(52g) Time-Dependent Stress and Multipotency Response of Glioma to Low Oxygen Tension in Vitro

Understanding the characteristics of the highly malignant brain tumor glioblastoma multiforme requires accurate in vitro models of in vivo human tumors. Established cell lines derived from these tumors, such as U87-MG, have been cultured for prolonged periods in poorly controlled culture environments and are suspected to no longer provide an accurate representation of actual cancers. Furthermore, a multipotent subpopulation of cancer cells commonly termed “cancer stem cells,” or CSCs, have recently been postulated to more accurately recapitulate the in vivo human tumors in vitro. Therefore, our current studies are focused on using bioprocessing principles to define well-controlled conditions that promote the growth of these more aggressive CSCs. One of particular interest is oxygen tension, since laboratory cell culture is normally performed at near-atmospheric partial pressure of oxygen. This is a poor model for in vivo tumors since the partial pressure of oxygen in the brain is much lower than the partial pressure of oxygen in atmospheric air. By inducing a “hypoxic” state which is more similar to the in vivo environment of tumors in the brain, we hypothesize that the total percentage of CSCs in a cell culture will increase. To test this hypothesis, we are studying established glioma cell lines and primary glioma cells in two culture apparatuses: typical cell culture flasks in an O­₂ controlled incubator and a liter-scale bioreactor. Multipotent characteristics and stress response were visualized by flow cytometric analysis of CSC-specific biomarkers, hypoxia-inducible factors, and stress-related proteins (e.g. CD133, HIF-1α, and HSP90, respectively). The relative time scales of multipotency emergence and stress response were determined by qRT-PCR analysis of related mRNA products. The correlation of these data with future development of cell culture conditions for selective growth of CSCs will be discussed.