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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.


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Range of morphologies observed for MCF-10CA1a cells grown in rBM for 20 days. Phase contrast micrographs of malignant MCF-10CA1a cells after 20 days in overlay three-dimensional culture which form large defined dense masses (A), more spread, less defined masses (B), highly vacuolar acini (C), and clusters of MCF-10CA1a cells that have failed to thrive in rBM (D). Scale bar, 100 μm.
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Figure 3: Range of morphologies observed for MCF-10CA1a cells grown in rBM for 20 days. Phase contrast micrographs of malignant MCF-10CA1a cells after 20 days in overlay three-dimensional culture which form large defined dense masses (A), more spread, less defined masses (B), highly vacuolar acini (C), and clusters of MCF-10CA1a cells that have failed to thrive in rBM (D). Scale bar, 100 μm.

Mentions: From day 12 to day 20 in three-dimensional culture, the MCF-10A acini did not appreciably change in size or shape, consistent with a cessation of proliferation [4,9]. Acini remained spherical with a smooth outer edge and when the peripheral edge of the acinus was in the focal plane, individual cells at that edge appeared of uniform size and were evenly spaced (arrow panel F). A few acini were no longer spatially separated, but were touching by these later time points (Figure 2E, F). They did not, however, appear to be fused into a single structure. The multi-acinar MCF-10AT structures continued to increase in size from day 16 to day 20 (Figure 2K, L). By days 16 to 20, MCF-10CA1a structures were larger and even more easily distinguishable from those of the MCF-10A and MCF-10AT (Figure 2Q, R). Since the growth of MCF10CA1a cells in three-dimensional culture was variable, a spectrum of typical morphologies observed at 20 days in three-dimensional culture is presented in Figure 3. Some masses of MCF-10CA1a cells were large and very dense with uneven edges (Figure 3A); whereas other masses lacked distinct edges, were less dense, and were more spread (Figure 3B). Some structures were very large with round, smooth outer edges and a vacuolar interior (Figure 3C). We also continued to see both masses of cells that did not appear organized as well as remnants of cells that failed to thrive in three-dimensional culture (Figure 3D).


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)

Range of morphologies observed for MCF-10CA1a cells grown in rBM for 20 days. Phase contrast micrographs of malignant MCF-10CA1a cells after 20 days in overlay three-dimensional culture which form large defined dense masses (A), more spread, less defined masses (B), highly vacuolar acini (C), and clusters of MCF-10CA1a cells that have failed to thrive in rBM (D). Scale bar, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Range of morphologies observed for MCF-10CA1a cells grown in rBM for 20 days. Phase contrast micrographs of malignant MCF-10CA1a cells after 20 days in overlay three-dimensional culture which form large defined dense masses (A), more spread, less defined masses (B), highly vacuolar acini (C), and clusters of MCF-10CA1a cells that have failed to thrive in rBM (D). Scale bar, 100 μm.
Mentions: From day 12 to day 20 in three-dimensional culture, the MCF-10A acini did not appreciably change in size or shape, consistent with a cessation of proliferation [4,9]. Acini remained spherical with a smooth outer edge and when the peripheral edge of the acinus was in the focal plane, individual cells at that edge appeared of uniform size and were evenly spaced (arrow panel F). A few acini were no longer spatially separated, but were touching by these later time points (Figure 2E, F). They did not, however, appear to be fused into a single structure. The multi-acinar MCF-10AT structures continued to increase in size from day 16 to day 20 (Figure 2K, L). By days 16 to 20, MCF-10CA1a structures were larger and even more easily distinguishable from those of the MCF-10A and MCF-10AT (Figure 2Q, R). Since the growth of MCF10CA1a cells in three-dimensional culture was variable, a spectrum of typical morphologies observed at 20 days in three-dimensional culture is presented in Figure 3. Some masses of MCF-10CA1a cells were large and very dense with uneven edges (Figure 3A); whereas other masses lacked distinct edges, were less dense, and were more spread (Figure 3B). Some structures were very large with round, smooth outer edges and a vacuolar interior (Figure 3C). We also continued to see both masses of cells that did not appear organized as well as remnants of cells that failed to thrive in three-dimensional culture (Figure 3D).

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