Name: a cancer cell spheroid assay to assess invasion in a 3d setting
Uploaded: 2015-11-20T15:00:00
Description: Watch this Scientific Journal Video about A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting at JoVE.com

Supported by NIH grants P30 CA051008 and T32 CA009686 (ATR).

1. Generation of Spheroids
<ol>
	<li>Prepare single-cell suspension for hanging drop cultures by detaching adherent cancer cell cultures of ~70% confluence using a PBS wash followed by exposure to 0.05% trypsin-EDTA solution.
	<ol>
		<li>Neutralize the trypsin solution with cell culture media and count the cells using an aliquot of the cell suspension. Note: The specific cell culture media will depend on the cell line being tested. Follow ATCC media recommendations, where the cell culture media is typically DMEM + 10% ...

<ol>
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This study evaluated the performance of a panel of cancer cell lines in a spheroid invasion assay (Table 1). Generally, we find that spheroid formation is enhanced in the more epithelial-looking cell lines, where the presence of cell-cell junctions promotes the formation of a spheroid-like architecture. Known cell lines that have undergone an epithelial-mesenchymal transition, like MDA-MB-231 cells do not form spheroids in the hanging drop culture most likely due to their reduced E-, N-, and P-cadherin e...

It is established that cancer cell motility is predictive of metastatic potential, given that such behavior facilitates invasion through the basement membrane and entry into the circulatory system1. Most research on motility has focused on how cells behave in a two-dimensional (2D) setting, though it is becoming widely recognized that the movement of cells in a three-dimensional (3D) matrix is more representative of how these same cells will actually behave in vivo2. 3D culture systems are increasingly used to study cellular behaviors that range from cell morphology, to growth kinetics and drug sensitivity3. The desire to monitor cancer cell invasion within the context of a 3D milieu has led to the synthesis of previously established techniques involving the generation of 3D cell aggregates (spheroids) via the hanging drop culture method4, followed by embedding these spheroids into a 3D extracellular matrix (ECM) composed of collagen and basement membrane materials5. This method seeks to improve upon these previously established techniques by providing a streamlined approach that can be easily utilized to compare invasion under a variety of experimental conditions.
More traditional ways to assess cell motility in vitro are the scratch-wound assay and the transwell assay6. The former assay depicts cell motility in a 2D setting, and is therefore independent of a variety of features critical for in vivo invasion, e.g. protease activity7. The transwell assay can better model cell invasion when the well inserts are coated with ECM substrates but, only a single parameter, i.e. the appearance of cells on the opposite membrane surface is measured, and many nuances of cell invasion are thus not readily observable. In contrast to these techniques, the cancer cell spheroid invasion assay (Figure 1) allows for the real-time monitoring of cell invasion in a setting that is not only physiologically relevant, but also permits important cell line-specific features to be visualized, such as individual vs. collective cell migration8. This method also affords advantages over standard 3D culture growth assays. The generation of cellular aggregates via the hanging drop method initially constrains cell movement, so that cells will be incentivized to invade after this constraint is lifted. Furthermore, once that constraint is lifted, cell egress will proceed in a uniform direction that can then be conveniently quantitated.
The most popular ECM materials used for cancer cell spheroids assays are Matrigel and type I collagen, where each of these components has important and distinct roles in influencing metastatic behavior. Matrigel is a secreted mixture of proteins produced by Engelbreth-Holm Swarm mouse sarcoma cells, and is enriched in basement membrane proteins such as laminin, entactin, and type IV collagen9. For this reason Matrigel is henceforth referred to as &#8220;basement membrane materials.&#8221; These basement membrane materials provide essential ligands needed for integrin adhesion during cancer cell invasion10 in addition to many other proteins that exert a range of effects on cell behavior11. In comparison, type I collagen, commonly prepared from acid digests of tendons and other dense collagenous structures12, is a much simpler matrix material that serves as a major structural element of the connective tissue and stroma supporting tissues and organs of the body. It has been demonstrated that the physical characteristics of collagen can regulate a number of features of cell motility; for example, the alignment of collagen fibrils at the tumor-stromal interface permits cancer cells to subsequently migrate along those fibrils when invading into the stroma13. In the assay presented here, both type I collagen and basement membrane materials are utilized as tools to study 3D cancer cell-stroma interactions.
The effect of inhibition or stimulation of pathways that control invasion can be monitored after the cells have been embedded in the 3D matrix. Cells can be pretreated during growth in the hanging drops or upon transfer to the 3D culture, depending on whether a lengthy treatment will be required to modulate invasion. For shorter treatments, it is recommended that the drug be mixed with the spheroid suspension after collection, as well as the media that will surround the 3D cultures, to facilitate adequate drug exposure to the cells. Next, normal or tumor-associated stromal cells can be admixed with the matrix material to evaluate their role in modulating tumor cell invasion, or to determine how paracrine and autocrine signaling influences cell behavior. This idea was shown in a study where the coculture of colon cancer and endothelial cells in hanging drops led to a vascular network within the spheroids14. Finally, the ECM constituents can also be altered, as cancer cell invasion is impacted by different substrates15. The method presented below will thus provide a framework for assessing cancer cell invasion under a variety of conditions. In general it was found that not all cell lines will create spheroids in the hanging drops and epithelial-looking cell lines typically form regular spheres.

<table>
	<tbody>
		<tr>
			<td>Matrigel Growth Factor Reduced</td>
			<td>Corning</td>
			<td>CB-40234</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I, Rat Tail, 100 mg</td>
			<td>Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Heat Inactivated</td>
			<td>Omega Scientific</td>
			<td>FB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-treated Dishes</td>
			<td>Corning Incorporated</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well TC-treated Plates</td>
			<td>NEST Biotechnlology</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX-71 Inverted Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and LNCaP cells, which were mantained in RPMI + 10% FBS.</td>
		</tr>
	</tbody>
</table>

a cancer cell spheroid assay to assess invasion in a 3d setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

Using the spheroid invasion assay (Figure 1), a panel of cancer cell lines was tested for their ability to form spheroids, as well as for the amount of cell egress exhibited after implantation in a 3D matrix consisting of basement membrane materials and type I collagen (Table 1). These results demonstrate that not every cancer cell line will create well-formed spheroids, where cell lines possessing an epithelial morphology in vitro tended to produce regular and smooth aggregates...

Watch this Scientific Journal Video about A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting at JoVE.com

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

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