Role of malignant ascites on human mesothelial cells and their gene expression profiles

Abstract

Background: Malignant ascites is often present at diagnostic in women with advanced ovarian cancer (OC) and its presence is associated with a worse outcome. Human peritoneal mesothelial cells (HPMCs) are key components of malignant ascites. Although the interplay between HPMCs and OC cells is believed to be critical for tumor progression, it has not been well characterized. The purpose of this study was to assess the effect of ascites on HPMCs and clarify the role of HPMCs in OC progression.

Methods: Human OC ascites and benign peritoneal fluids were assessed for their ability to stimulate HPMC proliferation. Conditioned medium from ascites- and benign fluid-stimulated HPMCs were compared for their ability to attenuate apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). We conducted a comparative analysis of global expression changes in ascites-stimulated HPMCs using Agilent oligonucleotide microarrays.

Results: As compared to benign peritoneal fluids, malignant ascites stimulated the proliferation of HPMCs. TRAIL-induced apoptosis was attenuated in OC cells exposed to conditioned medium from ascites-stimulated HPMCs as compared to OC cells exposed to conditioned medium from benign fluid-stimulated HPMCs. A total of 649 genes were differentially expressed in ascites-stimulated HPMCs. Based on a ratio of more than 1.5-fold and a P < 0.05, 484 genes were up-regulated and 165 genes were down-regulated in ascites-exposed HPMCs. Stimulation of HPMCs with OC ascites resulted in differential expression of genes mainly associated with the regulation of cell growth and proliferation, cell death, cell cycle and cell assembly and organization, compared to benign peritoneal fluids. Top networks up-regulated by OC ascites included Akt and NF-κB survival pathways whereas vascular endothelial growth factor (VEGF) pathway was down-regulated.

Conclusions: The results of this study not only provide evidence supporting the importance of the interplay between cancer cells and HPMCs but also define the role that the tumor environment plays in these interactions.

Figure 1

Characterization of HPMCs. (A) Phase contrast pictures of HPMCs (Meso-7 and Meso-9) cultured in 10% FBS (x 100 magnification). Bars 200 μm. (B) Immunofluorescence detection of MOC31 and calretinin in human ovarian cancer cells SKOV3 and HPMCs. The cells were fixed with cold methanol and stained with FITC-conjugated anti-MOC31 and Texas Red-conjugated anti-calretinin (x 1000 magnification). HPMCs stained positive for calretinin and negative for MOC31 confirming that they were mesothelial cells. Bars 30 μm. (C) HPMCs were cultured either with absence of FBS, 10% FBS or 10% malignant ascites (OVC508) and representation phase contrast images were taken (x 200 magnification). Bars 100 μm. (D) The relative expression of TGF-β1 was determined as described in Material & Methods for peritoneal benign fluids (OV370 and OV401) and malignant ascites (OVC346 and OVC508). The solid line indicates the cut-off intensity (750 FU) for a positive signal. *indicate P < 0.001, T-student test.

Figure 2

Effect of ascites on HPMC proliferation. (A) HPMCs were cultured either with 10% FBS, 10% benign peritoneal fluids or 10% malignant ascites in the presence or absence of hydroxyurea (HU) for 12 h and phase contrast images were taken (x 200 magnification). Bars 100 μm. (B-C) Phase contrast pictures of Meso-7 and Meso-9 cells cultured either with 10% benign fluid (OV370 or OV401) or 10% malignant ascites (OVC508 or OVC509). Bars 100 μM. (D) HPMC (meso-7 cells) were seeded and cell growth for up to 96 h was determined by XTT assay. (E) Determination of LPA levels in benign fluids or malignant ascites. There was no significant difference between levels of LPA in OV401, OVC508 and OVC509 (P > 0.05). Levels of LPA in OV370 were however significantly higher. *indicate P < 0.01, T-student test.

Figure 3

TRAIL-induced apoptosis in ascites-stimulated HPMCs. (A) Diagram of HPMC-priming assays. Ascites-stimulated or benign fluid-stimulated HPMCs were culture overnight (shown in yellow), washed TWICE and cultured in serum/hormone-free medium for 8 to 24 h to generate HPMC-conditioned medium (shown in pink) that were collected at either 12 h (B) or different time points (C). HPMC-conditioned medium was then added to CaOV3 tumor cells in the presence of TRAIL (25 ng/ml). TRAIL-induced apoptosis was measured in CaOV3 cells incubated with the indicated HPMC-conditioned medium overnight and expressed as fold increased relative to cells that were exposed to HPMC-conditioned medium but not to TRAIL. Data are expressed as means of triplicates from three independent experiments ± SD. * indicate P < 0.01.

Figure 4

Functional analysis for the dataset of differentially expressed genes (≥ 1.5-fold) in ascites-stimulated HPMC cells. Top functions that meet a P value cutoff of 0.05 are displayed. The orange line represents the cutoff value for significance. (A) Genes that were up-regulated and (B) genes down-regulated.

Figure 5

Network analysis of dynamic gene expression in ascites-stimulated HPMCs based on the common up-regulated (A) or down-regulated (B) (≥ 1.5-fold) gene expression list obtained following stimulation with all three malignant ascites. The top-scoring networks were merged and displayed graphically as node (gene/gene product) and edges (the biological relationships between the nodes). Nodes are displayed using various shapes that represent the functional class of the gene product (square, cytokine; vertical oval, transmembrane receptor; rectangle, nuclear receptor; diamond, enzyme; rhomboid, transporter; hexagon, translation factor; horizontal oval, transcription factor; circle, other). Edges are displayed with various labels that described the nature of relationship between the nodes: — binding only; → acts on. The length of an edge reflects the evidence supporting that node-to-node relationship, in that edges supported by article from literature are shorter. Dotted edges represent indirect interaction.

Figure 6

Correlation between gene expression data and quantitative PCR.(A) Levels of mRNA expression in ascites-stimlated HPMCs expressed as fold changes relative to benign fluiids OVC370- or OVC401-stimulated HPMCs. (B) Relative expression of IL-8 and BMP-2 mRNA in ascites-stimulated HPMCs compared to either benign fluid OV1081- or OVC370-stimulated HPMCs. * indicate P < 0.03.