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
<p class="jove_co...

<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+epithelial+cell:+influence+of+extracellular+matrix+composition+and+organization+during+development+and+tumorigenesis.">Mammary epithelial cell: influence of extracellular matrix composition and organization during development and tumorigenesis.</a> Int J Biochem Cell Biol. 39 (11), 1987-1994 (2007).</li><li>Graham, J. D., 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=DNA+replication+licensing+and+progenitor+numbers+are+increased+by+progesterone+in+normal+human+breast.">DNA replication licensing and progenitor numbers are increased by progesterone in normal human breast.</a> Endocrinology. 150 (7), 3318-3326 (2009).</li><li>Wang, J., 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=Comment+on+&#34;Progesterone/RANKL+is+a+major+regulatory+axis+in+the+human+breast&#34;.">Comment on &#34;Progesterone/RANKL is a major regulatory axis in the human breast&#34;.</a> Sci Transl Med. 5 (215), 215le214 (2013).</li><li>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=Progesterone+signalling+in+breast+cancer:+a+neglected+hormone+coming+into+the+limelight.">Progesterone signalling in breast cancer: a neglected hormone coming into the limelight.</a> Nat Rev Cancer. 13 (6), 385-396 (2013).</li><li>Xu, R., Boudreau, A., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture+and+function:+dynamic+reciprocity+via+extra-+and+intra-cellular+matrices.">Tissue architecture and function: dynamic reciprocity via extra- and intra-cellular matrices.</a> Cancer Metastasis Rev. 28 (1-2), 167-176 (2009).</li><li>Tanos, T., 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=Progesterone/RANKL+is+a+major+regulatory+axis+in+the+human+breast.">Progesterone/RANKL is a major regulatory axis in the human breast.</a> Sci Transl Med. 5 (182), 182ra155 (2013).</li><li>Yalcin-Ozuysal, O., 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=Antagonistic+roles+of+Notch+and+p63+in+controlling+mammary+epithelial+cell+fate.">Antagonistic roles of Notch and p63 in controlling mammary epithelial cell fate.</a> Cell Death Differ. 17 (10), 1600-1610 (2010).</li><li>Hines, W. C., Su, Y., Kuhn, I., Polyak, K., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorting+out+the+FACS:+a+devil+in+the+details.">Sorting out the FACS: a devil in the details.</a> Cell Rep. 6 (5), 779-781 (2014).</li></ol>

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 th...

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 border="0" cellpadding="0" cellspacing="0" width="877">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Name of Material/ Equipment</td>
			<td style="width:164px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:260px;">Comments/Description</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:335px;">Microlitre Centrifuge&#160;</td>
			<td style="width:164px;">Heraeus Biofuge Fresco</td>
			<td style="width:119px;">75005521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Centrifuge 5810R</td>
			<td style="width:164px;">Eppendorf</td>
			<td style="width:119px;">5810 000.327</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cryobox</td>
			<td style="width:164px;">Nalgene&#160;&#160;</td>
			<td style="width:119px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:335px;">Controlled Rate Freezer EF600 Grant</td>
			<td style="width:164px;">Asymptote&#160;</td>
			<td style="width:119px;">EF600</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:335px;">CO2 incubator</td>
			<td style="width:164px;">Hera Cell Heraeus</td>
			<td style="width:119px;">51022391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Roller Mixer SRT9D</td>
			<td style="width:164px;">Sturt</td>
			<td style="width:119px;">SRT9D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Histology Cassettes</td>
			<td style="width:164px;">Medizintechnik</td>
			<td style="width:119px;">81-0021-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cell Strainer</td>
			<td style="width:164px;">Falcon</td>
			<td style="width:119px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Strile Surgical Blade&#160;</td>
			<td style="width:164px;">Aesculap</td>
			<td>BB536&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Scalpel</td>
			<td style="width:164px;">Aesculap</td>
			<td style="width:119px;">BB084R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Forceps</td>
			<td style="width:164px;">Aesculap</td>
			<td style="width:119px;">BD047R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Scissors</td>
			<td style="width:164px;">Aesculap</td>
			<td style="width:119px;">BC374R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Paraffin base mold</td>
			<td style="width:164px;">SAKURA</td>
			<td style="width:119px;">4166</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Optimal Cutting Temperature (OCT) Cryomatrix</td>
			<td style="width:164px;">Thermoscientific</td>
			<td style="width:119px;">6769006</td>
			<td style="width:260px;">Fetal Bovine Serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Antibiotic/Antimycotic</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">DMEM F/12</td>
			<td style="width:164px;">Life Technologies</td>
			<td>11039-021</td>
			<td>Prewarm (37&#176;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Collagenase</td>
			<td style="width:164px;">Roche</td>
			<td style="width:119px;">11 088 793 001</td>
			<td>Prewarm (37&#176;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fetal Bovine Serum</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">10270</td>
			<td>Can be replaced with Fetal Calf Serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cell Blood Lysis Buffer</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">R7757</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Trypsin-EDTA</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Agarose</td>
			<td style="width:164px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Formaldehyde</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">F1635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Paraformaldehyde</td>
			<td style="width:164px;">Roth</td>
			<td style="width:119px;">335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Isopentane</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">M32631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ethanol</td>
			<td style="width:164px;">Merck</td>
			<td style="width:119px;">1009831000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cryogenic vials (5.0ml)&#160;</td>
			<td style="width:164px;">VWR, International</td>
			<td style="width:119px;">479-0820</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Ultra low attachment culture dish</td>
			<td style="width:164px;">Corning, NY 14831</td>
			<td style="width:119px;">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 dissocia...

Watch this Scientific Journal Video about An Ex vivo Model to Study Hormone Action in the Human Breast at JoVE.com

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 ...

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

Research

JoVE Journal

Medicine

11.3K vistas.  Ecole polytechnique fédérale de Lausanne.  Hemos desarrollado un nuevo modelo ex vivo para estudiar la acción de la hormona en el pecho humano. Se basa en microestructuras de tejido aisladas a partir de muestras de tejido de mama quirúrgicos que conservan la arquitectura del tejido, las interacciones intercelulares, y la señalización paracrina. 