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 with 127&#176; angles to facilitate liquid entrance and exit (Figure 1B). The channel used in this protocol was reported in a previous publication13.</li>
		<li>Wash the silicon wafer with ultrasonication (500 W/42 kHz) in acetone, isopropanol, and deionized water for 15 min each. Dry the wafer with pressurized nitrogen gas. Heat the silicon wafer on a hot plate at 250 &#176;C for 15 min to completely dry it.</li>
		<li>Spin-coat a layer of SU-8 2075 photoresist (125 &#181;m-thick) onto the silicon wafer with a spinning speed of 1,800 rpm. Bake the photoresist-coated wafer directly on a hot plate at 65 &#176;C and then at 95 &#176;C, to remove excess solvent, for 5 and 25 min, respectively (according to the SU-8 product manual).</li>
		<li>Repeat step 3 and spin-coat another layer of SU-8 2075 photoresist to obtain a final thickness of 250 &#181;m. Bake the photoresist-coated wafer directly on a hot plate at 65 &#176;C for 7 min and then at 95 &#176;C for 30 min. Slowly cool the wafer to room temperature. Do not cool with pressurized air.</li>
		<li>Align and put the photomask directly on top of the wafer. Expose it to UV light for 20 s to crosslink the pattern.</li>
		<li>Bake the wafer with photoresist at 65 &#176;C and 95 &#176;C on a hotplate for 5 and 12 min, respectively, to remove the solvent for polymerization (according to the SU-8 product manual). Afterwards, cool the wafer to room temperature.</li>
		<li>Remove the non-crosslinked photoresist by immersing the wafer in SU-8 developer for 25 min. Rinse the wafer with isopropanol and dry it with pressured air. 
		NOTE: If a milky solution was observed when rinsing with isopropanol, indicating incomplete development, immerse the wafer back into SU-8 developer for a longer development time.</li>
		<li>Place the wafer on a hot plate at 180 &#176;C for 10 min to heal the pattern from potential cracks. Turn off the hot plate and slowly cool down the wafer to room temperature by leaving it on the hot plate.</li>
		<li>Add a few drops of trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane in a vial cap. Put the vial cap in the vacuum desiccator. Place the wafer next to the vial cap to silanize the wafer for 10 min.</li>
	</ol>
	</li>
	<li>PDMS chip fabrication
	<ol>
		<li>Wrap the wafer with aluminum foil to create a holder.</li>
		<li>Mix the polydimethylsiloxane (PDMS) base and curing agent at a 10:1 mass ratio with a centrifugal mixer under the mixing and degas function. Pour the PDMS mixture slowly onto the wafer to a height of around 5 mm. Degas the PDMS under vacuum for 40 min. Cure the PDMS in an oven at 65 &#176;C for 2 h.</li>
		<li>Carefully peel the PDMS slab off the master and trim the PDMS along the channels until it is the size of a glass slide. Punch the PDMS with a 1-mm biopsy punch to create the inlets and outlets of the device.</li>
		<li>Clean the PDMS surface with pressurized air and adhesive tape to remove dust and PDMS debris.</li>
		<li>Place the PDMS slab, with the channel side facing upwards, into a plasma cleaner. Place a clean glass slide into the plasma cleaner alongside the PDMS slab.</li>
		<li>Treat the PDMS and glass slide under air plasma at high RF level for 1 min. Bind the PDMS to the glass slide to create an irreversible covalent bond.</li>
		<li>Place the PDMS chip on a hot plate at 150 &#176;C for 1 h to enhance the bonding strength and to return the hydrophobicity of the PDMS before use.</li>
	</ol>
	</li>
</ol>
2. Seeding the Microfluidic Chip with Primary Peritoneal Mesothelial Cells (HPMCs)
<ol>
	<li>Fibronectin coating of microfluidic channels
	<ol>
		<li>Sterilize the microfluidic channel with 30 &#181;L of 75% ethanol. Remove the ethanol and rinse twice with 30 &#181;L of sterilized phosphate-buffered saline (PBS) using a pipette tip. Thoroughly aspirate PBS in the channels. 
		NOTE: To minimize contamination, the microfluidic cell culture should be carefully conducted under fully aseptic conditions through the use of a laminar flow hood.</li>
		<li>Prepare a 10 &#181;g/mL fibronectin solution in serum-free M199:MCDB105 (1:1) medium. Pipette the solution up and down, with special care to avoid bubble formation. Do not vortex.</li>
		<li>Slowly fill up each channel with 30 &#181;L of fibronectin solution. Seal the channels with tape and incubate the chip overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Seeding HPMCs into the microfluidic channels
	<ol>
		<li>Culture HPMCs in 10% fetal bovine serum (FBS) culture medium (M199:MCDB105 supplemented with 10% FBS and 100 U/mL penicillin/streptomycin) under 5% CO2 at 37 &#176;C until 80% confluence.</li>
		<li>Rinse the HPMCs with 3 mL of PBS and trypsinize with 2 mL of 0.05% trypsin/0.01% EDTA (TE) solution for 30 s. Neutralize with 4 mL of 10% FBS culture medium and spin down the cells at 1,000 x g for 5 min.</li>
		<li>Resuspend the HPMCs in 2 mL of 10% FBS culture medium. Count the cell number with a hemocytometer and adjust the cell concentration to 3.5&#215;106/mL.</li>
		<li>Warm the fibronectin-coated microfluidic chip in a 37 &#176;C incubator for 5 min before use. Completely aspirate the fibronectin solution.</li>
		<li>Slowly pipette 30 &#181;L of HPMC suspension into each channel. Seal the channels with tape and place the devices in the CO2 incubator overnight.</li>
	</ol>
	</li>
</ol>
3. Ovarian Cancer Spheroid Formation and Co-culture
<ol>
	<li>Ovarian cancer spheroid formation
	<ol>
		<li>Maintain the human epithelial ovarian cancer cell line SKOV-3 in 5% FBS culture medium (M199:MCDB105 with 5% FBS and 100 U/mL penicillin/streptomycin) under 5% CO2 at 37 &#176;C before the experiments.</li>
		<li>Dissolve 0.5% agarose in sterilized water by boiling. Dispense 50 &#181;L of agarose solution into each well of 96-well plates. Leave the plates in the hood for 20 min to allow the agarose to solidify; solidified agarose coating the wells can prevent cell adhesion and indicates that the plates are ready for use.</li>
		<li>Rinse the SKOV-3 cells with 3 mL of PBS. Trypsinize with 2 mL of TE solution for 3 min. Neutralize with 4 mL of 5% FBS culture medium and spin down the cells at 1,000 x g for 5 min.</li>
		<li>Count the SKOV-3 cell number with a hemocytometer and resuspend the cells in 5% FBS culture medium at a density of 5,000 cells/mL. Transfer 200 &#181;L of cell suspension into each agarose-coated well and culture under 5% CO2 at 37 &#176;C for 48 h.</li>
	</ol>
	</li>
	<li>Cancer spheroid loading
	<ol>
		<li>Transfer cancer spheroids to a 50-mL centrifuge tube pre-wetted with 1 mL of serum-free medium and spin down at 750 x g for 5 min. 
		NOTE: Pre-wetted pipette tips and centrifuge tubes can prevent undesired spheroid attachment onto the centrifuge tubes.</li>
		<li>Prepare 2.5 &#181;g/mL of 5-chloromethylfluorescein diacetate (CMFDA) solution in serum-free M199:MCDB105 medium. Resuspend cancer spheroids in 1 mL of CMFDA solution and incubate at 37 &#176;C for 30 min. Centrifuge at 750 x g for 5 min.</li>
		<li>Rinse the cancer spheroids with 2 mL of serum-free medium and centrifuge at 750 x g for 5 min. Repeat this step twice to remove the remaining fluorescent dye from the medium. 
		NOTE: The fluorescent signal can be retained in spheroids for more than 96 h.</li>
		<li>Resuspend the cancer spheroids in serum-free M199:MCDB105 medium and count the spheroid number with a hemocytometer. Adjust the concentration to 2,000 spheroids/mL with serum-free medium.</li>
		<li>Prior to spheroid loading, slowly inject 100 &#181;L of serum-free M199:MCDB105 medium to wash away the non-adhered mesothelial cells inside the channels. 
		NOTE: A fast injection, aspiration, or bubble formation will lead to the detachment of the mesothelial monolayer.</li>
		<li>Load 30 &#181;L of fluorescent-labeled spheroid suspension into each channel. Place the microfluidic chip into the CO2 incubator.</li>
	</ol>
	</li>
	<li>Perfusion platform assembly and co-culture
	<ol>
		<li>Attach a syringe loaded with fresh, serum-free M199:MCDB105 medium to a 1 m-long tube.</li>
		<li>Place the syringe on the syringe pump and secure the plunger and the body of the syringe. Install the syringe pump in a horizontal position at the same height as the microfluidic chip inside the incubator. 
		NOTE: The number and size of syringes depend on the number of channels used and the duration of the experiment.</li>
		<li>Quickly infuse the whole tube with medium by pressing and holding the fast forward button until the medium overflows the tubing.</li>
		<li>Run the injection at the desired flow rate. Estimate the wall shear stress using the following equation: 
		<img alt="Equation 1" src="/files/ftp_upload/55337/eq1.jpg" /> 
		where &#964; represents the wall shear stress inside the chamber, &#490; = flow rate, &#956; = viscosity (0.01 g cms-1); h = flow chamber height, and w = flow chamber width.</li>
		<li>Connect the tubing to the inlet of the channel. Place the excess inlet tubing in the incubator to ensure that the medium is warmed before injected into the channels. To facilitate the connection between the tubing and the channel, appropriate connectors can be chosen. NOTE: Beware not to introduce any bubble to the syringe, tubing, and connections.</li>
		<li>Connect the outlet of the channel to a 50-mL conical tube to collect the outflow medium with a short output tube. 
		NOTE: The complete dynamic co-culture system is shown in Figure 1D.</li>
	</ol>
	</li>
</ol>
4. Observation of HPMCs and Cancer Spheroids after Perfusion
<ol>
	<li>Observe the HPMCs and cancer spheroids with a bright-field or fluorescence microscope and take pictures after 1 h of perfusion.</li>
</ol>

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The authors have nothing to disclose.

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 channel, the co-culture time, etc.). Other cell types in the intraperitoneal microenvironment, such as fibroblasts, endothelial cells, or immune cells, can also be included in the system in order to study the essential interactions between ovarian cancer spheroids and nearby cells. Other ECM components, such as laminin, vitronectin, or collagen, can also be used. One weakness of this protocol that may need to be considered is that, while the coupling of nutrient replenishing and shear stress is reminiscent of that seen in vivo, there is no separate control of nutrient replenishing and shear stress in our current system. Moreover, techniques such as on-chip trypsinization combined with fluorescence-activated cell sorting will be needed to separately collect ovarian cancer cells and HPMCs for further molecular studies about different types of cells.
This technique exhibits several advantages when compared with traditional approaches. First, 3D co-culture better mimics the in vivo intercellular communication between ovarian cancer cells and mesothelial cells. Second, cells are cultured under fluidic flow but are not in static condition, generating a cellular response that is potentially representative of the in vivo situation12,13. Furthermore, the fluid flow can be precisely controlled, mimicking the hydrodynamic conditions at different stages of tumor progression.
In summary, the dynamic 3D ovarian cancer-mesothelium co-culture model presented here provides a flexible framework for modeling tumor behavior in the peritoneal cavity. With this model, the interaction between cancer cells and mesothelial cells under a dynamic hydrodynamic condition can be comprehensively analyzed. It can greatly advance our understanding of the underlying mechanisms of metastatic progression and might also facilitate therapeutic development.

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>100 mm</td></tr><tr><td>SU-8 2075 photoresist&amp;nbsp;</td><td>Microchem</td><td></td><td></td></tr><tr><td>SU-8 developer&amp;nbsp;</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&amp;nbsp;</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>0.86 mm (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&amp;nbsp;</td><td>&amp;nbsp;LSP01-2A</td><td></td></tr><tr><td>Medium 199</td><td>Invitrogen</td><td>31100-035</td><td>Add 2.2 g/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&amp;nbsp;</td><td>Invitrogen</td><td>15070-063</td><td></td></tr><tr><td>Trypsin EDTA solution&amp;nbsp;</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&amp;nbsp;</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>CO&lt;sub&gt;2&lt;/sub&gt; 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&amp;nbsp;</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 coating failure. Through non-adherent suspension culture, multicellular spheroids with diameters of approximately 100 &#181;m, which are similar in size to those found in patients&#39; ascites, were generated and labeled with green fluorescent dye, CMFDA. The spheroids were then transferred to HPMC-coated microfluidic channels and remained in suspension. Cell morphologies were observed under the microscope and images were captured (Figure 2B). The fluorescent-labeled cancer spheroids can be easily differentiated from the mesothelial cells under a fluorescence microscope (Figure 2C).
<img alt="Figure 1" src="/files/ftp_upload/55337/55337fig1.jpg" /> 
Figure 1. A Dynamic 3D Peritoneal Microdevice for Modeling Ovarian Cancer Multicellular Spheroid Behaviors. (A) Schematic diagram of a microfluidic chip used to co-culture ovarian cancer spheroids with HPMCs under flow condition. (B) Schematic diagram showing the design of the microfluidic channels. (C) Photograph of a microfluidic chip used in the protocol, with its channels infused with dye solution for clear visualization. Bar = 1 cm. (D) Photograph showing the experimental setup. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55337/55337fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55337/55337fig2.jpg" /> 
Figure 2. Images of the Ovarian Cancer Spheroid-mesothelial Cell Dynamic Co-culture Model. (A) Image of the HPMC monolayer grown in the microfluidic channel. (B) Images of ovarian cancer spheroids in the microfluidic channel coated with peritoneal mesothelial cells under phase contrast microscopy (left panel) or (C) fluorescence microscopy (Green, CMFDA; right panel). Bar = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55337/55337fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

modeling-ovarian-cancer-multicellular-spheroid-behavior-dynamic-3d

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.7K Views.  University of Hong Kong.  Ovariële tumorprogressie te bestuderen in een fysiologisch relevante model multicellulaire sferoïden werden gekweekt in een micro-inrichting onder gesimuleerde fluïdumstroming. Deze dynamische 3D-model emuleert de intraperitoneale omgeving met de cellulaire en mechanische componenten, waar eierstokkanker metastase optreedt. 

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

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

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 ...

Medicine

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Cancer Research

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 ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

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 ...

Establishing a 3D Ex Vivo Model for Ovarian Cancer-Omentum Interaction

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

Author Spotlight: Advanced Ex Vivo Model for Investigating Cancer-Adipose Microenvironment Interaction 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Modeling Ovarian Cancer Metastasis in Peritoneal Cavity Lining: A Method To Establish an In Vitro 3D Model of the Peritoneal Lining

Source: Peters, P. N. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/53541">Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the development of&#160; an in vitro &#160;three-dimensional model of the lining of the peritoneal cavity. This organotypic culture can help to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Source: Peters, P. N. et al. Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity. J. Vis. ...

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

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