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Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties.

ABSTRACT: Normal human luminal and myoepithelial breast cells separately purified from a set of 10 reduction mammoplasties by using a double antibody magnetic affinity cell sorting and Dynabead immunomagnetic technique were used in two-dimensional gel proteome studies. A total of 43,302 proteins were detected across the 20 samples, and a master image for each cell type comprising a total of 1,738 unique proteins was derived. Differential analysis identified 170 proteins that were elevated 2-fold or more between the two breast cell types, and 51 of these were annotated by tandem mass spectrometry. Muscle-specific enzyme isoforms and contractile intermediate filaments including tropomyosin and smooth muscle (SM22) alpha protein were detected in the myoepithelial cells, and a large number of cytokeratin subclasses and isoforms characteristic of luminal cells were detected in this cell type. A further 134 nondifferentially regulated proteins were also annotated from the two breast cell types, making this the most extensive study to date of the protein expression map of the normal human breast and the basis for future studies of purified breast cancer cells.

SUBMITTER: Page MJ

PROVIDER: S-EPMC23001 | biostudies-literature | 1999 Oct

REPOSITORIES: biostudies-literature

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Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties.

Page M J MJ Amess B B Townsend R R RR Parekh R R Herath A A Brusten L L Zvelebil M J MJ Stein R C RC Waterfield M D MD Davies S C SC O'Hare M J MJ

Proceedings of the National Academy of Sciences of the United States of America 19991001 22

Normal human luminal and myoepithelial breast cells separately purified from a set of 10 reduction mammoplasties by using a double antibody magnetic affinity cell sorting and Dynabead immunomagnetic technique were used in two-dimensional gel proteome studies. A total of 43,302 proteins were detected across the 20 samples, and a master image for each cell type comprising a total of 1,738 unique proteins was derived. Differential analysis identified 170 proteins that were elevated 2-fold or more b ...[more]

PMID: 10535966

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Project description:Background3D modelling fulfils a critical role in research, allowing for complex cell behaviour and interactions to be studied in physiomimetic conditions. With tissue banks becoming established for a number of cancers, researchers now have access to primary patient cells, providing the perfect building blocks to recreate and interrogate intricate cellular systems in the laboratory. The ducts of the human breast are composed of an inner layer of luminal cells supported by an outer layer of myoepithelial cells. In early-stage ductal carcinoma in situ, cancerous luminal cells are confined to the ductal space by an intact myoepithelial layer. Understanding the relationship between myoepithelial and luminal cells in the development of cancer is critical for the development of new therapies and prognostic markers. This requires the generation of new models that allows for the manipulation of these two cell types in a physiological setting.MethodsUsing access to the Breast Cancer Now Tissue Bank, we isolated pure populations of myoepithelial and luminal cells from human reduction mammoplasty specimens and placed them into 2D culture. These cells were infected with lentiviral particles encoding either fluorescent proteins, to facilitate cell tracking, or an inducible human epidermal growth factor receptor 2 (HER2) expression construct. Myoepithelial and luminal cells were then recombined in collagen gels, and the resulting cellular structures were analysed by confocal microscopy. RESULTS: Myoepithelial and luminal cells isolated from reduction mammoplasty specimens can be grown separately in 2D culture and retain their differentiated state. When recombined in collagen gels, these cells reform into physiologically reflective bilayer structures. Inducible expression of HER2 in the luminal compartment, once the bilayer has formed, leads to robust luminal filling, recapitulating ductal carcinoma in situ, and can be blocked with anti-HER2 therapies.ConclusionsThis model allows for the interaction between myoepithelial and luminal cells to be investigated in an in-vitro environment and paves the way to study early events in breast cancer development with the potential to act as a powerful drug discovery platform.

Project description:IntroductionBreast cancer is thought to arise in mammary epithelial stem cells. There is, therefore, a large amount of interest in identifying these cells. The breast is a complex tissue consisting of two epithelial layers (an outer myoepithelial/basal layer and an inner luminal epithelial layer) as well as a large non-epithelial component (fibroblasts, endothelial cells, lymphocytes, adipocytes, neurons and myocytes). The definitive identification of a mammary epithelial stem cell population is critically dependent on its purity. To date, this has been hampered by the lack of suitable markers to separate out the two epithelial layers, and to remove contaminating non-epithelial cells.MethodsMouse mammary glands were dissociated and stained with CD24. Cells were sorted into separate populations based on CD24 expression and assessed for luminal epithelial and myoepithelial/basal markers by direct fluorescent microscopy and real time PCR. The stem/progenitor potential of these cell populations was assessed in vivo by cleared mammary fat pad transplantation.ResultsThree populations of CD24 expressing cells were identified: CD24Negative, CD24Low and CD24High. Staining of these cells with cytokeratin markers revealed that these populations correspond to non-epithelial, myoepithelial/basal and luminal epithelial cells, respectively. Cell identities were confirmed by quantitative PCR. Cleared mammary fat pad transplantation of these cell populations revealed that extensive mammary fat pad repopulation capacity segregates with the CD24Low cells, whilst CD24High cells have limited repopulation capacity.ConclusionDifferential staining of mammary epithelial cells for CD24 can be used to simultaneously isolate pure populations of non-epithelial, myoepithelial/basal and luminal epithelial cells. Furthermore, mammary fat pad repopulation capacity is enriched in the CD24Low population. As separation is achieved using a single marker, it will be possible to incorporate additional markers to further subdivide these populations. This will considerably facilitate the further analysis of mammary epithelial subpopulations, whilst ensuring high purity, which is key for understanding mammary epithelial stem cells in normal tissue biology and carcinogenesis.

Project description:Two lineages, epithelial, and myoepithelial cells are the main cell populations in the normal mammary gland and in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures obtained from fresh breast tissue are a powerful tool to study more reliably new aspects of mammary gland biology, including normal and pathological conditions. Nevertheless, the methods described to date have some technical problems in terms of cell viability and yield, which hamper work with primary mammary cells. Therefore, there is a need to optimize technology for the proper isolation of epithelial and myoepithelial cells. For this reason, we compared four methods in an effort to improve the isolation and primary cell culture of different cell populations of human mammary epithelium. The samples were obtained from healthy tissue of patients who had undergone mammoplasty or mastectomy surgery. We based our approaches on previously described methods, and incorporated additional steps to ameliorate technical efficiency and increase cell survival. We determined cell growth and viability by phase-contrast images, growth curve analysis and cell yield, and identified cell-lineage specific markers by flow cytometry and immunofluorescence in 3D cell cultures. These techniques allowed us to better evaluate the functional capabilities of these two main mammary lineages, using CD227/K19 (epithelial cells) and CD10/K14 (myoepithelial cells) antigens. Our results show that slow digestion at low enzymatic concentration combined with the differential centrifugation technique is the method that best fits the main goal of the present study: protocol efficiency and cell survival yield. In summary, we propose some guidelines to establish primary mammary epithelial cell lines more efficiently and to provide us with a strong research instrument to better understand the role of different epithelial cell types in the origin of breast cancer.

Dataset Information

Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties.

Publications

Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties.

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