Characterization of ovarian cancer ascites on cell invasion, proliferation, spheroid formation, and gene expression in an in vitro model of epithelial ovarian cancer

Abstract

At least one third of all cases of epithelial ovarian cancer are associated with the production of ascites, although its effect on tumor cell microenvironment remains poorly understood. This study addresses the effect of the heterologous acellular fraction of ovarian cancer-derived ascites on a cell line (OV-90) derived from the chemotherapy-naïve ovarian cancer patient. Ascites were assayed for their effect on cell invasion, growth, and spheroid formation. When compared to either no serum or 5% serum, ascites fell into one of two categories: stimulatory or inhibitory. RNA from OV-90 cells exposed to selected ascites were arrayed on an Affymetrix HG-U133A GeneChip. A supervised analysis identified a number of differentially expressed genes and quantitative polymerase chain reaction validation based on OV-90 cells exposed to 54 independent ascites demonstrated that stimulatory ascites affected the expression of ISGF3G, TRIB1, MKP1, RGS4, PLEC1, and MOSPD1 genes. In addition, TRIB1 expression was shown to independently correlate with prognosis when its expression was ascertained in an independent set of primary cultures established from ovarian ascites. The data support the validity of the strategy to uncover molecular events that are associated with tumor cell behavior and highlight the impact of ascites on the cellular and molecular parameters of ovarian cancer.

Keywords: Epithelial ovarian cancer; cell behavior; invasion; molecular profiling; ovarian ascites.

Figure 1

Effect of ascites on the invasion of the ovarian cancer cell line OV-90. The invasive potential of OV-90 (solid bars) was determined by its ability to invade a synthetic basement membrane after 24 hours compared to 5% FBS (%Invasion*). (A) Effects of 54 ascites on the invasive potential of the cell line. (B) Invasion profile of OV-90 with OSE medium in the absence or presence of 5% FBS or with 5% of those ascites selected for gene expression analysis.

Figure 2

Effect of ascites on the proliferation of the OV-90 ovarian cancer cell line. On day 0, 2 × 10⁵ cells were incubated in media supplemented either with no serum, with 5% FBS, or with 5% ascites and the cell growth was evaluated with a Trypan Blue exclusion assay at 24 and 48 hours. Note that, with one exception, there were no statistically significant differences in growth after 24 hours in the different tested conditions. The asterisk denotes a statistical significance (P < .05).

Figure 3

Effect of ascites on spheroid formation. Spheroids were formed using a modification of the hanging droplet method. Cells were incubated with OSE media supplemented either with no serum, with 5% FBS, or with 5% of the indicated ascites. Spheroid formation was monitored after 4 days. All pictures were taken at a magnification of × 100.

Figure 4

In vitro invasion assay with the ovarian cancer cell line OV-90. (A) Effect of simultaneous exposure to ascites that stimulate or inhibit the invasion of OV-90. Note that the inhibitory effect appears to be dominant. (B) Proteins of ascites A1369 and A1526 were inactivated by heating and their effect on the invasion of OV-90 was evaluated. Note that ascites no longer maintain their inhibitory effect.

Figure 5

Relation between TRIB1 expression and cumulative survival of patients with ovarian cancer in the context of concomitant ascites. The threshold was determined by ROC analysis. Kaplan-Meier graphical representation of survival curve illustrates the poor survival associated with a high expression of TRIB1 (P < .005).