Simultaneous triple staining for hypoxia, proliferation, and DNA content in murine tumours
- Mount Vernon Hospital, Middlesex (United Kingdom)
Hypoxia and proliferation rate are two important biological factors influencing the outcome of radiotherapy regimes for solid tumors. Hypoxic cells are more resistant to radiation than aerobic cells and a rapidly dividing tumor may repopulate faster during treatment. 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-recognisable side-chain, NITP, and proliferation was measured by bromodeoxyuridine (BrdUrd) incorporation. These parameters were related to cell cycle position by measuring total DNA content with 7-aminoactinomycin D. The data were analysed using single laser excitation on a benchtop flow cytometer. Simultaneous measurement of the three parameters shows the presence of cells which have incorporated BrdUrd and are also hypoxic by the criterion of NITP binding. The murine SaF tumor has a relatively 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 BrdUrd labelled population there is a range of hypoxia from 2.8 to 28.5% (mean 15.1%); this represents 0.7 to 6.6% of the total tumor population. There are approximately twice as many oxygenated cells than hypoxic cells actively in the cell cycle. In vivo tumors contain cells with S phase DNA content which do not incorporate BrdUrd. This cell population has equivalent proportions of hypoxic and oxic cells. However, there are up to 12-fold more hypoxic cells in the unlabelled S than the BrdUrd labelled population. These data show that proliferation and hypoxia can be measured simultaneously using flow cytometry and the technique may form the basis of a predictive assay for these two important biological determinants of radiotherapy outcome. 24 refs., 2 figs., 4 tabs.
- Sponsoring Organization:
- USDOE
- OSTI ID:
- 456823
- Journal Information:
- Cytometry, Journal Name: Cytometry Journal Issue: 4 Vol. 21; ISSN CYTODQ; ISSN 0196-4763
- Country of Publication:
- United States
- Language:
- English
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