| Ovarian cancer is the most common cancer of women, which is also a major cause of death in gynecological malignancies. Since there is no obvious clinical manifestations in early ovarian cancer, most patients has been already in late stages of peritoneal metastasis at the time of diagnosis. In advanced ovarian cancer, the production of a large amount of ascites exacerbates the pain symptoms in patients, and provides a survival environment for metastasis of cancer cells with drug resistance, which also determines the poor prognosis of ovarian cancer. Metastatic ovarian cancer cells exist mainly in the form of multi-cellular spheroids (MCSs) in the ascites of patients with advanced ovarian cancer. Increasing evidence has shown that the formation of MCSs is necessary for ovarian cancer cells to survive and metastasize after shedding from original tumor lesions. Therefore, it is of great clinical significance to establish a stable suspension MCS model of ovarian cancer cells because this will enable us to properly study the characteristics of tumor cells in the ascites of late stage ovarian cancer, especially in terms of resistance to chemotherapy drugs.MCSs are an important means for the anchorage-independent growth of tumor cells, and the maintenance and function of MCSs in suspension depends to large extent on intracellular adhesion molecules. E-cadherin, as an intercellular adhesion molecule, was initially believed to be a tumor suppressor. However, recent research has revealed that E-cadherin plays a more complicated role than just inhibiting the metastasis of tumor cells. In breast cancer, the down-regulation or loss of E-cadherin indicates tumor aggressiveness and poor prognosis, but the expression of E-cadherin is necessary for the adhesion and aggregation of cells in MCS suspensions. It is noteworthy that E-cadherin plays totally different roles in ovarian cancer compared to other types of cancers. The E-cadherin expression level is significantly higher in ovarian cancer cells than in normal ovarian epithelial cells, and it activates the PI3K/AKT and MEK/ERK signaling pathways by mediating cell-cell adhesion. This promotes the growth and proliferation of ovarian cancer cells indicating a possibly unique and critical function of E-cadherin in ovarian cancer cells in MCS suspensions. Nevertheless, the roles and mechanisms of E-cadherin in ovarian cancer cells remain uncertain.Objectives:Ovarian cancer is the deadliest of all gynecologic malignancies. Metastatic ovarian cancer cells exist mainly in the form of multi-cellular spheroids (MCSs) in the ascites of patients with advanced ovarian cancer. We hypothesized that E-cadherin, as an important cell-adhesion molecule, might play an important role in the formation and survival of MCSs. Therefore, we established a three-dimensional suspension culture model of ovarian cancer cells that express high levels of E-cadherin to investigate their growth, proliferation, and resistance to chemotherapeutic drugs.Methods:1. Ovarian cancer cell lines with different level of E-cadherin expression were compared for their ability to form stereoscopic suspension cultures in a poly2-hydroxylethyl methacrylate gel that inhibits cell adhesion;2. We testified the role of E-cadherin in suspended MCS in ovarian cancer cells by calcium deprivation experiments;3. We used CCK-8assays to analyze cell growth and proliferation in the cells growing in suspension.4. We used CCK-8assays to analyze cell resistance to the chemotherapeutic drug cisplatin in the cells growing in suspension.Results:1. A three-dimensional suspension culture model was successfully constructed. with the high E-cadherin-expressing ovarian cancer cell line SKOV-3. Compared to the cell suspension masses formed with low E-cadherin expression in OVCAR-3or by SKOV-3cells that did not express E-cadherin, the MCSs of high E-cadherin SKOV-3had larger volumes, tighter cellular connections, and longer survival times;2. After the addition of EDTA the intercellular connections between the suspended MCS became loose and the large cell mass fell apart into smaller masses. The EDTA-treated cells were finally digested with trypsin and this resulted in the small cell masses dissociating into individual cells or into mini cell masses containing only a few cells. In the control group, the MCSs digested with trypsin for10minutes remained as large cell masses, and this demonstrated a far weaker effect of trypsin compared to EDTA.3. The results showed that after72h of culture the proliferation activities of all three suspended cell masses were significantly lower than their respective controls grown as adherent cells (P<0.05). The optical density readings of SK-H-M at three different time points showed no obvious difference (P>0.05) nor did the readings of SK-N-M or OV-L-M cells (P>0.05). Meanwhile, at each one of the three observing time point, no significant difference in proliferation activity was observed among these three cell lines in suspension (P>0.05for all);4. The mortality of the suspended cells were all significantly lower than the respective adherent cells for all three cell lines after72h of cisplatin treatment (P<0.05) indicating a higher drug resistance to cisplatin in suspended cells than in adherent cells. In addition, the mortality of SK-H cells was significantly lower than SK-N cells and OV-L cells in both the suspended and adherent cultures respectively after72h of cisplatin treatment (P<0.05for both).Conclusions:1. A three-dimensional suspension culture model was successfully constructed with the high E-cadherin-expressing ovarian cancer cell line SKOV-3.;2. E-cadherin plays a vital role in MCS formation, maintenance, and drug resistance in ovarian cancer and could be a potential target for late-stage ovarian cancer treatment. |
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