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 α2, α3, αv, α6 and β1 interin was determined by flow cytometric analysis. Neutralizing antibodies against α2, β1 subunits and α2β1 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 α2 and diminution of α6 integrin subunits in spheroids
versus monolayer cells. No change in the expression of α3, αv and β1 subunits was evident. Conversely, except for αv integrin, a 1.5–7.5-fold decrease in α2, α3, α6 and β1 integrin subunit expression was observed in IOSE29 cells within 2 days. Neutralizing antibodies against α2, β1 subunits and α2β1 integrin inhibited disaggregation as well as activation of
MMPs in spheroids.

Conclusion: Our results suggest that enhanced expression of α2β1 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.

Epidermal growth factor-induced ovarian carcinoma cell migration is associated with JAK2/STAT3 signals and changes in the abundance and localization of α6β1 integrin

Peritoneal dissemination of ovarian carcinoma is mediated by epithelial–mesenchymal interconversions leading to the disruption of cell–cell contact and modulation of cell–extracellular matrix (ECM) interactions. The present study was designed to evaluate the effects of epidermal growth factor (EGF) as a modulator of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) signalling and changes in integrin expression during the process similar to EMT. A fibroblastic morphology with reduced intercellular cell contacts and increased cell motility was observed in ovarian cancer cell lines in response to EGF and was concomitant with the up regulation of EMT-associated N-cadherin and vimentin expression. These changes were accompanied by an increase in α2, α6 and β1 integrin subunits and activation of JAK2 and STAT3 signalling which was suppressed by a specific JAK2 inhibitor. Consistent with the suppression of STAT3 activity, N-cadherin and vimentin expression were abrogated and was coherent with the loss of cell motility and the expression of α6 and β1 integrin subunits. Neutralizing antibodies against α6 and β1 subunits inhibited cancer cell migration. A strong correlation between the expression of N-cadherin, vimentin and JAK2/STAT3 levels were detected in high-grade ovarian tumors and was consistent with the previously reported enhanced expression of α6 integrin subunit in advanced tumors [Ahmed N, Riley C, Oliva K, Rice G, Quinn M. Ascites induces modulation of α6β1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma. British Journal of Cancer 2005;92:1475–85]. Our data incorporating the clinical samples and the cancer cell lines is the first to demonstrate that JAK2/STAT3 pathway may be one of the downstream events in EMT-like process and α6β1 integrin-mediated signalling in ovarian carcinomas.

Multicellular spheroids in ovarian cancer metastases: Biology and pathology

Epithelial ovarian cancer (EOC) has a relatively high mortality rate (~55%). One of the presiding causes is that the current chemotherapeutic regimes are unable to achieve sustained remission, despite frequently producing a positive response at first treatment. One of the reasons that EOC is difficult to treat is that the mechanism of dissemination is unusual. EOC dissemination characteristically involves local invasion of pelvic and abdominal organs. Unlike many epithelial cancers, initial dissemination rarely requires the vasculature, although the vasculature is often implicated in the advanced stages of disease. Recently, it has become apparent that aggregates of malignant cells (spheroids) contained within malignant ascites represent a significant impediment to efficacious treatment of late stage EOC. In vivo, spheroids are present in the malignant ascites of EOC patients, while in vitro cultured spheroids are capable of tumorgenesis in vivo and display a reduced response to chemotherapeutic drugs when compared to monolayers. A major problem associated with the current generation of chemotherapy agents is that they do not address the anchorage and vascular-independent growth conditions associated with a 3-dimensional structure that has formed and/or grown in suspension. Thus, spheroid formation may represent a key component of platinum/taxanesensitive recurrence. If this is correct, a better understanding of spheroid biology may contribute to the identification of new treatment opportunities for the sustained treatment of metastatic EOC. This review article outlines the key biological features of spheroids, specifically discussing their role in EOC dissemination and chemo-response as well as providing insights into spheroid functionality.