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Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.

Wolf K, Mazo I, Leung H, Engelke K, von Andrian UH, Deryugina EI, Strongin AY, Bröcker EB, Friedl P - J. Cell Biol. (2003)

Bottom Line: This process, however, is only incompletely attenuated by protease inhibitor-based treatment, suggesting the existence of migratory compensation strategies.In three-dimensional collagen matrices, spindle-shaped proteolytically potent HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells exhibited a constitutive mesenchymal-type movement including the coclustering of beta 1 integrins and MT1-matrix metalloproteinase (MMP) at fiber bindings sites and the generation of tube-like proteolytic degradation tracks.Near-total inhibition of MMPs, serine proteases, cathepsins, and other proteases, however, induced a conversion toward spherical morphology at near undiminished migration rates.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Würzburg, 97080 Würzburg, Germany.

ABSTRACT
Invasive tumor dissemination in vitro and in vivo involves the proteolytic degradation of ECM barriers. This process, however, is only incompletely attenuated by protease inhibitor-based treatment, suggesting the existence of migratory compensation strategies. In three-dimensional collagen matrices, spindle-shaped proteolytically potent HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells exhibited a constitutive mesenchymal-type movement including the coclustering of beta 1 integrins and MT1-matrix metalloproteinase (MMP) at fiber bindings sites and the generation of tube-like proteolytic degradation tracks. Near-total inhibition of MMPs, serine proteases, cathepsins, and other proteases, however, induced a conversion toward spherical morphology at near undiminished migration rates. Sustained protease-independent migration resulted from a flexible amoeba-like shape change, i.e., propulsive squeezing through preexisting matrix gaps and formation of constriction rings in the absence of matrix degradation, concomitant loss of clustered beta 1 integrins and MT1-MMP from fiber binding sites, and a diffuse cortical distribution of the actin cytoskeleton. Acquisition of protease-independent amoeboid dissemination was confirmed for HT-1080 cells injected into the mouse dermis monitored by intravital multiphoton microscopy. In conclusion, the transition from proteolytic mesenchymal toward nonproteolytic amoeboid movement highlights a supramolecular plasticity mechanism in cell migration and further represents a putative escape mechanism in tumor cell dissemination after abrogation of pericellular proteolysis.

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Protease expression in HT-1080/MT1 cells and inhibition of collagenolysis. (A) mRNA expression of MMPs, ADAMs, cathepsins, and serine proteases detected by RT-PCR. Asterisks, proteases cleaving native type I collagen. (B) Degradation of 3D fibrillar collagen by HT1080/MT1 cells (500,000 cells) layered on top of a 1-mm-thick collagen matrix in the absence (1) or presence (2) of protease inhibitor cocktail. (B, 3) Confocal backscatter of lysis zone bordered by clumped collagen, as indicated by the square in (1). (B, 4) Negative control matrix overlaid with cell-free medium only. (C) Cell contact to FITC-labeled collagen fibers; confocal reflection (gray) and FITC fluorescence (green). Bar, 20 μm. (D) Migration-associated collagenolysis caused by HT1080/MT1 cells within 3D FITC–collagen lattices was quantified from the FITC release after 40 h of migration in the absence or presence of inhibitors. ***, P < 0.001; two-tailed t test for independent means, difference to control cells. As negative control, T cells did not release FITC above background levels (not depicted).
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fig2: Protease expression in HT-1080/MT1 cells and inhibition of collagenolysis. (A) mRNA expression of MMPs, ADAMs, cathepsins, and serine proteases detected by RT-PCR. Asterisks, proteases cleaving native type I collagen. (B) Degradation of 3D fibrillar collagen by HT1080/MT1 cells (500,000 cells) layered on top of a 1-mm-thick collagen matrix in the absence (1) or presence (2) of protease inhibitor cocktail. (B, 3) Confocal backscatter of lysis zone bordered by clumped collagen, as indicated by the square in (1). (B, 4) Negative control matrix overlaid with cell-free medium only. (C) Cell contact to FITC-labeled collagen fibers; confocal reflection (gray) and FITC fluorescence (green). Bar, 20 μm. (D) Migration-associated collagenolysis caused by HT1080/MT1 cells within 3D FITC–collagen lattices was quantified from the FITC release after 40 h of migration in the absence or presence of inhibitors. ***, P < 0.001; two-tailed t test for independent means, difference to control cells. As negative control, T cells did not release FITC above background levels (not depicted).

Mentions: Pericellular degradation of native or denatured collagens can be directly or indirectly provided by proteases from different classes, including MMPs, serine proteases (e.g., plasmin and urokinase-type plasminogen activator [uPA]), as well as cysteine and aspartic proteases (e.g., cathepsins) (Montcourrier et al., 1990; Aimes and Quigley, 1995; Ohuchi et al., 1997; Sassi et al., 2000). In addition to MT1-MMP, many of these proteases, including up to five other collagenases toward native type collagen (Fig. 2 A, asterisks), were expressed by HT-1080/MT1 cells, as detected by RT-PCR. Because collagenolytic redundancy was anticipated, we used a cocktail of broad-spectrum protease inhibitors to simultaneously target a wide spectrum of endoproteolytic activity (Table I). To maintain sufficient inhibitory activity within the matrix, the employed concentrations of protease inhibitors were orders of magnitudes higher than the known maximum inhibitory values, unless cell viability was dose limiting (Table I). Because most of the proteases detectable by RT-PCR escape visualization by zymography (Deryugina et al., 1997, 1998; Ntayi et al., 2001), inhibition of cell-dependent collagenolysis in situ was measured using the fibrillar collagen migration substrate as read-out. On a qualitative basis, structural breakdown of the matrix fibers caused by HT-1080/MT1 cells (Fig. 2 B, 1 and 3) was fully abrogated in the presence of protease inhibitor cocktail (Fig. 2 B, 2). This strong inhibitory effect was confirmed by a quantitative 3D fluorometric fluorescein (FITC) release assay, detecting the degradation of FITC-labeled collagen fibers (Fig. 2 C). Upon migration within FITC–collagen, HT-1080/MT1 cells released 20% of the total FITC content (Fig. 2 D), confirming the observed macroscopic (Fig. 2 B, 1) and microscopic collagen fiber degradation (Fig. 1 E; Fig. 2 B, 3). Protease inhibitor cocktail inhibited 95% of the cell-mediated FITC release generated by HT-1080/MT1 cells (Fig. 2 D) and to the same residual level in vector-transfected HT-1080/neo cells (unpublished data). Broad-range MMP inhibitor BB-2516 (marimastat) alone abrogated >80% of the FITC release in HT-1080/MT1 cells, suggesting a major contribution of MMPs to the degradation of collagen (Fig. 2 D).


Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.

Wolf K, Mazo I, Leung H, Engelke K, von Andrian UH, Deryugina EI, Strongin AY, Bröcker EB, Friedl P - J. Cell Biol. (2003)

Protease expression in HT-1080/MT1 cells and inhibition of collagenolysis. (A) mRNA expression of MMPs, ADAMs, cathepsins, and serine proteases detected by RT-PCR. Asterisks, proteases cleaving native type I collagen. (B) Degradation of 3D fibrillar collagen by HT1080/MT1 cells (500,000 cells) layered on top of a 1-mm-thick collagen matrix in the absence (1) or presence (2) of protease inhibitor cocktail. (B, 3) Confocal backscatter of lysis zone bordered by clumped collagen, as indicated by the square in (1). (B, 4) Negative control matrix overlaid with cell-free medium only. (C) Cell contact to FITC-labeled collagen fibers; confocal reflection (gray) and FITC fluorescence (green). Bar, 20 μm. (D) Migration-associated collagenolysis caused by HT1080/MT1 cells within 3D FITC–collagen lattices was quantified from the FITC release after 40 h of migration in the absence or presence of inhibitors. ***, P < 0.001; two-tailed t test for independent means, difference to control cells. As negative control, T cells did not release FITC above background levels (not depicted).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172637&req=5

fig2: Protease expression in HT-1080/MT1 cells and inhibition of collagenolysis. (A) mRNA expression of MMPs, ADAMs, cathepsins, and serine proteases detected by RT-PCR. Asterisks, proteases cleaving native type I collagen. (B) Degradation of 3D fibrillar collagen by HT1080/MT1 cells (500,000 cells) layered on top of a 1-mm-thick collagen matrix in the absence (1) or presence (2) of protease inhibitor cocktail. (B, 3) Confocal backscatter of lysis zone bordered by clumped collagen, as indicated by the square in (1). (B, 4) Negative control matrix overlaid with cell-free medium only. (C) Cell contact to FITC-labeled collagen fibers; confocal reflection (gray) and FITC fluorescence (green). Bar, 20 μm. (D) Migration-associated collagenolysis caused by HT1080/MT1 cells within 3D FITC–collagen lattices was quantified from the FITC release after 40 h of migration in the absence or presence of inhibitors. ***, P < 0.001; two-tailed t test for independent means, difference to control cells. As negative control, T cells did not release FITC above background levels (not depicted).
Mentions: Pericellular degradation of native or denatured collagens can be directly or indirectly provided by proteases from different classes, including MMPs, serine proteases (e.g., plasmin and urokinase-type plasminogen activator [uPA]), as well as cysteine and aspartic proteases (e.g., cathepsins) (Montcourrier et al., 1990; Aimes and Quigley, 1995; Ohuchi et al., 1997; Sassi et al., 2000). In addition to MT1-MMP, many of these proteases, including up to five other collagenases toward native type collagen (Fig. 2 A, asterisks), were expressed by HT-1080/MT1 cells, as detected by RT-PCR. Because collagenolytic redundancy was anticipated, we used a cocktail of broad-spectrum protease inhibitors to simultaneously target a wide spectrum of endoproteolytic activity (Table I). To maintain sufficient inhibitory activity within the matrix, the employed concentrations of protease inhibitors were orders of magnitudes higher than the known maximum inhibitory values, unless cell viability was dose limiting (Table I). Because most of the proteases detectable by RT-PCR escape visualization by zymography (Deryugina et al., 1997, 1998; Ntayi et al., 2001), inhibition of cell-dependent collagenolysis in situ was measured using the fibrillar collagen migration substrate as read-out. On a qualitative basis, structural breakdown of the matrix fibers caused by HT-1080/MT1 cells (Fig. 2 B, 1 and 3) was fully abrogated in the presence of protease inhibitor cocktail (Fig. 2 B, 2). This strong inhibitory effect was confirmed by a quantitative 3D fluorometric fluorescein (FITC) release assay, detecting the degradation of FITC-labeled collagen fibers (Fig. 2 C). Upon migration within FITC–collagen, HT-1080/MT1 cells released 20% of the total FITC content (Fig. 2 D), confirming the observed macroscopic (Fig. 2 B, 1) and microscopic collagen fiber degradation (Fig. 1 E; Fig. 2 B, 3). Protease inhibitor cocktail inhibited 95% of the cell-mediated FITC release generated by HT-1080/MT1 cells (Fig. 2 D) and to the same residual level in vector-transfected HT-1080/neo cells (unpublished data). Broad-range MMP inhibitor BB-2516 (marimastat) alone abrogated >80% of the FITC release in HT-1080/MT1 cells, suggesting a major contribution of MMPs to the degradation of collagen (Fig. 2 D).

Bottom Line: This process, however, is only incompletely attenuated by protease inhibitor-based treatment, suggesting the existence of migratory compensation strategies.In three-dimensional collagen matrices, spindle-shaped proteolytically potent HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells exhibited a constitutive mesenchymal-type movement including the coclustering of beta 1 integrins and MT1-matrix metalloproteinase (MMP) at fiber bindings sites and the generation of tube-like proteolytic degradation tracks.Near-total inhibition of MMPs, serine proteases, cathepsins, and other proteases, however, induced a conversion toward spherical morphology at near undiminished migration rates.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Würzburg, 97080 Würzburg, Germany.

ABSTRACT
Invasive tumor dissemination in vitro and in vivo involves the proteolytic degradation of ECM barriers. This process, however, is only incompletely attenuated by protease inhibitor-based treatment, suggesting the existence of migratory compensation strategies. In three-dimensional collagen matrices, spindle-shaped proteolytically potent HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells exhibited a constitutive mesenchymal-type movement including the coclustering of beta 1 integrins and MT1-matrix metalloproteinase (MMP) at fiber bindings sites and the generation of tube-like proteolytic degradation tracks. Near-total inhibition of MMPs, serine proteases, cathepsins, and other proteases, however, induced a conversion toward spherical morphology at near undiminished migration rates. Sustained protease-independent migration resulted from a flexible amoeba-like shape change, i.e., propulsive squeezing through preexisting matrix gaps and formation of constriction rings in the absence of matrix degradation, concomitant loss of clustered beta 1 integrins and MT1-MMP from fiber binding sites, and a diffuse cortical distribution of the actin cytoskeleton. Acquisition of protease-independent amoeboid dissemination was confirmed for HT-1080 cells injected into the mouse dermis monitored by intravital multiphoton microscopy. In conclusion, the transition from proteolytic mesenchymal toward nonproteolytic amoeboid movement highlights a supramolecular plasticity mechanism in cell migration and further represents a putative escape mechanism in tumor cell dissemination after abrogation of pericellular proteolysis.

Show MeSH
Related in: MedlinePlus