Ovarian cancer is the fifth leading cause of
cancer related deaths in the United States(1). Despite a positive initial response to
therapies, 70 to 90 percent of women with
ovarian cancer develop new
metastases, and the recurrence is often fatal(2). 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 cavity(3) (Figure 1, Movie 1). All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells(4-6) (Figure 2). However, mesothelial cells are absent from underneath peritoneal
tumor masses, as revealed by electron micrograph studies of excised human
tumor tissue sections(3,5-7) (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 cells(8), 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 monolayers(8-16), and was first described in a report showing that ovarian
tumor cells utilize an
integrin -dependent activation of
myosin and
traction force to promote the exclusion of the mesothelial cells from under a
tumor spheroid(17). 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.