Mesenchymal gene program–expressing ovarian cancer spheroids exhibit enhanced mesothelial clearance
Rachel A. Davidowitz, … , Ronny Drapkin, Joan S. Brugge
Rachel A. Davidowitz, … , Ronny Drapkin, Joan S. Brugge
Published April 24, 2014
Citation Information: J Clin Invest. 2014;124(6):2611-2625. https://doi.org/10.1172/JCI69815.
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Research Article

Mesenchymal gene program–expressing ovarian cancer spheroids exhibit enhanced mesothelial clearance

Abstract

Metastatic dissemination of ovarian tumors involves the invasion of tumor cell clusters into the mesothelial cell lining of peritoneal cavity organs; however, the tumor-specific factors that allow ovarian cancer cells to spread are unclear. We used an in vitro assay that models the initial step of ovarian cancer metastasis, clearance of the mesothelial cell layer, to examine the clearance ability of a large panel of both established and primary ovarian tumor cells. Comparison of the gene and protein expression profiles of clearance-competent and clearance-incompetent cells revealed that mesenchymal genes are enriched in tumor populations that display strong clearance activity, while epithelial genes are enriched in those with weak or undetectable activity. Overexpression of transcription factors SNAI1, TWIST1, and ZEB1, which regulate the epithelial-to-mesenchymal transition (EMT), promoted mesothelial clearance in cell lines with weak activity, while knockdown of the EMT-regulatory transcription factors TWIST1 and ZEB1 attenuated mesothelial clearance in ovarian cancer cell lines with strong activity. These findings provide important insights into the mechanisms associated with metastatic progression of ovarian cancer and suggest that inhibiting pathways that drive mesenchymal programs may suppress tumor cell invasion of peritoneal tissues.

Authors

Rachel A. Davidowitz, Laura M. Selfors, Marcin P. Iwanicki, Kevin M. Elias, Alison Karst, Huiying Piao, Tan A. Ince, Michael G. Drage, Judy Dering, Gottfried E. Konecny, Ursula Matulonis, Gordon B. Mills, Dennis J. Slamon, Ronny Drapkin, Joan S. Brugge

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Figure 1

Ovarian cancer cell line spheroids display differential clearance ability that correlates with epithelial and mesenchymal marker expression.

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Ovarian cancer cell line spheroids display differential clearance abilit...
(A) Representative images from mesothelial clearance assays of 2 clearance-competent or clearance-incompetent ovarian cancer cell lines. The extent of clearance of a ZT mesothelial monolayer (green) by OVCA433 or CAOV3 ovarian cancer spheroids (red) was imaged at 0, 4, and 8 hours after coincubation. (B) Quantification of clearance by ovarian tumor cell lines. Clearance area was measured in 20 established ovarian cancer cell lines by coculturing preformed multicellular spheroids with ZT mesothelial cell monolayers. After 8 hours of coincubation, the negative space created in the mesothelial monolayer by the ovarian cancer spheroid was measured and divided by the initial size of the ovarian cancer spheroid at time 0 to determine the normalized clearance area. Cell lines with a normalized clearance area >1 were classified as clearance competent and <1 were classified as clearance incompetent. >10 spheroids scored over 2 replicates. (C) Analysis of enrichment of mRNAs associated with EMT. Heat map showing mRNA expression of genes associated with the Taube EMT core signature and 3 additional transcription factors that are significantly (P < 0.05) differentially expressed in the clearance-competent and clearance-incompetent ovarian tumor cell lines. Ovarian cancer cell line data in the left column and manipulations to HMLE cells from Taube et al. (25) in the right column. Both data sets were log2 transformed for visualization. (D) Western blot analysis of E-cadherin and vimentin expression in the 20 established ovarian cancer cell lines. (E and F) Average (E) E-cadherin or (F) vimentin protein expression levels in clearance-competent and clearance-incompetent cell lines measured by densitometry:. Error bars denote SEM. *P < 0.05, Student’s t test. Scale bar: 100 μm.