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.
View: Text | PDF
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

×

Figure 2

OvCa cells induce fibronectin matrix assembly in stromal cells.

Options: View larger image (or click on image) Download as PowerPoint
OvCa cells induce fibronectin matrix assembly in stromal cells. (A) Fibr...
(A) Fibronectin protein expression in full mouse omentum after in vivo i.p. injection of HeyA8 OvCa cells for 48 hours. Immunoblot analysis was performed using a fibronectin-specific antibody. (B) Left: Ex vivo full human omentum adhesion, invasion, and proliferation assays with fluorescently labeled OvCa cells. Middle: Immunoblot analysis of fibronectin protein in the surface cells of the human omentum cultured alone (unbound) or with fluorescently labeled SKOV3ip1 cells (bound; after FACS). OD of fibronectin is shown normalized to GAPDH signal. Right: qRT-PCR analysis of FN1 mRNA expression. qRT-PCR was performed on mRNA using fibronectin specific probes (mean ± SEM; n = 5; 3 independent experiments). *P < 0.05, Student’s t test. (C) Fibronectin protein production and matrix secretion were analyzed in the 3D omental culture cocultured with SKOV3ip1 cells. Left: IF was performed using a fibronectin-specific antibody, and fibronectin production (green; IF with Triton-X 100) and fibronectin matrix secretion (red; IF without Triton-X 100) were analyzed. Right: Immunoblot analysis of fibronectin matrix production using DOC-soluble and -insoluble fractions of fibronectin (indicating a dense matrix) in SKOV3ip1 cells, 3D culture, or coculture. (D) Fibronectin matrix assembly by OvCa cells cultured on fibronectin. Fibronectin was seeded on a glass-bottomed plate, and SKOV3ip1, HeyA8, or HT1080 cells were added for 24 hours. IF for fibronectin (red) was performed; the formation of a dense fibronectin matrix was indicated by presence of fibrils. Scale bars: 50 μm.