Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Summary

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Abstract

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 μm x 700 μm fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Introduction

Genetically engineered mouse models are tools to study and understand how different genetic lesions contribute to the risk of developing breast cancer. For example, genetically engineered mice have shown that the combination of three factors: loss of the full-length breast cancer 1, early onset (Brca1) gene in mammary epithelial cells, tumor protein p53 (Tp53) germ-line haploinsufficiency, and mammary epithelial cell targeted up-regulated Estrogen receptor alpha (ERa) expression results in the development of mammary cancer in 100% of Brca1^{floxed (f)11/f11/Mouse Mammary Tumor Virus (MMTV)-Cre/p53+/-/tetracycline-operator (tet-op)-ER/MMTV-reve....}

Protocol

1. Overall Scheme

1. Generate primary cultures of preneoplastic mammary epithelial cells from mammary glands of genetically engineered and control wild-type mice.
2. Capture live-cell images every 15 min using Volocity image acquisition software (version 5.3.1, PerkinElmer, Waltham, MA) for up to 5 days.
3. View time-lapse images directly to assess timing and mechanism of epithelial cell colony formation, incidence of apoptosis, and phasing of morphological changes.
4. Convert Volocity generated .TIFF stacks to .JPG files using MetaMorph Microscopy Automation & Image Analysis Software (Molecular Devices, LLC Sunnyvale, CA) and rename digi....

Results

Epithelial and fibroblast cells can be distinguished by cell morphology. Epithelial cells have a cuboidal shape (Figure 1A-B) and form cell colonies (Figure 1A). Fibroblasts, a type of stromal cell, have an elongated morphology (Figure 1C).

Cells were rounded and floating at the onset of imaging (Figure 2A-D). After attachment to the plate they became flat and demonstrated a cuboidal-type appearance (Figure 2E, H

Discussion

Critical steps

It is important to ensure that mammary glands are harvested from mice of the same age to control for age-related variability in mammary epithelial cell behavior. When plating the cells, the same number of cells should be plated in each well for every experiment. Cells should be relatively sparse when plating so that cultures do not become confluent too quickly making it possible to follow multiple individual cells through serial images. It is essential that the plate be equil.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EpiCult-B Basal Medium Mouse	StemCell Technologies	05610
EpiCult-B Proliferation Supplements Mouse	StemCell Technologies	05612
recombinant human Epidermal Growth Factor (rhEGF)	StemCell Technologies	02633
Collagenase/Hyaluronidase	StemCell Technologies	07912
Disposable Scapels	Feather	2975#10
Hanks' Balanced Salt Solution	StemCell Technologies	37150
Ammonium Chloride	StemCell Technologies	07800
Tryspin-EDTA	StemCell Technologies	07901
Dispase	StemCell Technologies	07913
DNase I	StemCell Technologies	07900
40 μm cell strainer	StemCell Technologies	27305
FBS	StemCell Technologies	06100
PenStrep	Gibco	15140