Hypoxia and proliferation in murine tumour models

This thesis considers two of the contributory factors in the local failure of radiotherapy; hypoxia and rapidly proliferating cells. Model systems were developed from SaF murine cells to enable analysis of the effect of the bioreductive quinones, mitomycin C (MMC) and porfiromycin (POR), upon single cells, in vitro spheroids, in vivo peritoneal spheroids and subcutaneous tumours. A novel hypoxic probe, 7-(4'-(2- nitroimidazole-1-yl)-butyl)-theophylline (NITP) allows quantitation of hypoxia, by bioreductively binding to macromolecules in cells under low O2 conditions. In cells, NITP displayed progressive binding as oxygen decreased with concomitant increases in the cell kill of both MMC and POR. At extremes of O2 concentration (air and N2) MMC and POR had oxic:hypoxic differentials of 1.7 and 3.5 respectively at 10% SaF survival. As spheroids increased in size, (250, 400 and 600?m), their degree of hypoxia increased, (5.0, 15.8, 36.0%). MMC produced high cell kill with pronounced growth delay, whereas POR showed lower cell kill with slight growth delay increasing with spheroid size. Peritoneal spheroids showed little quantitative response to MMC or POR due to the variable nature of this in vivo tumour model. Subcutaneous tumours exhibited an exponential dose response, where surviving fraction was strongly correlated with hypoxia for the more hypoxia specific POR in contrast to the poor correlation between hypoxia and the effects of MMC and radiation. Tumour size, geometric mean diameter 4 to 12mm, had no effect on the amount of hypoxia in SaF or Rh tumours. Analysis of each phase of the cell cycle by DNA content showed that although the greatest population of hypoxic cells had G1 DNA content, the highest proportion of hypoxic cells resided in the population with G2/M DNA content. A flow cytometric technique to simultaneously measure hypoxia, proliferation rate (bromodeoxyuridine incorporation) and DNA content in a single sample was developed for murine tumours. This showed that cells recently hypoxic could enter the cell cycle and hypoxic cells tended to accumulate in G2 and G1. This technique could be used as the basis for a clinical test for proliferation and hypoxia in tumours once the NITP hypoxia marker has been approved for clinical evaluation