Hypoxia and rapamycin induced changes to the cell cycle of multicellular spheroids and human tumour xenografts leading to potential therapeutic advantage

The cell cycle is a tightly regulated process that is functioning optimally when all factors contribute at the precise time and level that is required. However, in tumours some of these pathways are malfunctioning and to study this, we are utilizing spheroids as our in vitro tumour model system. In this thesis, we hypothesize that spheroids once adequately characterized will provide an invaluable means to assess the potential for using cytostatic agents to minimize accelerated repopulation and target radioresistant cells during radiation therapy of multicellular systems. The general methods involved for the majority of the work included immunoblotting, flow cytometry, as well as clonogenic assays. We have studied differences in the cell cycle time of subpopulations within each spheroid, where cells near the necrotic center showed a prolonged cell cycle time. Cycle time required by the peripheral cell layers was more rapid suggesting that cell kinetic variations may likely be a result of the local microenvironmental conditions such as hypoxia. Cyclin B1 and D levels were measured in spheroid populations that were subjected to a range of anoxic to aerobic conditions where cyclin levels decreased with decreasing concentrations of oxygen. Interestingly, these observations illustrate that measuring cyclin levels can provide a quick and convenient index for proliferation rate. The cytostatic agent, rapamycin, was analyzed for its influence on cell cycle progression and its affect on cyclin levels. Using human WiDr tumour xenografts, we found that the combination of rapamycin treatment with radiation had additive therapeutic benefits. Accelerated repopulation has been established as a limitation in radiation therapy of some cancers. Therapeutic advances have led to using accelerated fractionation regimens in an attempt to counteract the tumours’ inherent ability to grow in the face of continued therapy. Since accelerated repopulation of irradiated tumours may be associated with the recruitment of quiescent cells into the cell cycle, rapamycin can potentially be used to control those cells. By incorporating rapamycin and concurrently administering radiation, we found a synergistic effect where the cell cycle time of the solid xenograft tumours were even slower than with either treatment alone.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat