Production of IL1-beta by ovarian cancer cells induces mesothelial cell beta1-integrin expression facilitating peritoneal dissemination

Abstract

Background: A crucial step in the metastatic spread of ovarian cancer (OC) is the adhesion and implantation of tumor cells to the peritoneal mesothelium. In order to study this step in the cascade, we derived a pro-metastatic human ovarian carcinoma cell line (MFOC3) from the non-metastatic FOC3 line.

Methods: Molecular profiling of the isogeneic lines identified differentially expressed genes, and investigation for a role in dissemination for specific factors was achieved by development of a co-culture adhesion assay utilizing monolayers of human mesothelial cells.

Results: After murine intraperitoneal inoculation, the FOC3 cell line formed no metastases, but the MFOC3 subline formed metastases in > 80% of SCID mice. MFOC3 cells also adhered 2-3 times more avidly to mesothelial monolayers. This adhesion was inhibited by neutralizing antibodies to IL-1β and enhanced by recombinant IL-1β (p < 0.01). IL-1β induced mesothelial cell β1-integrin, and an antibody to this subunit also inhibited the adhesion of MFOC3 to mesothelial cells in vitro and significantly reduced metastases in vivo. Immunohistochemical analysis of a cohort of 96 ovarian cancer cases showed that negative IL-1β expression was significantly associated with an improved overall survival rate.

Conclusions: These results suggest that a IL-1β/β1-integrin axis plays a role in ovarian tumor cell adhesion to mesothelia, a crucial step in ovarian cancer dissemination.

Figure 1

Representative appearance of intraperitoneal (i.p.) tumor implants in SCID mice. A. Macroscopic view of a peritoneal metastatic tumor after i.p. injection of MFOC3. Small round metastatic foci of extensive local growth (arrowhead) are present in the peritoneum, omentum and mesenterium. B. Microscopic view of a peritoneal metastatic tumor after i.p. injection of MFOC3. MFOC3 cells (arrowhead) grew on the peritoneum.

Figure 2

Validation of IL-1β expression and cell adhesion assay. A. RT-PCR analysis of IL-1beta was performed on FOC3 and MFOC3 mRNA using GAPDH as the internal control. B. Expression of IL-1β protein. Western blot analysis was performed on FOC3 and MFOC3 cell samples with anti- IL-1β, using β-actin as the loading control. C. Tumor cell adhesion assay. GFP-labeled FOC3 (▲) and MFOC3 (●) cells were added to the confluent mesothelial cell monolayer for 10, 20, 30 and 60 minutes and counted using fluorescent microscopy after washing away unattached cells. Each point represents the mean ± SD of at least three times separate experiments. * p < 0.05, ** p < 0.01.

Figure 3

A. FOC3 cell adhesion to mesothelial cells after pre-incubation with 1, 10, 20 or 100 ng/ml recombinant IL-1β. * p < 0.01 B. Inhibition of MFOC3 adhesion to mesothelial cells by 1, 10 or 100 ug/ml anti-IL-1β. Control (C) experiment was performed in the presence of 100 ug/ml mouse IgG. Data are expressed as the mean ± SD of three times separate experiments. * p < 0.05.

Figure 4

A. Expression of cell adhesion molecules on mesothelial cells (MCs). MCs were exposed to IL-1β (20 ng/ml) for 30 minutes, and expression of a panel of adhesion molecules was assessed by flow cytometry. Untreated control was set as 100%. Results are presented as median values ± median absolute deviation in relation to control. Data are representative of five experiments. * p < 0.01. B. Inhibition of MFOC3 adhesion to mesothelial cells by 1, 10 or 100 ug/ml anti-β1 integrin * p < 0.05. Control (C) experiment was performed in the presence of 100 ug/ml mouse IgG. C. Summary of the effects of anti-IL-1β (10 ug/ml) and β1 integrin (10 ug/ml) on the binding to mesothelial cells of FOC3 and MFOC3, and FOC3 with recombinant IL-1β (20 ng/ml). Open bars represent FOC3; shaded bars, MFOC3; solid bars, FOC3 with recombinant IL-1β. Data are expressed as the mean ± SD of three separate experiments.

Figure 5

IL-1β expression in ovarian tissues. A. High expression in a section of ovarian carcinoma. Immunoreactions appeared in both the nucleus and cytoplasm of tumor cells (x200). B. Low/no expression in a section of ovarian tumor cells (x200). C. Kaplan-Meier analysis of overall survival of 96 patients with ovarian cancer with IL-1β high expression (solid line; n = 31) versus low/no expression (dashed line; n = 65).