This work was supported by NIH grants R01 CA107469, R01 CA125577, and U01CA154224 to C.G. Kleer, the University of Michigan's Cancer Center Support.

1. Preparation of Materials and Culture Media
<ol>
	<li>Thaw growth factor reduced (GFR) Matrigel at 4 &#176;C O/N.</li>
	<li>Warm up complete media for required cell line at 37 &#176;C. MCF10A- ATCC specified media; HME- Ham's F-12 medium supplemented with 5% fetal bovine serum, 1 &#181;g/ml hydrocortisone, 5 &#181;g/ml insulin, 10 ng/ml epidermal growth factor, and 100 ng/ml cholera toxin; SUM149- Ham's F-12 media supplemented with 5% FBS, 2 &#181;g/ml hydrocortisone and 5 &#181;g/ml insulin; and MDA-MB-231-10% FBS-D...

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We have described here the three dimensional culture of breast epithelial cells on a reconstituted basement membrane and the usefulness of this system to differentiate between malignant vs. benign breast phenotype. This system can also be used to culture different cell types from other glandular tissues and has been reported to have been successfully used to culture prostate epithelial, bronchial epithelial, and thyroid epithelial cells, to name a few12-14. The importance of 3D culture lies in the fact that it...

Three dimensional culture (3D) of breast epithelial cells on reconstituted basement membrane is an important model system to study the complex phenotype and associated signaling of normal breast and breast cancer2,3. The functional unit of breast is the terminal duct lobular unit (TDLU) which consists of a small duct which branches into acini. The acini are highly organized structures, composed of two cell layers, epithelial and myoepithelial cells, which are surrounded by a basement membrane5. The most distinguishing features of normal acini are cellular polarization, attachment to the underlying basement membrane and specialized cell-cell contacts. This intricate organization is disrupted in invasive carcinomas. The widely used 2D cultures, where cells are grown as monolayers, do not allow for the formation of acini. Thus, the monolayer culture is lacking in providing a system to capture the intricate relationship between epithelial cells in normal breast and their deregulation in breast cancer. Functional monotypic epithelial cultures of breast cells have been developed in several laboratories2,3. 3D culture involves the growth of breast cells on Matrigel, which is a solubilized extract derived from Engelbreth-Holm-Swarm mouse sarcoma cells and is available commercially. Other 3D substrata like collagen I are also used. The breast cells can be cultured in 3D by 3D embedded assay, where cells are cultured embedded in Matrigel2. Alternatively, cells can be cultured by 3D on top assay, also called 3D overlay assay, which is cost effective as it requires lesser volume of Matrigel, and facilitates time lapse monitoring of colony formation by phase contrast imaging and is ideal for in situ imaging2-3. The 3D on top assay has been used to define the correlation between the acinar phenotype and gene expression profile7.
3D culture can be effectively used to distinguish normal and benign cells from invasive carcinoma. Normal and benign breast cells form growth arrested polarized structures with a well-defined lumen in the center on Matrigel whereas invasive carcinoma cells grow prolifically and haphazardly with no clearing of the lumen. E6/E7 immortalized human mammary epithelial cells and MCF10A cell line, which is a spontaneously immortalized cell line isolated from a 36 year old patient with fibrocystic changes, have been successfully used to recapture the benign breast phenotype in 3D3,8 and have served as a model system to study the oncogenic and tumor suppressor function of several molecules8,9. These benign cells can be engineered to manipulate gene(s) of interest by the introduction of lentivirus/retrovirus. If the gene(s) of interest is a tumor suppressor, shRNA mediated knockdown will lead to a malignant transformation whereas if the gene of interest is an oncogene overexpression in benign cells should show a malignant phenotype. In both cases the acinar structures formed in 3D can capture the malignant transformation.
Benign single cells plated in 3D start to proliferate and form round spherical structures. By day 5-8 this spherical aggregate of cells will differentiate into a surrounding polarized layer of cells that is in contact with the Matrigel, and an inner mass of less polarized cells, which lack contact with the Matrigel. In the next 10-15 days the inner cells will start to die by apoptosis forming a clear lumen. The malignant cells will grow haphazardly losing their polarity. Highly malignant cells usually form branching structures. These differences in phenotype can be captured by time lapse phase contrast imaging and by in situ immunostaining with relevant molecular markers. The most commonly used markers are alpha 6-integrin, a basal polarity marker, in combination with the proliferation marker phospho-histone 3 and the apoptotic marker cleaved caspase-3. The latter is important to determine whether there is lumen formation in the center of the acini, a feature of normal and benign breast cells. Other polarity and cell-cell contact markers include GM130, laminin, ERM, E-cadherin. Alternately the breast cancer cell lines can be engineered to manipulate the gene of interest. In this case reversion to a more growth arrested benign phenotype can be used as a read out to distinguish from the cancer phenotype.
3D culture can also be carefully used to delineate the signaling pathways involved in malignant transformation10-11. Using the above mentioned read-outs, pharmacological inhibitors, blocking antibodies or recombinant proteins can be used be pinpoint at the molecular signaling leading to malignant transformation. With continued efforts from various laboratories it is possible to extract the cells from 3D to isolate RNA and also prepare lysates for immunoblotting and immunoprecipitation. These strategies are described in a stepwise and detailed manner in the protocol.

<table border="0" cellpadding="0" cellspacing="0" width="585">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Growth Factor Reduced matrigel</td>
			<td style="width:83px;">BD biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;">Avoid repeat freeze thaw&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass hamber slides 8-well</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177402</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass chamber slides 2- well&#160;</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177380</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">goat anti mouse F(ab&#180;)2 fragment</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">115-006-006</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Normal goat serum</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorescent conjugated Alexa antibodies</td>
			<td style="width:83px;">Molecular probes</td>
			<td style="width:119px;"></td>
			<td>Please look at molecular probes website</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Prolong gold antifade reagent with DAPI</td>
			<td style="width:83px;">Molecular Probes</td>
			<td style="width:119px;">P36935</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">3D Culture Cell Harvesting kit</td>
			<td style="width:83px;">Trevigen</td>
			<td style="width:119px;">3448-020-k</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Apha-6-integrin</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">MAB1378</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-GM130</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610822</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-phospho-histone 3 (Ser10)FITC Conjugated</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">16-222</td>
			<td>1:100 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Cleaved caspase 3 (Asp 175)</td>
			<td style="width:83px;">Cell Signaling</td>
			<td style="width:119px;">9661s</td>
			<td>1:50 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-E-cadherin</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610181</td>
			<td>1:200 dilution for IF</td>
		</tr>
	</tbody>
</table>

three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

3D culture can be effectively used to differentiate malignant phenotype of human breast epithelial cells from the benign breast. Single benign cells plated on Matrigel grow as organized spherical structures that can easily be imaged as single round flat acini in one plane using phase contrast imaging. The single malignant cells plated on Matrigel grow haphazardly, show very high refractive index and are difficult to image in one plane. As shown in Figure 1, the benign HME and MCF10A cells form spherical ...

Watch this Scientific Journal Video about Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells at JoVE.com

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

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19.8K Views.  University of Michigan Comprehensive Cancer Center. Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.