Alteration in the hypoxic fraction of quiescent cell populations by hyperthermia at mild temperatures

Abstract

We investigated oxygenation of quiescent (Q) tumour cells in vivo by mild heat treatment. C3H/He mice bearing SCC VII tumours received BrdU continuously for 5 days via implanted mini-osmotic pumps, to label all proliferating (P) cells. The tumours were then irradiated after treatment, and were excised, minced and trypsinized. The tumour cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labelling was determined using immunofluorescence staining for BrdU. This MN frequency was then used to calculate the surviving fraction of unlabelled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total (P + Q) tumour cells. Thus, a cell survival curve could be determined for the cells not labelled with BrdU, which can be regarded as the Q cells in a tumour for all practical purposes. The MN frequency in total tumour cell population was determined from the irradiated tumours that were not pretreated with BrdU. Assays performed immediately after irradiation of both normally aerated and hypoxic tumours showed that Q cells contained higher hypoxic fractions than the total tumour cell population. Mild heat treatment (400°C, 60 min) before irradiation decreased the hypoxic fraction, even when it was combined with nicotinamide administration. In contrast, mild heating did not decrease the hypoxic fraction when the mice were placed in a circulating carbogen (95% 02/5% CO2) chamber. Therefore, mild heat treatment was thought to preferentially oxygenate the chronically hypoxic fraction.