Name: modeling ovarian cancer multicellular spheroid behavior in a dynamic 3d peritoneal microdevice
Uploaded: 2017-02-18T15:00:00
Description: Watch this Scientific Journal Video about Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice at JoVE.com

This work was supported by Hong Kong Research Grant Council (grants 17122014, C1013-15G, 719813E, and 17304514). A. S. T. Wong is a recipient of the Croucher Senior Research Fellowship.

1. Microfluidic Device Design and Fabrication
<ol>
	<li>Microfluidic master design
	<ol>
		<li>Design and draw the microfluidic channel pattern with any computer-aided design (CAD) software. 
		NOTE: Normally, the CAD drawing can be sent to a photomask company to produce the photomask. The microfluidic design consists of three identical parallel channels, each with the following dimensions: 4 mm &#215; 25 mm &#215; 250 &#181;m (width &#215; length &#215; height) and set 2 mm apart. Both channel ends were designed ...

<ol>
	<li>Jemal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cancer+statistics.">Global cancer statistics.</a> CA: Cancer J. Clin. 61, 69-90 (2011).</li><li>Burleson, K. M., et al. <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>Chau, W. K., Ip, C. K., Mak, A. S., Lai, H. C., Wong, A. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+tumor+vasculature:+An+emerging+concept+in+antiangiogenic+therapy.">Normalization of tumor vasculature: An emerging concept in antiangiogenic therapy.</a> Science. 307, 58-62 (2005).</li><li>Chang, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+cycle+arrest+induced+by+shear+stress:+Roles+of+integrins+and+Smad.">Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad.</a> Proc. Natl. Acad. Sci. USA. 105, 3927-3932 (2008).</li><li>Rutkowski, J. M., Swartz, M. 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+driving+force+for+change:+interstitial+flow+as+a+morphoregulator.">A driving force for change: interstitial flow as a morphoregulator.</a> Trends Cell Biol. 17, 44-50 (2007).</li><li>Rizvi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+induces+epithelial-mesenchymal+transition,+cellular+heterogeneity+and+biomarker+modulation+in+3D+ovarian+cancer+nodules.">Flow induces epithelial-mesenchymal transition, cellular heterogeneity and biomarker modulation in 3D ovarian cancer nodules.</a> Proc. Natl. Acad. Sci. USA. 110, E1974-E1983 (2013).</li><li>Ip, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stemness+and+chemoresistance+in+epithelial+ovarian+carcinoma+cells+under+shear+stress.">Stemness and chemoresistance in epithelial ovarian carcinoma cells under shear stress.</a> Sci. Rep. 6, 26788 (2016).</li><li>Avraham-Chakim, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid-flow+induced+wall+shear+stress+and+epithelial+ovarian+cancer+peritoneal+spreading.">Fluid-flow induced wall shear stress and epithelial ovarian cancer peritoneal spreading.</a> PloS one. 8, e60965 (2013).</li><li>Burkhalter, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peritoneal+mechanobiology+and+metastatic+success+in+epithelial+ovarian+cancer.">Peritoneal mechanobiology and metastatic success in epithelial ovarian cancer.</a> Faseb Journal. 26, (2012).</li><li>Lane, W. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel-plate+flow+chamber+and+continuous+flow+circuit+to+evaluate+endothelial+progenitor+cells+under+laminar+flow+shear+stress.">Parallel-plate flow chamber and continuous flow circuit to evaluate endothelial progenitor cells under laminar flow shear stress.</a> Journal of visualized experiments : JoVE. , (2012).</li><li>Botta, G. P., Manley, P., Miller, S., Lelkes, P. 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This assay offers a flexible and physiologically relevant model that can be incorporated with various biochemical and cell-based assays, including, but not limited to, adhesion assays, mesothelial clearance assays, and drug screening. It can be applied to the evaluation of the effect of the intraperitoneal microenvironment on cancer progression. However, several experimental conditions might need to be optimized, depending on the aims of the project (e.g., the number of HPMCs and cancer spheroids seeded per chan...

Ovarian cancer is the most lethal gynecological cancer and is characterized by widespread peritoneal dissemination and the formation of malignant ascites1. This extensive peritoneal metastasis represents a major clinical challenge and is associated with poor clinical outcomes. Unlike most solid carcinomas that metastasize via the blood, ovarian cancer primarily disseminates within the peritoneal cavity. Tumor cells exist as multicellular aggregates/spheroids during the process of metastasis2. The fact that suspension culture can enrich ovarian cancer stem/tumor-initiating cells further suggests that these spheroids may be associated with both tumor aggressiveness and enhanced chemoresistance3,4. There are differences in drug response between 2D and 3D cultures, which presumably have different molecular mechanisms5.
The essential interaction with the mesothelium constructs the primary microenvironment for ovarian tumor progression. These mesothelial cells lie on an extracellular matrix (ECM), where fibronectin is a ubiquitous constituent. A link between the increased expression of mesothelial cell-derived fibronectin and tumor progression has been shown. Fibronectin is abundantly present in malignant ascites6,7. Ovarian cancer cells are also able to induce the secretion of fibronectin from mesothelial cells in order to promote early ovarian cancer metastasis8.
Emerging evidence shows that mechanical stimuli, including shear stress, can modulate cell morphology, gene expression, and, thus, the phenotypes of tumor cells9,10,11. As malignant ascites develop and accumulate during tumor progression, ovarian tumor cells are exposed to fluid flow and the resulting shear stress. A number of groups, ours included, have shown the impact of shear stress on ovarian cancer progression, including cytoskeleton modifications, epithelial-to-mesenchymal transitions, and cancer stemness12,13,14,15. Thus, a physiologically relevant microenvironment is important for the investigation of tumor peritoneal metastasis. However, current in vitro hydrodynamic culture systems have limitations on mimicking and controlling a constant, low, physiologically relevant shear stress16,17,18,19. Conventional in vitro approaches focusing on either the cellular or mechanical environment are still limited in mimicking the complexity of the intraperitoneal microenvironment with proper physiological relevance.
Here, in order to engineer a new model of the peritoneum to overcome the limitations of conventional strategies and to advance the study of the intraperitoneal compartment in cancer metastasis, a 3D microfluidic-based platform with controlled fluid flow was designed. In this model, ovarian cancer spheroids were co-cultured with primary human peritoneal mesothelial cells in microfluidic chips under continuous fluidic flow (Figure1A). The mesothelial cells were plated on fibronectin. Non-adherent ovarian cancer spheroids were seeded into microfluidic channels with continuous flow medium perfused by a syringe pump. Both the 3D environment and the dynamic mechanical forces are very important factors of the metastatic cascade. This platform can be used for investigating the intraperitoneal microenvironment in terms of complex cellular and co-culture interactions, as well as with regard to dynamic mechanical cues.

<table>
	<tbody>
		<tr>
			<td>Silicon wafer</td>
			<td>University wafer</td>
			<td>#1196</td>
			<td>100mm</td>
		</tr>
		<tr>
			<td>SU-8 2075 photoresist&#160;</td>
			<td>Microchem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer&#160;</td>
			<td>Microchem</td>
			<td>108-65-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane</td>
			<td>Sigma</td>
			<td>448931</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>Dow Corning</td>
			<td>1673921</td>
			<td>Polydimethylsiloxane (PDMS) + curing agent kit</td>
		</tr>
		<tr>
			<td>Biopsy punch&#160;</td>
			<td>Miltex</td>
			<td>33-31AA</td>
			<td>1 mm diameter</td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>SCI</td>
			<td>BB31695-PE/5</td>
			<td>0.86mm (inner diameter)</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Terumo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Longer precision pump&#160;</td>
			<td>&#160;LSP01-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Invitrogen</td>
			<td>31100-035</td>
			<td>Add 2.2g/L sodium bicarbonate</td>
		</tr>
		<tr>
			<td>MCDB 105 Medium</td>
			<td>Sigma</td>
			<td>M6395</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30068.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin&#160;</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA solution&#160;</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>0.05% Trypsin -0.01% EDTA, phenol red</td>
		</tr>
		<tr>
			<td>Fibronectin human</td>
			<td>BD</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Invitrogen</td>
			<td>15510-027</td>
			<td></td>
		</tr>
		<tr>
			<td>5-chloromethylfluorescein diacetate</td>
			<td>Life technologies</td>
			<td>C7025</td>
			<td>Green CMFDA</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>SANYO</td>
			<td>MCO-18AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hitachi</td>
			<td>CT15RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Model: 80i or ECLIPSE Ti; software: SPOT</td>
		</tr>
		<tr>
			<td>SKOV-3&#160;</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. N Auersperg (University of British Columbia)</td>
		</tr>
	</tbody>
</table>

modeling ovarian cancer multicellular spheroid behavior in a dynamic 3d peritoneal microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

Using this protocol, a microfluidic platform was set up to model ovarian cancer spheroids with mesothelial cells under hydrodynamic conditions. Primary human peritoneal mesothelial cells were cultured in the microdevice for 16 h and observed under a bright-field microscope. As shown in Figure 2A, the channel bottom was successfully covered with a monolayer of HPMCs. It is important to note that bubble formation during fibronectin or HPMC patterning will lead to channel co...

Watch this Scientific Journal Video about Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice at JoVE.com

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

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