Tumor radiosensitivity: it's the subpopulations that count.

Primarily because of their different oxygen levels, not all the cells in solid tumors have equal sensitivity to ionizing radiation. Conventionally, radiation biologists and oncologists have classified tumor cells as being either well-oxygenated and radiosensitive or hypoxic and radioresistant. The hypoxic cells can arise in two different ways: by intermittent fluctuations in blood flow (the “acutely” hypoxic cells), and by the limited diffusion distance, of some 100–150 mm, of oxygen through tumor tissue (producing the “chronically” hypoxic cells). Both types of hypoxia should contribute to radioresistance, and in the past 5 years several investigators have indeed established such a correlation between hypoxia and local control following radiotherapy for head and neck and cervical cancers (1, 2). It has also been assumed that hypoxic cells will be resistant to certain chemotherapy drugs, not because of low oxygen levels per se but because these cells would be far from functioning blood vessels (and the source of the chemotherapy drug) and also because they would have a lower proliferation rate. Indeed, reduced proliferation as a function of distance from blood vessels was first shown over 30 years ago by Tannock using a transplanted mouse tumor, and by others since. Experimental evidence that cell killing by both radiation and anticancer drugs decreases as a function of distance from blood vessels in transplanted tumors has been obtained in studies by Durand (3). On pages 799 to 807 of this issue, Masunaga and colleagues (4) present data showing different sensitivities of the proliferating (P) and quiescent (Q) cells in transplanted SCCVII mouse tumors to radiation, to cisplatin, to the hypoxic cytotoxin tirapazamine (TPZ), and to mild hyperthermia (MHT). They also assessed the effect of the antiangiogenic drug TNP-470 on combinations of these agents. Most scientists are not too enthusiastic about what is often referred to as the “kitchen sink” approach, in which everything we could think of that might be useful is “thrown” at the tumor. This seems to be the case in the Masunaga study, in which the tumor may or may not get TNP-470, ionizing radiation, cisplatin, TPZ, mild hyperthermia (MHT), or various combinations of these five agents. However, the experiments have been well designed, and the micronucleus technique used, though not perfect, does allow some interesting conclusions to be made about the effects of these multiple agents on the quiescent as well as the proliferating cells in the tumors. The technique used is based on counting cells with micronuclei after the cells are dissociated from the tumor and cultured for 2 days in vitro. The micronuclei are formed from chromosome breaks and, as Revell showed many years ago, they correlate well with cell killing by radiation. Cells with micronuclei are destined to die; those without them live. This also seems to be true for other DNA-damaging agents such as TPZ and cisplatin. Thus, micronucleus frequency is a surrogate for cell kill. The advantage for using it over cell kill itself is that one can determine whether the tumor cells were proliferating or quiescent at the time of treatment. This is done by prolonged infusion prior to treatment of bromodeoxyuridine (BrdU), which distinguishes those cells that synthesized DNA during the infusion period (P cells) from those cells that did not (Q cells). The BrdUcontaining cells are subsequently recognized after tumor removal and dissociation of the cells using an antibody to BrdU. Thus, Masunaga and colleagues are able to distinguish lethally damaged cells (those with micronuclei) from viable cells, and whether the cells were proliferating or quiescent at the time of treatment. What are the conclusions of this work? First, nonproliferating (Q) cells are much more hypoxic than the population as a whole. This is seen both as a much-reduced response (micronucleus frequency) to g rays (Fig. 3) and by an increased response to TPZ (Fig. 2) of the Q cells compared to total cells. Based on radiation response, the authors estimate that the “hypoxic fraction” of Q cells in this tumor is 55% compared to about 15% for the population as a whole (including the Q cells). Another way of expressing this is that, for the most part, the hypoxic cells are nondividing and the aerobic cells are dividing. However, we have to remember that it is a continuum for both dividing/nondividing and aerobic/hypoxic cells, not an “all-or-nothing” state.