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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

The mutual effect of MDA-MB-231 and MDCK cells in a 3D co-culture. (A) Co-culture of MDA-MB-231 and MDCK cells (5:1 ratio), cultured for 8 days and stained for GFP showing the MDA-MB-231 cells (green), f-actin (red) labeling the MDCK cells, and nuclei (blue). The mass of MDCK cells without any MDA-MB-231 cells attached (arrowheads) maintains the typical honey-comb pattern of cytoskeletal organization, whereas the MDCK cell masses that are in contact with MDA-MB-231 cells show disorganised f-actin arrangement. (B) A similar culture to A, stained for GFP showing the MDA-MB-231 cells (green), β-catenin (red) highlighting the MDCK cells, and nuclei (blue). The masses of MDCK cells without any MDA-MB-231 cells attached (arrows) maintain the typical honey-comb pattern of cytoskeletal organisation suggesting localisation of β-catenin to the cell membrane, whereas the large MDCK mass with MDA-MB-231 cells attached show β-catenin in the cytoplasm as well as on the cell membrane. (C) Snapshots of a time-lapse movie (Additional files 8: Movie 8) showing co-culture of MDA-MB-231 (green) and MDCK (red) cells (5:1 ratio). Throughout the first 25 hours, the MDA-MB-231 cells interact solely with one of the MDCK masses and keep the second MDCK mass intact (arrow). Once a large number of MDA-MB-231 cells fully surround the first mass, they move onto the second MDCK mass (33 h 40 m time frame). (D) A snapshot showing co-culture of MDA-MB-231 (green) and MDCK (red) cells (5:1 ratio), cultured in 3D for 11 days. The MDCK cells that are surrounded by MDA-MB-231 cells (arrow) display a debris-like structure. (E) A snapshot of Additional file 9: Movie 9 showing MDA-MB-231 and MDCK cells co-cultured in 3D. The MDCK cells (arrow) are surrounded and engulfed by the MDA-MB-231 cells. Some red particles of MDCK debris are seen in the cytoplasm of the MDA-MB-231 cells. (F) Snapshots of MDA-MB-231 (green) and MDCK (red) cells (20:1 ratio) co-cultured in 3D for 11 days. The population is now dominated by MDA-MB-231 cells. Only a few pieces of red debris are visible. (G) Co-culture of MDA-MB-231 and MDCK cells (1:20 ratio) for 8 days, stained for MDA-MB-231 (green) and for smooth muscle actin (red) mainly labelling MDCK cells. The MDCK cells maintain spherical structures while the MDA-MB-231 cells distribute around them. (H) A similar culture to (G), stained for MDA-MB-231 (green) and for β-catenin (red) mainly labeling MDCK cells. MDA-MB-231 cells do not appear to affect β-catenin expression in the MDCK cells. (I) Snapshots of a time-lapse movie (Additional file 11: Movies 11) showing a mix of MDA-MB-231 (green) and MDCK (red) cells at a ratio of 5:1 cultured for 11 days in 3D. In this field, there are only a few MDA-MB-231 cells attached to the surface of the MDCK sphere. In the first 11 hours, the MDA-MB-231 cells move while stuck to the surface of the sphere, and then suddenly appear to explode at the 11:00 hour time frame. Scale bars; 50 μm. The indicated time; hour:min.
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Fig3: The mutual effect of MDA-MB-231 and MDCK cells in a 3D co-culture. (A) Co-culture of MDA-MB-231 and MDCK cells (5:1 ratio), cultured for 8 days and stained for GFP showing the MDA-MB-231 cells (green), f-actin (red) labeling the MDCK cells, and nuclei (blue). The mass of MDCK cells without any MDA-MB-231 cells attached (arrowheads) maintains the typical honey-comb pattern of cytoskeletal organization, whereas the MDCK cell masses that are in contact with MDA-MB-231 cells show disorganised f-actin arrangement. (B) A similar culture to A, stained for GFP showing the MDA-MB-231 cells (green), β-catenin (red) highlighting the MDCK cells, and nuclei (blue). The masses of MDCK cells without any MDA-MB-231 cells attached (arrows) maintain the typical honey-comb pattern of cytoskeletal organisation suggesting localisation of β-catenin to the cell membrane, whereas the large MDCK mass with MDA-MB-231 cells attached show β-catenin in the cytoplasm as well as on the cell membrane. (C) Snapshots of a time-lapse movie (Additional files 8: Movie 8) showing co-culture of MDA-MB-231 (green) and MDCK (red) cells (5:1 ratio). Throughout the first 25 hours, the MDA-MB-231 cells interact solely with one of the MDCK masses and keep the second MDCK mass intact (arrow). Once a large number of MDA-MB-231 cells fully surround the first mass, they move onto the second MDCK mass (33 h 40 m time frame). (D) A snapshot showing co-culture of MDA-MB-231 (green) and MDCK (red) cells (5:1 ratio), cultured in 3D for 11 days. The MDCK cells that are surrounded by MDA-MB-231 cells (arrow) display a debris-like structure. (E) A snapshot of Additional file 9: Movie 9 showing MDA-MB-231 and MDCK cells co-cultured in 3D. The MDCK cells (arrow) are surrounded and engulfed by the MDA-MB-231 cells. Some red particles of MDCK debris are seen in the cytoplasm of the MDA-MB-231 cells. (F) Snapshots of MDA-MB-231 (green) and MDCK (red) cells (20:1 ratio) co-cultured in 3D for 11 days. The population is now dominated by MDA-MB-231 cells. Only a few pieces of red debris are visible. (G) Co-culture of MDA-MB-231 and MDCK cells (1:20 ratio) for 8 days, stained for MDA-MB-231 (green) and for smooth muscle actin (red) mainly labelling MDCK cells. The MDCK cells maintain spherical structures while the MDA-MB-231 cells distribute around them. (H) A similar culture to (G), stained for MDA-MB-231 (green) and for β-catenin (red) mainly labeling MDCK cells. MDA-MB-231 cells do not appear to affect β-catenin expression in the MDCK cells. (I) Snapshots of a time-lapse movie (Additional file 11: Movies 11) showing a mix of MDA-MB-231 (green) and MDCK (red) cells at a ratio of 5:1 cultured for 11 days in 3D. In this field, there are only a few MDA-MB-231 cells attached to the surface of the MDCK sphere. In the first 11 hours, the MDA-MB-231 cells move while stuck to the surface of the sphere, and then suddenly appear to explode at the 11:00 hour time frame. Scale bars; 50 μm. The indicated time; hour:min.

Mentions: In MDA-MB-231 and MDCK 3D co-cultures of 8-12 days, many MDA-MB-231 cells were found attached to the surface of MDCK spheres, though not exclusively (Figure 2A, see also Figure 3A,B). Time-lapse microscopy revealed that the MDA-MB-231 cells move continuously whereas the MDCK cells were rather persistent in the form of acini in the 3D culture. The directed movement of MDA-MB-231 cells towards the MDCK cells was observed in the time-lapse analyses which showed protrusion of the MDA-MB-231 cells towards the MDCK cells, even from a distance (Figure 2D, Additional file 4: Movie 4). In other cases, a sphere of MDCK cells was attracted to a stream of MDA-MB-231 cells (Figure 2E, Additional file 5: Movie 5), or a group of MDA-MB-231 cells worked as a mass to restrain the MDCK spheres and surround them (Figure 2F, Additional file 6: Movie 6). As a result of the movement of MDA-MB-231 cells towards the MDCK, most of the MDCK spheres were surrounded by MDA-MB-231 cells after ten days of incubation. The surrounding effect of the MDA-MB-231 cells was prominent when a sufficient number of cells were in the local area. On the contrary, in a low-density culture where a small population of MDA-MB-231 cells was available, the MDA-MB-231 cells continued moving close to the MDCK sphere until a relatively large amount of MDA-MB-231 cells coalesced (Figure 2G, Additional file 7: Movie 7). In summary, MDA-MB-231 cells tend to surround MDCK cells when co-cultured in 3D, and this surrounding activity seems most efficient when a substantial number of MDA-MB-231 cells are present.Figure 3


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)

The mutual effect of MDA-MB-231 and MDCK cells in a 3D co-culture. (A) Co-culture of MDA-MB-231 and MDCK cells (5:1 ratio), cultured for 8 days and stained for GFP showing the MDA-MB-231 cells (green), f-actin (red) labeling the MDCK cells, and nuclei (blue). The mass of MDCK cells without any MDA-MB-231 cells attached (arrowheads) maintains the typical honey-comb pattern of cytoskeletal organization, whereas the MDCK cell masses that are in contact with MDA-MB-231 cells show disorganised f-actin arrangement. (B) A similar culture to A, stained for GFP showing the MDA-MB-231 cells (green), β-catenin (red) highlighting the MDCK cells, and nuclei (blue). The masses of MDCK cells without any MDA-MB-231 cells attached (arrows) maintain the typical honey-comb pattern of cytoskeletal organisation suggesting localisation of β-catenin to the cell membrane, whereas the large MDCK mass with MDA-MB-231 cells attached show β-catenin in the cytoplasm as well as on the cell membrane. (C) Snapshots of a time-lapse movie (Additional files 8: Movie 8) showing co-culture of MDA-MB-231 (green) and MDCK (red) cells (5:1 ratio). Throughout the first 25 hours, the MDA-MB-231 cells interact solely with one of the MDCK masses and keep the second MDCK mass intact (arrow). Once a large number of MDA-MB-231 cells fully surround the first mass, they move onto the second MDCK mass (33 h 40 m time frame). (D) A snapshot showing co-culture of MDA-MB-231 (green) and MDCK (red) cells (5:1 ratio), cultured in 3D for 11 days. The MDCK cells that are surrounded by MDA-MB-231 cells (arrow) display a debris-like structure. (E) A snapshot of Additional file 9: Movie 9 showing MDA-MB-231 and MDCK cells co-cultured in 3D. The MDCK cells (arrow) are surrounded and engulfed by the MDA-MB-231 cells. Some red particles of MDCK debris are seen in the cytoplasm of the MDA-MB-231 cells. (F) Snapshots of MDA-MB-231 (green) and MDCK (red) cells (20:1 ratio) co-cultured in 3D for 11 days. The population is now dominated by MDA-MB-231 cells. Only a few pieces of red debris are visible. (G) Co-culture of MDA-MB-231 and MDCK cells (1:20 ratio) for 8 days, stained for MDA-MB-231 (green) and for smooth muscle actin (red) mainly labelling MDCK cells. The MDCK cells maintain spherical structures while the MDA-MB-231 cells distribute around them. (H) A similar culture to (G), stained for MDA-MB-231 (green) and for β-catenin (red) mainly labeling MDCK cells. MDA-MB-231 cells do not appear to affect β-catenin expression in the MDCK cells. (I) Snapshots of a time-lapse movie (Additional file 11: Movies 11) showing a mix of MDA-MB-231 (green) and MDCK (red) cells at a ratio of 5:1 cultured for 11 days in 3D. In this field, there are only a few MDA-MB-231 cells attached to the surface of the MDCK sphere. In the first 11 hours, the MDA-MB-231 cells move while stuck to the surface of the sphere, and then suddenly appear to explode at the 11:00 hour time frame. Scale bars; 50 μm. The indicated time; hour:min.
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Fig3: The mutual effect of MDA-MB-231 and MDCK cells in a 3D co-culture. (A) Co-culture of MDA-MB-231 and MDCK cells (5:1 ratio), cultured for 8 days and stained for GFP showing the MDA-MB-231 cells (green), f-actin (red) labeling the MDCK cells, and nuclei (blue). The mass of MDCK cells without any MDA-MB-231 cells attached (arrowheads) maintains the typical honey-comb pattern of cytoskeletal organization, whereas the MDCK cell masses that are in contact with MDA-MB-231 cells show disorganised f-actin arrangement. (B) A similar culture to A, stained for GFP showing the MDA-MB-231 cells (green), β-catenin (red) highlighting the MDCK cells, and nuclei (blue). The masses of MDCK cells without any MDA-MB-231 cells attached (arrows) maintain the typical honey-comb pattern of cytoskeletal organisation suggesting localisation of β-catenin to the cell membrane, whereas the large MDCK mass with MDA-MB-231 cells attached show β-catenin in the cytoplasm as well as on the cell membrane. (C) Snapshots of a time-lapse movie (Additional files 8: Movie 8) showing co-culture of MDA-MB-231 (green) and MDCK (red) cells (5:1 ratio). Throughout the first 25 hours, the MDA-MB-231 cells interact solely with one of the MDCK masses and keep the second MDCK mass intact (arrow). Once a large number of MDA-MB-231 cells fully surround the first mass, they move onto the second MDCK mass (33 h 40 m time frame). (D) A snapshot showing co-culture of MDA-MB-231 (green) and MDCK (red) cells (5:1 ratio), cultured in 3D for 11 days. The MDCK cells that are surrounded by MDA-MB-231 cells (arrow) display a debris-like structure. (E) A snapshot of Additional file 9: Movie 9 showing MDA-MB-231 and MDCK cells co-cultured in 3D. The MDCK cells (arrow) are surrounded and engulfed by the MDA-MB-231 cells. Some red particles of MDCK debris are seen in the cytoplasm of the MDA-MB-231 cells. (F) Snapshots of MDA-MB-231 (green) and MDCK (red) cells (20:1 ratio) co-cultured in 3D for 11 days. The population is now dominated by MDA-MB-231 cells. Only a few pieces of red debris are visible. (G) Co-culture of MDA-MB-231 and MDCK cells (1:20 ratio) for 8 days, stained for MDA-MB-231 (green) and for smooth muscle actin (red) mainly labelling MDCK cells. The MDCK cells maintain spherical structures while the MDA-MB-231 cells distribute around them. (H) A similar culture to (G), stained for MDA-MB-231 (green) and for β-catenin (red) mainly labeling MDCK cells. MDA-MB-231 cells do not appear to affect β-catenin expression in the MDCK cells. (I) Snapshots of a time-lapse movie (Additional file 11: Movies 11) showing a mix of MDA-MB-231 (green) and MDCK (red) cells at a ratio of 5:1 cultured for 11 days in 3D. In this field, there are only a few MDA-MB-231 cells attached to the surface of the MDCK sphere. In the first 11 hours, the MDA-MB-231 cells move while stuck to the surface of the sphere, and then suddenly appear to explode at the 11:00 hour time frame. Scale bars; 50 μm. The indicated time; hour:min.
Mentions: In MDA-MB-231 and MDCK 3D co-cultures of 8-12 days, many MDA-MB-231 cells were found attached to the surface of MDCK spheres, though not exclusively (Figure 2A, see also Figure 3A,B). Time-lapse microscopy revealed that the MDA-MB-231 cells move continuously whereas the MDCK cells were rather persistent in the form of acini in the 3D culture. The directed movement of MDA-MB-231 cells towards the MDCK cells was observed in the time-lapse analyses which showed protrusion of the MDA-MB-231 cells towards the MDCK cells, even from a distance (Figure 2D, Additional file 4: Movie 4). In other cases, a sphere of MDCK cells was attracted to a stream of MDA-MB-231 cells (Figure 2E, Additional file 5: Movie 5), or a group of MDA-MB-231 cells worked as a mass to restrain the MDCK spheres and surround them (Figure 2F, Additional file 6: Movie 6). As a result of the movement of MDA-MB-231 cells towards the MDCK, most of the MDCK spheres were surrounded by MDA-MB-231 cells after ten days of incubation. The surrounding effect of the MDA-MB-231 cells was prominent when a sufficient number of cells were in the local area. On the contrary, in a low-density culture where a small population of MDA-MB-231 cells was available, the MDA-MB-231 cells continued moving close to the MDCK sphere until a relatively large amount of MDA-MB-231 cells coalesced (Figure 2G, Additional file 7: Movie 7). In summary, MDA-MB-231 cells tend to surround MDCK cells when co-cultured in 3D, and this surrounding activity seems most efficient when a substantial number of MDA-MB-231 cells are present.Figure 3

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