This work was conducted with funds to M.B. from the Canadian Institutes of Health Research (CIHR) grant MOP 107972. M.B. is a recipient of a CIHR New Investigator Salary Award. D.C. is a recipient of studentships from the Translational Breast Cancer Research Unit and the CIHR Strategic Training Program in Cancer Research and Technology Transfer, London Regional Cancer Program. C.G. is recipient of studentships from the Translational Breast Cancer Research Unit, London Regional Cancer Program and from the CIHR- Strategic Training Program in Cancer Research and Technology Transfer. SGD.

1. Three-dimensional Culture of Breast Cancer Cells in Basement Membrane Matrix (The Embedment Technique)
<ol>
	<li>Handling Matrigel basement membrane matrix: Thaw on ice overnight at 4 &#176;C. Basement membrane matrix is liquid at low temperature but solidifies at room temperature. Keep basement membrane matrix on ice (Figures 1A-B).</li>
	<li>Cover the Confocal No.1 glass-bottom dish with 50 &#956;l of basement membrane matrix by means of spreading the matrix using the tip of a P-200 Pipetman in a ...

<ol>
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M., 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+ultrastructure+of+MCF-10A+acini.">The ultrastructure of MCF-10A acini.</a> J Cell Physiol. 208, 141-148 (2006).</li><li>Lelievre, S. 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=Tissue+phenotype+depends+on+reciprocal+interactions+between+the+extracellular+matrix+and+the+structural+organization+of+the+nucleus.">Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus.</a> Proc Natl Acad Sci U S A. 95, 14711-14716 (1998).</li><li>Petersen, O. W., Ronnov-Jessen, L., Howlett, A. R., Bissell, M. 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M., 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=beta4+integrin-dependent+formation+of+polarized+three-dimensional+architecture+confers+resistance+to+apoptosis+in+normal+and+malignant+mammary+epithelium.">beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium.</a> Cancer Cell. 2, 205-216 (2002).</li><li>Cvetkovic, D., 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=KISS1R+Induces+Invasiveness+of+Estrogen+Receptor-Negative+Human+Mammary+Epithelial+and+Breast+Cancer+Cells.">KISS1R Induces Invasiveness of Estrogen Receptor-Negative Human Mammary Epithelial and Breast Cancer Cells.</a> Endocrinology. , 1999-2014 (2013).</li><li>Alemayehu, M., 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=beta-Arrestin2+regulates+lysophosphatidic+acid-induced+human+breast+tumor+cell+migration+and+invasion+via+Rap1+and+IQGAP1.">beta-Arrestin2 regulates lysophosphatidic acid-induced human breast tumor cell migration and invasion via Rap1 and IQGAP1.</a> PLoS One. , (2013).</li></ol>

The development of 3D cell culture techniques has allowed researchers to study the transformation of breast epithelial cells, allowing us to visualize the dramatic morphological changes. Besides analyzing cell invasion, the single or multicellular mammary epithelial spheroids can be used to assess changes in cellular adhesion, proliferation, size, and basal-apical polarity. In contrast to previously reported methodologies where the cells are overlaid with ECM8, our method embeds the cells in ECM18,19,24</...

Migration and invasion of individual or collective cells are two hallmarks of cancer, and required for the metastatic spread of cancer cells1-4. The ability of cancers cells to initiate metastasis depends on their capability to migrate and invade into the neighboring tissue using invadopodia to degrade the basement membrane of the cells. Invadopodia are dynamic actin-rich matrix degradation protrusions that enable degradation of the extracellular matrix through the release of matrix-degrading proteases5. Cancer cell invasion involves the degradation of the matrix followed by the migration of the cancer cells and this is accompanied by a reorganization of the three-dimensional (3D) matrix environment2. Thus, to penetrate through the matrix, a cell must transform its shape and interact with the extracellular matrix (ECM)2.
The maintenance of breast tissue integrity depends on tightly controlled tissue architecture since cell&#8211;ECM and cell&#8211;cell adhesion junctions influence gene expression and disruption of epithelial polarity can lead to the onset of cancer6-10. However, most in vitro migration and invasion assays such as transwell chamber assays or wound-scratch assays are two-dimensional (2D) and hence these neglect the intricate interactions between cells and their adjacent environment3,6,8,11-14. Considerable morphological and functional diversities including variations in cellular morphology, cellular differentiation, cell-matrix adhesions and gene expression patterns have been detected by culturing cells in 3D cultures that are commonly lacking in 2D assays2,6,8,11. Thus, uses of 3D assays are significantly beneficial in recapitulating a more physiological in vivo condition, leading to a better translation of ground-breaking findings in basic research to the clinic6-10. However, it should be noted, despite the many advantages gained with the use of 3D cultures, this model cannot capture all of the complexities of the in vivo tumor microenvironment that includes various cell types. However, it is possible to incorporate stromal cells into the 3D models (for example, fibroblasts, leukocytes, and macrophages) to study the effect of tumor-stromal interactions on cancer cell adhesion and invasion15-17.
Breast epithelial cells in culture grow most effectively when ECM proteins such as laminin and collagen are present. With this known, a commercially available matrix mixture has been derived from Engelbreth-Holm-Swarm (EHS) murine tumor and is known as Matrigel basement membrane matrix2,8. A number of techniques have been established to grow epithelial cells as 3D colonies in basement membrane matrix2,8. The 3D basement membrane matrix model is effective for establishing both malignant and non-malignant breast cell growth, resembling what is occurring in the in vivo environment18,19. MCF10A cells are non-malignant mammary epithelial cells. When grown in basement membrane matrix, these cells exhibit in vivo traits of normal breast cells and undergo controlled cell proliferation, cell polarization, and apoptosis to establish the lumen space8,12,20. Furthermore, the appearance of cell nuclei of MCF10A cells forming acini in 3D cultures more closely resemble those of mammary epithelial cells in tissue than those cultured in monolayer21. Studies by Bissell and colleagues were the first to reveal that malignant breast cells can be differentiated from non-malignant breast cells when grown in a laminin-rich surroundings, since the malignant cells display a highly disorganized phenotype, increased proliferation, decreased cell-to-cell adhesion, increased expression of mesenchymal markers and an increase in the number of invasive structure formed3,6,22.
Abnormalities of the cell environment can influence tumor formation20. The 3D culture method can be used to effectively study the communication that occurs between the tumor cells and their surrounding environment and determine how protein expression influences such communication14,20,21,23. This article provides a detailed methodology to grow MDA-MB-231 breast cancer cells in 3D cultures to analyze invasiveness, and to study the loss of epithelial morphology using an epithelial marker laminin, a component of the cell basement membrane18,19,24,25. The detailed procedures provide the ability to accurately and reproducibly quantify stellate (invasive) structure formation by any invasive cancer cell and is not limiting to the common breast cancer cell lines (such as MDA-MB-231, Hs578T, MCF-7, or T47D). Thus, this assay can serve as a platform for evaluating how protein expression in cells or treatment with pro- or anti-invasive compounds regulate extracellular matrix degradation, by single or multiple cells.

<table border="0" cellpadding="0" cellspacing="0" width="1062">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Material/ Equipment</td>
			<td style="width:265px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:355px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL tubes</td>
			<td>VWR</td>
			<td>CA10011-700</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100-mm culture dish BD353003</td>
			<td>VWR</td>
			<td>CABD353003</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Falcon tube</td>
			<td>VWR</td>
			<td>CA21008-918</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL filtered tips</td>
			<td>VWR</td>
			<td>10011-350</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-016</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-008</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35-mm glass-bottomed Confocal No.1 culture dishes&#160;</td>
			<td style="width:265px;">MatTek Corporation</td>
			<td>P35G-1.0-14-C</td>
			<td>Precooled before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>BioShop</td>
			<td>ALB003.100</td>
			<td>Used at 3% for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody, highly cross-adsorbed</td>
			<td>Life Technologies</td>
			<td>&#160;A11029</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td>Life Technologies</td>
			<td>&#160;A11011</td>
			<td>1:1200 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-Beta-Catenin&#160;</td>
			<td>BD Transduction Laboratories</td>
			<td>610153</td>
			<td>Mouse-monoclonal; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td>Used at 10% (v/v)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33258, Pentahydrate (bis-Benzimide) - 10 mg&#8260;mL Solution in Water</td>
			<td>Life Technologies</td>
			<td>H3569</td>
			<td>Used at 0.1% (1:10000 dilution)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InVivo Analyzer Suite&#160;</td>
			<td style="width:265px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kisspeptin-10 (KP-10)</td>
			<td style="width:265px;">EZ Biolabs</td>
			<td style="width:119px;">PT0512100601</td>
			<td>Used at 100nM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Laminin&#160;</td>
			<td>Cedarlane</td>
			<td>AB19012(CH)</td>
			<td>Rabbit-polyclonal full length human; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM-510 META laser scanning microscope&#160;</td>
			<td style="width:265px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>Used at 63X objective; oil immersion lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel phenol red free (BD356237)</td>
			<td style="width:265px;">VWR</td>
			<td>CACB356237</td>
			<td>&#160;Lot No.2180819; 10.4 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus IX-81 microscope&#160;</td>
			<td style="width:265px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Antibiotic (added to media; used at 0.01%)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL pipet&#160;</td>
			<td>VWR</td>
			<td>CA53300-523</td>
			<td>Sterile, disposeable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium with Glutamine</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td>Used for culturing of MDA-MB-231 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA (1X), Phenol Red</td>
			<td>Life Technologies</td>
			<td>25200-072</td>
			<td>Used to trypsinize MDA-MB-231 cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">MEGM (bullet kit): MEBM (CC3151)+Single quots (CC4136)</td>
			<td style="width:265px;">Lonza</td>
			<td style="width:119px;">CC-3150</td>
			<td>Used for culturing of MCF10A cells</td>
		</tr>
	</tbody>
</table>

quantification of breast cancer cell invasiveness using a three-dimensional (3d) model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

An example of the MDA-MB-231 cells invading in 3D matrix is illustrated in Figure 3C. The cells are embedded in matrix (Day 1), and start forming invasive (stellate) structures by Day 3, and completely invade into the matrix by Day 5 (Figure 3C). The number of stellate colonies formed are counted, and expressed as a percentage of total number of colonies per dish (invasive and non-invasive). Additionally, since measurements are done daily for the five days, the rate of invasion can also ...

Watch this Scientific Journal Video about Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model at JoVE.com

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

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15.7K vistas.  University of Western Ontario.  Este artículo proporciona metodologías detalladas para el uso de ensayos (3D) en tres dimensiones para cuantificar la invasión de células de cáncer de mama. En concreto, se discuten los procedimientos necesarios para configurar este tipo de ensayos, la cuantificación y el análisis de datos, así como los métodos para examinar la pérdida de integridad de la membrana que se produce cuando las células invaden. 