhibits leader bleb–based migration. A and B, montage of an A375-M2 cell treated with vehicle (A, DMSO) or simvastatin (B, 10 μm) expressing F-tractin–mEmerald and vimentin-FusionRed under PDMS. Arrows point to areas of collapsed vimentin in the cell body. C, quantitative evaluation of vimentin network collapse for vehicle-treated (n = 39), simvastatin-treated (n = 26), and pravastatin-treated (n = 27) cells. Statistical significance was determined by an ordinary one-way ANOVA followed by a post hoc multiple comparisons test. D, quantitative evaluation of instantaneous speeds for vehicle-treated (n = 28), simvastatin-treated (n = 19), and pravastatin-treated (n = 20) cells. Statistical significance was determined by F tests. Error is S.E. E, cell stiffness measurements for vehicle (n = 77), simvastatin (n = 63), and pravastatin (n = 62). Statistical significance was determined by a Kruskal-Wallis test followed by post hoc multiple comparisons. F, Western blots of endogenous p-RLC, RLC, and vimentin in drug-treated cells. G, percentage of live (drug-treated) cells after 5 h under PDMS. In Tukey box plots, + and lines denote the mean and median, respectively. All data are representative of at least three independent experiments. *, p ≤ 0.05; **, p ≤ 0.01; NS, not significant.

Figure 5.

Simvastatin impairs migration and proliferation of cells expressing high levels of vimentin. A and B, quantitative evaluation of transmigration for drug-treated A375-M2 (A) and A549 (B) cells through fibronectin-coated (10 μg/ml) polycarbonate filters with 8- or 12-μm pores. Statistical significance was determined by a two-tailed (unpaired) Student's t test. Error is S.E. C and D, growth curves for drug-treated A375-M2 (C) and A549 (D) cells on tissue culture plastic. Error is S.E. E, bright-field images of A375-M2 (top row) and A549 (bottom row) cells 1 day after treatment with vehicle, simvastatin, or pravastatin. All experiments were performed at least three times. *, p ≤ 0.05; **, p ≤ 0.01; NS, not significant.