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. 2011 Dec;3(12):1153-66.
doi: 10.1039/c1ib00073j. Epub 2011 Oct 13.

Collective epithelial cell invasion overcomes mechanical barriers of collagenous extracellular matrix by a narrow tube-like geometry and MMP14-dependent local softening

Jordi Alcaraz  1 , Hidetoshi Mori, Cyrus M Ghajar, Doug Brownfield, Roland Galgoczy, Mina J Bissell
Affiliations

Collective epithelial cell invasion overcomes mechanical barriers of collagenous extracellular matrix by a narrow tube-like geometry and MMP14-dependent local softening

Jordi Alcaraz et al. Integr Biol (Camb). 2011 Dec.

Abstract

Collective cell invasion (CCI) through interstitial collagenous extracellular matrix (ECM) is crucial to the initial stages of branching morphogenesis, and a hallmark of tissue repair and dissemination of certain tumors. The collagenous ECM acts as a mechanical barrier against CCI. However, the physical nature of this barrier and how it is overcome by cells remains incompletely understood. To address these questions, we performed theoretical and experimental analysis of mammary epithelial branching morphogenesis in 3D type I collagen (collagen-I) gels. We found that the mechanical resistance of collagen-I is largely due to its elastic rather than its viscous properties. We also identified two strategies utilized by mammary epithelial cells that can independently minimize ECM mechanical resistance during CCI. First, cells adopt a narrow tube-like geometry during invasion, which minimizes the elastic opposition from the ECM as revealed by theoretical modeling of the most frequent invasive shapes and sizes. Second, the stiffness of the collagenous ECM is reduced at invasive fronts due to its degradation by matrix metalloproteinases (MMPs), as indicated by direct measurements of collagen-I microelasticity by atomic force microscopy. Molecular techniques further specified that the membrane-bound MMP14 mediates degradation of collagen-I at invasive fronts. Thus, our findings reveal that MMP14 is necessary to efficiently reduce the physical restraints imposed by collagen-I during branching morphogenesis, and help our overall understanding of how forces are balanced between cells and their surrounding ECM to maintain collective geometry and mechanical stability during CCI.

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Figures

Fig. 1
Fig. 1
Morphometric analysis revealed the most frequent multicellular geometries during early invasion. (A) Representative phase-contrast image of a cluster of EpH4 cells in collagen-I (3 mg ml−1) induced to branch with FGF2 for 4 days. White arrow and stars point to invasive geometries labeled as cylindrical- or cone-like, respectively, by qualitative image examination. L, wbase and wtip were measured in each invasive body, whereas 2θ was measured on conical-like bodies only as shown in the image. Note that some branching structures are out of focus due to their large 3D size. Scale bar corresponds to 20 μm. (B–E) Normalized histograms of the ratio wtip/wbase (B), θ (C), wbase/w1 (D), and L/w1 (E) measured over ~200 invasive bodies. Details on the thresholds used to distinguish cones, cylinders and uncertain (mixed) geometries are given in the main text. For each image, w1 was taken as the minimum wtip. (F) Drawings illustrating the idealized most frequent cross-section invasive geometries based on combining one of the three most frequent widths (cylinder, sphere) or semiincluded angles (cone) with one of the most common and compatible tube lengths for cylinders (a–c), cones (d–f) and spheres (g–i) as found in the frequency plots (B–E). All lengths were scaled in units of the single cell width w1 to facilitate direct size comparisons. The asterisk (*) indicates those geometries theoretically permissive for lumen formation here and in Fig. 2 and 3. Likewise, the geometrical values and letters describing these idealized most frequent geometries were also used in Fig. 2 and 3.
Fig. 2
Fig. 2
Theoretical simulations of the impact of the most frequent invasive geometries on Fel and Fvis in the absence of ECM degradation. (A) Drawing illustrating the ECM-dependent elastic and viscous forces acting on the tip of an invasive body in the direction of invasion, and the relevant geometrical parameters (L, δ and v). (B–C) Theoretical ECM elastic resistance against invasion was quantified as either Fel (B) or keff (C) as a function of gel indentation up to 7 cell diameters for the three most frequent cylindrical (blue), conical (red) and spherical (gray) invasive geometries as described in Fig. 1F. (D) Likewise, the Translational Friction Coefficient was used to assess the theoretical ECM viscous resistance for cylinders (blue), ellipsoids (red, used as surrogate for a cone), and sphere (gray) as a function of L. Note that the ticks in the x-axis are given in units of 7 μm, which corresponds to the average diameter of single MECs (w1). Formulas used in these simulations are shown in the main text. The letters a–i on the right side of each plot (B–D) correspond to the letters used in Fig. 1F, and are also used in Fig. 3.
Fig. 3
Fig. 3
Theoretical simulations of the impact of the most frequent multicellular geometries during early invasion on their topology. (A) Drawings defining the relevant geometrical parameters used to simulate the topological parameters for a cone, a truncated cone, and a truncated sphere that best capture the most frequent invasive geometries. (B–D) Plots showing S, V and S/V as a function of L up to 7 cell diameters for the three most frequent geometries using the same color coding and x-axis ticks as in Fig. 2. The most frequent geometrical values described in Fig. 1F were considered in the topological calculations. Details on all the geometrical values and formulas used in these calculations are given in the main text. Note that only one curve is shown for the spherical geometry in B–D, since the values for L and R are not independent, but related by 2R = L + fR as shown in A. As in Fig. 2, w1 was taken as the average MEC diameter.
Fig. 4
Fig. 4
Assessment of local ECM degradation and softening at invasive fronts by AFM and confocal microscopy. (A) Representative bright-field (BF) images illustrating the spatial localization of the AFM measurements. Only the cell clusters closest to the gel surface (< 25 μm in depth) were considered, which correspond to cell clusters nearly in focus with the AFM cantilever (see methods for details). For each cell cluster, the tip of the AFM cantilever was positioned at the very edge of either an invasive tubule (top left) or a non-invasive cluster (bottom left), the corresponding E was measured, and normalized afterwards with respect to E measured far (> 100 μm) from the cell cluster (Efar) (right images). White arrow points to an invasive front (top left image) or an edge of a non-invasive cluster (bottom left image). Scale bars correspond to 40 μm. (B) Normalized E data obtained in the absence or presence of GM6001. *P < 0.05 between normalized data obtained at the cell cluster edge or far from it. (C) Confocal sections (left images) showing collagen filaments surrounding the middle of a cell cluster embedded in 3D collagen-I gel containing fluorescent DQ-Collagen and untreated (top images) or treated (bottom images) with GM6001. Corresponding BF and merged images are shown in the middle and right of each panel. The arrows (top left) point to an area void of DQ-Collagen fluorescence in the untreated cells, whereas such void areas were absent or randomly distributed in GM6001 treated cells. The full series of confocal sections are shown in Supplementary Fig. 3 of the ESI. Scale bars correspond to 20 μm.
Fig. 5
Fig. 5
Structural and elastic properties of cell-free collagen-I gels untreated or treated with exogenous collagenase. (A) Representative confocal sections of collagen filaments obtained by CRM corresponding to an untreated or a collagenase treated (up to 60 min) 3 mg ml−1 collagen-I gel. Scale bars correspond to 5 μm. (B) E of untreated or collagenase treated collagen-I gels measured by AFM. **P < 0.01. (C) E of different concentrations of cell-free collagen-I gels measured by AFM. Data were fitted to semiflexible polymer models corresponding to semi-diluted (red) or concentrated (blue) gels.
Fig. 6
Fig. 6
Effect of MMP14 increased or reduced expression on ECM mechanical integrity. (A–B) MMP14 overexpression experiments: EpH4 cells were stably transfected with MMP14. (A) Western-blotting showing increased MMP14 activity of transfected EpH4 cells with respect to control. (B) Representative epifluorescence images of control or MMP14 overexpressing cells stained with the live dye Calcein AM. Note that MMP14 overexpressing cells exhibited longer tubules. (C–D) Reduced MMP14 expression experiments: (C) PCR gel showing a reduction in MMP14 expression in EpH4 cells stably transfected with shRNA against MMP14 with respect to scrambled shRNA control. (D) Representative epifluorescence images of control or transfected cells stained with Calcein AM. Note the absence of branching structures in the MMP14 shRNA transfected cells. (E) Normalized E data of either MMP14 shRNA or MMP14 transfected EpH4 cells measured by AFM. *P < 0.05 between normalized E at the invasive front or far from it. All scale bars correspond to 200 μm.
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References

    1. Fleury V, Watanabe T, Nguyen T, Unbekandt M, Warburton D, Dejmek M, Nguyen MB, Lindner A, Schwartz L. In: Branching Morphogenesis. Davies JA, editor. Landes Bioscience; Georgetown: 2005. pp. 202–234.
    1. Lu P, Sternlicht MD, Werb Z. J Mammary Gland Biol Neoplasia. 2006;11:213–228. - PMC - PubMed
    1. Friedl P, Gilmour D. Nat Rev Mol Cell Biol. 2009;10:445–457. - PubMed
    1. Gjorevski N, Nelson CM. Wiley Interdiscip Rev Syst Biol Med. 2010;2:734–741. - PMC - PubMed
    1. Schedin P, Keely PJ. Cold Spring Harbor Perspect Biol. 2010;3:a003228. - PMC - PubMed

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