Effect of ovarian cancer ascites on cell migration and gene expression in an epithelial ovarian cancer in vitro model

Abstract

A third of patients with epithelial ovarian cancer (EOC) present ascites. The cellular fraction of ascites often consists of EOC cells, lymphocytes, and mesothelial cells, whereas the acellular fraction contains cytokines and angiogenic factors. Clinically, the presence of ascites correlates with intraperitoneal and retroperitoneal tumor spread. We have used OV-90, a tumorigenic EOC cell line derived from the malignant ascites of a chemonaive ovarian cancer patient, as a model to assess the effect of ascites on migration potential using an in vitro wound-healing assay. A recent report of an invasion assay described the effect of ascites on the invasion potential of the OV-90 cell line. Ascites sampled from 31 ovarian cancer patients were tested and compared with either 5% fetal bovine serum or no serum for their nonstimulatory or stimulatory effect on the migration potential of the OV-90 cell line. A supervised analysis of data generated by the Affymetrix HG-U133A GeneChip identified differentially expressed genes from OV-90 cells exposed to ascites that had either a nonstimulatory or a stimulatory effect on migration. Ten genes (IRS2, CTSD, NRAS, MLXIP, HMGCR, LAMP1, ETS2, NID1, SMARCD1, and CD44) were upregulated in OV-90 cells exposed to ascites, allowing a nonstimulatory effect on cell migration. These findings were validated by quantitative polymerase chain reaction. In addition, the gene expression of IRS2 and MLXIP each correlated with prognosis when their expression was assessed in an independent set of primary cultures established from ovarian ascites. This study revealed novel candidates that may play a role in ovarian cancer cell migration.

Figure 1

Effect of ascites on OV-90 cell migration. Migration was assessed by determining the ability of cells to migrate in a culture plate using a wound-healing assay in the presence of ascites compared with 5% FBS (% migration) after 54 hours of incubation. The symbol “0” indicates medium without ascites or FBS. (A) Effect of FBS and ascites on OV-90 cell migration. (B) Migration profile of OV-90 with OSE medium in the presence or absence of 5% FBS or with 5% of several ascites. (C) Effect of 31 ascites on OV-90 cell migration potential. (D) Migration profile of OV-90 with OSE medium in the presence or absence of 5% FBS or with 5% of ascites. *Statistical significance, P < .05.

Figure 2

Effect of heat-treated ascites in OV-90 cell migration assays in vitro. Both stimulatory (A2647 and A2839) and nonstimulatory (A2295(2) and A2090) ascites were heated at 100°C for 10 minutes to inactivate the proteins before adding to OV-90 cell culture media. The effect on cell migration was subsequently evaluated. *P < .05, **P < .01.

Figure 3

Relationship between IRS2 and MLXIP expression and cumulative survival of patients with ovarian cancer in the context of concomitant ascites. The cutoffs were determined by survival tree. Kaplan-Meier graphical representation of the survival curves illustrates the survival associated with candidate gene expression levels (log-rank).