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Increasingly transformed MCF-10A cells have a progressively tumor-like phenotype in three-dimensional basement membrane culture.

Imbalzano KM, Tatarkova I, Imbalzano AN, Nickerson JA - Cancer Cell Int. (2009)

Bottom Line: The pre-malignant MCF-10AT cells and malignant MCF-10CA1a lines were sequentially derived from the MCF-10A parental cell line first by expression of a constitutively active T24 H-Ras generating the MCF-10AT cell line.Features of tissue morphogenesis were assessed, including proliferation, basement membrane formation, polarization of alpha-6 beta-4 integrin to the basement membrane, formation of cell:cell junctions, and apoptosis for luminal clearance.The matched series of normal MCF-10A, pre-malignant MCF-10AT, and malignant MCF-10CA1a cells offers a unique opportunity to study the mechanisms of malignant progression both in a three-dimensional microenvironment and in the same cell background.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA. karen.imbalzano@umassmed.edu

ABSTRACT

Background: MCF-10A cells are near diploid and normal human mammary epithelial cells. In three-dimensional reconstituted basement membrane culture, they undergo a well-defined program of proliferation, differentiation, and growth arrest, forming acinar structures that recapitulate many aspects of mammary architecture in vivo. The pre-malignant MCF-10AT cells and malignant MCF-10CA1a lines were sequentially derived from the MCF-10A parental cell line first by expression of a constitutively active T24 H-Ras generating the MCF-10AT cell line. This was followed by repeated selection for increasingly aggressive tumor formation from cells recovered from xenograft tumors in immuno-compromised mice, generating the MCF-10CA1a cell line. When inoculated subcutaneously into the flanks of immuno-compromised mice, MCF-10AT cells occasionally form tumors, whereas MCF-10CA1a cells invariably form tumors with a shorter latency than MCF-10AT derived tumors.

Results: MCF-10AT cells grown in three-dimensional basement membrane culture form complex multi-acinar structures that produce a basement membrane but undergo delayed cell cycle arrest and have incomplete luminal development. MCF-10CA1a cells grown in three-dimensional basement membrane culture form large, hyper-proliferative masses, that retain few characteristics of MCF10A acini and more closely resemble tumors.

Conclusion: Here we report on the growth and differentiation properties of these three matched cell lines in three-dimensional basement membrane culture. Features of tissue morphogenesis were assessed, including proliferation, basement membrane formation, polarization of alpha-6 beta-4 integrin to the basement membrane, formation of cell:cell junctions, and apoptosis for luminal clearance. The matched series of normal MCF-10A, pre-malignant MCF-10AT, and malignant MCF-10CA1a cells offers a unique opportunity to study the mechanisms of malignant progression both in a three-dimensional microenvironment and in the same cell background.

No MeSH data available.


Related in: MedlinePlus

Altered morphology of MCF-10AT cells in grown for 20 days in embedded three-dimensional culture. Differential interference contrast (DIC) images (A, E) show that MCF-10AT cells grown under these conditions form irregular acini but not the multi-acinar structures seen in overlay cultures (see Figure 2). Nuclei were stained with Draq5 (B, F). Apoptosis, cell:cell junctions, and basement membrane formation were assessed by confocal microscopy with caspase-3 (C), cadherin (G), β4 integrin (D), and Laminin V (H) staining. Scale bars, 10 μm.
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Figure 9: Altered morphology of MCF-10AT cells in grown for 20 days in embedded three-dimensional culture. Differential interference contrast (DIC) images (A, E) show that MCF-10AT cells grown under these conditions form irregular acini but not the multi-acinar structures seen in overlay cultures (see Figure 2). Nuclei were stained with Draq5 (B, F). Apoptosis, cell:cell junctions, and basement membrane formation were assessed by confocal microscopy with caspase-3 (C), cadherin (G), β4 integrin (D), and Laminin V (H) staining. Scale bars, 10 μm.

Mentions: To this point, all observations were made from overlay cultures where cells were plated on a Matrigel layer and overlaid with media containing Matrigel. We subsequently compared characteristics of each cell line when three-dimensional cultures were established by embedding the cells in the lower layer of Matrigel and without including Matrigel in the overlay medium. MCF-10A acini were slightly smaller and more homogeneous in size than when grown in overlay culture, as was previously reported, [17], but otherwise showed no significant difference in apoptosis, basement membrane deposition, or the formation of cell:cell junctions (data not shown). Similarly, the malignant MCF-10CA1a cells showed little difference when the two culture methods were compared (data not shown). However, in embedded culture, MCF-10AT cells formed single acini with irregular edges (Figure 9), but did not form the multi-acinar structures seen in overlay three-dimensional cultures (Figure 2). As previously noted (Figure 5), there was no staining for the apoptosis marker, activated Caspase 3 (Figure 9C), while laminin V containing basement membrane deposition occurred at the basal surface (Figure 9H) with β4 integrin polarized to that surface (Figure 9D). Cadherin labeling indicated that cell:cell junctions formed in the embedded MCF-10AT cultures (Figure 9G), though we noted that cells at the basal surface of these structures consistently lacked cadherin staining.


Increasingly transformed MCF-10A cells have a progressively tumor-like phenotype in three-dimensional basement membrane culture.

Imbalzano KM, Tatarkova I, Imbalzano AN, Nickerson JA - Cancer Cell Int. (2009)

Altered morphology of MCF-10AT cells in grown for 20 days in embedded three-dimensional culture. Differential interference contrast (DIC) images (A, E) show that MCF-10AT cells grown under these conditions form irregular acini but not the multi-acinar structures seen in overlay cultures (see Figure 2). Nuclei were stained with Draq5 (B, F). Apoptosis, cell:cell junctions, and basement membrane formation were assessed by confocal microscopy with caspase-3 (C), cadherin (G), β4 integrin (D), and Laminin V (H) staining. Scale bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666639&req=5

Figure 9: Altered morphology of MCF-10AT cells in grown for 20 days in embedded three-dimensional culture. Differential interference contrast (DIC) images (A, E) show that MCF-10AT cells grown under these conditions form irregular acini but not the multi-acinar structures seen in overlay cultures (see Figure 2). Nuclei were stained with Draq5 (B, F). Apoptosis, cell:cell junctions, and basement membrane formation were assessed by confocal microscopy with caspase-3 (C), cadherin (G), β4 integrin (D), and Laminin V (H) staining. Scale bars, 10 μm.
Mentions: To this point, all observations were made from overlay cultures where cells were plated on a Matrigel layer and overlaid with media containing Matrigel. We subsequently compared characteristics of each cell line when three-dimensional cultures were established by embedding the cells in the lower layer of Matrigel and without including Matrigel in the overlay medium. MCF-10A acini were slightly smaller and more homogeneous in size than when grown in overlay culture, as was previously reported, [17], but otherwise showed no significant difference in apoptosis, basement membrane deposition, or the formation of cell:cell junctions (data not shown). Similarly, the malignant MCF-10CA1a cells showed little difference when the two culture methods were compared (data not shown). However, in embedded culture, MCF-10AT cells formed single acini with irregular edges (Figure 9), but did not form the multi-acinar structures seen in overlay three-dimensional cultures (Figure 2). As previously noted (Figure 5), there was no staining for the apoptosis marker, activated Caspase 3 (Figure 9C), while laminin V containing basement membrane deposition occurred at the basal surface (Figure 9H) with β4 integrin polarized to that surface (Figure 9D). Cadherin labeling indicated that cell:cell junctions formed in the embedded MCF-10AT cultures (Figure 9G), though we noted that cells at the basal surface of these structures consistently lacked cadherin staining.

Bottom Line: The pre-malignant MCF-10AT cells and malignant MCF-10CA1a lines were sequentially derived from the MCF-10A parental cell line first by expression of a constitutively active T24 H-Ras generating the MCF-10AT cell line.Features of tissue morphogenesis were assessed, including proliferation, basement membrane formation, polarization of alpha-6 beta-4 integrin to the basement membrane, formation of cell:cell junctions, and apoptosis for luminal clearance.The matched series of normal MCF-10A, pre-malignant MCF-10AT, and malignant MCF-10CA1a cells offers a unique opportunity to study the mechanisms of malignant progression both in a three-dimensional microenvironment and in the same cell background.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA. karen.imbalzano@umassmed.edu

ABSTRACT

Background: MCF-10A cells are near diploid and normal human mammary epithelial cells. In three-dimensional reconstituted basement membrane culture, they undergo a well-defined program of proliferation, differentiation, and growth arrest, forming acinar structures that recapitulate many aspects of mammary architecture in vivo. The pre-malignant MCF-10AT cells and malignant MCF-10CA1a lines were sequentially derived from the MCF-10A parental cell line first by expression of a constitutively active T24 H-Ras generating the MCF-10AT cell line. This was followed by repeated selection for increasingly aggressive tumor formation from cells recovered from xenograft tumors in immuno-compromised mice, generating the MCF-10CA1a cell line. When inoculated subcutaneously into the flanks of immuno-compromised mice, MCF-10AT cells occasionally form tumors, whereas MCF-10CA1a cells invariably form tumors with a shorter latency than MCF-10AT derived tumors.

Results: MCF-10AT cells grown in three-dimensional basement membrane culture form complex multi-acinar structures that produce a basement membrane but undergo delayed cell cycle arrest and have incomplete luminal development. MCF-10CA1a cells grown in three-dimensional basement membrane culture form large, hyper-proliferative masses, that retain few characteristics of MCF10A acini and more closely resemble tumors.

Conclusion: Here we report on the growth and differentiation properties of these three matched cell lines in three-dimensional basement membrane culture. Features of tissue morphogenesis were assessed, including proliferation, basement membrane formation, polarization of alpha-6 beta-4 integrin to the basement membrane, formation of cell:cell junctions, and apoptosis for luminal clearance. The matched series of normal MCF-10A, pre-malignant MCF-10AT, and malignant MCF-10CA1a cells offers a unique opportunity to study the mechanisms of malignant progression both in a three-dimensional microenvironment and in the same cell background.

No MeSH data available.


Related in: MedlinePlus