Introduction PEO1, PEO4 and PEO6 are cell lines derived from a single patient with high-grade serous ovarian cancer, the most common disease subtype, which illustrate disease progression. In cell culture-treated flat-bottom flasks, PEO1 and PEO4 form two-dimensional cellular aggregates and PEO6 form three-dimensional structures. This project aims to determine if differences in morphology, viability, proliferation, and metabolic activity exist between the three cell lines when grown in an ultra-low attachment plate more representative of in-vivo conditions.

Methods PEO1, PEO4 and PEO6 cells were grown in ultra-low attachment plates. Live/dead cell imaging, apoptosis and proliferation detection as well as ATP quantitation assays were performed using microscope imaging, cytometry and spectrophotometry methods.

Results The cell lines were morphologically different, mimicked the multilayered structure of in-vivo tumors and had a similar proliferation pattern. PEO1 displayed the highest aggregation level, PEO6 the highest compaction level, and PEO4 the lowest aggregation and compaction levels. All three cell lines were found to mimic poorly vascularized tumors by forming a multilayered structure with an outer layer of live cells and an inner core of apoptotic cells, but at different times. It was observed that PEO1, PEO4 and PEO6 cells proliferate mostly in the cell masses’ periphery. PEO6 cells produced a higher amount of ATP followed by PEO4 and then PEO1 cells after 4 and 7 days.

Conclusion/Implications Three-dimensional cell culture of established ovarian cancer cell lines in such environment likely will serve as a preclinical model of disease to provide experimental responses to therapeutic agents.