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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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 5

Genetic knockdown of Fn1 reduces metastasis.

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Genetic knockdown of Fn1 reduces metastasis. (A) Left: qRT-PCR analysis ...
(A) Left: qRT-PCR analysis for Fn1 on mRNA isolated from mouse mesothelial cells cultured from Fn1fl/fl mice treated for 72 hours with Ad-Cont or Ad-Cre, followed by treatment with TGF-β1 for 24 hours. Right: Functional assays investigating the effect of fibronectin knockdown in primary Fn1fl/fl mesothelial cells on ID8 mouse OvCa cell adhesion, invasion, and proliferation. (B–D) In vivo knockdown of fibronectin in omental surface cells of Fn1fl/fl mice. Ad-Cre or Ad-Cont was injected i.p. into Fn1fl/fl mice for 96 hours to downregulate fibronectin, followed by i.p. injection of 4 × 106 GFP-expressing ID8 OvCa cells. (B) 48 hours after injection, the mouse omentum was removed and digested, omental surface cells were collected by FACS, and qRT-PCR was performed for Fn1 on mRNA isolated from the cells. (C and D) Metastasis assay. (C) Fn1fl/fl or control C57BL/6 mice were injected with PBS, Ad-Cre, or Ad-Cont. (D) GFP-expressing ID8 cells were mixed with control IgG or 10 μg/ml murine α5 integrin antibody for 15 minutes prior to i.p. injection. 72 hours after ID8 cell injections, the mouse omentum was removed, imaged using fluorescent microscopy (right), and digested, and cancer cells were quantified using a fluorescence reader. Data are mean ± SEM (n = 3 [invasion and qRT-PCR assays] or 5 [other assays]). *P < 0.05, Student’s t test. Scale bars: 2 mm.