α2β1 integrin affects metastatic potential of ovarian carcinoma spheroids by supporting disaggregation and proteolysis

BACKGROUND Ovarian cancer is characterized by a wide-spread intra-abdominal metastases which represents a major clinical hurdle in the prognosis and management of the disease. A significant proportion of ovarian cancer cells in peritoneal ascites exist as multicellular aggregates or spheroids. We hypothesize that these cellular aggregates or spheroids are invasive with the capacity to survive and implant on the peritoneal surface. This study was designed to elucidate early inherent mechanism(s) of spheroid survival, growth and disaggregation required for peritoneal metastases METHODS In this study, we determined the growth pattern and adhesive capacity of ovarian cancer cell lines (HEY and OVHS1) grown as spheroids, using the well established liquid overlay technique, and compared them to a normal ovarian cell line (IOSE29) and cancer cells grown as a monolayer. The proteolytic capacity of these spheroids was compared with cells grown as a monolayer using a gelatin zymography assay to analyze secreted MMP-2/9 in conditioned serum-free medium. The disaggregation of cancer cell line spheroids was determined on extracellular matrices (ECM) such as laminin (LM), fibronectin (FN) and collagen (CI) and the expression of alpha2, alpha3, alphav, alpha6 and beta1 interin was determined by flow cytometric analysis. Neutralizing antibodies against alpha2, beta1 subunits and alpha2beta1 integrin was used to inhibit disaggregation as well as activation of MMPs in spheroids. RESULTS We demonstrate that ovarian cancer cell lines grown as spheroids can sustain growth for 10 days while the normal ovarian cell line failed to grow beyond 2 days. Compared to cells grown as a monolayer, cancer cells grown as spheroids demonstrated no change in adhesion for up to 4 days, while IOSE29 cells had a 2-4-fold loss of adhesion within 2 days. Cancer cell spheroids disaggregated on extracellular matrices (ECM) and demonstrated enhanced expression of secreted pro-MMP2 as well as activated MMP2/MMP9 with no such activation of MMP's observed in monolayer cells. Flow cytometric analysis demonstrated enhanced expression of alpha2 and diminution of alpha6 integrin subunits in spheroids versus monolayer cells. No change in the expression of alpha3, alphav and beta1 subunits was evident. Conversely, except for alphav integrin, a 1.5-7.5-fold decrease in alpha2, alpha3, alpha6 and beta1 integrin subunit expression was observed in IOSE29 cells within 2 days. Neutralizing antibodies against alpha2, beta1 subunits and alpha2beta1 integrin inhibited disaggregation as well as activation of MMPs in spheroids. CONCLUSION Our results suggest that enhanced expression of alpha2beta1 integrin may influence spheroid disaggregation and proteolysis responsible for the peritoneal dissemination of ovarian carcinoma. This may indicate a new therapeutic target for the suppression of the peritoneal metastasis associated with advanced ovarian carcinomas.