oa Role of cell adhesion molecules in invasion, anoikis resistance and drug resistance: An in vitro analysis using multiple phenotyping approach

Epithelial ovarian carcinoma (EOC) is an aggressive neoplasm that mainly metastasizes to organs of the peritoneal cavity. This event is mediated by molecular mechanisms that remain elusive. Cell adhesion molecules play a key role in tumor invasion, metastasis and drug resistance. The conventional in vitro two-dimensional cell culture models are not sufficient to explain the exact mechanism behind tumor invasion, migration and anoikis resistance events. The objective of the present study is to analyze the role of cell adhesion molecules in tumor invasion metastasis and drug resistance using multiple phenotyping approach. Five ovarian cancer cell lines PA1, SW626, CAOV3, SKOV3 and OVCAR3 were selected for the study. Cell line phenotyping was conducted using cultured cells in an anchorage independent method that utilized an ultra low attachment plate for mimicking anoikis resistance in vitro. Second type phenotyping was done for sorting highly invasive phenotype by selecting cells that can pass through Boyden chamber (8 micron pore size upper chamber membrane). Development of drug-resistant cell lines were achieved by growing cells in culture media containing standard chemotherapeutic agents such as Taxol and Carboplatin. Cells were selected according to their ability to attach different ECM and cell adhesion molecule coated chambers. A real time PCR array of 29 genes involved in cell adhesion, drug resistance and EMT (epithelial-mesenchymal transition) were also analyzed and gene expression analysis conducted. The invasive potential of PA1 (teratocarcinoma) seems to be higher than other cell lines followed by SKOV3 cells. PA1 cells form embryoid bodies while culturing in in anchorage independent condition and exhibit an elevated level of expression of myc and TGF beta. Cell viability assay also shows that PA1 is the most sensitive cell line against carboplatin and taxol. Gene expression levels of cell adhesion molecules were altered among the phenotypes. Anoikis resistant cells show altered levels of expression in EPCAM, Collagen 6, CD24, vimentin and N-cadherin. These results suggest the cell lines are heterogeneous in nature and three dimensional culture model mimics the in vivo tumor model for anoikis resistance and EMT. To conclude, this study shows selection of various phenotypes of heterogeneous cancer cell lines can help decipher the role of cell adhesion molecules in ovarian cancer invasion and drug resistance. These experiments will give an overall view of the role of cell adhesion molecules in different ovarian cancer types in vitro.