This study investigates the interaction between ovarian cancer spheroids and mesothelial cells during metastasis. The methodology involves imaging these interactions to understand the mechanisms of mesothelial clearance.
During ovarian cancer metastasis, malignant cells detach from the primary tumor site and start circulating within the peritoneal cavity. These cells can then form clusters or spheroids that adhere to the mesothelial cell monolayer lining the peritoneal cavity. As the tumor grows, it clears the underlying mesothelial cells to facilitate invasion.
To model mesothelial clearance, begin by seeding mesothelial cells expressing green fluorescent protein or GFP to a fibronectin-coated glass-bottom culture dish. The fibronectin coating provides a matrix that promotes cell attachment. Incubate the dish to allow mesothelial cells to spread and form a monolayer.
Next, transfer a suspension of ovarian cancer cell spheroids expressing red fluorescent protein onto the mesothelial monolayer. Image the culture for an extended duration using time-lapse fluorescence microscopy. The red-fluorescent spheroids will settle and attach to the green-fluorescent mesothelial monolayer.
The cancer cells exert force on the mesothelial cells, disrupting cell junctions and altering the mesothelial layer integrity. The force also displaces the underlying mesothelial cells, generating a hole. Choose an appropriate GFP filter setting to visualize the resultant hole as a black space in the image. Measure the space to quantify the extent of mesothelial clearance.
To begin this procedure, pre-coat the wells of a 6-well glass-bottom MatTek dish with fibronectin. Add 2 milliliters of fibronectin/PBS solution to each well of the dish and incubate at room temperature for 30 minutes. The green fluorescent protein-expressing mesothelial cells, for forming a mesothelial cell monolayer, are cultured in 10% base medium. Trypsinize a plate of mesothelial cells and spin down in a tabletop centrifuge at 900 RPM for three minutes.
Aspirate the supernatant and resuspend cells in 10% base medium. Adjust to the desired concentration with 10% base medium. When the 30-minute fibronectin incubation of the MatTek dish is complete, wash the wells with 2 milliliters of PBS. Aspirate the PBS and plate 6 times 10 to the fifth mesothelial cells per well in each well of the 6-well MatTek dish. Incubate the MatTek dish in a 37 degrees Celsius cell culture incubator overnight to allow the mesothelial cells to attach to the dish and form a monolayer.
Use a pipette to collect the ovarian cancer spheroids from the 96-well poly-HEMA coated plate. Aspirate the medium from one well of the 6-well MatTek dish containing a mesothelial cell monolayer. Wash once with 2 milliliters of PBS. Add all of the spheroids from the 96-well plate to one well of the MatTek dish. This is approximately three times 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.
Since we want to image only one spheroid per field of view, aggregation of the spheroids must be avoided. Rock the dish gently from side to side to distribute the spheroids evenly across the monolayer before they attach.
Place the MatTek dish on the stage of an inverted wide-field fluorescence microscope capable of performing time-lapse imaging for the duration of at least eight hours. Use a motorized stage to image multiple positions in the dish, with multiple spheroid intercalation events, in a single experiment. 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 plus spheroid/monolayer interactions every ten minutes for eight hours.
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