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.

Figure 8. Blocking α₅ or β₁ integrin prevents OvCa metastasis.

(A) In vitro testing of α₅ and β₁ integrin blocking antibodies. OvCa cell lines HeyA8 and SKOV3ip1 were mixed with antibodies (10 μg/ml) for 15 minutes, and adhesion (2 hours), invasion (24 hours), and proliferation (96 hours) to the 3D omental culture were measured (mean ± SEM; n = 3 [invasion] or 5 [adhesion and proliferation]; 3 independent experiments). *P < 0.01. (B) In vivo testing of α₅ and β₁ integrin blocking antibodies using the HeyA8 xenograft model of OvCa metastasis. HeyA8 cells were mixed with antibodies (10 μg/ml) and injected into the ovary (2.5 × 10⁴). Mice were treated 2 and 4 days after inoculation with i.p. antibody injection (10 mg/kg), and the number of metastatic tumors (left) and tumor weight (right) were determined 28 days after injection. Shown are individual data points and means of treatment groups (horizontal lines and numeric values). *P < 0.01, Student’s t test. (C) OvCa and mesothelial cell communication during the initial steps of OvCa metastasis (see text for details).

Figure 7. OvCa cells regulate fibronectin expression in mesothelial cells by a RAC1/SMAD3-dependent signaling pathway.

(A) Immunoblot and IF analysis using a phospho-SMAD2/3 antibody in primary human mesothelial cells cultured on plastic or cocultured with SKOV3ip1 cells. The primary human mesothelial cells were separated from SKOV3ip1 cells using FACS (top). (B) Immunoblot analysis for secreted fibronectin. Primary human mesothelial cells were transfected with control, SMAD3, or TGF-βRII siRNA, followed by treatment with or without SKOV3ip1 CM and the TGF-βRII antibody or the TGF-βRI kinase inhibitor II for 72 hours. OD of fibronectin is shown normalized to total protein signal. (C) Top: RAC1 activity assay. Immunoblot analysis for total and active RAC1 (pulldown with a GTP-specific antibody) in cell lysates of primary human mesothelial cells, SKOV3ip1 cells, or coculture. Bottom: IF of RAC1-GTP in mesothelial cells cultured on plastic or cocultured with SKOV3ip1 cells for 24 hours using antibody specific for RAC1-GTP. (D) Immunoblot analysis for fibronectin secreted by primary human mesothelial cells transfected with RAC1 siRNA, followed by treatment with SKOV3ip1 CM and TGF-βRI kinase inhibitor II. OD of fibronectin is shown normalized to total protein signal. (E) Immunoblot analysis for fibronectin in cell lysates of primary human mesothelial cells treated in combination with TGF-β1, TGF-βRI kinase inhibitor I, and/or a RAC1 inhibitor for 48 hours. Scale bars: 10 μm.

Figure 6. Tumor cells induce fibronectin expression in mesothelial cells through a TGF-β1–dependent signaling pathway.

(A) Left: qRT-PCR analysis for CDH1 and VIM. Primary human mesothelial cells were cultured on plastic or cocultured with the indicated OvCa cells for 48 hours followed by cell separation using FACS and extraction of RNA (mean ± SEM; n = 3; 3 independent experiments). *P < 0.05, **P < 0.01, Student’s t test. Right: Immunoblot analysis of fibronectin, E-cadherin, and vimentin in primary human mesothelial cells treated with or without SKOV3ip1 CM. (B and C) Immunoblots for fibronectin. (B) Primary human mesothelial cells were treated with SKOV3ip1 CM for 72 hours, and cell lysates were extracted. (C) Detection of fibronectin secreted by primary human mesothelial cells treated with or without SKOV3ip1 CM and the RHO kinase inhibitor, the RAS signaling activity inhibitor, the RAC-GEF interaction inhibitor, the TGF-βRI kinase inhibitor I, or the TGF-βRII blocking antibody.

Figure 5. Genetic knockdown of Fn1 reduces metastasis.

(A) Left: qRT-PCR analysis for Fn1 on mRNA isolated from mouse mesothelial cells cultured from Fn1^fl/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 Fn1^fl/fl mesothelial cells on ID8 mouse OvCa cell adhesion, invasion, and proliferation. (B–D) In vivo knockdown of fibronectin in omental surface cells of Fn1^fl/fl mice. Ad-Cre or Ad-Cont was injected i.p. into Fn1^fl/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) Fn1^fl/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.

Figure 4. Inhibition of fibronectin in mesothelial cells or omentum impairs OvCa cell adhesion, invasion, and proliferation.

(A) Fibronectin knockdown in primary human mesothelial cells. Immunoblot analysis of fibronectin in primary human mesothelial cells transfected with fibronectin-specific (FN), vitronectin-specific (Vn), or control (NC) siRNA. Functional assays investigating the effect of fibronectin knockdown in mesothelial cells on OvCa cell adhesion (30 minutes), invasion (24 hours), and proliferation (96 hours). Primary human mesothelial cells were transfected with fibronectin-targeted or control siRNA, followed by addition of fluorescently labeled SKOV3ip1 and HeyA8 OvCa cells, which were detected using a fluorescence reader (mean ± SEM; n = 5 [adhesion and proliferation], 3 [invasion]; 3 independent experiments). *P < 0.05, Student’s t test. (B) Fibronectin knockdown in pieces of full human omentum. Left: Transfection of fibronectin siRNA (specifically designed for in vivo use) in pieces of full human omentum (72 hours); FN1 mRNA downregulation was confirmed in detached surface cells (after scraping off surface cells of the omentum) using qRT-PCR. Right and bottom: Adhesion, invasion, and proliferation of OvCa cells was inhibited on full human omentum when fibronectin expression in omental surface cells decreased (mean ± SEM; n = 5; 3 independent experiments). *P < 0.05, Student’s t test.

Figure 3. OvCa cells stimulate fibronectin expression in mesothelial cells.

(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.

Figure 2. OvCa cells induce fibronectin matrix assembly in stromal cells.

(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.

Figure 1. Fibronectin is overexpressed in the stroma of omental metastases.

(A) Immunohistochemistry for fibronectin (FN) levels in the tumor and stromal compartments of omental metastases (n = 108) was analyzed in tumor sample cores using Aperio ImageScope and Spectrum software (see Supplemental Figure 9). Black dots, outliers; boxes, interquartile range (IQR); lines within boxes, median. ***P < 0.001, Wilcoxon rank test (median ± 1.5 IQR). 3 different tumor tissue cores from separate patients are shown. (B) Immunohistochemistry for fibronectin in tissue from a patient coincidentally detected with early, microscopic OvCa metastasis to the omentum (stage IIIA; representative sections of affected areas are shown). Arrowhead, mesothelial cells; arrows, OvCa cells. (C) Immunohistochemistry for fibronectin expression in omental tissues (n = 11) sampled from patients treated for benign disease and omental metastases (n = 43) removed from patients with serous papillary OvCa (mean ± SEM). *P < 0.05. (D) Immunoblot analysis of ECM extracted from omental tissues (n = 3) sampled from patients treated for benign disease and omental metastases removed from patients with serous papillary OvCa. rh-FN, recombinant human fibronectin. (E) Left: Adhesion (30 minutes) of OvCa lines SKOV3ip1 and HeyA8 to the isolated ECM in D. Right: Pretreatment of SKOV3ip1 cells for 30 minutes with integrin function blocking antibodies or peptides, followed by the adhesion assay to isolated ECM (mean ± SEM; n = 5; 3 independent experiments). mIgG, murine IgG control. *P < 0.05, Student’s t test. Scale bars: 100 μm.