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 fluorescen...

<ol>
	<li>Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CA Cancer J. Clin. 59, 225-249 (2009).</li><li>Ries, L. G., Melbert, D., Krapcho, M., Stinchcomb, D. G., Howlader, N., Horner, M. J., Mariotto, A., Miller, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SEER Cancer Statistics Review, 1975-2005. , (2007).</li><li>Burleson, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+carcinoma+ascites+spheroids+adhere+to+extracellular+matrix+components+and+mesothelial+cell+monolayers.">Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers.</a> Gynecol. Oncol. 93, 170-181 (2004).</li><li>Birbeck, M. S., Wheatley, D. 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Pathol. 154, 1525-1537 (1999).</li><li>Leroy-Dudal, J., Heyman, L., Gauduchon, P., Carreiras, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+human+ovarian+adenocarcinoma+IGROV1+cells+to+endothelial+cells+is+partly+mediated+by+the+alphav+integrins-vitronectin+adhesive+system+and+induces+an+alteration+of+endothelial+integrity.">Adhesion of human ovarian adenocarcinoma IGROV1 cells to endothelial cells is partly mediated by the alphav integrins-vitronectin adhesive system and induces an alteration of endothelial integrity.</a> Cell. Biol. Int. 29, 482-488 (2005).</li><li>Leroy-Dudal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmigration+of+human+ovarian+adenocarcinoma+cells+through+endothelial+extracellular+matrix+involves+alphav+integrins+and+the+participation+of+MMP2.">Transmigration of human ovarian adenocarcinoma cells through endothelial extracellular matrix involves alphav integrins and the participation of MMP2.</a> Int. J. Cancer. 114, 531-543 (2005).</li><li>Iwanicki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+spheroids+use+myosin-generated+force+to+clear+the+mesothelium.">Ovarian cancer spheroids use myosin-generated force to clear the mesothelium.</a> Cancer Discovery. 1, 144-157 (2011).</li><li>Folkman, J., Moscona, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cell+shape+in+growth+control.">Role of cell shape in growth control.</a> Nature. 273, 345-349 (1978).</li><li>Gregoire, L., Munkarah, A., Rabah, R., Morris, R. T., Lancaster, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+human+ovarian+surface+epithelial+cells:+a+potential+model+for+ovarian+carcinogenesis.">Organotypic culture of human ovarian surface epithelial cells: a potential model for ovarian carcinogenesis.</a> In Vitro Cell Dev. Biol. Anim. 34, 636-639 (1998).</li><li>Roberts, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+molecular+and+cellular+events+during+neoplastic+progression:+a+mouse+syngeneic+ovarian+cancer+model.">Sequential molecular and cellular events during neoplastic progression: a mouse syngeneic ovarian cancer model.</a> Neoplasia. 7, 944-956 (2005).</li><li>Okada, T., Okuno, H., Mitsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+in+vitro+assay+system+for+transendothelial+tumor+cell+invasion:+significance+of+E-selectin+and+alpha+3+integrin+in+the+transendothelial+invasion+by+HT1080+fibrosarcoma+cells.">A novel in vitro assay system for transendothelial tumor cell invasion: significance of E-selectin and alpha 3 integrin in the transendothelial invasion by HT1080 fibrosarcoma cells.</a> Clin. Exp. 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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 cel...

<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

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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.