Name: in vitro evaluation of oncogenic transformation in human mammary epithelial cells
Uploaded: 2020-09-24T14:30:00
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 d...

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Stampfer, M. R., Yaswen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+models+of+human+mammary+epithelial+cell+transformation.">Culture models of human mammary epithelial cell transformation.</a> Journal of Mammary Gland Biology and Neoplasia. 5 (4), 365-378 (2000).</li><li>Schinzel, A. C., Hahn, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+transformation+and+experimental+models+of+human+cancer.">Oncogenic transformation and experimental models of human cancer.</a> Frontiers in Bioscience : A Journal and Virtual Library. 13 (13), 71 (2008).</li><li>Balani, S., Nguyen, L. V., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+process+of+human+tumorigenesis.">Modeling the process of human tumorigenesis.</a> Nature Communications. 8 (1), 15422 (2017).</li><li>Bray, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cancer+statistics+2018:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA: A Cancer Journal for Clinicians. 68 (6), 394-424 (2018).</li><li>Ince, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+different+human+breast+epithelial+cell+types+leads+to+distinct+tumor+phenotypes.">Transformation of different human breast epithelial cell types leads to distinct tumor phenotypes.</a> Cancer Cell. 12 (2), 160-170 (2007).</li><li>Repull&#233;s, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+malignant+transformation+of+preneoplastic+and+normal+breast+primary+epithelial+cells.">Radiation-induced malignant transformation of preneoplastic and normal breast primary epithelial cells.</a> Molecular Cancer Research. , 1-13 (2019).</li><li>Weigelt, B., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+microenvironmental+influences+on+the+normal+mammary+gland+and+breast+cancer.">Unraveling the microenvironmental influences on the normal mammary gland and breast cancer.</a> Seminars in Cancer Biology. 18 (5), 311-321 (2008).</li><li>Paoli, P., Giannoni, E., Chiarugi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoikis+molecular+pathways+and+its+role+in+cancer+progression.">Anoikis molecular pathways and its role in cancer progression.</a> Biochimica et Biophysica Acta. 1833 (12), 3481-3498 (2013).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+oncogenesis+of+MCF-10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures.">Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> Methods. 30 (3), 256-268 (2003).</li><li>Borowicz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+soft+agar+colony+formation+assay.">The soft agar colony formation assay.</a> Journal of Visualized Experiments. (92), (2014).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Brocher, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BioVoxxel+Image+Processing+and+Analysis+Toolbox.">The BioVoxxel Image Processing and Analysis Toolbox.</a> European BioImage Analysis Symposium. 8 (2), 67112 (2015).</li><li>Torquato, S., Truskett, T. M., Debenedetti, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+random+close+packing+of+spheres+well+defined.">Is random close packing of spheres well defined.</a> Physical Review Letters. 84 (10), 2064-2067 (2000).</li><li>LaBarge, M. A., Garbe, J. C., Stampfer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+human+reduction+mammoplasty+and+mastectomy+tissues+for+cell+culture.">Processing of human reduction mammoplasty and mastectomy tissues for cell culture.</a> Journal of Visualized Experiments. (71), (2013).</li><li>Zubeldia-Plazaola, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoids+promote+transition+of+ductal+carcinoma+in+situ+to+invasive+ductal+carcinoma+by+inducing+myoepithelial+cell+apoptosis.">Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis.</a> Breast Cancer Research. 20 (1), 65 (2018).</li></ol>

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 &#956;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 &#956;l Pipette tips w/ filter</td>
			<td>Labclinics</td>
			<td>LAB1000ULFNL</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;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 &#956;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 &#186;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 &#956;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 &#186;C)</td>
			<td></td>
			<td></td>
			<td>Used to temper solutions prior to cell subculture</td>
		</tr>
		<tr>
			<td>Heater bath (42 &#186;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 &#186;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 &#38; 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 &#956;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 &#186;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 ...

Watch this Scientific Journal Video about In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells at JoVE.com

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 ...

This methodology uses the integration of different indicators to assess the degree of transformation of any cell model. This technique provides researchers with a set of time-consuming but simple-to-use tools for assessing cell transformation in vitro. This allows the method to be used in most laboratories.Demonstrating the procedure will be Teresa Anglada, a post-doctoral researcher from my laboratory, and Joan Repulles, a microscopy specialist. When the breast primary epithelial cell culture reaches 90%confluency, transfer the supernatant from the culture flask to a 15 milliliter tube containing two milliliters of fetal bovine serum and wash the cells with PBS. Treat the cells with one milliliter of trypsin for five minutes at 37 degrees Celsius.When the cells have detached, inactivate the trypsin with the reserved medium and collect the cells by centrifugation. Flick the tube to resuspend the pellet and add one to two milliliters of fresh medium for counting. After determining the cell concentration, add 300, 000 cells to a new tube and bring the final volume to five milliliters with additional fresh culture medium.Seed cells into a new T25 flask and return the cells to the cell culture incubator. Then use the formula to calculate the number of accumulated population doublings. To assess the proliferation rate of the cells, plot the accumulated number of population doublings for a specific time interval.An increased slope indicates an increased cell proliferation rate. To set up a 3D cell culture, rinse wells with a 1.9 square centimeter surface area with cold sterile PBS and coat the bottom of each well with 100 microliters of basement membrane matrix. Then place the plate in the cell culture incubator for about 20 minutes to allow the matrix to solidify.In the meantime, harvest mammary cells as just demonstrated and dilute the cells to a 400, 000 cells per milliliter of medium concentration. Mix the cells with fresh medium supplemented with 8%basement membrane matrix at a one-to-one ratio and add 500 microliters of the cells to each well of solidified matrix. Incubate the cells at 37 degrees Celsius for a few minutes, then add 500 microliters of medium supplemented with 4%basement membrane matrix to each well.Place the cells in the cell culture incubator for 14 days. The seeded cells will group and proliferate forming acini-like structures. For the immunofluorescent protein detection in the 3D cultured mammary cells, use a cutoff P200 pipette tip to remove the basement membrane matrix from each well and smear approximately 50 microliters of disaggregated matrix from each well onto individual glass slides.When the samples have completely dried, fix the cells with a one-to-one methanol-acetone solution at minus 20 degrees Celsius for 30 minutes. After fixing, block samples with blocking solution for two hours at room temperature. At the end of the incubation, add 30 microliters of the primary antibody working solution of interest and cover the slides with a strip of laboratory wrapping film for an overnight incubation at four degrees Celsius in a humid chamber.The next morning, wash the slides three times with PBS for one hour per wash and label the samples with the appropriate secondary antibody solution as just demonstrated. At the end of the incubation, wash the samples two times in PBS for two hours per wash before counter-staining with DAPI diluted in anti-fade mounting medium and covering each slide with a coverslip. Then analyze the fluorescent signal distribution for each acinus on a confocal microscope.To perform an anchorage-independent assay, first cover the bottom of the appropriate number of wells of a six-well plate with 1.5 milliliters of 0.6%agar in medium solution and let the agar solidify at room temperature. Next, prepare a five times 10 to the fourth cells per milliliter solution as demonstrated and filter the cells through a 40 micron strainer into a sterile 50 milliliter tube. Holding the tube at a 45 degree angle, add an equal volume of 0.6%agar in medium solution down the wall of the tube.And after thorough mixing, layer one milliliter of the cells in agar suspension to each well of the previously prepared six-well plate. Use an inverted microscope to confirm that the cells have been homogeneously dispersed throughout the agar. When the agar has completely solidified, carefully add one milliliter refresh medium to each well without disturbing the agar and incubate the cells for three weeks, changing the medium twice per week.At the end of the colony formation period, replace the medium with one milliliter of one milligram per milliliter MTT per well for a 24-hour incubation in the cell culture incubator. The next day, remove the MTT from each well and acquire images of each well on the inverted microscope. When all of the images have been acquired, open the images in ImageJ and click image, adjust, threshold, and apply to obtain a binary mask through thresholding of the original image.When well-delimited colonies have been obtained, select plugins and biovoxxel to launch the extended particle analyzer to identify the MTT-positive colonies. Over a specific interval of time, partially and fully transformed cells achieve a higher number of population doublings compared to non-transformed cells, indicating that the cell division rate is increased with the transformation process. Over the two-week 3D cell culture period, the cells aggregate and proliferate, increasing the acini size.The proper polarization of each acinus can be accurately assessed using protein immunofluorescent detection in conjunction with three-dimensional signal location by confocal microscopy. While all of the acini formed by non-transformed breast cells are properly organized, a loss of polarization is observed in acini formed by partially and fully transformed cells. After three weeks of embedded agar culture, cells with an anchorage independent growth capacity give rise to colonies composed of multiple cells.Only metabolically active cells are capable of cleaving the tetrazolium MTT ring, resulting in purple MTT formazan crystals after 24 hours. Measurement of the colony diameter by automatic image analysis allows the filtering of low proliferative colonies or individual cells. The number of colonies formed by non-transformed cells is negligible compared to those from partially and fully transformed cells.Colony size can also be used to discriminate between cells with different degrees of transformation. These assays can be used to screen cell lines prior to any inoculation as only those cell lines that have tested positive in vitro should be inoculated into mice.

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Research

JoVE Journal

Cancer Research

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&amp;#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Reconstruct Human Retinoblastoma In Vitro

Human RB is pediatric cancer, which is lethal if no treatment is administered. As RB originates from cone precursors, which is relatively rare in rodent models, meanwhile regarding the interspecies differences between humans and rodents, a disease model derived from humans is more beneficial for uncovering the mechanisms of human RB and seeking the targets of therapy. Herein, the protocol describes the generation of two gene-edited hESC lines with a biallelic RB1 point mutation (RB1Mut/Mut) and an RB1 knockout mutation (RB1-/-), respectively. During the process of retinal development, the formation of RB is observed. The RB cell lines are also established by segregating from the RB organoids. Altogether, by differentiating the gene-edited hESC lines into the retinal organoids using a 2D and 3D combined differentiation protocol, we have successfully reconstructed the human RB in a dish and identified its cone-precursor origin. It would provide a helpful disease model for observing the retinoblastoma genesis, proliferation, and growth as well as further developing novel therapeutic agents.

We describe a method for generating human retinoblastoma (RB) by introducing biallelic RB1 mutations in human embryonic stem cells (hESC). RB cell lines could also be successfully cultured using the isolated RB in a dish.

Human RB is pediatric cancer, which is lethal if no treatment is administered. As RB originates from cone precursors, which is relatively rare in rodent ...

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 ...

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 ...