Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Summary

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Abstract

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior¹. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex vivo system^2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D⁴. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved³. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D^6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Introduction

Three dimensional culture (3D) of breast epithelial cells on reconstituted basement membrane is an important model system to study the complex phenotype and associated signaling of normal breast and breast cancer^2,3. The functional unit of breast is the terminal duct lobular unit (TDLU) which consists of a small duct which branches into acini. The acini are highly organized structures, composed of two cell layers, epithelial and myoepithelial cells, which are surrounded by a basement membrane⁵. The most distinguishing features of normal acini are cellular polarization, attachment to the underlying basement membrane and specialized cell-cell conta....

Protocol

1. Preparation of Materials and Culture Media

1. Thaw growth factor reduced (GFR) Matrigel at 4 °C O/N.
2. Warm up complete media for required cell line at 37 °C. MCF10A- ATCC specified media; HME- Ham's F-12 medium supplemented with 5% fetal bovine serum, 1 µg/ml hydrocortisone, 5 µg/ml insulin, 10 ng/ml epidermal growth factor, and 100 ng/ml cholera toxin; SUM149- Ham's F-12 media supplemented with 5% FBS, 2 µg/ml hydrocortisone and 5 µg/ml insulin; and MDA-MB-231-10% FBS-DMEM
3. Precool an 8-well chamber slide on ice.
4. Prepare tissue culture hood and supplies.

2. Three Di....

Results

3D culture can be effectively used to differentiate malignant phenotype of human breast epithelial cells from the benign breast. Single benign cells plated on Matrigel grow as organized spherical structures that can easily be imaged as single round flat acini in one plane using phase contrast imaging. The single malignant cells plated on Matrigel grow haphazardly, show very high refractive index and are difficult to image in one plane. As shown in Figure 1, the benign HME and MCF10A cells form spherical .......

Discussion

We have described here the three dimensional culture of breast epithelial cells on a reconstituted basement membrane and the usefulness of this system to differentiate between malignant vs. benign breast phenotype. This system can also be used to culture different cell types from other glandular tissues and has been reported to have been successfully used to culture prostate epithelial, bronchial epithelial, and thyroid epithelial cells, to name a few^12-14. The importance of 3D culture lies in the fact that it.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Growth Factor Reduced Matrigel	BD Biosciences	354230	Avoid repeat freeze thaw
Lab-Tek glass hamber slides 8-well	Thermo Fisher Scientific	177402	Precool on ice
Lab-Tek glass chamber slides 2-well	Thermo Fisher Scientific	177380	Precool on ice
Goat anti mouse F(ab´)2 fragment	Jackson Immunoresearch	115-006-006
Normal goat serum	Jackson Immunoresearch	005-000-121
Fluorescent conjugated Alexa antibodies	Molecular Probes		Please look at molecular probes website
Prolong gold antifade reagent with DAPI	Molecular Probes	P36935
3D Culture Cell Harvesting kit	Trevigen	3448-020-k
Anti-Apha-6-integrin	Fisher Scientific	MAB1378	1:100 dilution for IF
Anti-GM130	BD Biosciences	610822	1:100 dilution for IF
Anti-phospho-histone 3 (Ser10)FITC Conjugated	Fisher Scientific	16-222	1:100 dilution for IF
Anti-Cleaved caspase 3 (Asp 175)	Cell Signaling	9661s	1:50 dilution for IF
Anti-E-cadherin	BD Biosciences	610181	1:200 dilution for IF