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% FBS. More information can be found in the Materials Table.</li>
		<li>Perform a dilution to allow for the seeding of 500-1,000 cells per 20 &#181;l drop of cell culture media.</li>
	</ol>
	</li>
	<li>Acquire 10 cm dish and add 5 ml of sterile PBS to the bottom. Note: This step protects the hanging drops from evaporation. Lower-cost &#8220;bacteriological grade&#8221; dishes can be used in place of cell culture grade dishes, as cells will not contact the culture surface.</li>
	<li>Use a multi-channel pipet to transfer 20 &#181;l droplets of the diluted cell suspension to the inner surface of the lid.
	<ol>
		<li>Pipet 40 drops (5 rows of 8 drops) onto the lid of the 10 cm dish.</li>
	</ol>
	</li>
	<li>Invert the lid and place over the culture dish. Incubate the hanging drop cultures at 37 &#176;C for 72 hr to generate spheroids.
	<ol>
		<li>Flip the lid in a confident, yet controlled manner. Note: Inverting the lid too fast or too slow may cause the droplets to shift. Some cell lines may form spheroids in 48 hr or less, while others may require more than 72 hr to produce compact aggregates.</li>
	</ol>
	</li>
</ol>
2. Embedding of Spheroids into 3D Matrix
<ol>
	<li>Thaw an aliquot of growth factor-reduced basement membrane materials at 4 &#176;C overnight before embedding the spheroids.
	<ol>
		<li>Optional: Layer wells with ECM in advance to prevent potential spheroid interaction with the tissue culture surface.
		<ol>
			<li>Pipet 200 &#181;l of ECM per well on a 24-well plate.</li>
			<li>Tilt the plate to ensure that the ECM covers the entire well surface.</li>
			<li>Carefully remove excess ECM with a pipet.</li>
			<li>Incubate the plate until the wells are dry: 3 hr at room temperature or overnight at 4 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Collect the spheroids by tilting the lid of the tissue culture dish and pooling the media. Transfer the media with the spheroids into a 1.5 ml microcentrifuge tube.</li>
	<li>Allow 10 min for the spheroids to settle at the bottom of the microcentrifuge tube. Spheroids should be visible by eye.</li>
	<li>Mix 100 &#181;l of the basement membrane materials with 100 &#181;l of cold (4 &#176;C) type I collagen in a separate pre-chilled tube. Keep the mixture at 4 &#176;C to prevent either gel from solidifying prematurely.
	<ol>
		<li>Use pre-chilled pipet tips if transferring small volumes.</li>
		<li>Give care when mixing the basement membrane materials and collagen type I to prevent air bubble formation. Note: Air bubbles may hinder imaging later in the protocol if they become embedded in the gel.</li>
	</ol>
	</li>
	<li>Aspirate the spheroids from the 40 &#181;l bottom portion of the microcentrifuge tube, and combine with the basement membrane materials/type I collagen mixture. Be careful to prevent air bubble formation when mixing. Note: The solution containing of 100 &#181;l basement membrane materials, 100 &#181;l collagen type I, and 40 &#181;l of the spheroids will create enough material for 4 independent 3D cultures. This can be scaled up or down.</li>
	<li>Pipet 40 &#181;l drops of the viscous mixture into the centers of wells on a 24-well plate. Keep the plate level to prevent the mixture from running into the side of the well. Note: A single column on the 24-well plate &#8211; 4 wells &#8211; is recommended for each condition to be tested.</li>
	<li>Place the plate into a 37 &#176;C incubator and leave undisturbed for 30 min. The 3D culture will polymerize during this period.</li>
	<li>Slowly submerge the 3D cultures in 1 ml of cell culture media.
	<ol>
		<li>Ensure that the media is warm when adding it into the wells to further promote gel polymerization.</li>
		<li>CRITICAL STEP: Gently add the media to the 3D cultures. Pipetting the media too quickly into the wells can lead to detachment of the 3D cultures from the tissue culture surface.</li>
	</ol>
	</li>
</ol>
3. Monitoring and Analyzing Spheroid Invasion
<ol>
	<li>Image invasion from the spheroids into the surrounding 3D matrix at time-points decided by the investigator. Acquire photographs using an inverted microscope with the 20x objective lens. Note: Ideal time-points will differ depending on the cell line being tested. More invasive cell lines will begin their egress from the spheroids shortly after plating and, therefore, take initial photographs within a couple of hours after plating. Generally, photographs are taken at 0 hr (after plating), 24 hr, and 48 hr. Invasion typically concludes 24 to 48 hr after plating.</li>
	<li>Quantitate invasive ability using image analysis software like Image J.
	<ol>
		<li>Analyze invasion as the cell distance from the edge of the spheroid.
		<ol>
			<li>Use the Straight Draw Tool to mark the radius and/or maximal invasive distance.</li>
			<li>Click &#8220;Analyze&#8221; in the top menu, and then click &#8220;Measure&#8221; to display the length measurement.</li>
		</ol>
		</li>
		<li>Analyze invasion as the total invasive area outside the spheroid.
		<ol>
			<li>Use the Freehand Draw Tool to trace the border of the spheroid and/or total invasive area.</li>
			<li>Click &#8220;Analyze&#8221; in the top menu, and then click &#8220;Measure&#8221; to display the area measurement.</li>
		</ol>
		</li>
		<li>Use a plugin like ROI Manager Tools to create measurements for a stack of images.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

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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32.2K Views.  Georgetown University. 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. 

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

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

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.

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.

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

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...