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

Summary

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.

Abstract

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.

Introduction

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 progression^1-3. 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 ....

Protocol

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.

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Results

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. T.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
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 (5.0 ml)	VWR, International	479-0820
Ultra low attachment culture dish	Corning, NY 14831	3471