The authors wish to thank Bofan Wu and Christian Raithel for technical assistance and Michael Rieger for his introduction to live-cell imaging. Supported by NCI, NIH RO1CA112176 (P.A.F.), NCI, NIH. R01CA89041-10S1 (P.A.F.), Deutscher Akademischer Austaush Dienst e.V. A/09/72227 Ref. 316 (R.E.N.), Department of Defense W81XWH-11-1-0074 (R.E.N.), WCU (World Class University) program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R31-10069) (P.A.F.), NIH IG20 RR025828-01 (Rodent Barrier Facility Equipment), and NIH NCI 5P30CA051008 (Microscopy and Imaging and Animal Shared Resources).

1. Overall Scheme
<ol>
	<li>Generate primary cultures of preneoplastic mammary epithelial cells from mammary glands of genetically engineered and control wild-type mice.</li>
	<li>Capture live-cell images every 15 min using Volocity image acquisition software (version 5.3.1, PerkinElmer, Waltham, MA) for up to 5 days.</li>
	<li>View time-lapse images directly to assess timing and mechanism of epithelial cell colony formation, incidence of apoptosis, and phasing of morphological changes.</li>
	<li>Convert Volocity gener...

<ol>
	<li>Jones, L. P., 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=Activation+of+estrogen+signaling+pathways+collaborates+with+loss+of+Brca1+to+promote+development+of+ERalpha-negative+and+ERalpha-positive+mammary+preneoplasia+and+cancer.">Activation of estrogen signaling pathways collaborates with loss of Brca1 to promote development of ERalpha-negative and ERalpha-positive mammary preneoplasia and cancer.</a> Oncogene. 27, 794-802 (2008).</li><li>Burga, L. N., 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=Altered+proliferation+and+differentiation+properties+of+primary+mammary+epithelial+cells+from+BRCA1+mutation+carriers.">Altered proliferation and differentiation properties of primary mammary epithelial cells from BRCA1 mutation carriers.</a> Cancer Res. 69, 1273-1278 (2009).</li><li>Li, M., Wagner, K., Furth, P. A., Ip, M., Asch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+primary+mammary+epithelial+cells+and+transfection+by+viral+and+nonviral+methods.">Preparation of primary mammary epithelial cells and transfection by viral and nonviral methods.</a> Methods in Mammary Gland Biology and Breast Cancer. , (2000).</li><li>Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+stem-cell-driven+regeneration+in+mammals.">Imaging stem-cell-driven regeneration in mammals.</a> Nature. 453, 345-351 (2008).</li><li>Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+single-cell+imaging+of+mammalian+stem+cells.">Long-term single-cell imaging of mammalian stem cells.</a> Nature Methods. 8, S30-S35 (2011).</li><li>Eilken, H. M., Nishikawa, S. -. I., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+single-cell+imaging+of+blood+generation+from+haemogenic+endothelium.">Continuous single-cell imaging of blood generation from haemogenic endothelium.</a> Nature. 457, 896-900 (2009).</li><li>Kokkaliaris, K. D., Loeffler, D., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+tracking+hematopoiesis+at+the+single-cell+level.">Advances in tracking hematopoiesis at the single-cell level.</a> Current opinion in hematology. , (2012).</li><li>Rieger, M. A., Hoppe, P. S., Smejkal, B. M., Eitelhuber, A. C., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+cytokines+can+instruct+lineage+choice.">Hematopoietic cytokines can instruct lineage choice.</a> Science. 325, 217-218 (2009).</li><li>Rieger, M. A., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instruction+of+lineage+choice+by+hematopoietic+cytokines.">Instruction of lineage choice by hematopoietic cytokines.</a> Cell Cycle. 8, 4019-4020 (2009).</li><li>Heinrich, C., 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=Generation+of+subtype-specific+neurons+from+postnatal+astroglia+of+the+mouse+cerebral+cortex.">Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex.</a> Nat. Protoc. 6, 214-228 (2011).</li></ol>

The authors declare that they have no competing financial interests.

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...

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 Brca1floxed (f)11/f11/Mouse Mammary Tumor Virus (MMTV)-Cre/p53+/-/tetracycline-operator (tet-op)-ER/MMTV-reverse tetracycline transactivator (rtTA mice by 12 months of age in comparison to the lower percentages reported in Brca1f11/f11/MMTV-Cre/p53+/- mice without ERa over-expression (~50-60%) and Brca1f11/f11/MMTV-Cre mice without Tp53 haploinsufficiency (&lt;5%).1
Dynamic time-lapse imaging of the behavior of preneoplastic primary mammary epithelial cells reveals differences in cell behavior that are less easily appreciated in static tissue sections. Alterations in proliferation and differentiation are observed in primary mammary cells from human BRCA1 mutation carriers.2 Creation of single-cell suspensions of primary mammary epithelial cells from normal and genetically engineered mice are generated through enzymatic disassociation of resected mammary gland tissue.3 Time-lapse images are viewed to assess mechanism and timing of cell colony appearance and incidence of morphological changes in cells including epithelial-mesenchymal transition (EMT) and apoptosis. Generation of cell fate maps, quantification of the length of time between cell divisions (cell lifetimes), and determination of patterns of cell division are facilitated by use of single-cell tracking. Timm's Tracking Tool (TTT) is publically available software used to generate single-cell fate maps. Its utility in elucidating mechanisms of cell fate has been established4,5 examining normal hematopoietic stem cell development6-9 and the generation of neurons.10

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>EpiCult-B Basal Medium Mouse</td>
 <td>StemCell Technologies</td>
 <td>05610</td>
 <td></td>
 </tr>
 <tr>
 <td>EpiCult-B Proliferation Supplements Mouse</td>
 <td>StemCell Technologies</td>
 <td>05612</td>
 <td></td>
 </tr>
 <tr>
 <td>recombinant human Epidermal Growth Factor (rhEGF)</td>
 <td>StemCell Technologies</td>
 <td>02633</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase/Hyaluronidase</td>
 <td>StemCell Technologies</td>
 <td>07912</td>
 <td></td>
 </tr>
 <tr>
 <td>Disposable Scapels</td>
 <td>Feather</td>
 <td>2975#10</td>
 <td></td>
 </tr>
 <tr>
 <td>Hanks' Balanced Salt Solution</td>
 <td>StemCell Technologies</td>
 <td>37150</td>
 <td></td>
 </tr>
 <tr>
 <td>Ammonium Chloride</td>
 <td>StemCell Technologies</td>
 <td>07800</td>
 <td></td>
 </tr>
 <tr>
 <td>Tryspin-EDTA</td>
 <td>StemCell Technologies</td>
 <td>07901</td>
 <td></td>
 </tr>
 <tr>
 <td>Dispase</td>
 <td>StemCell Technologies</td>
 <td>07913</td>
 <td></td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>StemCell Technologies</td>
 <td>07900</td>
 <td></td>
 </tr>
 <tr>
 <td>40 &mu;m cell strainer</td>
 <td>StemCell Technologies</td>
 <td>27305</td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>StemCell Technologies</td>
 <td>06100</td>
 <td></td>
 </tr>
 <tr>
 <td>PenStrep</td>
 <td>Gibco</td>
 <td>15140</td>
 <td></td>
 </tr>
 </tbody>
</table>

time-lapse imaging of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer

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 &mu;m x 700 &mu;m 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.

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</str...

Watch this Scientific Journal Video about Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer at JoVE.com

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

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.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

time-lapse-imaging-primary-preneoplastic-mammary-epithelial-cells

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11.7K 조회수.  Georgetown University. 시간 경과 영상은 특정 행동 매개 변수 독특한 유전 적 병변 사이에 상관 관계가 있는지 확인하기 위해 유방암 위험 유전자 변형 마우스 모델에서 파생 된 기본 preneoplastic 유방 상피 세포의 행동을 평가하는 데 사용됩니다.