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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

Expression of monocarboxylate transporters in a co-culture of MDA-MB-231 and MDCK cells and a monoculture of MCF7 in 2D and 3D. (A,B) MDA-MB-231 cells cultured alone in 3D, stained with anti-GFP (green, A) and anti-MCT1 (red, B) antibodies and with DAPI to visualize the nuclei (blue, A). The majority of MDA-MB-231 cells express MCT1, mostly on the cell membrane. (C,D) MDCK cells cultured alone in 3D, stained with anti-MCT1 (red, D) antibody and DAPI to visualize the nuclei (blue, C). The MDCK cells express MCT1, mainly on the cell membrane. (E,F) A co-culture of MDA-MB-231 and MDCK cells (1:1 ratio) in 3D, stained with anti-GFP (green, E) and anti-MCT1 (red, F) antibodies and with DAPI to visualize the nuclei (blue, E). E shows a mixed population of MDA-MB-231 (green) and MDCK cells in the sphere (no green). MCT1 is barely detectable in either cell types. (G,H) MCF7 cells cultured alone in 2D, stained with anti-GFP antibody labeling MCF7 cells (green, G), anti-MCT4 antibody (red, H) and DAPI to visualize the nuclei (blue, G). The MCF7 cells do not express MCT4 when cultured alone in 2D. (I,J) MCF7 cells cultured alone in 3D, stained with anti-GFP antibody for MCF7 (green, I), anti-MCT4 antibody (red, J) and DAPI to visualise the nuclei (blue, I). MCF7 cells strongly express MCT4 on their cell membrane when cultured in 3D. Scale bars; 50 μm.
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Fig5: Expression of monocarboxylate transporters in a co-culture of MDA-MB-231 and MDCK cells and a monoculture of MCF7 in 2D and 3D. (A,B) MDA-MB-231 cells cultured alone in 3D, stained with anti-GFP (green, A) and anti-MCT1 (red, B) antibodies and with DAPI to visualize the nuclei (blue, A). The majority of MDA-MB-231 cells express MCT1, mostly on the cell membrane. (C,D) MDCK cells cultured alone in 3D, stained with anti-MCT1 (red, D) antibody and DAPI to visualize the nuclei (blue, C). The MDCK cells express MCT1, mainly on the cell membrane. (E,F) A co-culture of MDA-MB-231 and MDCK cells (1:1 ratio) in 3D, stained with anti-GFP (green, E) and anti-MCT1 (red, F) antibodies and with DAPI to visualize the nuclei (blue, E). E shows a mixed population of MDA-MB-231 (green) and MDCK cells in the sphere (no green). MCT1 is barely detectable in either cell types. (G,H) MCF7 cells cultured alone in 2D, stained with anti-GFP antibody labeling MCF7 cells (green, G), anti-MCT4 antibody (red, H) and DAPI to visualize the nuclei (blue, G). The MCF7 cells do not express MCT4 when cultured alone in 2D. (I,J) MCF7 cells cultured alone in 3D, stained with anti-GFP antibody for MCF7 (green, I), anti-MCT4 antibody (red, J) and DAPI to visualise the nuclei (blue, I). MCF7 cells strongly express MCT4 on their cell membrane when cultured in 3D. Scale bars; 50 μm.

Mentions: Immunocytochemical analyses were carried out to identify cellular changes in MDA-MB-231 cells when co-cultured with MDCK cells. MDA-MB-231 and MDCK cells when co-cultured at a ratio of 1:1. At this ratio, a substantial amount of MDCK cells kept growing for more than a week without significant cell death, thus increasing the likelihood of interaction of the MDA-MB-231 cells with the MDCK cells. Among tested markers, a prominent change was observed in the expression of monocarboxylate transporter-1 (MCT1). MCTs are a family of plasma membrane proteins which function to transport monocarboxylates such as lactate and pyruvate between neighbouring cells, and are involved in the metabolism of cancer and the surrounding tissue [23,24]. Most cancer cells generate energy by glycolysis and produce monocarboxylates that are used to synthesise macromolecules required for rapid cell proliferation. In advanced cancer where the microenvironment promotes the growth of cancer, cancer cells intake additional lactate molecules from the surrounding cells. This increase in lactate uptake is manifested by changes in the expression of MCTs. MCT1 is elevated in glycolytic cancer cells whereas MCT4 is up-regulated in the surrounding fibroblast cells which fuel cancer growth [25,26]. The expression of MCT1 has also been found to correlate with the malignancy of cancer cells [27]. In the present study using 3D cultures, MDA-MB-231 cultured alone expressed MCT1 (Figure 5A,B). MDCK cultured alone also expressed MCT1 faintly (Figure 5C,D). When these two cell types were co-cultured, however, the MCT1 expression was diminished to an undetectable level in both MDA-MB-231 and MDCK (Figure 5E,F). Therefore co-culture with MDCK appears to alter the energy consumption pattern of MDA-MB-231 cells, whereby the intake of monocarboxylates is reduced, thus perhaps, mimicking the metabolism pattern of less malignant cells.Figure 5


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)

Expression of monocarboxylate transporters in a co-culture of MDA-MB-231 and MDCK cells and a monoculture of MCF7 in 2D and 3D. (A,B) MDA-MB-231 cells cultured alone in 3D, stained with anti-GFP (green, A) and anti-MCT1 (red, B) antibodies and with DAPI to visualize the nuclei (blue, A). The majority of MDA-MB-231 cells express MCT1, mostly on the cell membrane. (C,D) MDCK cells cultured alone in 3D, stained with anti-MCT1 (red, D) antibody and DAPI to visualize the nuclei (blue, C). The MDCK cells express MCT1, mainly on the cell membrane. (E,F) A co-culture of MDA-MB-231 and MDCK cells (1:1 ratio) in 3D, stained with anti-GFP (green, E) and anti-MCT1 (red, F) antibodies and with DAPI to visualize the nuclei (blue, E). E shows a mixed population of MDA-MB-231 (green) and MDCK cells in the sphere (no green). MCT1 is barely detectable in either cell types. (G,H) MCF7 cells cultured alone in 2D, stained with anti-GFP antibody labeling MCF7 cells (green, G), anti-MCT4 antibody (red, H) and DAPI to visualize the nuclei (blue, G). The MCF7 cells do not express MCT4 when cultured alone in 2D. (I,J) MCF7 cells cultured alone in 3D, stained with anti-GFP antibody for MCF7 (green, I), anti-MCT4 antibody (red, J) and DAPI to visualise the nuclei (blue, I). MCF7 cells strongly express MCT4 on their cell membrane when cultured in 3D. Scale bars; 50 μm.
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Related In: Results  -  Collection

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Fig5: Expression of monocarboxylate transporters in a co-culture of MDA-MB-231 and MDCK cells and a monoculture of MCF7 in 2D and 3D. (A,B) MDA-MB-231 cells cultured alone in 3D, stained with anti-GFP (green, A) and anti-MCT1 (red, B) antibodies and with DAPI to visualize the nuclei (blue, A). The majority of MDA-MB-231 cells express MCT1, mostly on the cell membrane. (C,D) MDCK cells cultured alone in 3D, stained with anti-MCT1 (red, D) antibody and DAPI to visualize the nuclei (blue, C). The MDCK cells express MCT1, mainly on the cell membrane. (E,F) A co-culture of MDA-MB-231 and MDCK cells (1:1 ratio) in 3D, stained with anti-GFP (green, E) and anti-MCT1 (red, F) antibodies and with DAPI to visualize the nuclei (blue, E). E shows a mixed population of MDA-MB-231 (green) and MDCK cells in the sphere (no green). MCT1 is barely detectable in either cell types. (G,H) MCF7 cells cultured alone in 2D, stained with anti-GFP antibody labeling MCF7 cells (green, G), anti-MCT4 antibody (red, H) and DAPI to visualize the nuclei (blue, G). The MCF7 cells do not express MCT4 when cultured alone in 2D. (I,J) MCF7 cells cultured alone in 3D, stained with anti-GFP antibody for MCF7 (green, I), anti-MCT4 antibody (red, J) and DAPI to visualise the nuclei (blue, I). MCF7 cells strongly express MCT4 on their cell membrane when cultured in 3D. Scale bars; 50 μm.
Mentions: Immunocytochemical analyses were carried out to identify cellular changes in MDA-MB-231 cells when co-cultured with MDCK cells. MDA-MB-231 and MDCK cells when co-cultured at a ratio of 1:1. At this ratio, a substantial amount of MDCK cells kept growing for more than a week without significant cell death, thus increasing the likelihood of interaction of the MDA-MB-231 cells with the MDCK cells. Among tested markers, a prominent change was observed in the expression of monocarboxylate transporter-1 (MCT1). MCTs are a family of plasma membrane proteins which function to transport monocarboxylates such as lactate and pyruvate between neighbouring cells, and are involved in the metabolism of cancer and the surrounding tissue [23,24]. Most cancer cells generate energy by glycolysis and produce monocarboxylates that are used to synthesise macromolecules required for rapid cell proliferation. In advanced cancer where the microenvironment promotes the growth of cancer, cancer cells intake additional lactate molecules from the surrounding cells. This increase in lactate uptake is manifested by changes in the expression of MCTs. MCT1 is elevated in glycolytic cancer cells whereas MCT4 is up-regulated in the surrounding fibroblast cells which fuel cancer growth [25,26]. The expression of MCT1 has also been found to correlate with the malignancy of cancer cells [27]. In the present study using 3D cultures, MDA-MB-231 cultured alone expressed MCT1 (Figure 5A,B). MDCK cultured alone also expressed MCT1 faintly (Figure 5C,D). When these two cell types were co-cultured, however, the MCT1 expression was diminished to an undetectable level in both MDA-MB-231 and MDCK (Figure 5E,F). Therefore co-culture with MDCK appears to alter the energy consumption pattern of MDA-MB-231 cells, whereby the intake of monocarboxylates is reduced, thus perhaps, mimicking the metabolism pattern of less malignant cells.Figure 5

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