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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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Related in: MedlinePlus

MDA-MB-231 cells form collectively invading chains in CIA.(A) Photos from a time-lapse video of MDA-MB-231 cells invading into Matrigel in CIA showing the formation of long invasion chains at the invading wound edge. Scale bar 50 µm. (B) Cells in a large finger-like chain at the leading edge of a CIA are shown fixed and stained with actin (red), and DNA (blue). Scale bar 20 µm. (C) Actin (red), N-WASP (green) and DAPI (blue) staining of a cell chain at the front of the invading area. White arrowheads indicate puncta of N-WASP co-localizing with filamentous actin. Scale bar 20 µm. (D) Image sequence showing an invading cell actively remodeling the matrix and generating what appear as micro-tunnels (white arrow). Scale bar 20 µm. (E) Staining of actin (red) and DNA (blue) of cell invasion chains in inverted invasion assay and visualized by confocal microscopy. Image showing collective cell invasion chains on single plane (left panel) and the side view of z-stack 3D projection (right panel). Scale bar 20 µm. See also Movies S1, S2, S3.
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pone-0030605-g001: MDA-MB-231 cells form collectively invading chains in CIA.(A) Photos from a time-lapse video of MDA-MB-231 cells invading into Matrigel in CIA showing the formation of long invasion chains at the invading wound edge. Scale bar 50 µm. (B) Cells in a large finger-like chain at the leading edge of a CIA are shown fixed and stained with actin (red), and DNA (blue). Scale bar 20 µm. (C) Actin (red), N-WASP (green) and DAPI (blue) staining of a cell chain at the front of the invading area. White arrowheads indicate puncta of N-WASP co-localizing with filamentous actin. Scale bar 20 µm. (D) Image sequence showing an invading cell actively remodeling the matrix and generating what appear as micro-tunnels (white arrow). Scale bar 20 µm. (E) Staining of actin (red) and DNA (blue) of cell invasion chains in inverted invasion assay and visualized by confocal microscopy. Image showing collective cell invasion chains on single plane (left panel) and the side view of z-stack 3D projection (right panel). Scale bar 20 µm. See also Movies S1, S2, S3.

Mentions: We have further developed and more extensively characterized the wound closure-based circular invasion assay as an effective method for visualizing cells during the invasion process [19]. As an alternative to wounding, we used a square, self-stick silicone cell stopper to create a uniform and neat central cell-free space without damaging or disrupting cells. MDA-MB-231 cells were seeded on a 35 µm glass-bottom dish with a stopper blocking the central area. After the cells attached to the glass bottom, the stopper was removed and a thin layer of Matrigel was applied on top of cells and allowed to set. Cells were generally invaded into the Matrigel for 16 hours or longer. As shown in Fig. 1A and B and Movie S1, MDA-MB-231 cells migrated into the central space, penetrating into the Matrigel while assuming an elongated shape near the invading front. Unlike traditional invasion assays, cells could be straightforwardly fixed and stained with antibodies to obtain high quality images. Fig. 1B shows phalloidin and DAPI labeling of leading edge invasion chains. Fig. 1C shows phalloidin stain of actin (red) and punctate appearance of endogenous N-WASP staining (green) with DAPI stain of DNA in blue. N-WASP and actin frequently co-localized to punctuate structures at the cell periphery and protrusions (Fig. 1C, white arrowheads). Invading cells formed cylinder-shape pseudopods and appeared to actively reshape the surrounding matrix by creation of tunnels resembling previously described micro-tunnels also termed SCITS (single-cell invasion tunnels) (Fig. 1D) [20]. Collective migration chains formed as following cells filled into the micro-tunnels (Fig. 1A–C, arrows indicate in C and Movie S1 and S2). These invasion chains resemble those that we and others have observed in 3D inverted invasion assays ([21] and Fig. 1E and Movie S3).


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)

MDA-MB-231 cells form collectively invading chains in CIA.(A) Photos from a time-lapse video of MDA-MB-231 cells invading into Matrigel in CIA showing the formation of long invasion chains at the invading wound edge. Scale bar 50 µm. (B) Cells in a large finger-like chain at the leading edge of a CIA are shown fixed and stained with actin (red), and DNA (blue). Scale bar 20 µm. (C) Actin (red), N-WASP (green) and DAPI (blue) staining of a cell chain at the front of the invading area. White arrowheads indicate puncta of N-WASP co-localizing with filamentous actin. Scale bar 20 µm. (D) Image sequence showing an invading cell actively remodeling the matrix and generating what appear as micro-tunnels (white arrow). Scale bar 20 µm. (E) Staining of actin (red) and DNA (blue) of cell invasion chains in inverted invasion assay and visualized by confocal microscopy. Image showing collective cell invasion chains on single plane (left panel) and the side view of z-stack 3D projection (right panel). Scale bar 20 µm. See also Movies S1, S2, S3.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0030605-g001: MDA-MB-231 cells form collectively invading chains in CIA.(A) Photos from a time-lapse video of MDA-MB-231 cells invading into Matrigel in CIA showing the formation of long invasion chains at the invading wound edge. Scale bar 50 µm. (B) Cells in a large finger-like chain at the leading edge of a CIA are shown fixed and stained with actin (red), and DNA (blue). Scale bar 20 µm. (C) Actin (red), N-WASP (green) and DAPI (blue) staining of a cell chain at the front of the invading area. White arrowheads indicate puncta of N-WASP co-localizing with filamentous actin. Scale bar 20 µm. (D) Image sequence showing an invading cell actively remodeling the matrix and generating what appear as micro-tunnels (white arrow). Scale bar 20 µm. (E) Staining of actin (red) and DNA (blue) of cell invasion chains in inverted invasion assay and visualized by confocal microscopy. Image showing collective cell invasion chains on single plane (left panel) and the side view of z-stack 3D projection (right panel). Scale bar 20 µm. See also Movies S1, S2, S3.
Mentions: We have further developed and more extensively characterized the wound closure-based circular invasion assay as an effective method for visualizing cells during the invasion process [19]. As an alternative to wounding, we used a square, self-stick silicone cell stopper to create a uniform and neat central cell-free space without damaging or disrupting cells. MDA-MB-231 cells were seeded on a 35 µm glass-bottom dish with a stopper blocking the central area. After the cells attached to the glass bottom, the stopper was removed and a thin layer of Matrigel was applied on top of cells and allowed to set. Cells were generally invaded into the Matrigel for 16 hours or longer. As shown in Fig. 1A and B and Movie S1, MDA-MB-231 cells migrated into the central space, penetrating into the Matrigel while assuming an elongated shape near the invading front. Unlike traditional invasion assays, cells could be straightforwardly fixed and stained with antibodies to obtain high quality images. Fig. 1B shows phalloidin and DAPI labeling of leading edge invasion chains. Fig. 1C shows phalloidin stain of actin (red) and punctate appearance of endogenous N-WASP staining (green) with DAPI stain of DNA in blue. N-WASP and actin frequently co-localized to punctuate structures at the cell periphery and protrusions (Fig. 1C, white arrowheads). Invading cells formed cylinder-shape pseudopods and appeared to actively reshape the surrounding matrix by creation of tunnels resembling previously described micro-tunnels also termed SCITS (single-cell invasion tunnels) (Fig. 1D) [20]. Collective migration chains formed as following cells filled into the micro-tunnels (Fig. 1A–C, arrows indicate in C and Movie S1 and S2). These invasion chains resemble those that we and others have observed in 3D inverted invasion assays ([21] and Fig. 1E and Movie S3).

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