Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion
Hilary A. Kenny, … , David Bowtell, Ernst Lengyel
Hilary A. Kenny, … , David Bowtell, Ernst Lengyel
Published September 9, 2014
Citation Information: J Clin Invest. 2014;124(10):4614-4628. https://doi.org/10.1172/JCI74778.
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Research Article

Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion

Abstract

Ovarian cancer (OvCa) metastasizes to organs in the abdominal cavity, such as the omentum, which are covered by a single layer of mesothelial cells. Mesothelial cells are generally thought to be “bystanders” to the metastatic process and simply displaced by OvCa cells to access the submesothelial extracellular matrix. Here, using organotypic 3D cultures, we found that primary human mesothelial cells secrete fibronectin in the presence of OvCa cells. Moreover, we evaluated the tumor stroma of 108 human omental metastases and determined that fibronectin was consistently overexpressed in these patients. Blocking fibronectin production in primary mesothelial cells in vitro or in murine models, either genetically (fibronectin 1 floxed mouse model) or via siRNA, decreased adhesion, invasion, proliferation, and metastasis of OvCa cells. Using a coculture model, we determined that OvCa cells secrete TGF-β1, which in turn activates a TGF-β receptor/RAC1/SMAD-dependent signaling pathway in the mesothelial cells that promotes a mesenchymal phenotype and transcriptional upregulation of fibronectin. Additionally, blocking α5 or β1 integrin function with antibodies reduced metastasis in an orthotopic preclinical model of OvCa metastasis. These findings indicate that cancer-associated mesothelial cells promote colonization during the initial steps of OvCa metastasis and suggest that mesothelial cells actively contribute to metastasis.

Authors

Hilary A. Kenny, Chun-Yi Chiang, Erin A. White, Elizabeth M. Schryver, Mohammed Habis, Iris L. Romero, Andras Ladanyi, Carla V. Penicka, Joshy George, Karl Matlin, Anthony Montag, Kristen Wroblewski, S. Diane Yamada, Andrew P. Mazar, David Bowtell, Ernst Lengyel

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Figure 3

OvCa cells stimulate fibronectin expression in mesothelial cells.

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OvCa cells stimulate fibronectin expression in mesothelial cells. (A) Le...
(A) Left: Coculture of fluorescently labeled OvCa cells and primary human mesothelial cells, followed by FACS. Middle: Immunoblot for fibronectin protein expression in cell lysates of primary human mesothelial cells grown on plastic or cocultured (48 hours) with the indicated OvCa cells, followed by separation using FACS. Right: Immunoblot for secreted levels of fibronectin (48 hours) in culture of SKOV3ip1 or primary human mesothelial cells on plastic, and after coculture. (B) IF analysis of fibronectin production (green) and fibronectin matrix secretion (red) in mesothelial cells cultured on plastic or cocultured with OvCa cells. (C) Left: FN1 mRNA expression. SKOV3ip1 or primary human mesothelial cells were cultured either on plastic or cocultured together (24 hours), followed by separation using FACS. mRNA was isolated, and qRT-PCR was performed using FN1-specific probes. Right: Primary human mesothelial cells were transfected with a luciferase reporter construct containing the full-length (1.2 kb) FN1 promoter (48 hours) and cultured on plastic or with SKOV3ip1 cells (24 hours). (D) Left: Immunoblot for fibronectin expression in primary human mesothelial cells after treatment with CM from SKOV3ip1 cells. Right: qRT-PCR analysis of FN1 mRNA expression in primary human mesothelial cells grown on plastic stimulated with or without SKOV3ip1 CM for 24 and 48 hours, or cocultured with SKOV3ip1 cells for 24 hours followed by separation of cells using FACS (mean ± SEM; n = 5; 3 independent experiments). *P < 0.05, **P < 0.01, Student’s t test. Scale bars: 50 μm.