Comparison of methods for the isolation of human breast epithelial and myoepithelial cells.

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

Comparison of methods for the isolation of human breast epithelial and myoepithelial cells

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

Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis

BACKGROUND: The microenvironment and stress factors like glucocorticoids have a strong influence on breast cancer progression but their role in the first stages of breast cancer and, particularly, in myoepithelial cell regulation remains unclear. Consequently, we investigated the role of glucocorticoids in ductal carcinoma in situ (DCIS) in breast cancer, focusing specially on myoepithelial cells. METHODS: To clarify the role of glucocorticoids at breast cancer onset, we evaluated the effects of cortisol and corticosterone on epithelial and myoepithelial cells using 2D and 3D in vitro and in vivo approaches and human samples. RESULTS: Glucocorticoids induce a reduction in laminin levels and favour the disruption of the basement membrane by promotion of myoepithelial cell apoptosis in vitro. In an in vivo stress murine model, increased corticosterone levels fostered the transition from DCIS to invasive ductal carcinoma (IDC) via myoepithelial cell apoptosis and disappearance of the basement membrane. RU486 is able to partially block the effects of cortisol in vitro and in vivo. We found that myoepithelial cell apoptosis is more frequent in patients with DCIS+IDC than in patients with DCIS. CONCLUSIONS: Our findings show that physiological stress, through increased glucocorticoid blood levels, promotes the transition from DCIS to IDC, particularly by inducing myoepithelial cell apoptosis. Since this would be a prerequisite for invasive features in patients with DCIS breast cancer, its clinical management could help to prevent breast cancer progression to IDC

Influence of stress and microenvironmental factors in the progression from breast ductal carcinoma in situ to invasiveness

[eng] One of the key events in the evolution of breast cancer is the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). The myoepithelial cell layer is disrupted in this transition allowing cancer epithelial cells to spread from the duct to the mammary stroma and thus, acquiring invasiveness. However, it is unknown which is the cause that triggers the rupture of the myoepithelial cell layer. Tumour microenvironment and in particular, the neuroendocrine factors released under stress situations, have an important role in the progression of breast cancer. Thus, the main aim of this work was to study how microenvironmental factors released under stress situations could influence the invasiveness of DCIS by affecting the integrity of the myoepithelial cell layer. Due to the fact that there are not commercially available human myoepithelial cells, the isolation of human mammary epithelial and myoepithelial cells from reduction mammoplasties was first optimized, selecting the methodology that provided with the better properties to both epithelial and myoepithelial cells, in terms of proliferation capabilities, lineage-specific markers and functionality. Then, as senescence appearance and loss of antigens are common limitations in the primary cultures of mammary epithelial cells, the optimization of the media was performed, selecting the best media concerning proliferation capabilities, lineage-specific markers, senescence and functionality of epithelial and myoepithelial cells. Then, as the necessary tools for the study of the effect of stress-related neuroendocrine factors had been achieved, 3D in vitro studies were performed with acinar structures formed by primary human epithelial and myoepithelial cells, which mimic the architecture found in the mammary gland. Different neuroendocrine factors (Substance P, Nerve Growth Factor, oxytocin, epinephrine, cortisol and corticosterone) were used in order to study how they affected the integrity of 3D acini. The results showed that glucocorticoids (cortisol and corticosterone) had the bigger impact in terms of acini formation and myoepithelial cell layer and BM disruption, traits that characterize the invasiveness of DCIS. In order to shed more light into the role of glucocorticoids in the fate of DCIS, in vivo studies were performed, subjecting mice to immobilization stress that increased the levels of corticosterone in blood. Tumour cells that had the ability to form DCIS in mice were inoculated and the effect of stress was studied. Stress caused an earlier transition of DCIS to IDC, since it triggered the disorganization of the ductal architecture, loss of the BM and the apoptosis of myoepithelial cells. Since the apoptosis of myoepithelial cells seemed to be an important characteristic in the invasiveness of DCIS, the fate of myoepithelial cells in patient samples with both DCIS alone or DCIS+IDC was studied, observing that the apoptosis of myoepithelial cells was a trait differentiating both sets of samples, since apoptotic myoepithelial cells were exclusively present in patient samples with DCIS+IDC.[spa] Uno de los eventos más importantes en la evolución del cáncer de mama es la transición del carcinoma ductal in situ (DCIS) al carcinoma ductal invasivo (IDC). La capa mioepitelial se rompe en esta transición, permitiendo que las células epiteliales cancerosas se propaguen desde el ducto al estroma mamario, adquiriendo invasividad. Sin embargo, se desconocen cuáles son las causas que desencadenan la ruptura de la capa mioepitelial. El microambiente tumoral y en particular, los factores neuroendocrinos secretados en situaciones de estrés tienen un papel importante en la progresión del cáncer de mama. Por ello, el objetivo principal de esta tesis es estudiar cómo los factores microambientales secretados en situaciones de estrés pueden influenciar en la invasión del DCIS afectando la integridad de la capa mioepitelial. Dado que no existen células mioepiteliales humanas comerciales, el primer objetivo del trabajo ha sido optimizar la metodología de obtención de las células epiteliales y mioepiteliales mamarias, partiendo de mamoplastias de reducción. Además, debido a que la senescencia y la pérdida de antígenos específicos es un problema en los cultivos primarios de células mamarias, el segundo objetivo ha sido optimizar el medio de crecimiento de las células epiteliales y mioepiteliales. Una vez obtenidas las herramientas necesarias, se realizaron cultivos 3D con las células mamarias primarias, obteniendo acinis formados por células epiteliales y mioepiteliales. El tratamiento con los factores neuroendocrinos demostró que los glucocorticoides (cortisol y corticosterona) causaban el mayor efecto en las estructuras ductales, ya que provocaban la apoptosis de las células mioepiteliales y la ruptura de la capa mioepitelial y la membrana basal. Los estudios in vivo con la inoculación en ratones de células cancerosas capaces de formar DCIS, demostraron que el estrés aumenta los niveles de glucocorticoides (corticosterona) y provoca un adelanto en la transición del DCIS al IDC, causando la pérdida de la membrana basal y la apoptosis de las células mioepiteliales. Además, dado que la apoptosis de las células mioepiteliales parece ser un rasgo característico en la invasión del DCIS, estudiamos dicha apoptosis en muestras de pacientes con DCIS y DCIS+IDC, mostrando que las células mioepiteliales apoptóticas son específicas en las pacientes con DCIS+IDC

Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures.

The ability to culture normal human mammary epithelial cells (HMEC) greatly facilitates experiments that seek to understand both normal mammary cell biology and the many differences between normal and abnormal human mammary epithelia. To maximize in vivo relevance, the primary cell culture conditions should maintain cells in states that resemble in vivo as much as possible. Towards this goal, we compared the properties of HMEC strains from two different reduction mammoplasty tissues that were grown in parallel using different media and culture conditions. Epithelial organoids were initiated into three different media: two commonly used serum-free-media, MCDB 170-type (e.g. MEGM) and WIT-P, and a low stress media, M87A. Growth, lineage heterogeneity, p16 protein expression, and population doublings to senescence were measured for each culture condition. MCDB 170 caused rapid senescence and loss of heterogeneity within 2 to 3 passages, but some cultures went through the 1 to 2 month process of selection to generate clonal finite post-selection post-stasis cells. WIT-P caused impressive expansion of luminal cells in 2nd passage followed by their near complete disappearance by passage 4 and senescence shortly thereafter. M87A supported as much as twice the number of population doublings compared to either serum-free medium, and luminal and myoepithelial cells were present for as many as 8 passages. Thus, of the three media compared, WIT-P and MCDB 170 imposed rapid senescence and loss of lineage heterogeneity, phenotypes consistent with cells maintained in high-stress conditions, while M87A supported cultures that maintained multiple lineages and robust growth for up to 60 population doublings. In conjunction with previous studies examining the molecular properties of cultures grown in these media, we conclude that M87A medium is most able to support long-term culture of multiple lineages similar to in vivo conditions, thereby facilitating investigations of normal HMEC biology relevant to the mammary gland in situ

Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures.

The ability to culture normal human mammary epithelial cells (HMEC) greatly facilitates experiments that seek to understand both normal mammary cell biology and the many differences between normal and abnormal human mammary epithelia. To maximize in vivo relevance, the primary cell culture conditions should maintain cells in states that resemble in vivo as much as possible. Towards this goal, we compared the properties of HMEC strains from two different reduction mammoplasty tissues that were grown in parallel using different media and culture conditions. Epithelial organoids were initiated into three different media: two commonly used serum-free-media, MCDB 170-type (e.g. MEGM) and WIT-P, and a low stress media, M87A. Growth, lineage heterogeneity, p16 protein expression, and population doublings to senescence were measured for each culture condition. MCDB 170 caused rapid senescence and loss of heterogeneity within 2 to 3 passages, but some cultures went through the 1 to 2 month process of selection to generate clonal finite post-selection post-stasis cells. WIT-P caused impressive expansion of luminal cells in 2nd passage followed by their near complete disappearance by passage 4 and senescence shortly thereafter. M87A supported as much as twice the number of population doublings compared to either serum-free medium, and luminal and myoepithelial cells were present for as many as 8 passages. Thus, of the three media compared, WIT-P and MCDB 170 imposed rapid senescence and loss of lineage heterogeneity, phenotypes consistent with cells maintained in high-stress conditions, while M87A supported cultures that maintained multiple lineages and robust growth for up to 60 population doublings. In conjunction with previous studies examining the molecular properties of cultures grown in these media, we conclude that M87A medium is most able to support long-term culture of multiple lineages similar to in vivo conditions, thereby facilitating investigations of normal HMEC biology relevant to the mammary gland in situ

Comparison of methods for the isolation of human breast epithelial and myoepithelial cells.

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

Additional file 2: of Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis

Tissue digestion and cell fraction separation protocol. (PDF 231 kb

Additional file 7: of Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis

Figure S4. Representative immunofluorescence images of MCF10DCIS xenografts in chicken embryo CAM membrane.a Hoechst in blue, p63 in red and laminin in green. b Merge of p63 and laminin double immunofluorescence images and zoom in showing an acini detail. Scale bar=20 µm. (PPT 4617 kb