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Evaluation of MCF10A as a Reliable Model for Normal Human Mammary Epithelial Cells.

Qu Y, Han B, Yu Y, Yao W, Bose S, Karlan BY, Giuliano AE, Cui X - PLoS ONE (2015)

Bottom Line: However, there is limited knowledge about whether MCF10A cells reliably represent normal human mammary cells.When grown in suspension culture, MCF10A cells showed low mammosphere-forming ability.Our results raise a question as to whether the commonly used MCF10A cell line is a suitable model for human mammary cell studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.

ABSTRACT
Breast cancer is the most common cancer in women and a leading cause of cancer-related deaths for women worldwide. Various cell models have been developed to study breast cancer tumorigenesis, metastasis, and drug sensitivity. The MCF10A human mammary epithelial cell line is a widely used in vitro model for studying normal breast cell function and transformation. However, there is limited knowledge about whether MCF10A cells reliably represent normal human mammary cells. MCF10A cells were grown in monolayer, suspension (mammosphere culture), three-dimensional (3D) "on-top" Matrigel, 3D "cell-embedded" Matrigel, or mixed Matrigel/collagen I gel. Suspension culture was performed with the MammoCult medium and low-attachment culture plates. Cells grown in 3D culture were fixed and subjected to either immunofluorescence staining or embedding and sectioning followed by immunohistochemistry and immunofluorescence staining. Cells or slides were stained for protein markers commonly used to identify mammary progenitor and epithelial cells. MCF10A cells expressed markers representing luminal, basal, and progenitor phenotypes in two-dimensional (2D) culture. When grown in suspension culture, MCF10A cells showed low mammosphere-forming ability. Cells in mammospheres and 3D culture expressed both luminal and basal markers. Surprisingly, the acinar structure formed by MCF10A cells in 3D culture was positive for both basal markers and the milk proteins β-casein and α-lactalbumin. MCF10A cells exhibit a unique differentiated phenotype in 3D culture which may not exist or be rare in normal human breast tissue. Our results raise a question as to whether the commonly used MCF10A cell line is a suitable model for human mammary cell studies.

No MeSH data available.


Related in: MedlinePlus

Immunofluorescence staining of the mammary cell markers in MCF10A cells in 2D culture.MCF10A cells grown in monolayer were fixed and subjected to immunofluorescence staining using the antibodies against basal cell (A), luminal cell (B), and breast tissue markers (C). Secondary antibodies were either AF488 (green)—or AF594 (red)-conjugated. DAPI was used for nuclear staining. Bars: 50μm. Original magnification: ×200.
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pone.0131285.g002: Immunofluorescence staining of the mammary cell markers in MCF10A cells in 2D culture.MCF10A cells grown in monolayer were fixed and subjected to immunofluorescence staining using the antibodies against basal cell (A), luminal cell (B), and breast tissue markers (C). Secondary antibodies were either AF488 (green)—or AF594 (red)-conjugated. DAPI was used for nuclear staining. Bars: 50μm. Original magnification: ×200.

Mentions: Cytokeratin (CK) expression patterns are commonly used to identify mammary epithelial cells. Myoepithelial cells typically express CK5/6, CK14, and CK17 [15] as well as other markers such as P63 [16], vimentin [17], and alpha smoothen actin (SMA) [18], whereas luminal cells are characterized by CK8, CK18 [19], and CK7 [20, 21], as well as other markers such as Mucin1 (Muc1) [22] and E-cadherin (E-cad) [23]. Hence, we used immunofluorescence staining to profile the expression of these basal and luminal markers in MCF10A cells As shown in Fig 2A and S2 Table, MCF10A cells strongly expressed vimentin, alpha smooth muscle Actin (SMA), and the basal-cadherin N-cadherin [24], suggesting a basal origin for MCF10A. Around 50% of cells displayed strong staining of CK5, whereas around 10% of cells expressed moderate levels of CK17. Phalloidin staining (F-actin) indicated stress fiber formation in MCF10A cells. Surprisingly, MCF10A cells exhibited completely negative staining of P63 and less than 1% of cells were positive for CK14.


Evaluation of MCF10A as a Reliable Model for Normal Human Mammary Epithelial Cells.

Qu Y, Han B, Yu Y, Yao W, Bose S, Karlan BY, Giuliano AE, Cui X - PLoS ONE (2015)

Immunofluorescence staining of the mammary cell markers in MCF10A cells in 2D culture.MCF10A cells grown in monolayer were fixed and subjected to immunofluorescence staining using the antibodies against basal cell (A), luminal cell (B), and breast tissue markers (C). Secondary antibodies were either AF488 (green)—or AF594 (red)-conjugated. DAPI was used for nuclear staining. Bars: 50μm. Original magnification: ×200.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493126&req=5

pone.0131285.g002: Immunofluorescence staining of the mammary cell markers in MCF10A cells in 2D culture.MCF10A cells grown in monolayer were fixed and subjected to immunofluorescence staining using the antibodies against basal cell (A), luminal cell (B), and breast tissue markers (C). Secondary antibodies were either AF488 (green)—or AF594 (red)-conjugated. DAPI was used for nuclear staining. Bars: 50μm. Original magnification: ×200.
Mentions: Cytokeratin (CK) expression patterns are commonly used to identify mammary epithelial cells. Myoepithelial cells typically express CK5/6, CK14, and CK17 [15] as well as other markers such as P63 [16], vimentin [17], and alpha smoothen actin (SMA) [18], whereas luminal cells are characterized by CK8, CK18 [19], and CK7 [20, 21], as well as other markers such as Mucin1 (Muc1) [22] and E-cadherin (E-cad) [23]. Hence, we used immunofluorescence staining to profile the expression of these basal and luminal markers in MCF10A cells As shown in Fig 2A and S2 Table, MCF10A cells strongly expressed vimentin, alpha smooth muscle Actin (SMA), and the basal-cadherin N-cadherin [24], suggesting a basal origin for MCF10A. Around 50% of cells displayed strong staining of CK5, whereas around 10% of cells expressed moderate levels of CK17. Phalloidin staining (F-actin) indicated stress fiber formation in MCF10A cells. Surprisingly, MCF10A cells exhibited completely negative staining of P63 and less than 1% of cells were positive for CK14.

Bottom Line: However, there is limited knowledge about whether MCF10A cells reliably represent normal human mammary cells.When grown in suspension culture, MCF10A cells showed low mammosphere-forming ability.Our results raise a question as to whether the commonly used MCF10A cell line is a suitable model for human mammary cell studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.

ABSTRACT
Breast cancer is the most common cancer in women and a leading cause of cancer-related deaths for women worldwide. Various cell models have been developed to study breast cancer tumorigenesis, metastasis, and drug sensitivity. The MCF10A human mammary epithelial cell line is a widely used in vitro model for studying normal breast cell function and transformation. However, there is limited knowledge about whether MCF10A cells reliably represent normal human mammary cells. MCF10A cells were grown in monolayer, suspension (mammosphere culture), three-dimensional (3D) "on-top" Matrigel, 3D "cell-embedded" Matrigel, or mixed Matrigel/collagen I gel. Suspension culture was performed with the MammoCult medium and low-attachment culture plates. Cells grown in 3D culture were fixed and subjected to either immunofluorescence staining or embedding and sectioning followed by immunohistochemistry and immunofluorescence staining. Cells or slides were stained for protein markers commonly used to identify mammary progenitor and epithelial cells. MCF10A cells expressed markers representing luminal, basal, and progenitor phenotypes in two-dimensional (2D) culture. When grown in suspension culture, MCF10A cells showed low mammosphere-forming ability. Cells in mammospheres and 3D culture expressed both luminal and basal markers. Surprisingly, the acinar structure formed by MCF10A cells in 3D culture was positive for both basal markers and the milk proteins β-casein and α-lactalbumin. MCF10A cells exhibit a unique differentiated phenotype in 3D culture which may not exist or be rare in normal human breast tissue. Our results raise a question as to whether the commonly used MCF10A cell line is a suitable model for human mammary cell studies.

No MeSH data available.


Related in: MedlinePlus