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 F&#233;d&#233;rale 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&#39;s Seventh Framework Program (FP7/2007-2013) and European Federation of Pharmaceutical Industries and Associations companies&#39; in kind contribution. The Web address of Innovative Medicines Initiative is <a href="http://www.imi.europa.eu/" target="_blank">http://www.imi.europa.eu/</a>.

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:
<ol>
	<li>Obtain human breast tissues under sterile conditions from reduction mammoplasty surgery and blood samples, if required. Tissue needs to be examined by pathologist who will sample to exclude presence of malignant lesions.</li>
	<li>Place the breast tissue under the hood on a sterile board and prepare scissors, surgical scalpel and forceps which are already sterilized with steam sterilizer. Make deep cut at different sites of the mammary tissue using a scalpel to open it. In human breast tissue look for white strands consisting of ducts and lobules, which are embedded in yellow adipose tissue. 
	NOTE: The ratio of the white parenchymal to the yellow adipose tissue differs among women; typically younger women have more epithelial tissue.</li>
	<li>Pick pieces of white material and hold them with forceps, then separate them gently from the adipose tissue with scissors. Avoid taking blood vessels.</li>
	<li>Rinse pieces of tissue in Phosphate-buffered saline (PBS) pH 7.2 to remove blood. Transfer the tissue to 250 ml plastic bottles containing F12 medium (Dulbecco&#8217;s Modified Eagle&#8217;s Medium F12) without phenol red with 2% penicillin/streptomycin (including 5,000 unit/ml penicillin and 5,000 g/ml streptomycin) and 1% antibiotic/amphotericin B (including 10,000 units/ml penicillin, 10,000 &#181;g/ml of streptomycin, and 25 &#181;g/ml of amphotericin B). Store tissues in the medium for transport to the laboratory.</li>
	<li>Prepare pieces of 3 - 5 mm diameter for RNA and protein extraction and flash freeze them in cryovials with cold (dry ice) isopentane. Transfer them to dry ice box for transportation to the laboratory.</li>
	<li>Put pieces of 3 - 5 mm into mold and cover it with a layer of Optimal Cutting Temperature (OCT) and then place the mold on the flat bottom of box filled with cold isopentane. After complete freezing transfer the mold to the dry ice box.</li>
	<li>Fix 5 pieces of 3 - 5 mm diameter in paraformaldehyde (PFA) 4% and another 5 pieces in formaldehyde (FA) 4% in PBS at RT histological analysis later on. Two different fixatives are recommended as some antibodies work better with one or the other of the two.</li>
</ol>
In the laboratory:
<ol start="8">
	<li>Document all information about the reduction mammoplasty, date of surgery and whether the samples are derived from left versus right breast.</li>
	<li>Place the frozen samples at -80&#176; C and transfer the fixed tissues into histology cassettes after 2 hr of fixation. Then put them either in PBS or 70% ethanol, for O/N storage at 4 &#176;C. On the next day, transfer the histology cassettes to histology facilities for paraffin embedding.</li>
</ol>
2. Tissue Digestion and Hormone Stimulation

<ol>
	<li>Transfer the bottles containing the tissue samples to a laminar flow cabinet in a P2 cell culture room. Place tissue pieces on sterile chopping board (29 x 19 cm). Hold the material with forceps and scrape the remaining fat and vessels away from the breast parenchyma (white part) with scalpel . Place the white material in PBS in a 10 cm culture dish.</li>
	<li>After removing the fatty material from all pieces and disposing them according to the safety rules of the laboratory, place the white parts containing the epithelial tissues back on the chopping board. Using opposing scalpels cut the tissue and chop it to pieces of up to 2 mm diameter.</li>
	<li>Place the minced material into tubes of adequate size: up to 1, 2.5 and 10 ml into a 5, 15 or 50 ml tubes, respectively. Typically a 5 ml tube yields enough material for paraffin embedment and hormone stimulations, and a 15 ml tube yields enough material for Fluorescence Activated Cell Sorting (FACS). Larger tubes are used for freezing aliquots.</li>
	<li>Prepare digestion master mix consisting of medium (DMEM/F12 without phenol red) with 1% penicillin/streptomycin/amphotericin B and collagenase A (Clostridiopeptidase A from Clostridium histolyticum). Dose collagenase according to digestion times.
	<ol>
		<li>For 12, 24, and 36 hr of incubation, use final concentration of collagenase of 1.5 mg/ml, 1.0 mg/ml and 0.5 mg/ml respectively. Add digestion master mix up to 4 times of the volume of the digested material to ensure adequate agitation and aeration. 
		NOTE: Depending on the experimental design and the duration of hormone stimulation, add hormones either during or after the enzymatic digestion step and microstructure recovery.</li>
	</ol>
	</li>
	<li>Add hormone(s) at desired concentration to test samples and the vehicle to the control. For stimulation with 17-&#946;-estradiol, and promegestone (R5020) use a final concentration of 20 nM. 
	NOTE: R5020 is a synthetic progesterone receptor agonist, which is more stable than natural progesterone in medium.
	<ol>
		<li>Place tubes on the roller mixer inside an incubator at 37 &#176;C and 40 rpm O/N. Before placing the samples on the mixer, resuspend them very well to ensure that all material distributes along the centrifuge tubes.</li>
	</ol>
	</li>
	<li>As digestion takes at least 12 hr (O/N), for hormone stimulation times of less than 12 hr, add the hormones after recovery of the microstructures (detailed under step 3). Add the hormones as you resuspend the microstructures and place the suspension on ultra-low attachment plates. Tissue microstructures can be kept on ultra-low attachment plates for up to 7 days in growth-factor-free medium preserving about 70% viability.</li>
	<li>For RNA and protein analysis, collect tissue microstructures by centrifugation at 400 x g for 5 min and flash freeze in centrifuge tubes.</li>
</ol>
3. Recovery of Tissue Microstructures
<ol>
	<li>Centrifuge the tube(s) at 400 x g for 5 min. Aspirate the fat from the top of the centrifuge tube and transfer the supernatant of the remaining aqueous supernatant, which is enriched in fibroblasts, to a new centrifuge tube and centrifuge at 1,250 x g for 5 min. 
	NOTE: As the breast tissue microstructures are very sticky, for all subsequent steps resuspend the pellet by gently agitating the tube. Pipetting is not recommended due to the risk of losing material.</li>
	<li>Resuspend the pellets enriched in microstructures and in fibroblasts derived from aqueous supernatant in PBS, 2% Fetal Calf Serum (FCS). Subsequently spin both tubes at 1,250 x g for 5 min and then discard the supernatants.</li>
	<li>Resuspend the pellets in 3 - 5 ml red cell blood lysis buffer and transfer them to clean tubes. Incubate them for 5 min at RT.</li>
	<li>Add 2x volume of PBS, 2% FCS and centrifuge them at 1,250 x g for 5 min. Wash the pellets with PBS, 2% FCS and centrifuge at 1,250 x g for 5 min. Repeat this step twice.</li>
</ol>
4. Samples for Flow Cytometry
<ol>
	<li>Add 1 - 2 ml trypsin 0.25% to the pellet and mix it by pipetting up and down very gently for 2 min with a 1,000 &#181;l pipetman to dissociate cells.</li>
	<li>Inhibit trypsin activity by adding 9 ml PBS, 2% FCS. Centrifuge at 450 x g for 5 min and resuspend the pellet in 10 ml PBS, 2% FCS.</li>
	<li>Transfer the cell suspension onto a 40 &#181;m cell strainer placed on top of a 50 ml centrifuge tube to prepare single cells. If a few microliters of cell suspension remain on top of the filter, help them pass through the filter by aspirating them from the other side of strainer with a pipet and add them to the filtered microstructures.</li>
	<li>Separate immune cells, fibroblasts and endothelial cells from microstructures with a cocktail of anti-CD45, anti-FAP and anti-CD31 antibodies and then sort them with FACS, based on pan-epithelial marker EpCAM and CD10 to separate luminal and basal cells respectively10.</li>
</ol>
5. Samples for Histology
<ol>
	<li>After washing the mechanically and enzymatically digested material as described above, fix pellets from 1.5 ml initial material in 1 ml of 4% PFA in PBS at RT for 30 min.</li>
	<li>Centrifuge at 1,250 x g for 5 min and wash with 1 - 2 ml PBS twice.</li>
	<li>Centrifuge at 16,000 x g for 10 min. During this time, prepare 1% multi-purpose agarose in Tris-Acetate-EDTA (TAE) buffer and heat the solution in the microwave oven until the solution comes to a boil.
	<ol>
		<li>Remove the solution from the microwave oven and swirl the flask gently to mix the solution and resuspend any remaining agarose particles. Cool agarose solution to 50 &#176;C and pour 1 ml in a paraffin base mold sized 31 &#215; 23 &#215; 13.5 mm.</li>
	</ol>
	</li>
	<li>Discard the supernatant and place the pellets on top of the solidified layer (1 - 3 mm) of agarose using a spatula with a flat end. Cover them all with an additional layer of agarose (40 &#176;C).</li>
	<li>After the agarose solidifies, usually within 5 min, put the agarose blocks into the histology cassettes for paraffin embedment. Along with the agarose samples place a white office paper in the cassette with all information about the reduction mammoplasty number, experimental condition, and date written in small letters in pencil; this writing withstands all subsequent treatment steps.</li>
	<li>Put the cassettes in PBS O/N, or in 70% ethanol for storage over the weekend, at 4 &#176;C. We have not noticed any difference between the two storage solutions. Then transfer histology cassettes to histology facilities for paraffin embedding.</li>
</ol>
6. Freezing Microstructures (Future Culturing)
<ol>
	<li>Resuspend tissue microstructures in freezing medium consisting of FCS with 10% dimethyl sulfoxide (DMSO) and freeze 1 ml aliquots in cryovials. The number of obtained cryovials depends on the amount of primary material obtained from reduction mammoplasty.</li>
	<li>Freeze the cryovials containing tissue microstructures using a controlled rate freezer with the following program: 5 min dwelling at 4 &#176;C, decreasing the temperature to -7 &#176;C with rate of 6 &#176;C/min, decreasing the temperature to -12 &#176;C with rate of 6 &#176;C/min, 10 min dwelling, decreasing the temperature to -40 &#176;C with rate of 6 &#176;C/min, decreasing in temperature to -80 &#176;C with rate of 6 &#176;C/min. Then transfer the cryovials to -80&#176;C freezer.</li>
	<li>Next day, transfer the cryovials from -80&#176;C to liquid nitrogen tank for long storage.</li>
</ol>

<ol>
	<li>Beral, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+and+hormone-replacement+therapy+in+the+Million+Women+Study.">Breast cancer and hormone-replacement therapy in the Million Women Study.</a> Lancet. 362 (9382), 419-427 (2003).</li><li>MacMahon, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+at+first+birth+and+breast+cancer+risk.">Age at first birth and breast cancer risk.</a> Bull World Health Organ. 43 (2), 209-221 (1970).</li><li>Pike, M. C., Krailo, M. D., Henderson, B. E., Casagrande, J. T., Hoel, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Hormonal'+risk+factors,+'breast+tissue+age'+and+the+age-incidence+of+breast+cancer.">'Hormonal' risk factors, 'breast tissue age' and the age-incidence of breast cancer.</a> Nature. 303 (5920), 767-770 (1983).</li><li>Brisken, C., O'Malley, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone+action+in+the+mammary+gland.">Hormone action in the mammary gland.</a> Cold Spring Harb Perspect Biol. 2 (12), a003178 (2011).</li><li>Levenson, A. S., Jordan, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF-7:+the+first+hormone-responsive+breast+cancer+cell+line.">MCF-7: the first hormone-responsive breast cancer cell line.</a> Cancer Res. 57 (15), 3071-3078 (1997).</li><li>Tanos, T., Rojo, L. J., Echeverria, P., Brisken, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+and+PR+signaling+nodes+during+mammary+gland+development.">ER and PR signaling nodes during mammary gland development.</a> Breast Cancer Res. 14 (4), 210 (2012).</li><li>Roskelley, C., Desprez, P., Bissell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix-dependent+tissue-specific+gene+expression+in+mammary+epithelial+cells+requires+both+physical+and+biochemical+signal+transduction.">Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction.</a> Proc Natl Acad Sci U S A. 91 (26), 12378-12382 (1994).</li><li>Kass, L., Erler, J. T., Dembo, M., Weaver, V. 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The authors have nothing to disclose.

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&#8217;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.

A correction was made to <a href="/video/52436">An Ex Vivo Model to Study Hormone Action in the Human Breast</a>. There was an error with the author, Marie Shamsheddin&#39;s, name. The author&#39;s name has been corrected to:
Marie Shamseddin
instead of:
Marie Shamsheddin

A correction to the author list has been made for the article <a href="/video/52436">An Ex Vivo Model to Study Hormone Action in the Human Breast</a>.

Erratum: An Ex Vivo Model to Study Hormone Action in the Human Breast

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.&#160; 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.&#160; 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.

<table><tbody><tr><td>Microlitre Centrifuge</td><td>Heraeus Biofuge Fresco</td><td>75005521</td><td></td></tr><tr><td>Centrifuge 5810R</td><td>Eppendorf</td><td>5810 000.327</td><td></td></tr><tr><td>Cryobox</td><td>Nalgene&amp;nbsp;&amp;nbsp;</td><td>5100-0001</td><td></td></tr><tr><td>Controlled Rate Freezer EF600 Grant</td><td>Asymptote&amp;nbsp;</td><td>EF600</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Hera Cell Heraeus</td><td>51022391</td><td></td></tr><tr><td>Roller Mixer SRT9D</td><td>Sturt</td><td>SRT9D</td><td></td></tr><tr><td>Histology Cassettes</td><td>Medizintechnik</td><td>81-0021-00</td><td></td></tr><tr><td>Cell Strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>Strile Surgical Blade&amp;nbsp;</td><td>Aesculap</td><td>BB536&amp;nbsp;</td><td></td></tr><tr><td>Scalpel</td><td>Aesculap</td><td>BB084R</td><td></td></tr><tr><td>Forceps</td><td>Aesculap</td><td>BD047R</td><td></td></tr><tr><td>Scissors</td><td>Aesculap</td><td>BC374R</td><td></td></tr><tr><td>Paraffin base mold</td><td>SAKURA</td><td>4166</td><td></td></tr><tr><td>Optimal Cutting Temperature (OCT) Cryomatrix</td><td>Thermoscientific</td><td>6769006</td><td></td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>Antibiotic/Antimycotic</td><td>Life Technologies</td><td>15240-062</td><td></td></tr><tr><td>DMEM F/12</td><td>Life Technologies</td><td>11039-021</td><td>Prewarm (37 &amp;deg;C)</td></tr><tr><td>Collagenase</td><td>Roche</td><td>11 088 793 001</td><td>Prewarm (37 &amp;deg;C)</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>10270</td><td>Can be replaced with Fetal Calf Serum</td></tr><tr><td>Cell Blood Lysis Buffer</td><td>Sigma</td><td>R7757</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16500-500</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma</td><td>F1635</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Roth</td><td>335</td><td></td></tr><tr><td>Isopentane</td><td>Sigma</td><td>M32631</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>1009831000</td><td></td></tr><tr><td>Cryogenic vials (5.0 ml)</td><td>VWR, International</td><td>479-0820</td><td></td></tr><tr><td>Ultra low attachment culture dish</td><td>Corning, NY 14831</td><td>3471</td><td></td></tr></tbody></table>

an ex vivo model to study hormone action in the human breast

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.

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&#8217; 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 &#916;Np63 (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.
<img alt="Figure 1" src="/files/ftp_upload/52436/52436fig1.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/52436/52436fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52436/52436fig2.jpg" /> 
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 &#956;m. (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 &#916;Np63 and counterstained with hematoxylin. Arrowheads point to the inner, luminal cells, arrows point to &#916;Np63 positive myoepithelial cells. Scale bar: 40 &#956;m. <a href="https://www.jove.com/files/ftp_upload/52436/52436fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52436/52436fig3.jpg" /> 
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 &#177; SD of triplicates. RNA isolation and qRT-PCR were performed as described previously14. Primer sequences: Wnt-4: forward 5&#8242;- GTGGCCTTCTCACAGTCGTT-3&#8242; and reverse 5&#8242;- ACCTCACAGGAGCCTGACAC-3&#8242;, HPRT: forward 5&#8242;-GACCAGTCAACAGGGGACAT-3&#8242; and reverse 5&#8242;- CCTGACCAAGGAAAGCAAAG-3&#8242;. <a href="https://www.jove.com/files/ftp_upload/52436/52436fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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An Ex vivo Model to Study Hormone Action in the Human Breast

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.

An Ex vivo Model to Study Hormone Action in the Human Breast

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 ...

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Nanyang Technological University

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JoVE Journal

Medicine

11.3K Views.  Ecole polytechnique fédérale de Lausanne. 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.

Video: An Ex vivo Model to Study Hormone Action in the Human Breast

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Cancer Research

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Bioengineering

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

An Ex vivo Model to Study Hormone Action in the Human Breast

Summary

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Chapters in this video

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche