P-349 Impact of an Aurora kinase inhibitor on AML cells and mouse ovarian follicles in vitro

Abstract

Abstract Study question Can an aurora kinase B/C inhibitor suppress AML cell line proliferation while not impacting on mouse follicles in vitro? Summary answer Exposure to an aurora kinase inhibitor at a predetermined cytotoxic concentration suppressed AML cell proliferation but had no impact on subsequent follicle health in vitro. What is known already There is a risk that cryopreserved ovarian tissue from young women with a leukaemia diagnosis harbours leukemic cells, which on grafting could transfer disease. Novel chemotherapy agents, specifically aurora kinase inhibitors [ e.g. GSK 1070916 (GSK) ], target proteins involved in chromosomal alignment and segregation during mitosis and meiosis. It is anticipated that aurora kinase inhibitors would have an impact on the development of all somatic cells, including granulosa cells proliferating during follicle growth, however a previous study using ovarian tissue pieces indicated no impact on follicles. Study design, size, duration Primary and secondary follicles (diameter ≤ 100 µm) were manually dissected from adult mouse ovaries and embedded in an extracellular scaffold together with >1,000 AML cells. Individual follicles and AML cells were cultured for 7 days followed by exposure to GSK for 24 hours. GSK was removed by washing and the culture continued for another two days at which time cells were assessed for normal morphology and survival with live/dead staining (Calcein AM/ Ethidium Homodimer-1). Participants/materials, setting, methods The leukemic cell line OC1-AML-3 (DSMZ) was cultured in RPMI-1640 +10% FCS and routinely passaged as recommended. Cell proliferation and cytotoxicity were assessed using the Alamar Blue assay. 20% DMSO was used as a positive control in the cytotoxicity assays and live/dead evaluation. Follicles were cultured in αMEM, ITS, FSH, ascorbic acid and 10% FCS under oil in 5%CO2 : 95% air. Survival and growth were evaluated in two extracellular scaffolds; UltiMatrix and Hystem™-HP. Main results and the role of chance Follicle survival and growth was better (p < 0.05) in UltiMatrix (84.7%; 61/72) compared to Hystem™-HP (72.0%;77/107) with an average increase of 20 µm in diameter at day 7. When OCI-AML-3 cells (0.7 x105/ml) were exposed to a range of concentrations of GSK (10nM to 10µM) for 24hr in an in vitro cytotoxicity assessment, survival was reduced to 0% at a concentration of 10µM. The 20% DMSO positive control also resulted in 0% survival. However, in the 3- dimensional scaffold culture, exposure to 10 µM GSK for 24hr resulted in a mean survival of OCI-AML-3 cells of 27% (100 cells counted/ well) which was similar to the 20% DMSO positive control (32%) and significantly different to the negative (no GSK) control (95%; p < 0.01). Follicles exposed to GSK appeared alive, emitting a strong green fluorescence for both oocyte and granulosa cells, although some of the surface follicular cells were dead. Live/dead staining of GSK exposed follicles was similar to follicles not exposed to GSK. In contrast, the majority of the follicular cells were dead following exposure to 20% DMSO. Limitations, reasons for caution Due to whole mount assessment of the follicles for live/dead staining, it is difficult to determine whether the oocyte is alive. In scaffold culture some of the free cells present may be of follicular origin, potentially resulting in overestimation of the proportion of AML cells alive after GSK treatment. Wider implications of the findings This 3- dimensional culture system, incorporating a follicle and a leukemic cell line within a scaffold provides a good model to assess the impact of chemotherapy on both cell types and could be applicable for human follicles. Trial registration number Not applicable