The detection and modification of the hypoxic fraction in quiescent cell populations in murine solid tumours

Mice bearing SCC VII or EMT6/KU tumours were irradiated after receiving 10 injections of 5-bromo-2'-deoxyuridine (BUdR) to label all proliferating tumour cells, and the tumours were then excised and trypsinized. The tumour cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BUdR labelling was determined using immunofluorescence staining to BUdR. This MN frequency was then used to calculate the surviving fraction of unlabelled cells from the regression line for the relation between MN frequency and the surviving fraction of all tumour cells. Thus a cell survival curve could be determined for cells not labelled by BUdR, which can be regarded as quiescent tumour cells for all practical purposes. Assays performed immediately after irradiation of both normally aerated and hypoxic tumours showed that quiescent cells contained higher hypoxic fractions than the tumour cells as a whole. Furthermore, administration of nicotinamide before irradiation or the placement of mice in a circulating carbogen (95% O2, 5% CO2) chamber for 30 min before and during irradiation altered the acutely and chronically hypoxic fractions of the proliferating and quiescent tumour cell populations in a way which depended on the tumour system. Combined nicotinamide and carbogen therapy was shown to have a large potential to sensitize cells to low-dose radiation in vivo. In addition, this assay method appears to be useful for determining the size of the hypoxic fraction of quiescent tumour cells in murine solid tumours.