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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

Mammosphere culture of MCF10A cells.(A) MCF10A cells subjected to serial dilution and mammosphere formation assays. Each group was triplicated and the number of spheres was counted and plotted as mean ± SD. The mammospheres formed by MCF10A cells were fixed and embedded for histological analysis. Serial sections were cut for immunofluorescence staining against stem/progenitor markers (B), basal markers (C), luminal markers (D), and breast tissue markers (E). 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.g004: Mammosphere culture of MCF10A cells.(A) MCF10A cells subjected to serial dilution and mammosphere formation assays. Each group was triplicated and the number of spheres was counted and plotted as mean ± SD. The mammospheres formed by MCF10A cells were fixed and embedded for histological analysis. Serial sections were cut for immunofluorescence staining against stem/progenitor markers (B), basal markers (C), luminal markers (D), and breast tissue markers (E). Secondary antibodies were either AF488 (green)—or AF594 (red)—conjugated. DAPI was used for nuclear staining. Bars: 50μm. Original magnification: ×200.

Mentions: Next, we characterized the self-renewal ability of MCF10A cells by using a mammosphere culture method, which has been used to quantify both stem cell/early progenitor activity and self-renewal in the normal mammary tissue [11]. To test the stem/progenitor property of MCF10A cells, we performed mammosphere assays using a serial dilution to seed 500 to 10,000 cells per well. The number of formed spheres after 14 days was positively correlated with the initial cell number (R2 = 0.9576) (Fig 4A). Of note, the mammosphere formation rate of MCF10A cells was (0.16 ± 0.04) %. These data suggest that MCF10A cells have a low ability to form mammosphere although stem/progenitor markers are highly expressed. However, when we sorted MCF10A cells by using common stem cell markers (S1 File) such as CD49f, ALDH1A3, and CD44, the CD44+/CD24- and CD49f+ populations were found to yield more mammospheres than the CD44+/CD24+ and CD49f- populations, respectively, whereas the ALDH1A3+ and ALDH1A3- populations did not show a difference (S2 Fig). Our data suggest that these individual markers may be sufficient for distinguishing the whole stem-like population in MCF10A cells. Alternatively, as suggested by recent reports [34, 35], these commonly used stem cell markers may not represent stem-like cells in the MCF10A cell line.


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)

Mammosphere culture of MCF10A cells.(A) MCF10A cells subjected to serial dilution and mammosphere formation assays. Each group was triplicated and the number of spheres was counted and plotted as mean ± SD. The mammospheres formed by MCF10A cells were fixed and embedded for histological analysis. Serial sections were cut for immunofluorescence staining against stem/progenitor markers (B), basal markers (C), luminal markers (D), and breast tissue markers (E). 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.g004: Mammosphere culture of MCF10A cells.(A) MCF10A cells subjected to serial dilution and mammosphere formation assays. Each group was triplicated and the number of spheres was counted and plotted as mean ± SD. The mammospheres formed by MCF10A cells were fixed and embedded for histological analysis. Serial sections were cut for immunofluorescence staining against stem/progenitor markers (B), basal markers (C), luminal markers (D), and breast tissue markers (E). Secondary antibodies were either AF488 (green)—or AF594 (red)—conjugated. DAPI was used for nuclear staining. Bars: 50μm. Original magnification: ×200.
Mentions: Next, we characterized the self-renewal ability of MCF10A cells by using a mammosphere culture method, which has been used to quantify both stem cell/early progenitor activity and self-renewal in the normal mammary tissue [11]. To test the stem/progenitor property of MCF10A cells, we performed mammosphere assays using a serial dilution to seed 500 to 10,000 cells per well. The number of formed spheres after 14 days was positively correlated with the initial cell number (R2 = 0.9576) (Fig 4A). Of note, the mammosphere formation rate of MCF10A cells was (0.16 ± 0.04) %. These data suggest that MCF10A cells have a low ability to form mammosphere although stem/progenitor markers are highly expressed. However, when we sorted MCF10A cells by using common stem cell markers (S1 File) such as CD49f, ALDH1A3, and CD44, the CD44+/CD24- and CD49f+ populations were found to yield more mammospheres than the CD44+/CD24+ and CD49f- populations, respectively, whereas the ALDH1A3+ and ALDH1A3- populations did not show a difference (S2 Fig). Our data suggest that these individual markers may be sufficient for distinguishing the whole stem-like population in MCF10A cells. Alternatively, as suggested by recent reports [34, 35], these commonly used stem cell markers may not represent stem-like cells in the MCF10A cell line.

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