We would like to thank the Nikon Imaging Center at Harvard Medical School, specifically Jennifer Waters, Lara Petrak and Wendy Salmon, for training and the use of their timelapse microscopes. We would also like to thank Rosa Ng and Achim Besser for valuable discussions. This work was supported by NIH Grant 5695837 (to M. Iwanicki) and GM064346 to JSB; by a grant from Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (to JSB).

1. Ovarian Cancer Cell Spheroid Formation
<ol>
 <li>RFP-expressing ovarian cancer cells are cultured in 10% Base Medium (a custom cell culture medium containing a 50:50 mixture of 199 and MCDB105, 10% inactivated fetal bovine serum and 1% pen-strep). To express RFP in unlabeled ovarian cancer cells, transfect the cells with a plasmid containing RFP and select for cells expressing RFP. Alternatively, viral vectors can be used to transiently express fluorescent proteins, or cells can be pre-incubate with a red fluorescent cell tracker dye (Invitrogen).</li>
 <li>Prior to forming of ovarian cancer spheroids, it is necessary to prepare low-adhesion 96 well round bottom culture dishes. To produce the low-adhesion culture plates, 30&mu;l poly-HEMA (6mg polyhydroxyethylmethacrylate in 1 ml 95% EtOH) solution is added to each well of a 96 well Corning cell culture dish. The 96 well plates are incubated in a 37&deg;C non-humidified incubator to evaporate the ethanol, leaving a film of poly-HEMA on each well. This poly-HEMA film prevents cells from attaching to the bottom of the well, forcing the cells to grow in suspension18. [Alternatively, Ultra-Low Attachment culture plates (Corning) can be used instead of poly-HEMA coated dishes.]</li>
 <li>After the low-adhesion culture plates are prepared, trypsinize a plate of ovarian cancer cells, pellet the cells in a tabletop centrifuge (Heraeus) at 900 RCF for 3 minutes, aspirate the supernatant and re-suspend in 10% Base Medium.</li>
 <li>Count the cells using a hemocytometer.</li>
 <li>Adjust the concentration of cells such that there are 100 cells per 50&mu;l of 10% Base Medium.</li>
 <li>Add 50&mu;l of the uniformly suspended diluted cell suspension to each well of the 96 well poly-HEMA coated culture dish.</li>
 <li>Incubate the 96 well plate in a 37&deg;C cell culture incubator for 16 hours (this amount of time should be increased or decreased depending on the amount of time it takes for a particular cell line to form multicellular spheroids or desired experimental conditions) to allow the ovarian cancer cells to cluster together, forming a single multicellular spheroid in each well. Some tumor cells can undergo apoptosis during this period, so it is important to choose a time prior to induction of apoptosis.</li>
</ol>
2. Mesothelial Cell Monolayer Formation
<ol>
 <li>In a cell culture hood, pre-coat the wells of a 6 well glass-bottom MatTek dish with fibronectin by adding 2mL of a 5&mu;g fibronectin/ mL PBS solution to each well of the dish and incubating at room temperature for 30 minutes. The optical quality of the glass-bottoms in MatTek dishes allow for high-resolution microscopic imaging.</li>
 <li>GFP-expressing mesothelial cells are cultured in 10% Base Medium. Trypsinize a plate of mesothelial cells, spin down in a tabletop centrifuge (Heraeus) at 900RPM for 3 minutes, aspirate the supernatant, and re-suspend in 10% Base Medium. The mesothelial cells used here were already expressing GFP when they were obtained, but unlabeled mesothelial cells can be produced by transfecting with a plasmid containing GFP cDNA, or preincubating the cells in a green fluorescent cell tracker dye (Invitrogen).</li>
 <li>After the 30-minute fibronectin incubation (in step 2.1), wash the wells of the MatTek dish with 2mL PBS.</li>
 <li>Aspirate the PBS and plate 6 x105 mesothelial cell per well in each well of the 6 well MatTek dish. Incubate the MatTek dish in a 37&deg;C cell culture incubator overnight to allow the mesothelial cells to attach to the dish and form a monolayer.</li>
</ol>
3. Mesothelial Cell Clearance Assay
<ol>
 <li>Use a pipet to collect the ovarian cancer spheroids from the 96 well poly-HEMA coated plate.</li>
 <li>Aspirate the medium from one well of the 6 well MatTek dish containing a mesothelial cell monolayer. Wash once with 2mL PBS. Add all of the spheroids from the 96 well plate to one well of the MatTek dish (~3x the number of spheroids that are going to be imaged to account for spheroids landing on the part of the dish that cannot be imaged).</li>
 <li>Place the MatTek dish on the stage of an inverted widefield fluorescence microscope capable of performing time-lapse imaging for the duration of at least 8 hours. Use a motorized stage to image multiple positions in the dish, with multiple spheroid intercalation events, in a single experiment. We use a Nikon Ti-E Inverted Motorized Widefield Fluorescence time-lapse microscope with integrated Perfect Focus System and low [20x-0.75 numerical aperture (NA)] magnification/NA differential interference contrast (DIC) optics, a Nikon halogen transilluminator with 0.52 NA long working distance (LWD) condenser, Nikon fast (&lt;100-millisecond switching time) excitation and emission filters (GFP Ex 480/40, Em 525/50, RFP-mCherry Ex 575/50 Em 640/50), Sutter fast transmitted and epifluorescence light path Smart Shutters, a Nikon linear-encoded motorized stage, a Hamamatsu ORCA-AG cooled charge-coupled device (CCD) camera, a custom-built microscope incubation chamber with temperature and CO2 control, Nikon NIS-Elements AR software version 3, and a TMC vibration isolation table.</li>
 <li>The ovarian cancer cell spheroids will settle to the bottom of dish and attach to the mesothelial cell monolayer. Collect GFP, RFP and phase images of 20+ spheroid/monolayer interactions, every 10 minutes, for 8 hours.</li>
 <li>The RFP-expressing ovarian cancer cell spheroids will invade into the GFP-expressing mesothelial cell monolayer creating a hole in the monolayer. After 8 hours, measure the sizes of the holes by tracing the black holes in the GFP images using Elements software (or another suitable software such as image J) . Normalize the hole size to the initial spheroid size by dividing the hole size at 8 hours by the size of the spheroid in the corresponding RFP image at time zero. In this example, the hole size was only measured once, but it can be measured multiple times throughout the eight hour experiment to better understand the dynamics of intercalation.</li>
</ol>
4. Representative Results
In this example, we compared the mesothelial clearance ability of OVCA433 ovarian cancer cell spheroids that have attenuated expression of talin-1 to control OVCA433 spheroids. OVCA433 spheroids from each group were added to a MatTek dish containing ZT mesothelial cell monolayers. Six spheroids from each group were imaged every 10 minutes for eight hours (Figure 4, Movie 3, Movie 4). The holes produced in the monolayer by the spreading spheroids were measured and six positions from each group were averaged. Figure 4 shows that the average clearance area created by talin 1 knockdown spheroids was significantly smaller than the average area created by control spheroids, suggesting that talin is required for mesothelial clearance by OVCA433 ovarian cancer spheroids.
<img alt="Figure 1" src="/files/ftp_upload/3888/3888fig1.jpg" /> 
Figure 1. Ovarian Cancer Metastasis. Primary ovarian tumors develop either from the ovarian surface epithelium or fallopian tubes. Tumor cells/ clusters break off from the primary tumor and collect in the peritoneal cavity. Tumor cells can then aggregate to form multicellular spheroids. Spheroids then attach to the mesothelial cell monolayers lining the peritoneal cavity. The mesothelial cells are excluded from underneath the attached ovarian cancer spheroid, allowing the spheroids to gain access to the underlying basement membrane.
Movie 1. Ovarian Cancer Metastasis. <a href="https://www.jove.com/files/ftp_upload/3888/Movie_1_Lower_Quality.mp4">Click here to view movie</a>.
<img alt="Figure 2" src="/files/ftp_upload/3888/3888fig2.jpg" /> 
Figure 2. Mesothelial cells line the surface of human peritoneal tissue and are excluded from underneath ovarian cancer cell implants.
<img alt="Figure 3" src="/files/ftp_upload/3888/3888fig3.jpg" /> 
Figure 3. Mesothelial Clearance Assay. Ovarian cancer spheroids are formed by incubating 100 RFP-expressing ovarian cancer cells per well in a poly-HEMA coated 96 well round bottom culture dish at 37&deg;C for 16 hours. Poly-HEMA prevents the cells from attaching to the culture dish, allowing the cells to remain in suspension and adhere to one another to form a single cluster per well. Mesothelial cell monolayers are prepared by plating 6x105 mesothelial cells per well in a fibronectin coated 6 well MatTek dish and incubating the plate at 37&deg;C for 16 hours. The spheroids are then transferred to the MatTek dish with the mesothelial monolayer and the two cell populations are imaged every 10 minutes for 8 hours using a Nikon Ti-E Inverted Motorized Widefield Fluorescence time-lapse microscope and Elements software.
Movie 2. Mesothelial Clearance Assay. <a href="https://www.jove.com/files/ftp_upload/3888/Movie_2_lower_quality.mp4">Click here to view movie</a>.
<img alt="Figure 4" src="/files/ftp_upload/3888/3888fig4.jpg" /> 
Figure 4. Attenuation of talin 1 expression in OVCA433 spheroids decreases mesothelial clearance ability. OVCA433 spheroids (red) with and without attenuated expression of talin 1 were allowed to attach to and invade into a ZT mesothelial monolayer (green). The two cell populations were imaged every 10 minutes for 8 hours using a Nikon Ti-E Inverted Motorized Widefield Fluorescence time-lapse microscope and elements software. The graph shows that talin 1 attenuation significantly decreases mesothelial cell clearance (Quantile plot with green bars at the means).
Movie 3. Control OVCA433 spheroids (red) invading into a mesothelial monolayer (green). <a href="https://www.jove.com/files/ftp_upload/3888/Movie_3.mov">Click here to view movie</a>.
Movie 4. Attenuation of talin 1 expression in OVCA433 spheroids (red) decreases mesothelial (green) clearance ability. <a href="https://www.jove.com/files/ftp_upload/3888/Movie_4.mov">Click here to view movie</a>.

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We have nothing to disclose.

The "Mesothelial Clearance Assay" presented here uses time-lapse microscopy to monitor the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers, in great spatial and temporal detail. Previously, several groups8-14 had used endpoint assays to show that ovarian cancer cells attach to and invade into mesothelial cell monolayers. This assay is unique in that it uses fluorescently labeled cells to distinguish tumor cells from mesothelial cells, so that the dynamics of these two cell populations can be monitored throughout the assay. The process of intercalation can be visualized in real time and the rate of mesothelial clearance can be quantitatively measured over time. The use of timelapse microscopy allows one to closely monitor the dynamics of the interaction between the two cell populations under different experimental conditions. Additionally, a small percentage of either the mesothelial cells or the ovarian cancer cells can be labeled with a third fluorescent marker to monitor the dynamics of individual cells within the population. By tracking individual cells over time, the directionality and rate of migration can be calculated. To perform higher resolution analyses of mesothelial clearance, total internal reflection fluorescence (TIRF) microscopy can be used. If focal adhesions are labeled in the mesothelial cells, the dissociation of mesothelial adhesions by protrusive extensions of the tumor cells can be monitored, as described in our previous publication17. 
This assay can be used to compare the invasion ability of ovarian cancer cell spheroids that have been genetically or pharmacologically modified to elucidate the molecular mechanisms by which ovarian cancer cell spheroids clear the mesothelial monolayer or to identify small molecule inhibitors of the process. Furthermore, the assay requires a very small number of ovarian cancer cells, so primary tumor cells from fluid exudates can be used (see limitations below) if labeled by preincubating the cells with cytotracker dyes (Invitrogen). The assay is also amenable to high throughput analysis. To perform high throughput genetic or pharmacological studies, the ovarian cancer cells can be transfected with different siRNA vectors or treated with different pharmacological inhibitors in each well of the 96-well plate. Mesothelial cell monolayers can be plated in 96 well glass-bottom culture dishes, and the spheroids can be transferred 1:1 from the poly-HEMA coated plates to the monolayer-containing plates. All of these steps can be optimized for use with screening robots so that hundreds of siRNAs or inhibitors can screened at one time. 
One of the strengths of this assay is to be able to model the force-dependent intercalation of ovarian cancer cells into the mesothelial monolayer. Our lab used traction force microscopy (TFM) to determine whether mechanical force regulates mesothelial clearance17. We found that overexpression of &alpha;5 integrin increased the contractility of cells plated on a fibronectin-coated substrate, while RNAi-mediated knockdown of talin, or myosin II decreased cell contractility17. Since downregulation of &alpha;5 integrin, talin, or myosin II in the ovarian cancer cell spheroids also decreased mesothelial clearance, our TFM measurements support the idea that mesothelial clearance in an event dependent on cellular contractile forces, in which cells with higher contractile forces caused greater mesothelial clearance. Therefore, the mesothelial clearance assay can be used to further understand intercalation events, associated with ovarian spheroid metastasis, that are dependent on mechanical forces. 
This assay has a few limitations to consider. First, in order to form the multicellular spheroids, the cells must be cultured in suspension for at least 6 hours. If the cells are unable to survive without matrix contact their clearance ability will be compromised. Second, it is advantageous to use ovarian cancer cells that form uniform, compact multicellular spheroids. If the ovarian cancer cells only form loose clusters, they may break apart in the transfer from the polyHEMA-coated plate to the dish containing the mesothelial monolayer, creating spheroids of different shapes and sizes that will add variability to the data. Third, if the cells used are heterogeneous, this will add additional variability to the sizes of holes created in the monolayer. It is important to use multiple replicate wells (10-20/sample) due to the variability in extent of intercalation after eight hours. In the assays described here, well-established ovarian cancer (OVCA433) and mesothelial (ZT) cell lines were used. To make this assay more clinically relevant, primary ovarian cancer cells, from the ascites fluid of patients, can be used. It would be interesting to determine if in vitro mesothelial cell clearance ability correlates with clinical outcome. The limitations above are particularly important to consider when using primary samples, as the number of primary cells available is a limiting factor. Additionally, it is important to check the integrity of the mesothelial monolayer before performing this assay. The mesothelial monolayer can be fixed and stained for cell-cell junction proteins to ensure that mesothelial cell junctions are intact.
Finally, the mesothelial clearance assay can be easily modified to answer specific experimental questions. Here, we used fibronectin as the ECM component that allows the ovarian and mesothelial cells to adhere to the glass-bottom culture dishes, however, other ECM components can be used including collagen and laminin. Furthermore, other cell types that are found under the basement membrane, including fibroblasts, can be added to this experimental system, to assess the role of these cell types in mesothelial clearance9,19,20. Lastly, interactions of other types of tumor cells (e.g. pancreatic, breast, etc) with mesothelial cells can also be modeled using this assay. And it is feasible to study interactions between cancer cells and an endothelial monolayer, using this assay, to mimic intravasation or extravasation (Similar assays have been described in: 15,16,21-27 ).

<table border="1">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>OVCA433 Ovarian Cancer Cells</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Gift from Dr. Dennis Slamon</td>
 </tr>
 <tr>
 <td>ZT Mesothelial Cells</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Gift from Dr. Tan Ince </td>
 </tr>
 <tr>
 <td>Medium 199</td>
 <td>Gibco</td>
 <td>19950</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MCDB105</td>
 <td>Cell Applications Inc.</td>
 <td>117-500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FBS-heat inactivated</td>
 <td>Gibco</td>
 <td>10082</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pen-Strep</td>
 <td>Gibco</td>
 <td>15070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>96 well plates</td>
 <td>Corning Costar</td>
 <td>3799</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polyhydroxyethylmethacrylate (poly-HEMA)</td>
 <td>Sigma Aldrich</td>
 <td>192066-25G</td>
 <td>For poly-HEMA solution dissolve 6mg poly-HEMA powder in 1ml of 95% EtOH</td>
 </tr>
 <tr>
 <td>EtOH</td>
 <td>Pharmco-aaper</td>
 <td>111ACS200</td>
 <td>Dilute to 95% in dH20</td>
 </tr>
 <tr>
 <td>Cell culture hood</td>
 <td>Nuaire</td>
 <td>NU-425-300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tissue culture incubator</td>
 <td>Thermo Scientific</td>
 <td>3110</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>incubator for poly-HEMA plates</td>
 <td>Labline Instruments</td>
 <td>Imperial III 305</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tabletop centrifuge</td>
 <td>Heraeus</td>
 <td>75003429/01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>6 well glass-bottom dish</td>
 <td>MatTek corp.</td>
 <td>P06G-1.5-20-F</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fibronectin</td>
 <td>Sigma</td>
 <td>F1141-1MG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Cellgro</td>
 <td>21-040-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Timelapse Microscope:</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>Ti-E Inverted Motorized Fluorescence time-lapse microscope with integrated Perfect Focus System</td>
 </tr>
 <tr>
 <td>Lens</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>20X-0.75 numerical apeture</td>
 </tr>
 <tr>
 <td>Halogen transilluminator</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>0.52 NA long working distance condenser</td>
 </tr>
 <tr>
 <td>Excitation and emission filters</td>
 <td>Chroma single pass filters in Nikon housing</td>
 <td>&nbsp;</td>
 <td>GFP Ex 480/40, Em 525/50 
RFP-mCherry Ex 575/50 Em 640/50</td>
 </tr>
 <tr>
 <td>Transmitted and Epifluoresce light path </td>
 <td>Sutter</td>
 <td>&nbsp;</td>
 <td>Smart Shutters</td>
 </tr>
 <tr>
 <td>Linear-encoded motorized stage</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cooled charged-coupled device camera</td>
 <td>Hamamatsu</td>
 <td>ORCA-AG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscope incubation chamber with temperature and CO2 control</td>
 <td>custom-built</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vibration isolation table</td>
 <td>TMC</td>
 <td></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NIS-Elements software</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>Version 3</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

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.

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 ...

in-vitro-mesothelial-clearance-assay-that-models-early-steps-ovarian

Universidad San Sebastian

Research

JoVE Journal

Medicine

14.4K Views.  Harvard Medical School. 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.

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

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

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 ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

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 ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

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 ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...