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The ability to determine the proportion of hypoxic cells within a tumour during treatment could lead to a more rational assessment of benefit when planning and executing radiotherapy. Until recently the only method available to determine the hypoxic fraction has been an indirect radiobiological assessment. This is applicable to experimental tumour systems but not to human patients. We have therefore sought a more direct and rapid quantitative method for detecting hypoxic cells which could eventually be applicable in the clinic. The method is based on the production of fluorescence in hypoxic cells which can be detected by microscopy or by using a flow cytometer.The rationale for the present work was based on the observations that:—(1) with certain nitroaromatic compounds the quantum efficiencies of fluorescence for the nitroreduction products (amines) are at least four orders of magnitude greater than that of the parent compound (McGlynn et al, 1969); (2) the amine is only produced under hypoxic conditions by cellular enzymes, because the initial reduction step to the nitro radical-anion, an obligate intermediate, is rapidly reversed in the presence of oxygen (Mason, 1982); (3) the reduction products formed from the bioreductive metabolism of misonidazole, metronidazole and nitrofurazone bind intracellularly in hypoxic cells.Radiolabelling of misonidazole or its analogues, followed by measurement of bound label using scintillation counting (Verghese et al, 1967), autoradiography (Chapman et al, 1981), or (potentially) external gamma scanning (Rasey etal, 1982) have been proposed as the basis for clinical detection and imaging of hypoxic cells in tumours.

Two compounds, nitroakridin 3582 (NA) and a 3-nitronaphthalimide (DM113), have been tested as potential fluorescent markers for hypoxic cells. Cellular fluorescence in three cell lines (V79–379A, WHF1B, EMT6) was measured by flow cytometry and high-performance liquid chromatography (HPLC) after incubating the cells with the drugs for various times in air or hypoxia. In all three cell lines, both drugs showed greater fluorescence in hypoxic than in oxic cells. There were, however, differences between the cell lines in respect of the magnitudes of hypoxic and oxic cell fluorescence and in the ratio of hypoxic to oxic fluorescence. Differences in hypoxic cell fluorescence were due to differences in the rate and extent of nitroreduction. Drug uptake and DNA content per cell were relatively unimportant factors in determining the magnitude of fluorescence. There was not a good correlation between cytotoxicity of the drugs and hypoxic fluorescence. Nitroakridin was more toxic to hypoxic than to aerated cells but the reverse was true for DM113. The dependence of fluorescence and radiosensitivity on oxygen concentration were compared for the three cell lines and only small differences between the “K”-curves for the two end points were found.Two problems with the present compounds which should be addressed in designing future fluorogenic compounds as hypoxic markers were oxic cell fluorescence and leakage of fluorescent products from hypoxic cells.

The response of a murine tumour to single doses of X rays has been measured using three different assays—animal cure, cell survival in vitroafter irradiation in vivo, and tumour growth delay. The dose to cure 50% of the animals, the TCD50, was 79.0 Gy. This was not affected by clamping the tumours to render all the cells hypoxic at the time of irradiation, implying that most of the cells in the tumour were hypoxic already. The enhancement ratio for the hypoxic cell sensitizer Ro-07-0582 was 2.1. The cell survival assay gave an enhancement ratio of 1.6 and an hypoxic fraction of 5%. The discrepancy in the estimates of the hypoxic fraction can be explained by the ability of the naturally hypoxic cells, but not the oxic ones, to recover from potentially lethal damage in vivo. Neither the cell survival assay nor the growth delay assay agreed with the TCD50assay as to the effect of the hypoxic cell sensitizer, even allowing for recovery from potentially lethal damage. It is doubtful whether the measured survival curve would predict the measured TCD50.

A comparison has been made of the effects of the 2-nitroimidazole Ro-07-0582 on tumour growth delay after irradiation and tumour cell survival in vitroafter irradiation in vivo. This compound has previously been shown to be a specific sensitizer of hypoxic cells. A dose of 1 mg/g body weight gave an enhancement ratio of 2-2 for both growth delay and cell survival in a system where high pressure oxygen has been shown to have no effect. However, while the hypoxic fraction in the tumour was estimated to be less then 10% from the growth delay curves, the survival curves gave a value in excess of 50%. This discrepancy probably reflects differences in the response of cells left in situ or removed and assayed in vitro.

The causes of failure after radiotherapy or chemotherapy may be related to physical or biological factors. The physical factors will include geographic miss in radiotherapy. The biological reasons may be inadequate drug delivery in chemotherapy or resistance of nutritionally deprived or hypoxic cells, a capacity to repair damage or rapid proliferation of the tumour cells so that repopulation may occur during treatment. There is much current interest in repopulation and it would be of value to identify those tumours which have rapid cell proliferation for, in these cases, a change in drug scheduling or shortening the overall treatment time with radiotherapy may lead to increased success by overcoming the effect of repopulation (Fowler, 1985).We have previously described a method for labelling proliferating cells in human tumours by measuring the uptake of bromodeoxyuridine (BrdUrd) into cells synthesizing DNA, following an intravenous injection, and the potential applications of this procedure to the measurement of proliferation rates in human tumours (Begg et al, 1985; Wilson et al, 1985). In this preliminary communication we report on three human tumours where kinetic studies have been performed and demonstrate the potential advantages of the technique.

We have compared the effects of mixed fractionation schedules with X rays and neutrons on growth delay of a murine tumour and skin reactions in mice. The schedules were five daily fractions of X rays, neutrons or mixtures (NNXXX, XXXNN or NXXXN). For clamped tumours or skin all three mixed schedules had the same effect. In contrast, for undamped tumours giving the neutrons first (NNXXX) was more effective than the other two mixed schedules. This represented a true therapeutic gain and implies that if neutrons are used clinically as only part of a course of fractionated radiotherapy, they should be given at the beginning rather than at the end of treatment.

Bromodeoxyuridine (BrdU) incorporation and flow cytometry have been used to measure the kinetics of V79 cells growing at different temperatures in vitroand cells of the murine Sa F tumour growing in vivo. By simultaneously measuring total DNA content and BrdU incorporation in individual cells at different times after pulse labelling with BrdU, it is a simple procedure to quantify the movement of cells through the cell cycle. The method has the advantage of speed (2 × 104or more cells are analysed within a day or so of the experiment) and the ability to analyse the results in different ways. A G2 block in tumour cell progression in vivois readily detected after a dose as low as 2 Gy. The neutron relative biological effectiveness for this G2 block is probably larger than that for tumour growth delay.

Computer simulations have been made of four radiotherapy fractionation regimes either in current use or proposed for clinical trials of misonidazole. A variety of cell-survival parameters and reoxygenation patterns have been used. The models allow the relative importance of repair capacity, reoxygenation rate, and dose per fraction to be assessed for these four schedules in the presence or absence of misonidazole.Unlike hyperbaric oxygen, the dose of misonidazole and the fractionation scheme to be used are critically interdependent, because the total drug dose is limited to 12 g/m2by its neurotoxicity, regardless of the extent to which it is fractionated.The largest sensitizing effect is always demonstrated with six fractions, each given with 2 g/m2of misonidazole. In the absence of reoxygenation a sensitizer enhancement ratio of 1.7 is predicted, but this falls to 1.1–1.2 if extensive reoxygenation occurs. Less sensitization is observed with 30 fractions, each with 0.4 g/m2of drug.However, for clinical use, the important question is which treatment kills the maximum number of tumour cells. Many of the simulations predict a marked disadvantage of reducing the fraction number for X rays alone. The circumstances in which this disadvantage is offset by the large SER values with a six-fraction schedule are few. The model calculations suggest that many small fractions, each with a low drug dose, are safest unless the clinician has some prior knowledge that a change in fraction number is not disadvantageous.

Recently, Wheldon (1979) and Zeitz and McDonald (1978) have considered the effects of different fractionated irradiation schedules on a model tumour having properties similar to melanoma cells in culture. Using essentially similar assumptions, they came to different conclusions as to which of the four different schedules gave the lowest survival. Zeitz and McDonald (1978) considered the response of a totally oxic population of cells using the cell-survival data of Dewey (1971). They considered three forms of the equation for cell survival and showed that a multi-target model with initial slope gave the best fit to Dewey's data. However, regardless of the exact form of the cell-survival curve chosen, the greatest extent of cell kill was achieved when a few large radiation doses were used (3 × 12.00 Gy), and the least when a large number of small fractions were used (31 × 2.00 Gy). However, they demonstrated that when a large number of small radiation doses are used large differences in the estimates of cell kill can be obtained, depending on the exact form chosen for the cell survival equation. This is because survival is being determined by the shoulder region of the survival curve and that region is the least well described experimentally.

The ability of acutely hypoxic tumour cells to recover from sublethal damage following irradiation in vivoand in vitrohas been measured using a single tumour system. The methods of assay were tumour growth delay, local tumour control and tumour cell survival in vitrofollowing treatment in vivoor in vitro. Tumours in vivoor cells in vitrorendered acutely hypoxic during irradiation were irradiated with either single doses or two doses 24 hours apart. Cells left in situhad a greater capacity for recovery than those treated either in vivoor in vitroand then assayed in vitro. It is suggested that tumours may not show a systematically reduced capacity for recovery relative to normal tissues, unless chronically hypoxic tumour cells have a reduced capacity for recovery and determine the response. However, the results imply that deductions as to the ability of tumour cells to recover from sublethal damage (whether chronically hypoxic or not), which rely on in vitroassays, may underestimate the extent of recovery.

Ehrlich mouse ascites cells have been used as a biological dosimeter to check physical methods of measurement of central axis depth doses of a beam of fast neutrons. The cells were irradiated in suspension in Perspex tubes, using air or nitrogen bubbling. Irradiations with fast neutrons under aerobic conditions were carried out at four depths in a tissue-equivalent phantom and in the absence of the phantom. The relative intensities of the radiation under these conditions agreed well with measurements using a special ionisation chamber, provided allowance was made for accompanying ? radiation. The oxygen enhancement ratio was found to be 2·78 with 250 kV X rays and 1·80 with the fast neutron beam. No signicant change of OER was observed for irradiations at 8·7 cm deep in the phantom.