Mesothelial Clearance Assay: An In Vitro Technique to Quantify the Invasion Ability of Ovarian Cancer Spheroids into Mesothelial Cell Monolayers

1. Ovarian Cancer Cell Spheroid Formation

1. 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 express fluorescent proteins transiently, or cells can be pre-incubated with a red fluorescent cell tracker dye (Invitrogen).
2. Prior to forming ovarian cancer spheroids, it is necessary to prepare low-adhesion 96-well round-bottom culture dishes. To produce low-adhesion culture plates, add 30 μL poly-HEMA (6mg polyhydroxyethylmethacrylate in 1 ml 95% EtOH) solution to each well of a 96-well cell culture dish. The 96-well plates are incubated in a 37 °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 suspension. [Alternatively, Ultra-Low Attachment culture plates (Corning) can be used.]
3. Next, trypsinize ovarian cancer cells, pellet the cells in a tabletop centrifuge (Heraeus) at 900 RCF for 3 minutes. Aspirate the supernatant and resuspend in 10% Base Medium.
4. Count the cells using a hemocytometer.
5. Adjust the concentration of cells such that there are 100 cells per 50 μL of 10% Base Medium.
6. Add 50 μL of the uniformly suspended diluted cell suspension to each well of the 96-well poly-HEMA coated culture dish.
7. Incubate the 96-well plate in a 37 °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 essential to choose a time before induction of apoptosis.

2. Mesothelial Cell Monolayer Formation

1. In a cell culture hood, pre-coat the wells of a 6-well glass-bottom MatTek dish with fibronectin by adding 2 mL of a 5 μg 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 allows for high-resolution microscopic imaging.
2. GFP-expressing mesothelial cells are cultured in 10% Base Medium. Trypsinize a plate of mesothelial cells, spin down in a tabletop centrifuge (Heraeus) at 900 RPM 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 pre-incubating the cells in a green fluorescent cell tracker dye (Invitrogen).
3. After the 30-minute fibronectin incubation (in step 2.1), wash the wells of the MatTek dish with 2 mL PBS.
4. 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 °C cell culture incubator overnight to allow the mesothelial cells to attach to the dish and form a monolayer.

3. Mesothelial Cell Clearance Assay

1. Use a pipette to collect the ovarian cancer spheroids from the 96-well poly-HEMA coated plate.
2. Aspirate the medium from one well of the 6-well MatTek dish containing a mesothelial cell monolayer. Wash once with 2 mL PBS. Add all 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).
3. 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 (<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.
4. The ovarian cancer cell spheroids will settle to the bottom of the 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.
5. 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 understand the dynamics of intercalation better.