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Direct measurement of vertical forces shows correlation between mechanical activity and proteolytic ability of invadopodia

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Science Advances  11 Mar 2020:
Vol. 6, no. 11, eaax6912
DOI: 10.1126/sciadv.aax6912
  • Fig. 1 ERISM for measuring the mechanical activity of invadopodia formed by UM-SCC-1 cancer cells.

    (A) Schematic sketch of the ERISM measurement, illustrating how invadopodia forces against an elastic microcavity, formed by two gold films sandwiching a soft elastomer, lead to local changes in the thickness and resonance wavelengths of the microcavity. The microcavity substrate is coated with fluorescent gelatin (green line) to distinguish mature (degrading) from immature (nondegrading) invadopodia. (B) Phase-contrast image of UM-SCC-1 cell attached to the microcavity surface. (C) Coregistered monochromatic reflectance image of the microcavity, for illumination with 676-nm light. (D) ERISM thickness map obtained from a series of reflectance images, showing the substrate deformation induced by the same cell. Black arrows indicate three local indentation features. (E) ERISM stress map and (F) ERISM stress map after application of Fourier band-pass filter. (G) Lateral stress profile along the black and red dashed lines in (F). (H) Epifluorescence image of the same cell after immunostaining for cortactin. Scale bar, 10 μm.

  • Fig. 2 Correlation of invadopodial force with matrix degradation.

    (A and B) Phase-contrast images of two representative UM-SCC-1 cells with immature and mature invadopodia, respectively. (C and D) Fourier-filtered ERISM stress map of same cells, with arrows indicating representative invadopodia in each cell. (E and F) Epifluorescence image of fluorescent gelatin film on the surface of the ERISM substrate. Pink lines indicate the outline of cells. The maturity of invadopodia was classified by analysis of local gelatin fluorescence. (G) Comparison between force exerted by mature (n = 66) and immature (n = 92) invadopodia. Each data point represents the mean force exerted by an invadopodium over a 1-hour time frame. Plots indicate data (diamonds), median (center), mean (open square), Q1/Q3 (box), and ±1.5 SD (whiskers). As data in groups were not normally distributed, groups were compared using the Mann-Whitney U test (***P < 0.001). Scale bars, 10 μm.

  • Fig. 3 Dynamics of mechanical activity of invadopodia.

    (A) Phase-contrast image and (B) Fourier-filtered ERISM stress map of a typical UM-SCC-1 cell. (C) Time lapse of the invadopodium indicated by the green rectangle in (B), imaged every 2 min. (D) Temporal evolution of force exerted by a single invadopodium (gray line) and background force in a region without invadopodia (red line). Force measurements were taken every 10 s; recording started 2 hours after cell seeding. (E) Overall duration of protrusive activity and (F) mean duration of fast oscillation cycles in force for multiple invadopodia (3 individual experiments, 5 cells, and 93 invadopodia, of which 45 were classified as mature, 8 as immature, and the remaining not classified). Plots indicate data (diamonds), median (center), mean (open square), Q1/Q3 (box), and ±1.5 SD (whiskers). (G) Time evolution of forces exerted by three immature (top) and three mature (bottom) invadopodia. Force measurements were taken every 2.5 min; recording started 3 hours after cell seeding. Scale bars, 20 μm (A) and 1 μm (C).

  • Fig. 4 Impact of overexpression of the miR-375 on the mechanical activity of UM-SCC-1 cells.

    (A and B) Phase-contrast, (C and D) ERISM, and (E and F) Fourier-filtered ERISM images of representative UM-SCC-1 control cell (left column) and miR-375–overexpressing cell (right column). (G) Comparison of total volume by which cells indent into the ERISM substrate. Control, n = 13 cells; miR-375, n = 16 cells. As data in groups were not normally distributed, groups were compared using the Mann-Whitney U test (P = 0.983). (H) Comparison of the number of protruding invadopodia formed per cell (mixed population of mature and immature invadopodia). Control, n = 9 cells; miR-375, n = 6 cells. Two-sample t test (P = 0.005). (I) Comparison of force exerted by invadopodia from both cell types. Control, n = 106 invadopodia from eight cells; miR-375, n = 47 invadopodia from three cells. As data in groups were not normally distributed, groups were compared using the Mann-Whitney U test (P = 1.41 × 10−6). Statistical tests: not significant (n.s.), P > 0.05; **P < 0.01; ***P < 0.001. Scale bars, 20 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/11/eaax6912/DC1

    Fig. S1. AFM measurement of thickness and apparent stiffness of ERISM substrate and gelatin coating.

    Fig. S2. ERISM and immunostaining for actin, cortactin, and Tks5 in UM-SCC-1 cells.

    Fig. S3. Simultaneous imaging of gelatin matrix degradation and invadopodia force.

    Fig. S4. Time dependence of invadopodia force within a single cell.

    Fig. S5. Long-term measurement of invadopodia forces.

    Fig. S6. Fourier transform–based analysis of the characteristic period of force oscillation in invadopodia.

    Movie S1. Temporal evolution of invadopodia force for a typical cell with immature invadopodia.

    Movie S2. Temporal evolution of invadopodia force for a typical cell with predominantly mature invadopodia.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. AFM measurement of thickness and apparent stiffness of ERISM substrate and gelatin coating.
    • Fig. S2. ERISM and immunostaining for actin, cortactin, and Tks5 in UM-SCC-1 cells.
    • Fig. S3. Simultaneous imaging of gelatin matrix degradation and invadopodia force.
    • Fig. S4. Time dependence of invadopodia force within a single cell.
    • Fig. S5. Long-term measurement of invadopodia forces.
    • Fig. S6. Fourier transform–based analysis of the characteristic period of force oscillation in invadopodia.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Temporal evolution of invadopodia force for a typical cell with immature invadopodia.
    • Movie S2 (.avi format). Temporal evolution of invadopodia force for a typical cell with predominantly mature invadopodia.

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