We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.
The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.
Information about the mutational landscape in breast cancer is increasing at rapid pace. Less attention has been paid to systemic factors that influence breast cancer development. Exposure to reproductive hormones has a major impact on disease progression1-3. Yet, the mechanisms by which reproductive hormones impinge on the human breast are poorly understood. Work with genetically engineered mouse models has revealed that they involve cell intrinsic and paracrine signaling through several downstream effectors4.
The limited knowledge about hormone action in the human breast is largely attributable to a lack of adequate models. Most work on the mechanisms of estrogen receptor (ER) and progesterone receptor (PR) signaling has been performed with hormone receptor positive breast cancer cell lines, such as MCF-7 and T47D. These were derived from pleural effusions from patients with advanced breast cancer who had already received multiple treatments5. The biological relevance of findings in such simple in vitro models of the human breast is questionable and target genes identified in these in vitro models are differ from target genes that are identified in animal models6. When primary human breast epithelial cells are cultured in vitro they tend to lose hormone receptor expression and hence hormone response7,8. This problem can be circumventd by sophisticated 3D approaches using matrigel. In this way, C. Clarke and colleagues succeeded in establishing breast epithelial cells that maintained hormone receptor expression and showed a proliferative response to progesterone stimulation9. Yet, two important in vivo progesterone receptor target genes, Wnt-4 and RANKL, were not induced upon progesterone stimulation in this system9. This approach was recently taken on further with in vitro hormone pretreatment and RANKL induction was achieved10. A caveat remains that matrigel has activities that are batch-dependent, is expensive, and demands an experimental design that is apt for small cell numbers only.
Based on the finding that in vivo ER and PR signaling are largely mediated by paracrine interactions11, we argued that intercellular interactions need to be maintained. Another important factor that is lost as tissues are dissociated to single cells for in vitro culture are the interactions of the epithelial cells with the extracellular matrix; yet these are critical for epithelial differentiation and their disruption is important in tumorigenesis12. With this in mind, we established a method to isolate breast tissue microstructures from fresh surgical discard material13. The breast parenchyma, consisting of a two-layered epithelium with inner luminal and outer myoepithelial cells, is dissected away from adipose tissue and subjected to mechanical and enzymatic dissociation. After washing and centrifugation, fragments of milk ducts are obtained that retain close interactions with many stromal cells. These tissue microstructures remain hormone responsive. The model was validated in clinical specimens13. As such, the present procedure can help to study hormone action in the breast in a biologically and clinically relevant context.
General considerations: Ahead of time, set up an ethics protocol, prepare questionnaires for patients, and see to training the clinical personnel. Before using material from reduction mammoplasty surgery, procure authorizations, and ensure patient consent. Tissue may be infectious and needs to be handled accordingly. This protocol was approved by the ethical committee of ISREC – Swiss Institute for Experimental Cancer Research.
1. Tissue Recovery, Processing and Bio-banking
To ensure optimal sample quality for bio-banking, a number of steps are carried out at the hospital prior to transport to the laboratory.
At the hospital:
In the laboratory:
2. Tissue Digestion and Hormone Stimulation
3. Recovery of Tissue Microstructures
4. Samples for Flow Cytometry
5. Samples for Histology
6. Freezing Microstructures (Future Culturing)
To study the role of estrogens and progesterone and to better understand their molecular functions in the human breast, we collect fresh human breast tissue specimens from patients undergoing reduction mammoplasties (Figure 1) after obtaining their informed consent. We also obtain the patients’ medical and reproductive history, as well as a blood sample to determine serum progesterone levels at the time of surgery. Tissue from fresh reduction mammoplasties is mechanically and enzymatically dissociated. The resulting tissue microstructures have ducts and lobules characteristic of the breast tissue of origin (Figure 2A). Immunohistochemical analysis of agarose and paraffin-embedded tissue microstructures stained for the myoepithelial marker ΔNp63 (Figure 2B) reveal that microstructures retain not only the morphological characteristics of the normal breast tissue but also the molecular profile of the cell populations.
To assess the hormone response, tissue microstructures were treated either with the synthetic progesterone receptor agonist promegestone (R5020) or ethanol control. To enrich for the hormone receptor positive luminal cells flow cytometry was performed based on epithelial (EpCAM) and myoepithelial marker (CALLA; Figure 3A) after depletion for endothelial, fibroblasts and immune cells with a cocktail of anti CD31, anti-FAP and anti-CD45 antibodies. qRT-PCR analysis of EpCAM+, CD10- cells showed up regulation of the progesterone target gene Wnt-4 (Figure 3B) in the luminal cell enriched population from the R5020 exposed tissue microstructures.
Figure 1. Breast tissue specimen. Photograph of freshly dissected reduction mammoplasty sample. Arrows point to white tissue strands that contain the breast parenchyma, i.e., milk ducts and terminal ductal lobular units. Embedded within abundant yellow adipose tissue. While the color of the fat does is consistent between patients, the proportion of adipose tissue, generally the largest part of the breast tissue in terms of volume, varies. Scale bar: 5 mm. Please click here to view a larger version of this figure.
Figure 2. Tissue microstructures. (A) Bright field image of microstructures cultured on a low attachment plate for 48 hr after mechanical dissociation and enzymatic digestion. Scale bar: 40 μm. (B) Micrograph of a histological section on tissue microstructures embedded in agarose and paraffin after 3 days in culture. The section was subjected to immunohistochemical staining for the myoepithelial marker ΔNp63 and counterstained with hematoxylin. Arrowheads point to the inner, luminal cells, arrows point to ΔNp63 positive myoepithelial cells. Scale bar: 40 μm. Please click here to view a larger version of this figure.
Figure 3. Flow cytometry sorting separation and RNA analysis of tissue microstructures. (A) Separation of the different cell populations from tissue microstructure treated with R5020 or ethanol by flow cytometry. Tissue microstructures were dissociated and immunodepleted for immune cells, fibroblasts, and endothelial cells with a cocktail of anti-CD45, anti-FAP, and anti-CD31 antibodies. Cells were labelled with antibodies against Epithelial Cell Adhesion Molecule (EpCAM) (clone HEA-125) to enrich for the luminal cell population (green) and Common Acute Lymphoblastic Leukemia Antigen (CD10/CALLA) for myoepithelial cells (Clone SS2/36). A representative scatter blot is shown. (B) Bar graph showing relative Wnt-4 mRNA expression levels in the luminal subpopulation from tissue microstructures (green dot cloud, panel A) induced with ethanol or R5050 for 24 hr. Relative mRNA expression levels of the progesterone target gene, Wnt-4 normalized against mRNA levels of hypoxanthine-guanine phosphoribosyltransferase (HPRT). Data shown represent the mean ± SD of triplicates. RNA isolation and qRT-PCR were performed as described previously14. Primer sequences: Wnt-4: forward 5′– GTGGCCTTCTCACAGTCGTT-3′ and reverse 5′– ACCTCACAGGAGCCTGACAC-3′, HPRT: forward 5′-GACCAGTCAACAGGGGACAT-3′ and reverse 5′– CCTGACCAAGGAAAGCAAAG-3′. Please click here to view a larger version of this figure.
The ex vivo culture described here provides breast tissue microstructures containing intact ducts and lobules, along with other cell types normally found in the human female breast. In the processing of the human breast tissue usually obtained from reduction mammoplasties, removal of adipose tissue, mechanical and enzymatic digestion of the stromal matrix, and lysis of red blood cells enriches for milk duct fragments and terminal ductal lobular units. Gentle enzymatic digestion at the right concentration, for the right duration is essential to ensure that tissue microstructures remain intact, retain multiple cell types and largely preserve their extracellular matrices. Attention should also be paid to processing the tissue quickly once removed from the patient. Patient samples vary substantially and mechanical and enzymatic digestion may need to be prolonged, and/or extra enzyme needs to be added when tissue is particularly high in collagen content.
Although microstructures remain 90% viable for up to 6 days, the model has the limitations for long-term hormone stimulations. As different cell types have different half-lives, the cellular composition is likely to change over time. While the tissue microstructures preserve infiltrating immune cells, these are not replenished as the tissue is removed from the body’s circulation. Furthermore, a solid pipeline with the clinical partners who provide the surgical specimens is required as large numbers of samples need to be analyzed because of inter patient variation. Tissue microstructures can be frozen for later use. However, to what extent freezing and thawing affect cell viability, possibly differentially for different cell types, and how this may alter hormone response, has not been characterized. Planning of experiments can be difficult as for any mammoplasty the amount of tissue microstructures that will be obtained is hard to anticipate.
To our knowledge, the present ex vivo model presents the first model to study physiologic hormone action in the human breast. While sophisticated 3D culture systems for primary breast epithelial cells have been developed, this ex vivo system preserves the tissue architecture and its cellular complexity over several days. As a result, not only the response in the hormone receptor positive target cells can be analyzed but the events downstream of paracrine signaling in other cell types become amenable to study. The microstructures are kept in growth factor-free medium throughout the duration of the experiments. Hence, there is no confounding extrinsic activation of signaling cascades. As such, the breast tissue microstructures offer the possibility to address questions in a setting that reflects more closely the biological complexities of the human breast.
The ex vivo system can be used to assess the response of the breast to natural and synthetic hormones. This is very important in light of an increasing incidence of breast cancer. Furthermore, drugs, small molecules, peptides, growth factors, and cytokines can be applied and their effects on cell proliferation, apoptosis and signaling be studied.
The present detailed description is meant to facilitate the use of this method by other investigators and to help minimize inter laboratory variation. As this approach relies on patient samples, it is of utmost importance that a standardized method is used so that results can begin to be compared between different laboratories15 and a better appreciation of inter patient variability becomes possible. The authors appreciate feedback and will be happy to integrate improvements into revised versions of this protocol. The work with human samples is very challenging and the authors hope to stimulate more translational research with interesting biological samples.
The authors have nothing to disclose.
The authors thank M. Fiche of University Hospital of Lausanne for providing the mammoplasty photograph, M. Wirth and A. Ayyanan of Swiss Institute for Experimental Cancer Research, National Center of Competence in Research Molecular Oncology, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne for technical assistance and R. Clarke of University of Manchester for critical comments. The research leading to these results has received support from SNF3100A0-112090, Oncosuisse 531817, and the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115188, resources of which are composed of financial contribution from the European Union's Seventh Framework Program (FP7/2007-2013) and European Federation of Pharmaceutical Industries and Associations companies' in kind contribution. The Web address of Innovative Medicines Initiative is http://www.imi.europa.eu/.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Microlitre Centrifuge | Heraeus Biofuge Fresco | 75005521 | |
Centrifuge 5810R | Eppendorf | 5810 000.327 | |
Cryobox | Nalgene | 5100-0001 | |
Controlled Rate Freezer EF600 Grant | Asymptote | EF600 | |
CO2 incubator | Hera Cell Heraeus | 51022391 | |
Roller Mixer SRT9D | Sturt | SRT9D | |
Histology Cassettes | Medizintechnik | 81-0021-00 | |
Cell Strainer | Falcon | 352340 | |
Strile Surgical Blade | Aesculap | BB536 | |
Scalpel | Aesculap | BB084R | |
Forceps | Aesculap | BD047R | |
Scissors | Aesculap | BC374R | |
Paraffin base mold | SAKURA | 4166 | |
Optimal Cutting Temperature (OCT) Cryomatrix | Thermoscientific | 6769006 | Fetal Bovine Serum |
Penicillin/Streptomycin | Life Technologies | 15070-063 | |
Antibiotic/Antimycotic | Life Technologies | 15240-062 | |
DMEM F/12 | Life Technologies | 11039-021 | Prewarm (37°C) |
Collagenase | Roche | 11 088 793 001 | Prewarm (37°C) |
Fetal Bovine Serum | Life Technologies | 10270 | Can be replaced with Fetal Calf Serum |
Cell Blood Lysis Buffer | Sigma | R7757 | |
Dimethyl sulfoxide (DMSO) | Sigma | D2650 | |
Trypsin-EDTA | Life Technologies | 15400-054 | |
Agarose | Invitrogen | 16500-500 | |
Formaldehyde | Sigma | F1635 | |
Paraformaldehyde | Roth | 335 | |
Isopentane | Sigma | M32631 | |
Ethanol | Merck | 1009831000 | |
Cryogenic vials (5.0ml) | VWR, International | 479-0820 | |
Ultra low attachment culture dish | Corning, NY 14831 | 3471 |