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 spiral pattern (Figure 1C). Use caution when spreading the basement membrane matrix to avoid the formation of air bubbles. Likewise, avoid spreading the matrix to close to the borders of the glass-bottom dish to prevent meniscus formation. If inexperienced handling basement membrane matrix, then precooled the dish, P-200 Pipetman and pipettes can be on ice (alternatively left overnight in a fridge at 4 &#176;C). This step offers additional time to spread the basement membrane matrix before solidification. Place the dish(es) in a cell culture incubator (at 37 &#176;C with 5% CO2) for at least 30 min to enable the basement membrane matrix to solidify (Figure 1D).</li>
	<li>While the matrix is undergoing solidification, trypsinize a 70-80% confluent 100-mm plate of cells. Once the cells have begun lifting off of the plate, resuspend them in 10 ml of RPMI 1640 medium containing 10% (v/v) fetal bovine serum (FBS), to inactivate the trypsin (Figure 1E). Then transfer the resuspended cells into a 15 ml conical tube (Figure 1F).</li>
	<li>Spin the cells (present in conical tube) at 100 x g for 3 min in a dedicated cell culture centrifuge (Figures 1G-H).</li>
	<li>While the cells are being spun down, aliquot 50 &#956;l of matrix into an 1 ml microcentrifuge tube (note: each glass-bottomed dish is allocated one microcentrifuge tube; hence, if the experiment requires 3 dishes, then it follows that 3 microcentrifuge tubes are necessary as well), and then place the tube on ice (Figure 1B).</li>
	<li>Aspirate the medium from the conical tube from step 1.4, whilst leaving the pellet undisturbed (Figure 1I). Resuspend cells (that have been spun down) in 1 ml of FBS-supplemented RPMI medium (Figure 1J).</li>
	<li>Once the cells are counted using a hemocytometer (Figure 1K), or a cell particle counter aliquot 2.5 x 104 cells into the microcentrifuge tube and top it off using appropriate media so as to obtain total volume of 50 &#956;l (Figure 1L).</li>
	<li>Mix the cells from step 1.7 (25,000 cells in 50 &#956;l) with the matrix-containing microcentrifuge tube from step 1.5 in a 1:1 ratio; final volume will be 100 &#956;l (Figure 1M).</li>
	<li>Gently plate 100 &#956;l of the matrix:cell mixture from step 1.8 onto the solidified basement membrane matrix-coated dish from step 1.2 (Figure 1N). This allows for cells to be embedded in basement membrane matrix.</li>
	<li>Transfer the dish(es) into the cell culture incubator (at 37 &#176;C with 5% CO2) and allow the matrix:cell mixture to solidify for at least 30 min.</li>
	<li>Once the matrix:cell mixture is solidified, add 2 ml of FBS-supplemented RPMI media to the dish (Figure 1O) and take the dish back the incubator where it will be stored for the remainder of the experiment (Figure 1P).</li>
	<li>Change media every day for a period of 5&#160;days (or as per required for an assay; the duration of the assay for MDA-MB-231 cells is 5&#160;days in length).</li>
	<li>Using a light microscope, take differential interference contrast (DIC) images of the MDA-MB-231 colonies suspended in basement membrane matrix (Figure 3A). Image 20 representative areas at 10X objective once a day for five days to determine colony morphogenesis (Figure 3C).</li>
	<li>Analyze images blindly to determine cell colony stellate formation (Figure 3B). A colony is deemed to be stellate if one or more projections from the spheroid of cells are perceived. To determine the percentage of invasive stellate colonies, divide the number of stellate colonies by the total number of cell colonies per acquired image, and then average the stellate colonies percentages of the 20 images for each day.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig1highres.jpg" width="500" /> 
Figure 1. Three-dimensional culture of MDA-MB-231 breast cancer cells in basement membrane matrix (the embedment technique). A-B) A schematic of the experimental setup (performed in the fume hood). C) The well of the glass-bottomed dish coated with 50 &#956;l of basement membrane matrix. D) Dish(es) placed in a cell culture incubator (at 37 &#176;C with 5% CO2) to permit the matrix to solidify for at least 30 min. E) Cells were trypsinized. F) Cells were resuspended into a 15 ml conical tube. G-H) Cells (present in conical tube) were spun down at 100 x g for 3 min&#160;in a tissue culture centrifuge. I) Cell pellet. J) Cells (that have been spun down) were resuspended in 1 ml of FBS-supplemented RPMI medium. K) Cells were counted using a hemocytometer. L) 2.5 x 104 cells aliquoted into the microcentrifuge tube and topped off using appropriate media so as to obtain total volume of 50 &#956;l. M) Mix cells from step 1.7 (25,000 cells in 50 &#956;l) with the matrix-containing microcentrifuge tube from step 1.5 in a 1:1 ratio; final volume will be 100 &#956;l. N) Gently plate 100 &#956;l of the matrix: cell mixture from step 8 onto the solidified matrix-coated dish from step 1.2. O) Once the matrix:cell mixture is solidified, add 2 ml of FBS-supplemented RPMI media to the dish. P) Place the dish in the incubator where it will be stored for the remainder of the experiment.
2. Examination of Morphogenic Features of 3D Cultures with Immunofluorescence
<ol>
	<li>Set up an ice tray, cold phosphate buffered solution (PBS), 20% acetone: 80% methanol (fixative solution), and 3% bovine serum albumin (BSA; blocking solution) (Figure 2A) before taking out the dish(es) from the incubator.</li>
	<li>Place the dish(es) on the ice tray and aspirate media, then wash 3x&#160;with 2 ml of cold PBS (Figure 2B).</li>
	<li>Once the last PBS wash is aspirated, add 2 ml of 20% acetone: 80% methanol solution into the dish(es) (Figure 2C) to fix the cells for 20 min at 4 &#176;C (either on ice, or in a refrigerator) (Figure 2D).</li>
	<li>Once the 20 min of fixation is terminated, bring the dishes back at room temperature, aspirate the fixative, and subsequently wash 3x&#160;with PBS. Once the final PBS wash is aspirated, add 2 ml of 3% BSA to the dish(es) to block for at least 30 min at room temperature (Figure 2E).</li>
	<li>During the blocking period prepare dilutions of primary (laminin V 1:100) and secondary antibodies (at appropriate dilution) dissolved in 3% bovine serum albumin (BSA) blocking buffer. Please note that 400-500 &#956;l the &#8216;BSA + primary antibody&#8217; solution should be added directly onto the matrix: cell mixture.</li>
	<li>Once the 30 min of blocking have expired, add the primary antibodies and incubate for at least 1 hr at room temperature (Figure 2F). Please note that no PBS washes should be performed following the 30 min blocking period.</li>
	<li>Upon completion of step 2.6, remove the &#8216;BSA + primary antibody&#8217; solution, and wash 3x&#160;with PBS.</li>
	<li>Add the secondary antibody dissolved in 3% BSA (at appropriate dilution) directly onto the matrix: cell mixture, cover the dishes and incubate for 1 hr at room temperature (Figure 2G).</li>
	<li>Remove the &#8216;BSA + secondary antibody&#8217; solution, and wash 3x&#160;with PBS.</li>
	<li>Add 2 ml of Hoechst 33258 (1:10,000) dissolved in PBS for nuclei staining, and incubate for 5 min under aluminum foil (or cover with an opaque container to limit photobleaching) (Figure 2H).</li>
	<li>Once the 5 min of incubation with Hoechst 33258t have expired, wash the dish(es) 5x&#160;with PBS.</li>
	<li>Add mounting medium directly onto the matrix: cell mixture in the confocal dish and cover with glass coverslip carefully to prevent inducing any disruptions to the integrity of the colonies in the basement membrane matrix:cell mixture (Figure 2I).</li>
	<li>Allow the dish(es) to dry overnight (or 24 hr) at room temperature (Figure 2I). Once dry, the dish(es) can be stored at -20 &#176;C, but it is highly recommended that they should be imaged as quickly as possible.</li>
	<li>Acquire images using a fluorescent microscope with appropriate laser wavelengths of antibodies used (Figures 3D-E).</li>
</ol>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig2highres.jpg" width="500" /> 
Figure 2. Examination of 3D cultures by immunofluorescence. A) A schematic of the experimental setup. B) Dish(es) placed on the ice tray and media aspirated; subsequently cells were washed three times with 2 ml of cold PBS. C) 2 ml of 20% acetone: 80% methanol solution added into the dish(es). D) Fix the cells for 20 min&#160;at 4 &#176;C (either on ice, or in a refrigerator). E) 2 ml of 3% BSA added to the dish(es) to block for at least 30 min&#160;at room temperature. F) Once the 30 min&#160;of blocking have expired, add the primary antibodies and incubate for at least 1 hr at room temperature. G) Secondary antibodies dissolved in 3% BSA (at appropriate dilution) were added directly onto the basement membrane matrix:cell mixture; subsequently dishes covered and incubated for 1 hr at room temperature. H) 2 ml of Hoechst (1:10,000; dissolved in PBS) added to the dishe(es) and cells incubated for 5 min&#160;under aluminum foil. I) Mounting medium added directly onto the matrix: cell mixture in the confocal dish and the dish is covered with glass coverslip. Dish(es) left to dry overnight (or 24 hr) at room temperature.

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We have nothing to disclose.

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 ...

quantification-breast-cancer-cell-invasiveness-using-three

Research

JoVE Journal

Medicine

15.7K ビュー.  University of Western Ontario. この資料は、乳癌細胞の浸潤を定量化するための3次元（3D）のアッセイの使用のための詳細な方法論を提供する。具体的には、細胞が侵入したときに発生する膜の完全性の喪失を検査するためにこのようなアッセイ、定量化、およびデータ分析だけでなく、メソッドを設定するために必要な手順を説明します。 

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

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

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.

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 ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.