Name: in vitro evaluation of oncogenic transformation in human mammary epithelial cells
Uploaded: 2020-09-24T14:30:00.000+00:00
Duration: 9 min 44 s
Description: Watch this Scientific Journal Video about In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells at JoVE.com

The AG laboratory is funded by the Spanish Nuclear Safety Council. T.A. and A.G. are members of a research group recognized by Generalitat de Catalunya (2017-SGR-503). MT holds a contract funded by the Scientific Foundation Asociaci&#243;n Espa&#241;ola Contra el C&#225;ncer [AECC-INVES19022TERR]. G.F. contract is funded by a grant from Cellex Foundation.

Human samples used in the following experiments were obtained from reduction mammoplasties carried out at Cl&#237;nica Pilar Sant Jordi (Barcelona) under standard procedure consent. All procedures are performed in a Class II Biological Safety Cabinet unless otherwise stated.
1. In vitro culture of human mammary epithelial cells and growth curve plot build-up
<ol>
	<li>In vitro culture of breast primary epithelial cells (BPECs): cell passaging 
	NOTE: For BPEC derivation and cell culture follow instructions described by Ince et al., 20076.
	<ol>
		<li>Medium preparation.
		<ol>
			<li>Supplement WIT basal defined medium with P or T supplements, provided by the manufacturer, depending on whether primary or transformed BPECs are cultured.</li>
			<li>Add cholera toxin to the supplemented WIT medium to a final concentration of 100 ng/mL for primary or 25 ng/mL for transformed BPECs. 
			CAUTION: Cholera toxin is fatal if swallowed. Use personal protective equipment. Avoid its release to the environment.</li>
		</ol>
		</li>
		<li>Cell culture maintenance and passaging. 
		NOTE: For the following steps keep in mind that cells are growing in a T25 flask. Nonetheless, volumes can be adapted to other cell culture formats maintaining the proportionality in terms of surface area.
		<ol>
			<li>Check on the cell confluency every day. When the culture is 90% confluent, perform cell passaging.</li>
			<li>Acquire 1x PBS, 3x trypsin, medium and a 15 mL conical tube containing 2 mL of Fetal Bovine Serum (FBS) for each flask.</li>
			<li>Remove the medium from the flask and keep it in the 15 mL conical tube containing FBS.</li>
			<li>Rinse cells with 1x PBS.</li>
			<li>Detach cells from the surface by adding 1 mL of 3x trypsin. Incubate for 5 min at 37 &#730;C.</li>
			<li>Check if cells have been detached. Apply vigorous shaking if cells are not completely detached.</li>
			<li>Inactivate trypsin by adding the reserved medium supplemented with FBS.</li>
			<li>Harvest the cellular suspension and place it in the 15 mL conical tube.</li>
			<li>Centrifuge at 500 x g&#160;for 5 min, eliminate the supernatant, and resuspend the pelleted cells by flicking the bottom of the tube with a finger.</li>
			<li>Add 1&#8211;2 mL of fresh media to the pellet and measure the cell concentration using an automatic cell counter or a hemocytometer. This data will later be used to calculate the population doublings and draw the growth curve. Seed 12,000 cells/cm2 (e.g., 300,000 cells for a T25 flask) in modified cell culture surface flasks (see Table of Materials). 
			NOTE: Dilute the cell suspension solution if the concentration is too high to ensure proper quantification.</li>
			<li>Add medium to a final volume of 5 mL and incubate the cells at 37 &#730;C and 5% CO2 atmosphere.</li>
			<li>Replace the cell culture medium every 48 h.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Population doubling calculation and data visualization
	<ol>
		<li>Using data of the cell count obtained in the step 1.1.2.10, apply the following formula to obtain the accumulated population doubling (PD) values: 
		<img alt="Equation 1" src="/files/ftp_upload/61716/61716equ01.jpg" /> 
		Where, PDi denotes the number of population doublings achieved by the cells until the previous subculture (it refers to the PD accumulated on the previous subculture), Nh is the number of harvested cells, and Ns is the number of seeded cells.</li>
		<li>Represent data for a specific interval of time using an XY graph where the number of days in culture (x-axis) and the accumulated PD (y-axis) are represented.</li>
		<li>Get the best-fit line and the fitting equation: 
		<img alt="Equation 2" src="/files/ftp_upload/61716/61716equ02.jpg" /> 
		NOTE: An increased slope (b) means an increased proliferation rate.</li>
	</ol>
	</li>
</ol>
2. Three-dimensional (3D) culture in basement membrane matrix and immunofluorescent protein detection
<ol>
	<li>3D culture in basement membrane matrix 
	NOTE: This protocol has been adapted from Debnath et al., 200310 and is optimized for 24 well plates (see Table of Materials).
	<ol>
		<li>Prepare the material a day before the experiment: pre-chill basement membrane matrix overnight at 4 &#730;C and let pipette tips, microcentrifuge tubes, and well plates cool in the freezer. 
		NOTE: The matrix must be kept at -20 &#730;C for long-term storage. Make aliquots to avoid multiple freeze-thaw cycles.</li>
		<li>On the day of the experiment, place pre-cooled material on ice.</li>
		<li>Rinse wells with cold sterile 1x PBS in order to reduce surface tension.</li>
		<li>Cover the bottom of each well with 100 &#181;L of basement membrane matrix. 
		NOTE: Dispense the matrix slowly and spread it throughout the well; it is crucial to avoid bubble formation in the bottom layer to avert monolayer cell culture growth.</li>
		<li>Place the plate in the incubator, at 37 &#730;C, to let the matrix layer solidify. 
		NOTE: It usually takes about 20 min to solidify.</li>
		<li>Meanwhile, trypsinize cells as explained previously in step 1.1. Centrifuge the cells at 500 x g for 5 min and resuspend in medium. Prepare a 400,000 cells/mL suspension and gently disaggregate any cell clump by pipetting.</li>
		<li>Prepare the medium with 8% basement membrane matrix and mix 1:1 (v/v) with cellular suspension to obtain a 200,000 cells/mL solution in 4% matrix. 
		NOTE: Calculate the amount of medium needed to avoid matrix unnecessary waste.</li>
		<li>Place 500 &#181;L of cell suspension in matrix solution on top of the already solidified matrix layer to seed a total amount of 100,000 cells in medium with 4% basement membrane matrix.</li>
		<li>Incubate cells at 37 &#730;C for a few minutes and then, add 500 &#181;L of the medium with 4% basement membrane matrix. Incubate the cells at 37 &#730;C in an incubator with 5% CO2 for 14 days. Seeded cells will group and proliferate to originate the acini-like structures. 
		NOTE: Cell motility and aggregation can be monitored by time-lapse during the 3D formation process. Use image analysis software (e.g., Fiji/ImageJ or Imaris) to evaluate these events. The number and size of the acini depend on the aggregation process and the proliferation rate and might vary between cell types. Adjust basement membrane matrix concentration and seeded cells to obtain desired 3D structures.</li>
		<li>Add 500 &#181;L of the medium with 4% basement membrane matrix 2&#8211;3 times per week. 
		NOTE: Avoid disturbance of the layers carrying the plate gently during manipulation.</li>
		<li>If desired, the number and size of acini can be measured during the culture period. To do so, take random pictures at different times after seeding using a phase contrast or DIC inverted microscope. Use image analysis software to measure the diameter of 100&#8211;200 3D structures.</li>
	</ol>
	</li>
	<li>Immunostaining 
	NOTE: Sterile conditions are not required during this part of the protocol.
	<ol>
		<li>Remove the culture medium.</li>
		<li>Tear the basement membrane matrix using a p200 pipette tip with the end cut off. Place ~50 &#181;L of disaggregated matrix on top of a glass slide and smear it in an area of 1&#8211;2 cm2.</li>
		<li>Let the sample dry completely at room temperature or use a heating plate at 37 &#730;C to accelerate the process. Fix samples with methanol:acetone (1:1, v/v) at -20 &#730;C for 30 min. 
		NOTE: Fluorescent signal of previous markers, such as that of fluorescent proteins expressed by the cells, will be erased. 
		CAUTION: Methanol is flammable, toxic if inhaled, swallowed or in case it comes in contact with skin. Wear personal protective equipment and work inside a fume hood.</li>
		<li>Discard the fixation solution and remove the excess, if any, by reclining the slide on filter paper. 
		NOTE: The protocol can be paused here. Once dry, slides can be stored at -20 &#730;C for several months.</li>
		<li>Block samples epitopes with 5% normal goat serum and 0.1% triton-X-100 in 1x PBS (blocking solution) for 2 h at room temperature.</li>
		<li>Meanwhile, prepare antibodies working solutions by diluting primary or secondary antibodies at the desired concentration in blocking solution. 
		NOTE: Antibody concentration must be accurately adjusted depending on the cell type and the antibody reference. As a guide, to identify cells from luminal and myoepithelial lineages in BPEC, primary anti-Cytokeratin 14 and anti-Claudin-IV antibodies (see Table of Materials) can be used. The recommended working solution concentration is 1:100 for these primary antibodies and 1:500 for anti-Mouse and anti-Rabbit secondary antibodies (see Table of Materials).</li>
		<li>Add 30 &#181;L of primary antibodies working solution and cover it with a strip of laboratory wrapping film to avoid evaporation. Incubate overnight at 4 &#730;C in a humid chamber.</li>
		<li>Wash three times with 1x PBS for 1 h each.</li>
		<li>Repeat step 2.2.7 for secondary antibodies. Incubation should be performed in darkness.</li>
		<li>Wash with 1x PBS for 2 h. 
		NOTE: Adjust antibodies concentration, incubation time, and washing hardness to improve the signal/noise ratio for specific samples.</li>
		<li>Remove the remaining PBS and, once dry, counterstain with DAPI at 0.25 &#181;g/mL diluted in antifade mounting medium. Cover slides with a coverslip by letting it settle without applying pressure. Seal with nail polish. 
		NOTE: Samples can be stored at 4 &#730;C for several weeks. For long-term storage, keep them at -20 &#730;C.</li>
		<li>Analyze fluorescent signal distribution for each acinus using a confocal microscope. 
		NOTE: Confocal microscope configuration must be accurately determined depending on the equipment used and the antibodies applied to the sample. As a guide, with the equipment and reagents&#160;detailed in the Table of Materials, use a 40x objective and the following laser and detector settings: for DAPI use excitation with a 405 laser (3%&#8211;5%), detection with a PMT detector (800V, Offset: -9) and a spectral band from 410 nm to 500 nm; for A488 (Claudin-IV) use excitation with a 488 laser (7%&#8211;10%), detection with a PMT detector (800V, Offset: -20) and a spectral band from 490 nm to 550 nm; and for Cy3 (Cytokeratin 14) use excitation with a 555 laser (2%&#8211;10%), detection with a PMT detector (800V, Offset: -35) and a spectral band from 560 nm to 600 nm.</li>
	</ol>
	</li>
</ol>
3. Anchorage-independent assay, MTT staining and automatic colony quantification
<ol>
	<li>Anchorage-independent assay: agar and cellular suspension plating 
	NOTE: Protocol has been adapted from Borowicz et al., 201411 to perform experiments in BPECs.
	<ol>
		<li>Prepare a 1.2% agar solution diluted in ultrapure water in a sterile bottle. Autoclave the solution and maintain it at 42 &#730;C during the experiment. The agar solution can be stored at 4 &#730;C; when required, heat the agar solution until it is liquid again. 
		CAUTION: Use heat-resistant gloves to avoid burn after autoclave. 
		NOTE: From now on, sterile conditions must be maintained.</li>
		<li>Prepare a 0.6% agar solution by mixing 1:1 (v/v) complete pre-warmed medium with 1.2% agar solution. Maintain at 42 &#730;C to avoid premature solidification. 
		NOTE: Medium can be previously double supplemented to obtain a fully supplemented 0.6% agar + medium solution once mixed.</li>
		<li>Cover the bottom of a 35 mm well with 1.5 mL of 0.6% agar in medium solution and let it solidify at room temperature. Make sure that the bottom of the plate is completely covered before agar solidification, otherwise, cells may adhere to the plate and grow in monolayer. 
		NOTE: Adherent and non-adherent surface plates can be used.</li>
		<li>Meanwhile, trypsinize cells and, once centrifuged and resuspended in medium, prepare a 50,000 cells/ml solution and gently disaggregate any cell clump by pipetting repeatedly.</li>
		<li>Prepare a 0.3% agar + cell suspension in the medium at a final concentration of 25,000 cells/mL. 
		NOTE: Optimal cell concentration may differ among cell types. Try different concentrations until individualized colonies are formed.
		<ol>
			<li>Place a 40 &#181;m strainer filter on top of a 50 mL sterile tube and filter the 50,000 cells/mL solution letting it drop into the bottom of the tube.</li>
			<li>Remove the filter from the 50 mL sterile tube, tilt the cell-containing tube to a&#160;45&#730; angle&#160;and drop the same volume of 0.6% agar + medium solution pouring it through the internal wall of the tube. This will allow the agar solution to cool down just enough to not damage the cells and avoid its premature solidification.</li>
		</ol>
		</li>
		<li>Homogenize the mixture and deposit 1 mL of 0.3% agar + cell suspension in the medium (containing 25,000 cells) on top of the previously solidified bottom agar layer.</li>
		<li>Visualize seeded cells using an inverted microscope to make sure that the cells are individualized. Otherwise, the experiment should be repeated.</li>
		<li>Wait until the agar layer is completely solidified, then carefully add 1 mL of the fresh medium on top without disturbing the delicate agar layers beneath.</li>
		<li>Incubate the cells at 37 &#730;C and 5% CO2 in an incubator for 3 weeks. 
		NOTE: The time required for colony formation can vary among different cell types, but usually 3 weeks are sufficient.</li>
		<li>Change medium twice per week. To do so, gently tilt the plate toward you, aspirate medium in the lower corner, and add 1 mL of fresh medium. 
		NOTE: Avoid touching the agar layers as they easily detach from the plate.</li>
	</ol>
	</li>
	<li>MTT staining
	<ol>
		<li>Prepare Thiazolyl Blue Tetrazolium Bromide (MTT) stock solution at 6 mg/mL in ultrapure water in a sterile bottle and filter solution using 0.2 &#181;m filters. This MTT solution can be stored for up to 6 months at -20 &#730;C. 
		CAUTION: MTT may cause irritation and is suspected of causing genetic defects. Use safety glasses, gloves, and a respiratory filter. 
		NOTE: Avoid repeated freeze-thaw cycles.</li>
		<li>Prepare a working solution of MTT at 1 mg/mL by diluting the stock solution with sterile ultrapure water.</li>
		<li>Once the colony formation period has concluded, remove the medium from the plate and add 1 mL of 1 mg/mL MTT to each well.</li>
		<li>Incubate for 24 h in the incubator. Remove the MTT solution by aspirating it gently. Plates can be stored at 4 &#730;C for several weeks. 
		NOTE: Avoid light exposure to prevent non-specific crystal formation.</li>
	</ol>
	</li>
	<li>Colony quantification
	<ol>
		<li>Obtain images of each plate using an inverted microscope. Adjust the magnification in order to acquire the maximum field of view with fewer images and still being able to detect small colonies (usually 4x or 10x objectives). 
		NOTE: Make sure that images present a homogeneous background. Neither phase-contrast nor differential interference contrast is required since non-stained colonies will not be quantified.</li>
		<li>Upload images to ImageJ/Fiji software12 to count the number of colonies and the area of each MTT-positive colony. 
		NOTE: A script for automatic quantification is provided as a Supplementary File. To execute the code, paste it to the macro editor (Plugins | New | Macro) and follow the instructions.
		<ol>
			<li>Obtain a binary mask through thresholding the original image (Image | Adjust | Threshold) to obtain well-delimited colonies (Figure 1). 
			NOTE: 8- or 16-bit images are usually required to perform this step. The &#8220;Minimum threshold&#8221; method is recommended.</li>
			<li>Run the Extended Particle Analyzer from Biovoxxel plugin13 (Plugins | BioVoxxel | Extended Particle Analyzer) to identify MTT positive colonies (Figure 2). Initial guiding conditions: Size (&#181;m2) = 250&#8211;Infinity; Solidity = 0.75&#8211;1.00.</li>
		</ol>
		</li>
		<li>Estimate the average diameter (D) from each colony area value (A) according to the formula: 
		<img alt="Equation 3" src="/files/ftp_upload/61716/61716equ03.jpg" /></li>
		<li>Filter results by excluding low proliferative colonies (e.g., low diameter).
		<ol>
			<li>Choose a minimum number of divisions per week (m) to be considered (e.g., 1).</li>
			<li>Estimate the radius of a colony (R) with n cells according to the following formula: 
			<img alt="Equation 4" src="/files/ftp_upload/61716/61716equ04.jpg" /> 
			Where, r is the average radius of individual cells in suspension, n is the number of cells forming a colony that suffered m divisions each week during w weeks in culture. In exponential growth: n = 2(m*w). &#961; is the Packaging efficiency. Note that, in random movement, packaging efficiency is ~0.64 and the densest possible packing fraction for identical spheres is 0.7414.</li>
			<li>Discard all colonies that present a diameter lower than 2R as their cells have not achieved the minimum number of divisions considered in step 3.3.4.1.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The experimental protocols described in this paper provide useful tools to assess the oncogenic transformation of in vitro cultured cells. Each technique evaluates specific aspects of the transformation process, and thus, special attention must be paid when drawing conclusions from a single analysis. Growth curves build-up is an approach that demands information already available when culturing cells for other purposes. That makes this technique cheaper and easier to apply compared to other cell proliferation assays. How...

Multiple successive events are required for neoplasm development. In 2011, Hanahan and Weinberg described 10 capabilities that enable transformed cells&#8217; growth, survival, and dissemination: the so-called &#8220;Hallmarks of Cancer&#8221;1. The methodology described here compiles three different tools to evaluate in vitro cellular transformation by focusing on some of the tumoral cells&#8217; distinctive features. These techniques assess the cell proliferation rate, the behavior of cells when cultured in 3D and their capacity to form colonies with anchorage independence.
Cell models are crucial to test hypothesis in vitro. Different approaches have been developed to generate experimental models of cellular transformation for the study of cancer2,3,4. Since breast cancer is the most common cancer among women worldwide and is responsible for approximately 15% of cancer deaths&#160;among women5, providing suitable cellular models of mammary epithelial cells is of utmost importance for further investigation. In this article, we have illustrated the potential of three techniques to evaluate cellular transformation using an experimental model of Breast Primary Epithelial Cells (BPECs) transformation initially described by Ince and colleagues in 20076 and later implemented in our laboratory7. This experimental model is based on the sequential alteration of three targeted genes (SV40 Large T and small t antigens herein referred to as Ttag, hTERT, and HRAS) to the genome of non-transformed BPECs. Moreover, the method used for BPECs derivation favors the maintenance of mammary epithelial cells with luminal or myoepithelial markers, resulting in a heterogeneous cell culture that retains some of the mammary gland physiological traits.
In the mammary gland, luminal mammary epithelial cells, which are responsible for milk production, are located near the lumen, whereas myoepithelial cells are disposed around luminal cells and take care of contraction movements leading the milk to the nipple. The loss of proper organization between these cell lineages is a feature of tumoral transformation8 that can be assessed in vitro after immunofluorescent detection of distinctive lineage markers in 3D cell cultures. Another major characteristic of tumoral cells is their capacity to grow without attachment to other cells and to the extracellular matrix1. When healthy cells are forced to grow in suspension, mechanisms such as anoikis &#8210; a type of cell death induced in response to detachment from the extracellular matrix &#8210; are activated9. The evasion of cell death is one of the distinctive hallmarks of cancer and thus, transformed cells are capable to inactivate anoikis and survive in an anchor-independent manner. This capacity can be evaluated in vitro with the anchorage-independent assay using soft agar. Furthermore, an inherent feature of tumoral tissues is their sustained proliferative signaling capacity, which can be easily monitored under in vitro conditions by measuring the increase of cell number along time, not only in suspension assays but also by monitoring the growth rate of monolayer adherent cultures.
Despite the best model to test tumorigenic potential is the inoculation of tumoral cells in murine models and evaluation of tumor development in situ, it is important to minimize the number of animals employed in experimental procedures as much as possible. Therefore, having suitable tests to assess transformation in vitro is a top priority. Here, we provide a set of tools to evaluate the tumorigenic potential of partially and fully transformed breast epithelial cells that can be easily implemented in most of the laboratories that work with cellular transformation models.

<table><tbody><tr><td>1 ml Serological Pipettes</td><td>Labclinics</td><td>PLC91001</td><td></td></tr><tr><td>1.5 ml Eppendorfs</td><td>Thermo Fisher Scientific</td><td>3451</td><td>Dark eppendorfs are preferred for MTT long-term storage</td></tr><tr><td>10 &amp;mu;l Pipette tips w/o filter</td><td>Biologix</td><td>20-0010</td><td></td></tr><tr><td>100 ml glass bottle</td><td></td><td></td><td>With cap, autoclavable</td></tr><tr><td>1000 &amp;mu;l Pipette tips w/ filter</td><td>Labclinics</td><td>LAB1000ULFNL</td><td></td></tr><tr><td>1000 &amp;mu;l Pipette tips w/o filter</td><td>Biologix</td><td>20-1000</td><td></td></tr><tr><td>15 ml Conical tubes</td><td>VWR</td><td>525-0400</td><td></td></tr><tr><td>2 ml Serological Pipettes</td><td>Labclinics</td><td>PLC91002</td><td></td></tr><tr><td>200 &amp;mu;l Pipette tips w/ filter</td><td>Labclinics</td><td>FTR200-96</td><td></td></tr><tr><td>5 ml Serological Pipettes</td><td>Labclinics</td><td>PLC91005</td><td></td></tr><tr><td>50 ml Conical Tubes</td><td>VWR</td><td>525-0304</td><td></td></tr><tr><td>Acetone</td><td>PanReac AppliChem</td><td>211007</td><td>Used for 3D structure fixation prior to immunofluorescent labelling</td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A1296</td><td>Used for anchorage assay</td></tr><tr><td>Anti-Claudin 4 antibody</td><td>Abcam</td><td>15104, RRID:AB_301650</td><td>Working dilution 1:100, host: rabbit</td></tr><tr><td>Anti-Cytokeratin 14 [RCK107] antibody</td><td>Abcam</td><td>9220, RRID:AB_307087</td><td>Working dilution 1:100, host: mouse</td></tr><tr><td>Anti-mouse Cyanine Cy3 antibody</td><td>Jackson ImmunoResearch Inc.</td><td>115-165-146, RRID:AB_2338690</td><td>Working dilution 1:500, host: goat</td></tr><tr><td>Anti-rabbit Alexa Fluor 488 antibody</td><td>Thermo Fisher Scientific</td><td>A-11034, RRID:AB_2576217</td><td>Working dilution 1:500, host: goat</td></tr><tr><td>Autoclave</td><td></td><td></td><td></td></tr><tr><td>BioVoxxel Toolbox</td><td></td><td>RRID:SCR_015825</td><td></td></tr><tr><td>Cell culture 24-well Plate</td><td>Labclinics</td><td>PLC30024</td><td>Used for 3D cultures in Matrigel. Flat Bottom</td></tr><tr><td>Cell culture 6-well Plate</td><td>Labclinics</td><td>PLC30006</td><td>Used for anchorage assay</td></tr><tr><td>Cell incubator (37 &amp;ordm;C and 5 % CO2)</td><td></td><td></td><td></td></tr><tr><td>Cell Strainers</td><td>Fisherbrand</td><td>11587522</td><td>Mesh size: 40 &amp;mu;m</td></tr><tr><td>CellSense software</td><td>Olympus</td><td></td><td>Used to image acquisition</td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Cholera Toxin from Vibrio cholerae</td><td>Sigma-Aldrich</td><td>C8052</td><td>Used to supplement cell culture medium</td></tr><tr><td>Class II Biological Safety Cabinet</td><td>Herasafe</td><td>HAEREUS HS12</td><td></td></tr><tr><td>Confocal inverted Microscope</td><td>Leica</td><td>TCS SP5</td><td></td></tr><tr><td>Cover glasses</td><td>Witeg Labortechnik GmbH</td><td>4600122</td><td>22 X 22 mm, thickness 0.13 - 0.17 mm</td></tr><tr><td>DAPI</td><td></td><td></td><td>2-(4-amidinophenyl)-1H -indole-6-carboxamidine</td></tr><tr><td>Fetal Bovine Serum</td><td>Biowest</td><td>S1810</td><td>Used to inactivate trypsine action</td></tr><tr><td>Fiji software (ImageJ)</td><td>National Institutes of Health</td><td>RRID:SCR_002285</td><td>Free download, no license needed</td></tr><tr><td>Glass Pasteur Pipettes</td><td></td><td></td><td></td></tr><tr><td>Glass slides</td><td>Fisherbrand</td><td>11844782</td><td></td></tr><tr><td>Goat Serum</td><td>Biowest</td><td>S2000</td><td>Used for immunofluorescence of 3D structures</td></tr><tr><td>Heat-Resistant Gloves</td><td></td><td></td><td>Used for agar manipulation after autoclave</td></tr><tr><td>Heater bath (37 &amp;ordm;C)</td><td></td><td></td><td>Used to temper solutions prior to cell subculture</td></tr><tr><td>Heater bath (42 &amp;ordm;C)</td><td></td><td></td><td>Used to keep agar warm</td></tr><tr><td>Heating plate</td><td></td><td></td><td>Used for Matrigel dehydration</td></tr><tr><td>Humid chamber</td><td></td><td></td><td>Used for the incubation of antibodies during immunofluorescence</td></tr><tr><td>Ice</td><td></td><td></td><td>Used during Matrigel manipulation</td></tr><tr><td>Ice-box</td><td></td><td></td><td></td></tr><tr><td>Inverted Optic Microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>Matrigel Matrix</td><td>Becton Dickinson</td><td>354234</td><td>Store at -20 &amp;ordm;C and keep cold when in use. Referred to as basement membrane matrix</td></tr><tr><td>Methanol</td><td>PanReac AppliChem</td><td>131091</td><td>Used for 3D structure fixation prior to immunofluorescent labelling</td></tr><tr><td>Micropipette</td><td></td><td></td><td>p1000, p200 and p10</td></tr><tr><td>Microsoft Office Excel</td><td>Microsoft</td><td>RRID:SCR_016137</td><td>Used to calculate population doubling and to obtain growth rate equation</td></tr><tr><td>MilliQ water</td><td></td><td></td><td>Referred to as ultrapure water</td></tr><tr><td>Nail Polish</td><td></td><td></td><td>Used to seal samples after mounting</td></tr><tr><td>Parafilm M</td><td>Bemis</td><td>PM-999</td><td>Used to cover antibody solution during incubation</td></tr><tr><td>PBS pH 7.4 (w/o calcium &amp;amp; magnesium)</td><td>Gibco</td><td>10010-056</td><td>Sterile. Used for cell subculture</td></tr><tr><td>PBS tablets</td><td>Sigma-Aldrich</td><td>P4417</td><td>Dilute in milliQ water. No sterility required. Used for immunofluorescence</td></tr><tr><td>Pipette Aid</td><td></td><td></td><td></td></tr><tr><td>Primaria T25 flasks</td><td>Corning</td><td>353808</td><td>Used for BPEC culture</td></tr><tr><td>Scepter Automated Cell Counter</td><td>Millipore</td><td>PHCC20060</td><td>Alternatively, use an haemocytometer</td></tr><tr><td>Scissors</td><td></td><td></td><td>Used to cut pipette tips and parafilm</td></tr><tr><td>Sterile filters 0.22 &amp;mu;m</td><td>Millipore</td><td>SLGP033RS</td><td>Used to filter MTT solution</td></tr><tr><td>Thiazolyl Blue Tetrazolium Bromide (MTT)</td><td>Sigma-Aldrich</td><td>M2128</td><td>Store at -20 &amp;ordm;C</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Used for immunofluorescence of 3D structures</td></tr><tr><td>Trypsin-EDTA 10X</td><td>Biowest</td><td>X0930</td><td>Dilute in PBS to obtain 3X solution</td></tr><tr><td>Vectashield Antifade Mounting Medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>WIT-P-NC Culture Medium</td><td>Stemgent</td><td>00-0051</td><td>Used for primary BPEC culture</td></tr><tr><td>WIT-T Culture Medium</td><td>Stemgent</td><td>00-0047</td><td>Used for transformed BPEC culture</td></tr></tbody></table>

in vitro evaluation of oncogenic transformation in human mammary epithelial cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

An experimental model of cellular transformation with the introduction of three genetic elements in BPECs was chosen to generate representative results of oncogenic transformation6,7 (Figure 3). Non-transformed BPECs (N) were derived from disease-free breast tissue as described by Ince and colleagues6 and cultured following the protocol indicated here. After overcoming STASIS (stress ...

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In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

in-vitro-evaluation-oncogenic-transformation-human-mammary-epithelial

Research

JoVE Journal

Cancer Research

4.4K Views.  Universitat Autònoma de Barcelona. Dit protocol biedt experimentele in vitro tools om de transformatie van menselijke borstcellen te evalueren. Gedetailleerde stappen naar de follow-up cel proliferatie tarief, verankering-onafhankelijke groeicapaciteit, en de distributie van cellijnen in 3D-culturen met kelder membraan matrix worden beschreven.

Video: In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Medicine

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

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Developmental Biology

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 ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Bioengineering

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 ...

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play a major role in mammary gland function and are the origin of most mammary tumors. Introducing genes of interest into mouse mammary epithelial cells is a critical step in evaluating gene function in epithelial cells and generating mouse mammary tumor models. This goal can be accomplished through the intraductal injection of a viral vector carrying the genes of interest into the mouse mammary ductal tree. The injected virus subsequently infects mammary epithelial cells, bringing in the genes of interest. The viral vector can be lentiviral, retroviral, adenoviral, or adenovirus-associated viral (AAV). This study demonstrates how a gene of interest is delivered into mammary epithelial cells through mouse mammary intraductal injection of a viral vector. A lentivirus carrying GFP is used to show stable expression of a delivered gene, and a retrovirus carrying Erbb2 (HER2/Neu) is used to demonstrate oncogene-induced atypical hyperplastic lesions and mammary tumors.

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells.

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

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