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 (Video) | JoVE 

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.

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 ...

Research

JoVE Journal

Cancer Research

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Sectioning Mammary Gland Whole Mounts for Lesion Identification (Video) | JoVE 

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

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells (Video) | JoVE 

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

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function (Video) | JoVE 

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

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis (Video) | JoVE 

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

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

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

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

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad (Video) | JoVE 

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

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1Î± Nanoparticles (Video) | JoVE 

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

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research (Video) | JoVE 

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 ...

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures (Video) | JoVE 

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Reconstruct Human Retinoblastoma In Vitro

Reconstruct Human Retinoblastoma In Vitro (Video) | JoVE 

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 ...