Simultaneous triple staining for hypoxia, proliferation, and DNA content in murine tumours

Abstract

Hypoxia and proliferation rate are two important biological factors influencing the outcome of radiotherapy regimes for solid tumours. Hypoxic cells are more resistant to radiation than aerobic cells and a rapidly dividing tumour may repopulate faster during treatment. Clinical trials 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-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 bench top 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 tumour 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 tumour population. There are approximately twice as many oxygenated cells than hypoxic cells actively in the cell cycle. In vivo tumows 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. C? 1995 Wiley-Liss, Inc.Keyterms: Three colour, flow cytometry, unlabelled S phase, solid tumours, BrdUrd, NITP Hypoxia and proliferation are two of the most important biological factors that may affect the outcome of radiotherapy treatment of solid tumours. It has been recognised for many years that hypoxia has a limiting effect on radiotherapy treatment; hypoxic cells are three times as resistant to radiation than their well-oxygenated counterparts ( 12).A number of strategies have been proposed to overcome hypoxia in solid tumours including the use of hyperbaric oxygen, radiation sensitizers, oxygen or carbogen breathing, and blood transfusions of anaemic patients. Many of the trials involving chemical radiation sensitizers gave disappointing results for several reasons which included lack of patient selection, small numbers of patients in individual trials, and poor tolerance of some of the radiation-sensitizing drugs. For these reasons the results of these trials have been conflicting, such that the importance of hypoxia has not been fully appreciated. However, Overgaard ( 19...

Cell cycle distribution of hypoxia and progression of hypoxic tumour cells in vivo

“…1% normal goat serum (PNT) with 25 pl of IgG fluorescein isothiocyanate-conjugated goat anti-rabbit antiserum (Sigma) for 1 h. Cells were washed in 5 ml of PBS and the pellet resuspended in 2 ml of PBS containing 1 mg ml-' RNAase and 10 jug ml-' propidium iodide. The method for simultaneous measurement of hypoxia proliferation and DNA content has been reported previously (Webster et al, 1995). Briefly, 106 ethanol-fixed cells were washed in PBS, centrifuged for 5 min at 2000 r.p.m.…”

“…Markers set on the SaF DNA histogram represent; Ml, diploid host cells; M2, aneuploid tumour cells; M3, Gl; M4, S; M5, G/M DNA precursor bromodeoxyuridine (BrdUrd). We have developed a novel three-colour staining flow cytometry method to measure the BrdUrd, NITP and DNA content of tumour cells, so that the interaction of these three parameters can be investigated (Webster et al, 1995). In this paper, we describe the cell cycle distribution of hypoxia in three murine tumour models by flow cytometry and follow the progress of hypoxic cells through the cell cycle by simultaneous evaluation of hypoxia (NITP), proliferation (BrdUrd) and DNA content.…”

Multiparameter analysis of vasculature, perfusion and proliferation in human tumour xenografts

“…Hypoxic cells can be identified directly by bioreductive chemical probes, bound markers with immuno-recognizable side-chains, e.g. 7-(4'-(2-nitroimidazole-1 -yl)-butyl)-theophylline (NITP) (Webster et al, 1995). The metabolism of these compounds involves the generation of a free radical that is so reactive towards oxygen that further metabolism is inhibited in well-oxygenated cells.…”

Dynamics of Tumor Hypoxia Measured with Bioreductive Hypoxic Cell Markers

“…For instance, radioactively labeled 2-nitroimidazoles can be detected by noninvasive methods such as PET (142)(143)(144), MRS (71,136) and SPECT (144) or by autoradiography (138). Antibodies raised against reduced and bound bioreductive markers can be used for detection of hypoxic cells by means of three different immunoassays; the enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM) or immunohistochemistry (IHC) of frozen or paraffin-embedded sections (132,133,(145)(146)(147)(148)(149)(150)(151)(152). Table 2 shows an overview of the assays that are suitable for detection of the most common 2-nitroimidazoles.…”

“…However, repeated short episodes of hypoxia may also be detected, which could explain the presence of pimonidazole binding near functional, perfused blood vessels within 30-60 min after injection of the hypoxia marker (152). Similarly, tumor cells labeled with both a bioreductive hypoxia marker and a subsequently injected S-phase marker (indicating replicative DNA synthesis) may be transiently hypoxic (149,150,162), although such overlap could also be present in intermediate hypoxic areas.…”