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JoVE Encyclopedia of Experiments
Encyclopedia of Experiments: Cancer Research

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



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.

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