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Cells assemble invadopodia-like structures and invade into matrigel in a matrix metalloprotease dependent manner in the circular invasion assay.

Yu X, Machesky LM - PLoS ONE (2012)

Bottom Line: The ability of tumor cells to invade is one of the hallmarks of the metastatic phenotype.We have extended the characterization of the circular invasion assay and found that it provides a simple and amenable system to study cell invasion in matrix in an environment that closely mimics 3D invasion.Furthermore, it allows detailed microscopic analysis of both live and fixed cells during the invasion process.

View Article: PubMed Central - PubMed

Affiliation: The Beatson Institute for Cancer Research, Glasgow University College of Medical Veterinary and Life Sciences, Glasgow, United Kingdom.

ABSTRACT
The ability of tumor cells to invade is one of the hallmarks of the metastatic phenotype. To elucidate the mechanisms by which tumor cells acquire an invasive phenotype, in vitro assays have been developed that mimic the process of cancer cell invasion through basement membrane or in the stroma. We have extended the characterization of the circular invasion assay and found that it provides a simple and amenable system to study cell invasion in matrix in an environment that closely mimics 3D invasion. Furthermore, it allows detailed microscopic analysis of both live and fixed cells during the invasion process. We find that cells invade in a protease dependent manner in this assay and that they assemble focal adhesions and invadopodia that resemble structures visualized in 3D embedded cells. We propose that this is a useful assay for routine and medium throughput analysis of invasion of cancer cells in vitro and the study of cells migrating in a 3D environment.

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Invadopodia protrude into the matrix in CIA.(A) MDA-MB-231 cells completely embedded in collagen I also show similar elongated morphology as in CIA. Staining of N-WASP or cortactin (green) and actin (red) show that N-WASP and filamentous actin containing structures resembling invadopodia (white arrowheads) are also present in cells migrating in a pure 3D environment. Scale bar 20 µm. (B) Z-stack projection showing invadopodia-like structures (arrowheads) localize to various positions, including the front of the invading pseudopods facing upward into the Matrigel and at the periphery and dorsal surface. Staining shows N-WASP (green), actin (red) and DAPI (blue) or cortactin (blue). See also Movie S8.
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pone-0030605-g006: Invadopodia protrude into the matrix in CIA.(A) MDA-MB-231 cells completely embedded in collagen I also show similar elongated morphology as in CIA. Staining of N-WASP or cortactin (green) and actin (red) show that N-WASP and filamentous actin containing structures resembling invadopodia (white arrowheads) are also present in cells migrating in a pure 3D environment. Scale bar 20 µm. (B) Z-stack projection showing invadopodia-like structures (arrowheads) localize to various positions, including the front of the invading pseudopods facing upward into the Matrigel and at the periphery and dorsal surface. Staining shows N-WASP (green), actin (red) and DAPI (blue) or cortactin (blue). See also Movie S8.

Mentions: One of the important features of invadopodia is that they have matrix-degrading capability [7], [8]. To test whether the puncta observed in CIA were likely to be bona-fide invadopodia, we tested for protease localization with fluorescently tagged MT1-MMP and for protease activity using DQ collagen I, a quenched fluorescent collagen that becomes brighter upon cleavage. mCherry-MT1-MMP puncta co-localized with GFP-N-WASP near the edges of pseudopods facing into the empty space being invaded (Fig. 5A). We also observed DQ collagen fluorescence in the pericellular space, which was concentrated largely near bright F-actin containing protrusions (Fig. 5B). These F-actin protrusions with collagen degradation activity frequently contained multiple invadopodia-like puncta containing N-WASP and Arp2/3 complex, suggesting focal degradation activity does happen at these sites. We cannot rule out that some of the small puncta may be vesicles in transit to sites of matrix degradation, as cortactin, N-WASP and Arp2/3 complex can also localize to intracellular vesicles, as does MT1-MMP [27], [28], [29], [30]. Similar dot-like structures with co-localization of N-WASP, cortactin and actin are also observed in cells embedded in pure 3D collagen or Matrigel environment (Fig. 6A, white arrowheads).


Cells assemble invadopodia-like structures and invade into matrigel in a matrix metalloprotease dependent manner in the circular invasion assay.

Yu X, Machesky LM - PLoS ONE (2012)

Invadopodia protrude into the matrix in CIA.(A) MDA-MB-231 cells completely embedded in collagen I also show similar elongated morphology as in CIA. Staining of N-WASP or cortactin (green) and actin (red) show that N-WASP and filamentous actin containing structures resembling invadopodia (white arrowheads) are also present in cells migrating in a pure 3D environment. Scale bar 20 µm. (B) Z-stack projection showing invadopodia-like structures (arrowheads) localize to various positions, including the front of the invading pseudopods facing upward into the Matrigel and at the periphery and dorsal surface. Staining shows N-WASP (green), actin (red) and DAPI (blue) or cortactin (blue). See also Movie S8.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3275555&req=5

pone-0030605-g006: Invadopodia protrude into the matrix in CIA.(A) MDA-MB-231 cells completely embedded in collagen I also show similar elongated morphology as in CIA. Staining of N-WASP or cortactin (green) and actin (red) show that N-WASP and filamentous actin containing structures resembling invadopodia (white arrowheads) are also present in cells migrating in a pure 3D environment. Scale bar 20 µm. (B) Z-stack projection showing invadopodia-like structures (arrowheads) localize to various positions, including the front of the invading pseudopods facing upward into the Matrigel and at the periphery and dorsal surface. Staining shows N-WASP (green), actin (red) and DAPI (blue) or cortactin (blue). See also Movie S8.
Mentions: One of the important features of invadopodia is that they have matrix-degrading capability [7], [8]. To test whether the puncta observed in CIA were likely to be bona-fide invadopodia, we tested for protease localization with fluorescently tagged MT1-MMP and for protease activity using DQ collagen I, a quenched fluorescent collagen that becomes brighter upon cleavage. mCherry-MT1-MMP puncta co-localized with GFP-N-WASP near the edges of pseudopods facing into the empty space being invaded (Fig. 5A). We also observed DQ collagen fluorescence in the pericellular space, which was concentrated largely near bright F-actin containing protrusions (Fig. 5B). These F-actin protrusions with collagen degradation activity frequently contained multiple invadopodia-like puncta containing N-WASP and Arp2/3 complex, suggesting focal degradation activity does happen at these sites. We cannot rule out that some of the small puncta may be vesicles in transit to sites of matrix degradation, as cortactin, N-WASP and Arp2/3 complex can also localize to intracellular vesicles, as does MT1-MMP [27], [28], [29], [30]. Similar dot-like structures with co-localization of N-WASP, cortactin and actin are also observed in cells embedded in pure 3D collagen or Matrigel environment (Fig. 6A, white arrowheads).

Bottom Line: The ability of tumor cells to invade is one of the hallmarks of the metastatic phenotype.We have extended the characterization of the circular invasion assay and found that it provides a simple and amenable system to study cell invasion in matrix in an environment that closely mimics 3D invasion.Furthermore, it allows detailed microscopic analysis of both live and fixed cells during the invasion process.

View Article: PubMed Central - PubMed

Affiliation: The Beatson Institute for Cancer Research, Glasgow University College of Medical Veterinary and Life Sciences, Glasgow, United Kingdom.

ABSTRACT
The ability of tumor cells to invade is one of the hallmarks of the metastatic phenotype. To elucidate the mechanisms by which tumor cells acquire an invasive phenotype, in vitro assays have been developed that mimic the process of cancer cell invasion through basement membrane or in the stroma. We have extended the characterization of the circular invasion assay and found that it provides a simple and amenable system to study cell invasion in matrix in an environment that closely mimics 3D invasion. Furthermore, it allows detailed microscopic analysis of both live and fixed cells during the invasion process. We find that cells invade in a protease dependent manner in this assay and that they assemble focal adhesions and invadopodia that resemble structures visualized in 3D embedded cells. We propose that this is a useful assay for routine and medium throughput analysis of invasion of cancer cells in vitro and the study of cells migrating in a 3D environment.

Show MeSH
Related in: MedlinePlus