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Dynamic and influential interaction of cancer cells with normal epithelial cells in 3D culture.

Ivers LP, Cummings B, Owolabi F, Welzel K, Klinger R, Saitoh S, O'Connor D, Fujita Y, Scholz D, Itasaki N - Cancer Cell Int. (2014)

Bottom Line: However, when there was a relatively large population of normal epithelial cells, the MDA-MB-231 cells did not engulf the epithelial spheres effectively, despite repeated contacts.MDA-MB-231 cells co-cultured with a large number of normal epithelial cells showed reduced expression of monocarboxylate transporter-1, suggesting a change in the cell metabolism.A decreased level of gelatin-digesting ability as well as reduced production of matrix metaroproteinase-2 was also observed.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine and Medical Science, University College Dublin, Dublin, 4 Ireland.

ABSTRACT

Background: The cancer microenvironment has a strong impact on the growth and dynamics of cancer cells. Conventional 2D culture systems, however, do not reflect in vivo conditions, impeding detailed studies of cancer cell dynamics. This work aims to establish a method to reveal the interaction of cancer and normal epithelial cells using 3D time-lapse.

Methods: GFP-labelled breast cancer cells, MDA-MB-231, were co-cultured with mCherry-labelled non-cancerous epithelial cells, MDCK, in a gel matrix. In the 3D culture, the epithelial cells establish a spherical morphology (epithelial sphere) thus providing cancer cells with accessibility to the basal surface of epithelia, similar to the in vivo condition. Cell movement was monitored using time-lapse analyses. Ultrastructural, immunocytochemical and protein expression analyses were also performed following the time-lapse study.

Results: In contrast to the 2D culture system, whereby most MDA-MB-231 cells exhibit spindle-shaped morphology as single cells, in the 3D culture the MDA-MB-231 cells were found to be single cells or else formed aggregates, both of which were motile. The single MDA-MB-231 cells exhibited both round and spindle shapes, with dynamic changes from one shape to the other, visible within a matter of hours. When co-cultured with epithelial cells, the MDA-MB-231 cells displayed a strong attraction to the epithelial spheres, and proceeded to surround and engulf the epithelial cell mass. The surrounded epithelial cells were eventually destroyed, becoming debris, and were taken into the MDA-MB-231 cells. However, when there was a relatively large population of normal epithelial cells, the MDA-MB-231 cells did not engulf the epithelial spheres effectively, despite repeated contacts. MDA-MB-231 cells co-cultured with a large number of normal epithelial cells showed reduced expression of monocarboxylate transporter-1, suggesting a change in the cell metabolism. A decreased level of gelatin-digesting ability as well as reduced production of matrix metaroproteinase-2 was also observed.

Conclusions: This culture method is a powerful technique to investigate cancer cell dynamics and cellular changes in response to the microenvironment. The method can be useful for various aspects such as; different combinations of cancer and non-cancer cell types, addressing the organ-specific affinity of cancer cells to host cells, and monitoring the cellular response to anti-cancer drugs.

No MeSH data available.


Related in: MedlinePlus

Dynamic movement of MDA-MB-231 cells in 3D culture. All MDA-MB-231 cells are GFP-labelled. (A-B) MDA-MB-231 cells cultured in 2D on a plastic dish (A) or in 3D in Geltrex matrix (B and B’, two examples). In 2D (A), almost all of the cells exhibit spindle-shaped morphology and are dissociated from each other. In contrast, the cells cultured in 3D (B, B’) are either spindle-shaped or formed aggregates. (C) Snapshots of a time-lapse movie (Additional file 1: Movie 1) of MDA-MB-231 cells cultured in 3D, showing the cells in the aggregate dissociating and migrating into the matrix. Arrows indicate cells dissociating from the aggregate. These snapshots show an enlargement of the upper area of Additional file 1: Movie 1. (D) Snapshots of a time-lapse movie (Additional file 2: Movie 2) of MDA-MB-231 cells showing elongated cells assembling and forming aggregates. Arrows indicate spindle cells becoming round and forming aggregates. These snapshots show an enlargement of the left-hand side of Additional file 2: Movie 2. (E) Snapshots of a time-lapse movie (Additional file 3: Movie 3) of MDA-MB-231 cells showing small aggregates changing location. Two aggregates are indicated by the arrow or arrowhead to follow the translocation. These snapshots show an enlargement of the centre area of Additional file 3: Movie 3. The indicated time in (C-E); hour:min. (F) 3D cultured MDA-MB-231 cells immunostained with vimentin (red) and GFP (green), showing that all cells of various cell shapes are vimentin-positive. Scale bars; A,C-F, 50 μm; B and B’, 20 μm.
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Fig1: Dynamic movement of MDA-MB-231 cells in 3D culture. All MDA-MB-231 cells are GFP-labelled. (A-B) MDA-MB-231 cells cultured in 2D on a plastic dish (A) or in 3D in Geltrex matrix (B and B’, two examples). In 2D (A), almost all of the cells exhibit spindle-shaped morphology and are dissociated from each other. In contrast, the cells cultured in 3D (B, B’) are either spindle-shaped or formed aggregates. (C) Snapshots of a time-lapse movie (Additional file 1: Movie 1) of MDA-MB-231 cells cultured in 3D, showing the cells in the aggregate dissociating and migrating into the matrix. Arrows indicate cells dissociating from the aggregate. These snapshots show an enlargement of the upper area of Additional file 1: Movie 1. (D) Snapshots of a time-lapse movie (Additional file 2: Movie 2) of MDA-MB-231 cells showing elongated cells assembling and forming aggregates. Arrows indicate spindle cells becoming round and forming aggregates. These snapshots show an enlargement of the left-hand side of Additional file 2: Movie 2. (E) Snapshots of a time-lapse movie (Additional file 3: Movie 3) of MDA-MB-231 cells showing small aggregates changing location. Two aggregates are indicated by the arrow or arrowhead to follow the translocation. These snapshots show an enlargement of the centre area of Additional file 3: Movie 3. The indicated time in (C-E); hour:min. (F) 3D cultured MDA-MB-231 cells immunostained with vimentin (red) and GFP (green), showing that all cells of various cell shapes are vimentin-positive. Scale bars; A,C-F, 50 μm; B and B’, 20 μm.

Mentions: The breast cancer cells MDA-MB-231 exhibit a spindle-shaped morphology when cultured in 2D [17] (Figure 1A). In contrast, when cultured in 3D, using a reconstituted basement membrane matrix Geltrex®, they either form aggregates as previously described in a similar matrix [18] or distribute in a dissociated manner with various morphologies including very elongated shapes and round shapes (Figure 1B,B’). Therefore, in 3D culture MDA-MB-231 exhibit a wide variety of cell morphologies which are not normally seen in 2D culture. Time-lapse microscopy revealed that their cell morphology is dynamic, such that some cells in an aggregate dissociate and spread into the matrix (Figure 1C; Additional file 1: Movie 1) while others exhibiting elongated shapes assemble and form aggregates (Figure 1D; Additional file 2: Movie 2). The spindle-shaped cells are motile as expected (Additional file 1: Movie 1, Additional file 2: Movie 2). Notably, the round-shaped cells also move very actively as single cells or within the aggregates (Additional file 3: Movie 3). Furthermore, the aggregates themselves are able to move and change location in the matrix while spinning around their centre (Figure 1E; Additional file 3: Movie 3). Both the spindle-shaped cells and those in the aggregates are vimentin positive (Figure 1F), reflecting their motility and active changes in cell morphology [19]. Thus 3D culture reveals the morphological diversity and dynamics of MDA-MB-231 cells.Figure 1


Dynamic and influential interaction of cancer cells with normal epithelial cells in 3D culture.

Ivers LP, Cummings B, Owolabi F, Welzel K, Klinger R, Saitoh S, O'Connor D, Fujita Y, Scholz D, Itasaki N - Cancer Cell Int. (2014)

Dynamic movement of MDA-MB-231 cells in 3D culture. All MDA-MB-231 cells are GFP-labelled. (A-B) MDA-MB-231 cells cultured in 2D on a plastic dish (A) or in 3D in Geltrex matrix (B and B’, two examples). In 2D (A), almost all of the cells exhibit spindle-shaped morphology and are dissociated from each other. In contrast, the cells cultured in 3D (B, B’) are either spindle-shaped or formed aggregates. (C) Snapshots of a time-lapse movie (Additional file 1: Movie 1) of MDA-MB-231 cells cultured in 3D, showing the cells in the aggregate dissociating and migrating into the matrix. Arrows indicate cells dissociating from the aggregate. These snapshots show an enlargement of the upper area of Additional file 1: Movie 1. (D) Snapshots of a time-lapse movie (Additional file 2: Movie 2) of MDA-MB-231 cells showing elongated cells assembling and forming aggregates. Arrows indicate spindle cells becoming round and forming aggregates. These snapshots show an enlargement of the left-hand side of Additional file 2: Movie 2. (E) Snapshots of a time-lapse movie (Additional file 3: Movie 3) of MDA-MB-231 cells showing small aggregates changing location. Two aggregates are indicated by the arrow or arrowhead to follow the translocation. These snapshots show an enlargement of the centre area of Additional file 3: Movie 3. The indicated time in (C-E); hour:min. (F) 3D cultured MDA-MB-231 cells immunostained with vimentin (red) and GFP (green), showing that all cells of various cell shapes are vimentin-positive. Scale bars; A,C-F, 50 μm; B and B’, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4221723&req=5

Fig1: Dynamic movement of MDA-MB-231 cells in 3D culture. All MDA-MB-231 cells are GFP-labelled. (A-B) MDA-MB-231 cells cultured in 2D on a plastic dish (A) or in 3D in Geltrex matrix (B and B’, two examples). In 2D (A), almost all of the cells exhibit spindle-shaped morphology and are dissociated from each other. In contrast, the cells cultured in 3D (B, B’) are either spindle-shaped or formed aggregates. (C) Snapshots of a time-lapse movie (Additional file 1: Movie 1) of MDA-MB-231 cells cultured in 3D, showing the cells in the aggregate dissociating and migrating into the matrix. Arrows indicate cells dissociating from the aggregate. These snapshots show an enlargement of the upper area of Additional file 1: Movie 1. (D) Snapshots of a time-lapse movie (Additional file 2: Movie 2) of MDA-MB-231 cells showing elongated cells assembling and forming aggregates. Arrows indicate spindle cells becoming round and forming aggregates. These snapshots show an enlargement of the left-hand side of Additional file 2: Movie 2. (E) Snapshots of a time-lapse movie (Additional file 3: Movie 3) of MDA-MB-231 cells showing small aggregates changing location. Two aggregates are indicated by the arrow or arrowhead to follow the translocation. These snapshots show an enlargement of the centre area of Additional file 3: Movie 3. The indicated time in (C-E); hour:min. (F) 3D cultured MDA-MB-231 cells immunostained with vimentin (red) and GFP (green), showing that all cells of various cell shapes are vimentin-positive. Scale bars; A,C-F, 50 μm; B and B’, 20 μm.
Mentions: The breast cancer cells MDA-MB-231 exhibit a spindle-shaped morphology when cultured in 2D [17] (Figure 1A). In contrast, when cultured in 3D, using a reconstituted basement membrane matrix Geltrex®, they either form aggregates as previously described in a similar matrix [18] or distribute in a dissociated manner with various morphologies including very elongated shapes and round shapes (Figure 1B,B’). Therefore, in 3D culture MDA-MB-231 exhibit a wide variety of cell morphologies which are not normally seen in 2D culture. Time-lapse microscopy revealed that their cell morphology is dynamic, such that some cells in an aggregate dissociate and spread into the matrix (Figure 1C; Additional file 1: Movie 1) while others exhibiting elongated shapes assemble and form aggregates (Figure 1D; Additional file 2: Movie 2). The spindle-shaped cells are motile as expected (Additional file 1: Movie 1, Additional file 2: Movie 2). Notably, the round-shaped cells also move very actively as single cells or within the aggregates (Additional file 3: Movie 3). Furthermore, the aggregates themselves are able to move and change location in the matrix while spinning around their centre (Figure 1E; Additional file 3: Movie 3). Both the spindle-shaped cells and those in the aggregates are vimentin positive (Figure 1F), reflecting their motility and active changes in cell morphology [19]. Thus 3D culture reveals the morphological diversity and dynamics of MDA-MB-231 cells.Figure 1

Bottom Line: However, when there was a relatively large population of normal epithelial cells, the MDA-MB-231 cells did not engulf the epithelial spheres effectively, despite repeated contacts.MDA-MB-231 cells co-cultured with a large number of normal epithelial cells showed reduced expression of monocarboxylate transporter-1, suggesting a change in the cell metabolism.A decreased level of gelatin-digesting ability as well as reduced production of matrix metaroproteinase-2 was also observed.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine and Medical Science, University College Dublin, Dublin, 4 Ireland.

ABSTRACT

Background: The cancer microenvironment has a strong impact on the growth and dynamics of cancer cells. Conventional 2D culture systems, however, do not reflect in vivo conditions, impeding detailed studies of cancer cell dynamics. This work aims to establish a method to reveal the interaction of cancer and normal epithelial cells using 3D time-lapse.

Methods: GFP-labelled breast cancer cells, MDA-MB-231, were co-cultured with mCherry-labelled non-cancerous epithelial cells, MDCK, in a gel matrix. In the 3D culture, the epithelial cells establish a spherical morphology (epithelial sphere) thus providing cancer cells with accessibility to the basal surface of epithelia, similar to the in vivo condition. Cell movement was monitored using time-lapse analyses. Ultrastructural, immunocytochemical and protein expression analyses were also performed following the time-lapse study.

Results: In contrast to the 2D culture system, whereby most MDA-MB-231 cells exhibit spindle-shaped morphology as single cells, in the 3D culture the MDA-MB-231 cells were found to be single cells or else formed aggregates, both of which were motile. The single MDA-MB-231 cells exhibited both round and spindle shapes, with dynamic changes from one shape to the other, visible within a matter of hours. When co-cultured with epithelial cells, the MDA-MB-231 cells displayed a strong attraction to the epithelial spheres, and proceeded to surround and engulf the epithelial cell mass. The surrounded epithelial cells were eventually destroyed, becoming debris, and were taken into the MDA-MB-231 cells. However, when there was a relatively large population of normal epithelial cells, the MDA-MB-231 cells did not engulf the epithelial spheres effectively, despite repeated contacts. MDA-MB-231 cells co-cultured with a large number of normal epithelial cells showed reduced expression of monocarboxylate transporter-1, suggesting a change in the cell metabolism. A decreased level of gelatin-digesting ability as well as reduced production of matrix metaroproteinase-2 was also observed.

Conclusions: This culture method is a powerful technique to investigate cancer cell dynamics and cellular changes in response to the microenvironment. The method can be useful for various aspects such as; different combinations of cancer and non-cancer cell types, addressing the organ-specific affinity of cancer cells to host cells, and monitoring the cellular response to anti-cancer drugs.

No MeSH data available.


Related in: MedlinePlus