This work is supported by the Joseph Alexander Foundation, the ASBMB Undergraduate Research Award, 2013-2014, and a Science Award Grant, Marymount Manhattan College, 2012-2013.

Notes: Conduct all work with cells and cell culture reagents in a Class II biosafety cabinet (BSC)9. MMT cells are classified as Biosafety Level I, as they pose low to moderate biological risk. Apply proper cleaning and decontamination procedures to the BSC between uses (e.g., ultraviolet light, 70% ethanol wipe down).
1. Grow MMT cells
<ol>
	<li>Grow cells in 10 cm tissue culture dishes containing 10 ml of nutrient rich media that consists of Eagles Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 1% Antimycotic/Antibiotic (10 units penicillin, 100 &#181;g streptomycin, 0.25 &#181;g amphotericin B). Cultivate cells in a humidified 5% CO2 chamber, at 37 &#176;C. Plate cells at a density of 3.6 x 106 cells/cm2 (see Counting Cells in step 2).</li>
	<li>Replace half of the cell culture media with fresh media every 48 hrs unless otherwise indicated. Check cell viability and morphology by examination with an inverted phase contrast light microscope.
	<ol>
		<li>Note and characterize cells for size, structure, shape, organization and estimated number under at 100 - 200X magnification. At this density, the cells should appear in one plane, not clumped atop each other and in close proximity. Each cell consists of a thick, spherical core with thin, long, branch-like extensions expanding from it that come to a point (see Figure 1).</li>
	</ol>
	</li>
	<li>Subdivide cells when they reach a cell density of 7.2 x 106 cells/cm2. Cells exhibit a doubling rate of 1.8 x 106 cells/cm2 per 24 hrs. Designate cells Passage X (Px) to denote the number of times they have been split.
	<ol>
		<li>At generation 3 (i.e., after three passages, P3), harvest, count, and plate the cells onto a 24 well tissue culture plate at a density of 3.6 x 106 cells/cm2.</li>
	</ol>
	</li>
	<li>View cells every day and record digital photomicrographs. Note and describe cell morphology (i.e., size, shape, light reflective properties). Determine cell doubling time by counting cells regularly and relating elapsed time to cell number.</li>
</ol>
2. Count MMT Cells
Note: Count cells to determine if cells need to be subcultured, to set up for an experiment or to determine cell viability. There are two methods presented here.
<ol>
	<li>Use of a hemocytometer, a standard technique to count cells.
	<ol>
		<li>Obtain a bright-line hemocytometer, a 0.2% solution of trypan blue dye, a compound light microscope, sterile test tubes, pipettes and a manual cell counter.</li>
		<li>Under the BSC, use a 10 ml pipette to remove cells from a 10 cm tissue culture dish. If cells are grown on a 24 well plate use a 1 ml pipette or 1,000 &#181;l micropipette to remove the media.</li>
		<li>Draw cell media into the pipette, place the tip of the pipette against the bottom of the dish and expel the media while sliding the pipette tip across the plate. Repeat 4 - 5 times to help dislodge cells, dissociate clumps and lead to a more accurate cell count.</li>
		<li>Place the resulting cell suspension in a 15 ml sterile tube (or 1 ml micro centrifuge or other small volume tube). Otherwise, leave the cells in the original tissue culture plate/well.</li>
		<li>Under the BSC, combine 10 &#181;l of the cell suspension with 10 &#181;l of the trypan blue solution (1:1 ratio) in a non-sterile micro centrifuge or other small volume tube. 
		Note: Trypan blue is a vital stain that is not absorbed by healthy viable cells. When cells are damaged or dead, trypan blue can enter the cell allowing dead cells to be counted (aka dye exclusion method). Therefore under a microscope, dead cells appear dark purple while viable cells are a bright and light in color.</li>
		<li>Incubate at RT for 5 min.</li>
		<li>Place the cover slip on the hemocytometer. Apply 10 &#181;l of the trypan blue-cell suspension mixture into the groove. View the hemocytometer at 100 - 200X magnification. A four-corner grid is apparent (see Figure 2).</li>
		<li>Count the number of viable cells (unstained) versus total cells in each gridded area. Average the four grids and multiply by 1 x 104 to obtain cells/ml.
		<ol>
			<li>Extrapolate the total number of cells per dish (or cells/cm2). Use this number to either determine the correct volume of the cell suspension to use to seed a new tissue culture plate at the desired density of 3.6 x 106 cells/cm2&#160;or determine total number of viable cells on the plate or in the well. 
			Note: For additional guidance on the use of a hemocytometer, see10.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Use software and a digital camera to count viable cells.
	<ol>
		<li>Obtain a digital camera, its accompanying software, a 0.4% solution of trypan blue dye, a compound light microscope, sterile test tubes, pipettes and a computer. 
		Note: A Moticam 2,000 digital camera with accompanying Motic Software is employed here. Any comparable camera and software should be sufficient. Ensure that the digital camera software has been calibrated in advance (according to manufacturer&#8217;s protocol).</li>
		<li>Under the BSC, use a 1 ml pipet or 1,000 &#181;l micropipette to remove the media from the MMT cells growing in a 24 well tissue culture dish. Wash the adherent cells by gently applying a small amount (approximately 500 &#181;l) of un-supplemented (i.e., EMEM without any supplements) fresh media to the plate then removing it. Apply 10 &#181;l of 0.2% trypan blue (made by diluting 1:1 with un-supplemented EMEM) directly to the well.</li>
		<li>Incubate the plate at RT for 5 min.</li>
		<li>View the plate under the microscope at 100 - 200X magnification with a digital camera attached.&#160;Plate may be washed with unsupplemented media if staining with 2% trypan blue is too dark.</li>
		<li>Open the software on the computer and verify that the software is calibrated to the objective used via the settings tab. An image of the FOV should appear.</li>
		<li>Select the Grid from the Measure tab. A grid of squares approximately 0.005 cm in width will appear. Select Grid Info to confirm the area of each square.</li>
		<li>Choose a specific area of the grid to count cells (i.e., 5 squares x 9 squares). Select Rectangle from the Measure tab. Click the corner of a square and drag your cursor to encompass the squares of choice. 
		Note: A shade of green will cover the squares of choice and a white box will appear in the corner that provides the width, height, area, and perimeter of the section of the grid chosen (see Figure 3).</li>
		<li>Count the number of viable cells within the determined area. Do the same for two other locations in the same well or plate by moving the plate under the microscope.</li>
		<li>Be sure that the measured area is the same and the magnification has not changed. Calculate the average number of viable cells within the area. Using the area of the well (for a 24 well plate, one well has an area of 2 cm2, for a 100 mM dish the area is 78.6 cm2), extrapolate the number of viable cells from the area delineated in the grid to the total number of cells within the well (cells/cm2).</li>
	</ol>
	</li>
</ol>
3. Treat MMT Cells with anti-proliferative Agents
<ol>
	<li>Prepare solutions of the selected anti-proliferative therapeutic agents (tamoxifen,&#160;curcumin and metformin) and optional drug, aspirin under the BSC.
	<ol>
		<li>Dissolve curcumin and tamoxifen in 100% ethanol to generate a stock concentration of 27 mM. Dissolve metformin and aspirin in unsupplemented EMEM to generate a stock concentration of 500 mM and 15 mM, respectively.</li>
	</ol>
	</li>
	<li>Establish a Dose Response.
	<ol>
		<li>Treat MMT cells with the three anti-proliferative therapeutic agents (tamoxifen, curcumin and metformin) and optional drug (aspirin) at varying concentrations for 96 hrs to generate a dose response curve. Initially administer all drugs at a range of concentrations based on published reports1,11-16 and then at concentrations larger or smaller than those published. 
		Note: A dose response determines the minimum concentration of a drug necessary to produce the desired results. Here the desired result is a reduction in cell proliferation as compared to the control.
		<ol>
			<li>For tamoxifen and curcumin, use concentrations (and corresponding volumes) of 0.054 mM (1 &#181;l), 0.108 mM (2 &#181;l), 0.162 mM (3 &#181;l) and 0.216 mM (4 &#181;l).</li>
			<li>For metformin, use concentrations (and corresponding volumes) of 2 mM (2 &#181;l), 4 mM (4 &#181;l), 6 mM (6 &#181;l), 8 mM (8 &#181;l) and 10 mM (10 &#181;l).</li>
			<li>For aspirin, use concentrations (and corresponding volumes) of 0.030 mM (1 &#181;l), 0.060 mM (2 &#181;l), 0.099 mM (3.3 &#181;l), 0.150 mM (5 &#181;l), and 0.216 mM (6.7 &#181;l).</li>
		</ol>
		</li>
		<li>Split MMT cells from the 10 cm dish onto a 24 well plate at a concentration of 3.6 x 106 cells/cm2. Determine initial cell concentration by both cell-counting methods (Step 2). Call this new 24 well plate of cells &#8220;Day Split&#8221;. </li>
		<li>24 hrs after cell plating, treat the MMT cells with each of the anti-proliferative therapeutic agents, tamoxifen, curcumin, metformin and aspirin, at the concentrations described in Step 3.2.1. Refer to this as &#8220;Day 0&#8221;.
		<ol>
			<li>As each well can hold a maximum of 500 &#181;l of media, use micropipettes with sterile micropipette tips and a new tip each time a new well is treated to avoid cross contamination. Use two wells as controls: cells grown in the absence of any drug treatment (negative control) and cells grown in the presence 100% ethanol (solvent control).</li>
		</ol>
		</li>
		<li>Grow cells and re-administer drugs at the described concentrations when the cells are fed every other day (see Step 1.2) for 96 hrs (until Day 4). On Days 1 - 4 of treatment, observe the cells under the microscope and count using the method in Step 2.2 (see Figure 4).</li>
		<li>Repeat the experiment at least three times.</li>
		<li>Determine optimal concentration of each drug by graphing the relationship between cell viability and drug dosage over the length of the experiment (see Figure 5).</li>
	</ol>
	</li>
	<li>Establish a Time Course.
	<ol>
		<li>Treat MMT cells with the three anti-proliferative therapeutic agents (tamoxifen, curcumin and metformin) at a fixed concentration for varying time periods. Use the optimal concentration identified through the Dose Response experiments (see Step 3.2).
		<ol>
			<li>Use the following concentrations: 0.216 mM tamoxifen, 0.216 mM curcumin and 10 mM metformin. 
			Note: A time course determines the amount of time necessary for a drug to produce its optimal desired result. Here, the desired result is a reduction in cell proliferation as compared to the control.</li>
		</ol>
		</li>
		<li>Split MMT cells from the 10 cm dish onto a 24 well plate at a concentration of 3.6 x 106 cells/cm2. Determine initial cell concentration by both cell-counting methods (Step 2). Call this new 24 well plate of cells &#8220;Day Split&#8221;. </li>
		<li>24&#160;hrs after cell plating, treat the MMT cells with each of the anti-proliferative therapeutic agents, tamoxifen, curcumin, metformin and aspirin, at the optimal concentrations identified in Step 3.2. Refer to this as &#8220;Day 0&#8221;.
		<ol>
			<li>As each well can hold a maximum of 500 &#181;l of media, use micropipettes with sterile micropipette tips and a new tip each time a new well is treated to avoid cross contamination.</li>
			<li>Use two wells as controls: cells grown in the absence of any drug treatment (negative control) and cells grown in the presence 100% ethanol (solvent control).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Grow cells and re-administer drugs at the selected concentration when the cells are fed every other day (see Step 1.2) for 96 hrs (until Day 4). On Days 1 - 4 of treatment, observe the cells under the microscope and count using the method in Step 2.2 (see Figure 4).</li>
	<li>Repeat the experiment at least three times.</li>
	<li>Determine optimal exposure time of MMT cells to each drug by graphing the relationship between cell viability and length (time) of drug treatment at the selected concentration (see Figure 6).</li>
</ol>
4. The Lab Module
Note: The following is a 5-day lab schedule for the lab module. Figure 7 is a flow chart of what the schedule would entail within the 5 days. For this activity to be completed in five days either a time course or a dose response curve is generated. There is not sufficient time to generate both curves. A dose response experiment is described below. A time course experiment can be easily interchanged
<ol>
	<li>Split the class into groups: have one group establish a dose response and the other a time course choosing a concentration in the middle of the proposed dose response concentrations.
	<ol>
		<li>Maintain sufficient cultures and drug stocks at appropriate concentrations. Unless otherwise noted, perform all work under the BSC.</li>
	</ol>
	</li>
	<li>Day 1
	<ol>
		<li>Direct students to make media and grow cells (as in step 1).</li>
		<li>As students obtain a stock of MMT cells on a 10 cm plate, direct the students to prepare cell culture media and give a tutorial in the use of the hemocytometer, digital microscopy and the digital camera microscopy software (as in step 2).</li>
		<li>Calibrate the software to the objectives used on the microscope (e.g., 4X, 10X, 20X) now using manufacturer&#8217;s protocol.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Have students confirm total cell number and cell viability (as in step 2).</li>
		<li>Have students count cells on the 100 mm dish using both the microscopy software and the hemocytometer methods, to confirm similar results are obtained.</li>
		<li>Direct students to obtain a 24 well plate and split cells from the 100 mm dish to the 24 well plates to the desired starting cell plating density (as in step 1). This is considered Day Split. Place the 24 well plate in a cell culture incubator for 24 hrs.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Grow MMT cells for 24 hrs on a 24 well plate. Have students observe cells under microscope and count using microscopy software (as in step 2).</li>
		<li>Give the student/team samples of cancer treatment drug stocks. Have them prepare and dilute the original stock solution (as in step 3). Add the various concentrations of the drugs to their cells to establish a dose response curve. This is called Day 0.</li>
		<li>Incubate cells (step 1.2) with the drugs for maximum of 48 hrs.</li>
	</ol>
	</li>
	<li>Day 4
	<ol>
		<li>Have students observe treated cells after 24 hrs. This is Day 1.</li>
		<li>Count each well using microscopy software. Record photographs of each well so that counting can be conducted outside the lab (as in step 2).</li>
		<li>Incubate cells (step 1.2) with the drugs for another 24 hrs. 
		Note: Instructor provides additional tutorials and reviews of data analysis and graphical presentation. Students learn to create figures, plots, and statistical analysis for the following day of data collection.</li>
	</ol>
	</li>
	<li>Day 5
	<ol>
		<li>Have students treated cells after 48 hrs, Day 2.</li>
		<li>Count each well using microscopy software. Record photographs of each well so that counting can be conducted outside the lab (as in step 2).</li>
		<li>Harvest and collect cells for counting by the traditional hemocytometer method as a means of verifying student ability to effectively use the microscopy software method (as in step 2). 
		Note: Collect data and draw conclusions or revise hypotheses, accordingly.</li>
	</ol>
	</li>
</ol>

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The authors received no financial or comparable support from Motic.

A lab module is presented that aims to teach a variety of topics in cell biology through the advanced techniques of animal cell culture. The module achieves this by analyzing the effects of a number of anti-proliferative chemicals on the replication of cells that model human breast cancer. The primary assay relies on the fundamental technique of cell counting and introduces a novel way to count cells using microscopy software. The activities comprising the module can be conducted with instruments and equipment available ...

Often in undergraduate general biology courses, the topics of cell cycle regulation and cancer are touched upon but not explored in detail because the breadth of content in these courses leaves little time for depth. In addition, undergraduate biology students are not typically exposed to the advanced techniques associated with animal cell culture. To help students develop a deeper understanding of these concepts, while applying and analyzing what they have learned, a laboratory activity was developed as a modification of the Walter Reed Army Institute of Research (WRAIR) extended laboratory activity1. The lab module uses a step-wise, experimental strategy that includes growing and characterizing a cancer cell model, developing and executing cell counting methods, establishing optimal time course and dosages for treating cells with anti-proliferative agents, and identifying aberrant cell-signaling pathways. The experiment also allows for open-ended inquiry.
Most of the techniques required for this activity can be accomplished in a typical biology-teaching laboratory. The activity starts with students characterizing the morphology and growth rate of the mouse mammary tumor (MMT) cell line, a model for human breast cancer2&#160;.&#160;Breast cancer was chosen as the model cancer because of its prevalence in the population, its familiarity to college-aged students, and the widespread data available. The MMT cell line was specifically selected because it is easily obtainable, well characterized, has a short doubling time and is easy to grow. In addition, MMT cells are estrogen-dependent which is consistent with most female breast cancers. Students then identify aberrant cell-signaling pathways in the MMT cells by treating the cells with chemotherapy drugs whose mechanism of action is well established.The concentration of the drugs and length of the treatments are varied allowing students to evaluate the effect of these variables on the rate of cell division. The key assay for this activity is the determination of cell viability, which simply requires cell counting, using one of two methods. Each method depends on strong microscopy skills. Students determine cell viability by using a standard, hemocytometer method and a novel photomicroscopy method and propose. Based on their findings, they can propose and test modifications to the activity. Students then represent their data and interpret the results to refine their hypothesis and devise new experimental strategies.
This laboratory activity is suited for freshman or sophomore level students majoring in the biological sciences. It is condensed into a one-week lab module that can be completed in a first year, general biology or second year, cellular/molecular biology course. Skills needed for proper completion of the activity include basic arithmetic and algebra, familiarity with an array of core laboratory skills (e.g., pipetting, solution making, sterile technique), data analysis, basic light microscopy and time management, along with instructor knowledge of cell culture and spreadsheet software. Reagents required include an animal cell line model for cancer (e.g., mouse mammary tumor cells, MMT2), chemotherapy agents (e.g., tamoxifen, curcumin, metformin, and aspirin), trypan blue and cell culture media (e.g., Eagle&#39;s Minimum Essential Medium; EMEM) with appropriate supplements (e.g., donor horse and fetal bovine serum). Instruments needed include an inverted light microscope with digital camera attachment, computer, 100 mm and 24&#160;well tissue culture plates, CO2 incubator (or equivalent), biosafety cabinet (BSC; Class II), hemocytometer, and digital microscopy software.
There are good examples of specific lab activities that rely on animal cell culture to teach undergraduate students about concepts in cell biology3. However many require supplies or techniques that are not easily accessible (e.g., radioactive isotopes, live animal tissue, advanced imaging equipment1,4,5), describe protocols that are quite advanced (e.g., suitable for a 400 level course6), or require multi-week or semester long projects6,7. The lab activity described here is straightforward and can be conducted in a single week with common lab equipment.
In summary, this lab module effectively introduces or reinforces the concepts of cell cycle, cellular signaling pathways and cancer while teaching basic and advanced lab skills, experimental data analysis, the method of animal cell culture and the scientific process. The laboratory module is simple and economically accessible and provides both flexibility and opportunity for open-ended inquiry. The activity encourages student creativity by providing a template experimental strategy that acts as a guide but not a recipe. Most importantly, the activity satisfies all learning domains of Blooms Taxonomy8 as it requires remembering, understanding, applying, analyzing, evaluating and creating by engaging students in a process that pulls them out of the textbook and into the world of scientific research.

<table><tbody><tr><td>Tissue Culture Hood</td><td>ESCO Labculture Reliant</td><td></td><td>Class II Type A2 Biological Safety Cabinet</td></tr><tr><td>Waterjactor CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>CEDCO</td><td>Model 1510</td><td></td></tr><tr><td>Bright-line Hemocytometer</td><td>American Optical</td><td></td><td>with two separate grids</td></tr><tr><td>Motic Images Plus</td><td></td><td></td><td>Mac OSX Verison 2.0 or higher</td></tr><tr><td>Gilson Pipetman</td><td>Rainin instrument co. inc</td><td></td><td>P-20D, P-200D, P-1000D</td></tr><tr><td>CK30/CK40 Culture Microscope</td><td>Olympus</td><td></td><td>4 objective inverted light microscope with camera</td></tr><tr><td>200 &amp;#956;l Pipet tips</td><td>MidSci</td><td>40200C</td><td></td></tr><tr><td>1,000 &amp;#956;l Pipet tips</td><td>MidSci</td><td>AVR4</td><td></td></tr><tr><td>10 ml Seriological Pipets</td><td>TPP</td><td>TP94010</td><td></td></tr><tr><td>24 well plates</td><td>CoStar- Tissue Culture Cluster</td><td>3524</td><td>24 wells, 16 mm well diameter, radiation sterilized</td></tr><tr><td>Trypan Blue Solution 0.4%</td><td>Sigma</td><td>T8154</td><td>100 ml, cell culture tested non-haz</td></tr><tr><td>Bright-line Hemacytometer replacement coverslip, non-haz</td><td>Sigma</td><td>Z375357</td><td></td></tr><tr><td>Mouse Mammary Tumor(MMT) cells</td><td>ATCC</td><td>CCL-51</td><td></td></tr><tr><td>Eagle Minimum Essentail Medium (EMEM)</td><td>ATCC</td><td>30-2003</td><td>500 ml</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F0926</td><td>500 ml</td></tr><tr><td>Metformin Hydrochloride</td><td>Sigma</td><td>PHR1084</td><td>500 mg</td></tr><tr><td>Tamoxifen</td><td>Sigma</td><td>T5648</td><td>white or white-yellow powder</td></tr><tr><td>Curmumin</td><td>Sigma</td><td>C1386</td><td>yellow-orange powder</td></tr><tr><td>Aspirin</td><td>Sigma</td><td>A2093</td><td>meets USP testing specifications</td></tr></tbody></table>

using mouse mammary tumor cells to teach core biology concepts: a simple lab module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

Growing MMT cells and comparing counting methods.
Mouse mammary tumor cells were successfully grown and characterized (Figure 1) and a novel cell counting method developed using Motic Software, a digital camera-associated software program for a microscope. This new cell counting method was compared to a traditional counting method employing a hemocytometer (Figure 2) and was shown to be equally accurate in determining cell number (Table 1</str...

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Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

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13.0K Views.  Marymount Manhattan College. A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Video: Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

Cancer Research

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

Genetics

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 ...

Medicine

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.

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 ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

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 ...

Guided Tumor Inoculation and Multimodal Imaging in Mice

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Author Spotlight: Integrating High-Resolution Intravital Imaging and MRI to Enhance Stereotactic Body Radiation Therapy Planning

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

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Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

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

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

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

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

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis