SIMULTANEOUS TRIPLE STAINING FOR HYPOXIA, PROLIFERATION, AND DNA CONTENT IN MURINE TUMORS

Hypoxia and proliferation rate are two important biological factors in fluencing the outcome of radiotherapy regimes for solid tumours, Hypox ic cells are more resistant to radiation than aerobic cells and a rapi dly dividing tumour may repopulate faster during treatment, Clinical t rials are underway to assess the importance of both these parameters, In this article we describe a method to simultaneously measure hypoxia and proliferation using multiparameter flow cytometry, Hypoxic cells were detected using a bioreductively bound marker with an immuno-recog nisable side-chain, NITP and proliferation was measured by bromodeoxyu ridine (BrdUrd) incorporation, These parameters were related to cell c ycle position by measuring total DNA content with 7-aminoactinomycin D . The data were analysed using single laser excitation on a bench top now cytometer. Simultaneous measurement of the three parameters shows the presence of cells which have incorporated BrdUrd and are also hypo xic by the criterion of MTP binding, The murine SaF tumour has a relat ively constant aneuploid labelling index of 24%. However, the level of aneuploid hypoxia was variable ranging from 0.8 to 40.9% with a mean value of 15.6%. Within the Br-dUrd labelled population there is a rang e of hypoxia from 2.8 to 28.5% (mean 15.1%); this represents 0.7 to 6. 6% of the total tumour population, There are approximately twice as ma ny oxygenated cells than hypoxic cells actively in the cell cycle, In vivo tumours contain tells with S phase DNA content which do not incor porate BrdUrd. This cell population has equivalent proportions of hypo xic and oxic cells, However, there are up to 12-fold more hypoxic cell s in the unlabelled S than the BrdUrd labelled population, These data show that proliferation and hypoxia can be measured simultaneously usi ng now cytometry and the technique may form the basis of a predictive assay for these two important biological determinants of radiotherapy outcome. (C) 1995 Wiley-Liss, Inc.