In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Summary

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.

Abstract

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 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 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 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 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.

Protocol

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 transiently express fluorescent proteins, or cells can be pre-incubate with a red fluorescent cell tracker dye (Invitrogen).
2. Prior to forming of ovarian cancer spheroids, it is necessary to prepare low-adhesion 96 well round bottom ....

Discussion

The "Mesothelial Clearance Assay" presented here uses time-lapse microscopy to monitor the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers, in great spatial and temporal detail. Previously, several groups^8-14 had used endpoint assays to show that ovarian cancer cells attach to and invade into mesothelial cell monolayers. This assay is unique in that it uses fluorescently labeled cells to distinguish tumor cells from mesothelial cells, so that the dynamics of these two cel.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
OVCA433 Ovarian Cancer Cells			Gift from Dr. Dennis Slamon
ZT Mesothelial Cells			Gift from Dr. Tan Ince
Medium 199	GIBCO, by Life Technologies	19950
MCDB105	Cell Applications Inc.	117-500
FBS-heat inactivated	GIBCO, by Life Technologies	10082
Pen-Strep	GIBCO, by Life Technologies	15070
96 well plates	Corning	3799
Polyhydroxyethylmethacrylate (poly-HEMA)	Sigma-Aldrich	192066-25G	For poly-HEMA solution dissolve 6mg poly-HEMA powder in 1ml of 95% EtOH
EtOH	Pharmco-AAPER	111ACS200	Dilute to 95% in dH20
Cell culture hood	Nuaire	NU-425-300
Tissue culture incubator	Thermo Fisher Scientific, Inc.	3110
incubator for poly-HEMA plates	Labline Instruments	Imperial III 305
Tabletop centrifuge	Heraeus Instruments	75003429/01
6 well glass-bottom dish	MatTek Corp.	P06G-1.5-20-F
Fibronectin	Sigma-Aldrich	F1141-1MG
PBS	Cellgro	21-040-CV
Microscope	Nikon Instruments		Ti-E Inverted Motorized Fluorescence time-lapse microscope with integrated Perfect Focus System
Lens	Nikon Instruments		20X-0.75 numerical apeture
Halogen transilluminator	Nikon Instruments		0.52 NA long working distance condenser
Excitation and emission filters	Chroma Technology Corp.		GFP Ex 480/40, Em 525/50 RFP-mCherry Ex 575/50 Em 640/50
Transmitted and Epifluoresce light path	Sutter Instrument Co.		Smart Shutters
Linear-encoded motorized stage	Nikon Instruments
Cooled charged-coupled device camera	Hamamatsu Corp.	ORCA-AG
Microscope incubation chamber with temperature and CO2 control	Custom Made
Vibration isolation table	TMC
NIS-Elements software	Nikon Instruments		Version 3