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 generated .TIFF stacks to .JPG files using MetaMorph Microscopy Automation &amp; Image Analysis Software (Molecular Devices, LLC Sunnyvale, CA) and rename digital image files (THE Rename 2.1.6, <a href="http://www.softpedia.com/get/System/File-Management/THE-Rename.shtml" target="_blank">http://www.softpedia.com/get/System/File-Management/THE-Rename.shtml</a>) to enable compatibility with Timm's Tracking Tool software (TTT, <a href="http://www.helmholtz-muenchen.de/en/isf/hematopoiesis/software-download/index.html" target="_blank">http://www.helmholtz-muenchen.de/en/isf/hematopoiesis/software-download/index.html</a>).</li>
	<li>Using TTT, generate cell fate maps for determination of the time between cell divisions (cell lifetimes) and patterns of cell division (symmetric versus asymmetric).</li>
	<li>Analyze quantitative data from steps 3 and 5 above to determine if there are statistically significant differences between different genetically engineered mouse models and/or wild-type controls.</li>
</ol>
2. Generation of Primary Mammary Epithelial Cell Cultures
<ol>
	<li>Prepare Complete Media for isolation of single mammary epithelial cells following a variation of manufacturer's instructions (EpiCult-B Mouse Medium Kit, Stemcell Technologies, Vancouver, BC). Combine 450 ml Epi-Cult-B medium, 50 ml Epi-Cult B proliferation supplement, 5% Fetal Bovine Serum (FBS), 50 &mu;g/ml Penicillin/Streptomycin (PenStrep) and 10 ng/ml Epidermal Growth Factor (EGF).</li>
	<li>Prepare Dissociation Media by combining 1 part of a 10x Collagenase/Hyaluronidase mixture with 9 parts Complete Media from 2.1.</li>
	<li>Euthanize mouse and immediately proceed to necropsy. Place mouse on its back on a styrofoam necropsy platform and secure all four limbs so ventral skin is taut. Saturate ventral skin and hair, including that overlying the limbs, with 70% ethanol. Expose #2/3 (thoracic) and #4/5 (inguinal) mammary glands by making a midline incision through the skin (do not enter the peritoneum) that is extended into a Y-incision through the medial skin of each front limb and an upside down Y incision through the medial skin of each rear limb.</li>
	<li>Using blunt dissection, separate the skin from underlyingperitoneum, pull back on both sides of the skin until it is taut, and secure it to the styrofoam necropsy platform using pins. Starting from the outer side, use blunt dissection with judicious use of dissection scissors to isolate the intact inguinal and/or thoracic mammary glands from underlying connective tissue and muscle. Place no more than two mammary glands in a 10 cm sterile Petri dish and move to tissue culture hood.</li>
	<li>In tissue culture hood, examine glands for mammary lymph nodes that are identified as small, well-circumscribed nodules with a yellowish color. Remove lymph nodes from the surrounding gland using sterile scalpel and forceps. Mince mammary tissue into ~1 mm3 cubes using two sterile scalpels, one in each hand.</li>
	<li>Place minced mammary tissue in 5 ml Dissociation Media, mix by gently pipetting up and down 2-3 times using a 10 ml pipette, and incubate overnight (up to ~16 hr) in a 37 &deg;C incubator with 5% CO2 in a 50 ml conical tube with loosened cap.</li>
	<li>The next morning, prepare a cold mixture of 1 part Hanks' Balanced Salt Solution (HBSS) containing 2% FBS and 4 parts buffered Ammonium Chloride Solution to promote red blood cell lysis (HFAmCl) as well as cold HBSS supplemented with 2% FBS (HF). Pre-warm Trypsin-EDTA (0.25%), 5 mg/ml Dispase in Hank's Balanced Salt Solution Modified, 1 mg/ml DNase I, and Complete Media.</li>
	<li>Remove the conical centrifuge tube with the mammary tissue from the incubator and gently pulse vortex 2-3 sec two times. Centrifuge the tube at 450 &times; g for 5 min (room temperature or 4 &deg;C), and discard the supernatant.</li>
	<li>Resuspend the pellet in a minimum of 10 ml HFAmCl and centrifuge at 450 &times; g for 5 min (room temperature or 4 &deg;C) and discard supernatant.</li>
	<li>Add 3 ml Trypsin-EDTA to the pellet and mix first with a 5 ml pipette and then with a P1000 micropipettor until stringy (1-3 min). Add 10 ml HF, centrifuge at 450 &times; g for 5 min (room temperature or 4 &deg;C) and discard supernatant.</li>
	<li>Add 2 ml of 5 mg/ml Dispase and 200 &mu;l of 1 mg/ml DNase I to the pellet and mix with a P1000 micropipettor for 1 min. Sample should be cloudy but not stringy. If stringy, add 100 &mu;l of DNase I and mix again.</li>
	<li>Add 10 ml cold HF, strain through a 40 &mu;m cell strainer and centrifuge at 450 &times; g for 5 min (room temperature or 4 &deg;C) and discard supernatant.</li>
	<li>Resuspend the final pellet in Complete Media. Quantify the total number of cells (using a hemocytometer or Coulter Counter). Two mammary glands yield approximately 1 x 106 to 1 x 107 cells. Plate primary epithelial cells in a flat-bottomed 6-well plate at a density of 1.5 x 105 cells per well.</li>
</ol>
3. Live-cell Imaging
<ol>
	<li>Immediately after plating the cells, place the 6-well plate securely on a microscope stage within a 5% CO2/saturated humidity/37 &deg;C temperature incubation chamber. Adjust the condenser for Kohler illumination and center the phase rings. For the experiments presented here, an inverted Nikon Eclipse TE300/PerkinElmer Spinning Disc Confocal microscope system in widefield phase contrast mode with a 10x objective lens was used.</li>
	<li>Open image acquisition software, create and name a library for the time-lapse images, and save to a location with sufficient space for large files. The experiments presented here utilized Volocity image acquisition software (version 5.3.1, PerkinElmer, Waltham, MA).</li>
	<li>Allow the plate to equilibrate in the microscope incubator for a minimum of 15 min. Lower the lenses to avoid stage-lens interference. Under the "Stage" heading, select "Calibrate Stage." Reacquire the focal plane, set Z=zero under the x, y, z tab and mark imaging points by selecting "Add point" under the "Stage" heading. Place points in the middle of each well of the 6-well plate for imaging as phase contrast imaging may be distorted near the periphery of plastic wells. Arrange points in a square in 4 x 4 fields (650 &mu;m x 700 &mu;m fields), each with a small overlap (about 5%). Save the selected points under "Stage."</li>
	<li>Under "Stage" select "Make focus map" and follow the prompts to set the focus of each point. Set image acquisition timing to capture 4 pictures per hour (every 15 min). This can be adjusted depending on the requirements of the specific experiment. Save the focus map under "Stage".</li>
	<li>Right click on the right side tool bar "Image Acquisition" and save the imaging settings. Press the record button to begin the imaging. After 1 hr, check the focus of the images to see if any adjustment is needed.</li>
	<li>Monitor and continue live imaging for 5 days, ending when cells become confluent.</li>
</ol>
4. Direct Viewing of Time-lapse Images to Assess Timing and Mechanism of Initial Epithelial Cell Colony Formation, Incidence of Apoptosis, and Phasing of Morphological Changes
<ol>
	<li>Direct viewing of time-lapse images can be performing using any compatible video viewing software (e.g. <a href="http://www.real.com/" target="_blank">http://www.real.com/</a>realplayer or other freeware or commercially available software). Images can be viewed in .TIFF format at this step or after conversion to .JPG files (see Step 5).</li>
	<li>To determine mechanism of initial colony formation, start with a single-image stack representing one imaging site on the plate. In initial frames, cells will be floating. Follow serial images to determine when the first epithelial cell adheres to the plate. At this step, epithelial cells can be identified and differentiated from fibroblasts by their more cuboidal morphology. Follow this single epithelial cell through serial images and follow its fate over the subsequent 24 hr. Record if it becomes surrounded by additional epithelial cells or undergoes cell division or apoptosis. If surrounded by epithelial cells, the mechanism of colony formation can be determined directly by viewing if the surrounding cells are derived from floating cells that then aggregate with the initial adherent cell(s) or from division of the initial adherent cell. Record the number of colonies formed by cell aggregation versus cell division during the first 24 hr.</li>
	<li>To determine the number of initially adherent cells that undergo apoptosis over a specific time period, follow serial images for the appearance of classic features of apoptosis developing in a cell that has been adherent (<a href="http://www.alsa.org/research/about-als-research/cell-death-and-apoptosis.html" target="_blank">http://www.alsa.org/research/about-als-research/cell-death-and-apoptosis.html</a>), and the number of apoptotic cells identified during the entire duration of the imaging or within specific time frames recorded.</li>
	<li>Over time, epithelial cells in culture alter their morphology from an initial cuboidal shape to a more elongated appearance consistent with EMT. Follow serial images to determine the day/hour after plating when this occurs.</li>
</ol>
5. Image File Modification
<ol>
	<li>Through the Volocity software, export the image files to TIFF stacks. Here MetaMorph Microscopy Automation &amp; Image Analysis Software (Molecular Devices, LLC Sunnyvale, CA) was used to open the TIFF stack files and they were then saved in MetaMorph as single .JPG images.</li>
	<li>Rename the image folders and files according to the requirements for Timm's Tracking Tool software (<a href="http://www.helmholtz-muenchen.de/fileadmin/ISF/PDF/Haematopoese/publications/TTT_Operating_Instructions_July_2009_.pdf" target="_blank">http://www.helmholtz-muenchen.de/fileadmin/ISF/PDF/Haematopoese/publications/TTT_Operating_Instructions_July_2009_.pdf</a>). Use a freeware program like THE Rename 2.1.6 (<a href="http://www.softpedia.com/get/System/File-Management/THE-Rename.shtml" target="_blank">http://www.softpedia.com/get/System/File-Management/THE-Rename.shtml</a>) to rename files in groups. Create one folder containing all folders and files of the experiment and name the folder (e.g. EXPERIMENTNAME).</li>
	<li>Create a subfolder for each point/position where the culture was imaged. Name the subfolders: EXPERIMENTNAME_pPOSITIONNUMBER. The position number should have 3 digits. Example: 072511RN_p001. Put all the image files of that individual position in the subfolder.</li>
	<li>Add the timepoint (using 5 digits) and channel number (since there is only one bright field channel, use "0") at the end of each file name in this format: 
	EXPERIMENTNAME_pPOSITIONNUMBER_tTIMEPOINT_wCHANNELNUMBER.jpgExample: 072511RN_p001_t00001_w0.jpg</li>
	<li>Generate a log file for each position by first downloading the free program TTTlogfileconverter from the TTT website. Start the TTTlogfileconverter program and select the experiment folder. Input an image interval (in seconds). For a picture taken every 15 min, enter 900 sec. Then press the green covert log files button.</li>
</ol>
6. Generation of Cell Fate Maps of Single Cells Using TTT
<ol>
	<li>Create a folder called TTTexport and a subfolder called TTTfiles where all cell tracking data will be stored following TTT software instructions (<a href="http://www.helmholtz-muenchen.de/fileadmin/ISF/PDF/Haematopoese/publications/TTT_Operating_Instructions_July_2009_.pdf" target="_blank">http://www.helmholtz-muenchen.de/fileadmin/ISF/PDF/Haematopoese/publications/TTT_Operating_Instructions_July_2009_.pdf</a>).</li>
	<li>Start the TTT program, select user initials, and click "Continue."</li>
	<li>Press the "Set NAS," select the user created "TTTexport" folder, and click Ok. In the browser, select an experiment folder, select a position folder, and then press "Load position" button.</li>
	<li>Set ocular factor to "10x." Load the number of images required (loading all images is recommended for first time).</li>
	<li>In cell editor window, select "New colony" under the File menu. In the Movie window, press the "Track cell" or F2 to begin tracking a cell.</li>
	<li>Identify a cell in the Movie window and use mouse to place cursor over the cell. A circle with the number of the cell should appear after F2 has been clicked. Use the "0" key on the number pad of the keyboard to track the placement of the cell and advance to the next picture. Move the cursor to follow the placement of each cell through each frame. To delete a track and return to the previous image press "Del" on the number pad. To move forward frames without tracking the cell press "3," and to move backward without tracking press "1" on the number pad.</li>
	<li>To mark a cell division click the "Division" button in the Movie window. To mark cell death click the "Cell death" button in the Movie window. Once a division has occurred, the daughter cells can be tracked in the same cell fate map by right clicking on the circle symbol of the designated daughter cell in the Cell editor window to start tracking mode automatically.</li>
	<li>Press F10 to save the tree. Each tree will save in the designated output folder (TTTExport). To start a new cell fate map, select "New colony" under the "File" then "Open" menu in the Cell editor window.</li>
	<li>Tabulate cell lifetime data after gathering from "Cell data" tab in the Cell editor window.</li>
</ol>
7. Statistical Analyses
<ol>
	<li>Use either Student's t-test (if comparing two genotypes) or ANOVA (if comparing &gt; two genotypes) to determine if differences in parametric data such as numbers of initial cell colonies, cell lifetimes or hours until the appearance of EMT between different genotypes are statistically significant. Apoptotic frequency can be compared as the number/total number plated cells using Student's t-test or ANOVA or with Mann-Whitney or Kruskal-Wallis when derived as a percentage of adherent cells.</li>
	<li>Perform power tests using data from analyses of initial experiments to determine how many experimental replicates need to performed and cell fate maps generated from each replicate for adequate statistical power using available freeware (e.g. <a href="http://statpages.org/" target="_blank">http://statpages.org/</a>). In the experiments presented here, cell cultures derived from three mice per genotype were sufficient to determine if there were statistically significant differences in cell colony formation and cell lifetimes when specific genetic modifications were made.</li>
</ol>

<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 Views.  Georgetown University. 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.

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

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). 
The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. 
The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.