In! J Rudiarion Oncolq~ Biol ’ Pergamon Press Ltd., 1979.
l Radiation
PhyJ Vol 5. pp. 1103.1109 Printed m the U.S.A
Sensitivity:
FACTS
0306.3016179/0701-,103/$02.CKWO
Facts and Models
AND MODELS
APPLIED
ROBERT Radiobiology
Research
Division,
TO TUMOR
RADIOTHERAPYt
F. KALLMAN, Ph.D.
Department of Radiology, Stanford Stanford, CA 94305, U.S.A.
University
School of Medicine,
The therapeutic effectiveness of radiation, i.e. the preferential elimination of tumor cells relative to normal cells, lies in the ability of radiation to alter tumor physiology during the course of treatment, not in inherent radiosensitivity differences between normal and tumor cells. The SRs, reoxygenation, repair, repopulation, redistribution, and recruitment are all initiated by irradiation, but only reoxygenation is tumor-specific. Comprehensive models of the response of tumors to irradiation must therefore take account of all these factors, but much more information is required before realistic and accurate models can be formulated. Radiation curability,
Reoxygenation,
Repair, Repopulation,
Redistribution,
Recruitment.
provements in radiotherapeutic effectiveness might be achieved. The post-irradiation events which must be taken into account in assembling facts and models relevant to tumor radiotherapy may conveniently be referred to and grouped under 5 R’s. Four of these have been discussed elsewhere25,52; they are reoxygenation, repair, repopulation, and redistribution. The fifth is recruitment; although perhaps it may be a special kind of redistribution, it is of sufficient importance to justify separate consideration. The first R listed, reoxygenation, is solely a property of tumors. The other 4 occur in normal as well as malignant tissues.
Although this symposium is designed to explore radiation sensitivity, this paper is concerned less with radiation sensitivity per se than with radiation curability, for it is the latter which is sought in the application of radiation to cancers in humans. It will be the thesis of this presentation that the effectiveness of radiation as a therapeutic modality lies not in any greater inherent sensitivity of cancer cells to radiation, but in the manner in which radiation is applied. It has been repeatedly demonstrated empirically that tumor cure or control may be brought about with high frequency when the radiation is administered by a number of fractionation or protraction schedules, all of which extend over intervals from several days to several weeks. Thus, it seems obvious that therapeutic effectiveness rests in the ability of radiation to alter the physiology of tumors, both within cells and between cells, during the course of treatment. In a typical fractionated regimen, each dose has 2 major kinds of effect: (a) to inflict damage that is lethal to cells without further extrinsic perturbation, and (b) to perturb individual cells or their organized “community” so they will be more efficiently destroyed by a subsequent treatment. The first class of events is the subject of most of the other papers in this symposium, and it is the latter class that is the principal concern of this paper. Indeed, it is through the understanding and, hopefully, successful manipulation of these post-irradiation events that useful models might be conceived and that im-
This term was introduced49 to denote the reacquisition of radiosensitivity by cells which have survived irradiation because they were hypoxic at the time of exposure. As it has been shown experimentally that hypoxic cells that survive irradiation in a tumor become better oxygenated afterwards, the term has proved to be appropriate. The oxygen concentration that is necessary to ensure maximum radiation effectiveness in mammalian cells is low relative to the degree of oxygenation that obtains throughout a typical mammal. Essentially complete sensitivity is obtained at intracellular oxygen concentrations of approximately 70 PM (corresponding to a partial pressure of 40 mm Hg), and less damage is produced
tThis research was supported by Public Health Service Research Grants Nos. CA-03353, CA-20527, and CA-10372,
awarded by the National Cancer Institute, Education and Welfare.
REOXYGENATION
1103
Depts of Health,
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Oncology
0 Biology
0 Physics
with lower oxygen concentrations.” When the extent of cell killing is measured for cells irradiated in the presence as compared with the absence of oxygen, oxygen enhancement ratios (OERs) are usually found to be 3.0 or slightly lower. This kind of OER has been demonstrated repeatedly both in vitro and in vivo, and because many tumors have either a limited or grossly inadequate blood supply, it is reasonable to suspect that radioresistant tumors contain significant numbers of clonogenic cells that are not killed by the usual therapeutic radiation regimens simply because these cells are hypoxic and therefore radioresistant. Many tumors have been found to consist of cords of healthy-looking tumor cells surrounding necrotic cells, or vice versa.42-44,47 The survival curves of many experimental solid tumors irradiated in viuo have a characteristic shape first described by Powers and Tolmach.75 There is a bi-phasic pattern with an initial portion similar to the survival curves of well oxygenated cells (in vitro), and a resistant tail portion with a slope similar to those of hypoxic cells in vitro or ‘of cells in tumors made hypoxic in vim. The vertical displacement of the resistant tail reflects the proportion of hypoxic cells in the tumor as well as the intrinsic radiosensitivity of these tumor cells; the proportion of hypoxic cells in the tumor can be estimated from such a survival curve by the procedure popularized by Hewitt and Wilson.*’ Because it is impossible to measure quantitative levels of tumor oxygenation and reoxygenation directly, virtually all data available on this subject are from indirect measurements: analyses of tumor-cell survival curves or 50% tumor control doses (TCD,,‘s). In the vast majority of experimental animal tumors, the proportion of clonogenic cells which have survival curves characteristic of hypoxic cells, i.e. the “hypoxic fraction”, is between 0.12 and 0.35.14 Well-oxygenated tumor cells are preferentially killed by radiation, as reflected by the hypoxic fraction of the surviving, clonogenic tumor cells, which has been shown to approach 1.0 when it is measured immediately after irradiation. At least 4 different patterns of reoxygenation occur in experimental tumors (Fig. 1). Although these different kinetic patterns may be influenced by the different methods used in tracing reoxygenation, it is more likely that these differences are characteristic of different kinds of tumors. Except reoxygenation is prompt and for the osteosarcoma,48 rapid. The effective proportion of hypoxic cells begins to decrease immediately after irradiation. In some cases, evidence suggests that the proportion of hypoxic cells does not simply decrease to its preirradiation value where it might remain indefinitely or until a subsequent radiation exposure; in some tumors, there is a suggestion of a second rise in the
July 1979, Volume 5, No. 7
Days after
rradmtion
Fig. 1. Reoxygenation kinetics of 4 experimental tumors following a conditioning irradiation (1000 or 1500 rad) at 0 time. The data are as follows: A, Thomlinson;46 0, Howes;*’
0,
van Putten;
*, Kallman.23
From
Kallman.z3
hypoxic fraction, possibly a consequence of radiation damage to the vasculature.6 In other tumors, the hypoxic fraction eventually falls to values significantly lower than those of the same tumors without irradiation, possibly because of extensive shrinkage or improved microcirculation.*’ We2’ have studied the speed of reoxygenation in 3 different mouse tumors, using the same technique to follow this phenomenon; our data demonstrate that the kinetics of reoxygenation in a tumor is a function of its radiation history. In contrast to the pattern that is seen after a single dose of IOOOrad, wherein reoxygenation is prompt and virtually completed by about 12 hr, it does not start until at least 12 hr after 2000rad. Although this is a provocative finding, it may not be directly applicable to human tumors undergoing fractionated radiotherapy by the customary daily irradiation regimens, because a single dose of 2000rad is not equivalent to the customary fractionated course to the same or even higher total doses. In fact, earlier evidence suggests that reoxygenation is not noticeably inhibited in tumors exposed to large total doses when these are accumulated by daily treatment with 200 rad fractions.49 Understanding the mechanism(s) responsible for reoxygenation is necessary for the formulation of realistic models that can account for the radiobiological findings and, hopefully, predict the effects of novel radiation treatments. Although possible mechanisms have been suggested,*’ the mechanism, or combination of mechanisms, responsible for reoxygenation remains to be established. Hypoxic cells limit the effectiveness of single doses of radiation in eradicating the cells of solid tumors in animals and probably in people as well. The greater therapeutic effectiveness of fractionated radiotherapy, relative to large single radiation treatments, probably results largely from reoxygenation,
Facts
and models applied to tumor radiotherapy
as the reoxygenation induced by each radiation treatment tends to ensure that more, formerly hypoxic, cells would be treated in an oxic environment in subsequent fractions, and therefore would be sterilized. However, not all fractionation schedules are equally successful in eliminating hypoxic cells in experimental rat and mouse tumors. The therapeutic ratios of different regimens vary significantly as a result of differences in reoxygenation and repopulation (see below) between fractions.‘0333 Similarly, there is evidence that the problem of hypoxic cells is not completely eliminated for all human tumors by reoxygenation occurring during the conventional courses of fractionated radiotherapy, and that residual, chronically hypoxic cells may limit the curability of certain human tumors by present therapeutic regimens.3 There are several means that might be employed to enhance the killing of hypoxic cells during radiotherapy; they include using an adjuvant to radiotherapy, such as a chemical agent such as the electron affinic nitroimidazole, misonidazole (Ro-07-0582), which will selectively radiosensitize hypoxic cells in a tumor;’ radiation therapy also could be combined with a second modality, such as hyperthermia,14 that is selectively toxic to hypoxic cells. These approaches are currently under intensive investigation and appear promising, but considerably more must be learned before data generated by such experiments can either be used reliably in the clinic or applied in more meaningful modeling studies. REPAIR For general purposes, the definition of repair includes all of the events which lead to the return or recovery of a biological system to its preirradiated state, after it has been injured by exposure to radiation or other therapeutic modalities. Because other papers in this symposium deal with specific aspects of repair, this discussion will include only those questions that are especially relevant to tumors. Two broad classes of phenomena are important here: the repair of sublethal damage (SLD) and of potentially lethal damage (PLD). SLD refers to injury which is insufficient to kill a cell and which may be repaired, usually in hours, if no further insult is received. As long as this damage is not completely repaired, additional injury may add to it and kill. Because of this, a dose of X-rays is less effective when it is subdivided into several fractions separated by time than when it is delivered in a single exposure. In the manner originally described by Elkind and Sutton,7.9 SLD is detected by comparing the survival of cells exposed to single treatments with survival of cells exposed to 2 or more treatments. The environment of a tumor, both extra- and in-
0
R. F. KALLMAN
1105
tracellular, is of paramount importance in determining the fate of potentially lethal damage. Some of the injury experienced by irradiated cells may become lethal under certain conditions of growth or metabolism, but it may be repaired under other conditions and thus be non-lethal. Such PLD is detected not by examining the consequences of a second radiation exposure but by tracing cell survival when the postirradiation environment is altered.34 Both SLD and PLD repair occur in uico-in tumors’7,3’.37 and in normal tissuess’.54 as well. In addition to the importance of oxygenation status to the response of tumor cells to radiation, the condition of tumor cells with respect to cycling, or generative, activity is of major importance. In a typical established tumor, usually only a minority of the potentially clonogenic cells is actively proliferating; many if not most of the remaining cells are viable, look normal, and are either temporarily or permanently non-proliferating.‘* Do all tumor cells share the same capacities to repair radiation damage, whether they are oxygenated or non-oxygenated, or proliferating or non-proliferating? It is common to use, as a model for proliferating tumor cells, exponentially growing cultures of mammalian cells of various types. The laboratory model generally used to simulate non-proliferating tumor cells is the plateau phase characteristic of cells maintained in vitro.16 Well-oxygenated, exponentially growing mammalian cells in vitro have repeatedly been shown to accumulate SLD and be capable of SLD repair. At moderate levels of hypoxia, both accumulation and repair of SLD occur, while it has been reported*’ that severely hypoxic cells are less able to accumulate SLD and totally anoxic cells are unable to accumulate SLD. This, however, is not universally accepted, as others’.*’ have found conflicting results. These discrepancies relate to the repair of SLD as well. If it were possible to confirm that all severely hypoxic tumor cells are incapable of normal SLD repair, this would have obvious relevance to a comprehensive model of tumor radiosensitivity. It would constitute another tumor-specific phenomenon similar to reoxygenation, as normal tissue cells are reasonably adequately oxygenated. On the other hand, tumor cells in exponential growth would be incapable of PLD repair if indeed they resemble exponentially growing mammalian cells in culture. When these cells cease proliferating, however, they should be able to repair PLD if they resemble their in uitro counterparts.‘s.34 Unlike SLD repair, PLD repair involves a change in the slope of the survival curve and is essentially independent of the shoulder. Several experimental observations in vitro suggest that non-cycling cells in solid tumors should be especially capable of PLD
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repair: (I) intercellular contact appears to increase the capacity for PLD repair; cells in dense monolayers exhibit 2-5 times as much PLD repair as the same cells irradiated after exposure in suspension. (2) Repair of PLD is enhanced when the irradiated cells are held in a nonoptimum nutrient medium which supports proliferation only poorly, and is greater still in a medium which does not support proliferation at all.‘5,70 (3) Cultured plateau-phase cells are capable of extensive PLD repair under conditions of moderate hypoxia.lh It is clear then that these repair properties must be incorporated into any model which would accurately take account of important determinants of tumor radiation sensitivity. All subpopulations of cells within a tumor, uniquely determined by oxygenation status, proliferation status, nutrient status, or other important physiological conditions, must be characterized by their abilities to repair PLD and SLD. REDISTRIBUTION The clonogenic cells of a tumor may be classified into either of 2 compartments: actively proliferating, P, or non-proliferating (or, perhaps, extremely slowly proliferating), Q. By redistribution, we refer to changes within the P compartment in the distribution and numbers of cells among the phases of the cell generation cycle. Movement of cells from the Q to the P compartment is a special case of redistribution, termed recruitment, which will be discussed below. It was both understood and expected that the P cell compartment would be altered by radiation, or by any other kind of experimental perturbation, after the classical findings of Terasima and Tolmach4’ and later, Sinclair and Morton:38 cellular radiosensitivity undergoes pronounced and regular changes as cells progress through the generation cycle, the wellknown “age-response function”. Terasima and Tolmach4’ also reported the important observation that the duration of the division delay experienced by irradiated cells depends not only on radiation dose but also on cell age. This, coupled with the differential killing of cells at more sensitive stages, supports the expectation that an exponentially growing and therefore asynchronous population of cells will be effectively, albeit temporarily, synchronized by moderate radiation exposure, as cells tend to progress beyond the transition point as a cohort when they recover from the reversible division delay lesion. This kind of expected redistribution was suggested by the experiments of Elkind” in which the survival of Chinese hamster cells in vitro was examined at 37°C and 24°C as a function of time between 2 doses of X-rays. If cells were held at 24°C between treatthrough the cell cycle was ments, progression effectively inhibited; and although SLD repair pro-
July 1979, Volume 5, NO. 7
ceeded normally, the subsequent fall and rise in survival observed at 37°C did not occur, presumably because redistribution could not be expressed at this temperature. Thus, redistribution leads to a state of partial growth synchrony among surviving cycling cells, and this should appear shortly after irradiation. It should be evidenced by changes in net cellular radiosensitivity as the surviving cohort continues to progress through the cell cycle. Because of the great heterogeneity which is characteristic of virtually all cell populations and the possibly stochastic nature of the control of growth and the transition from one phase of the cycle to the next, synchrony would not be expected to persist more than a short time-approximately one average cycle time for the cells in question. Evidence that these expectations are most reasonable and that indeed redistribution occurs in the cells of tumors and normal tissues has been provided.24.‘6 The data in Fig. 2 illustrate the cyclic fluctuation in radiosensitivity of the clonogenic cells of the EMT6 mouse tumor. Tumors were irradiated in situ with a first dose of 300 rad, and at 2-hr intervals thereafter they were exposed to a second dose of 600rad. To avoid complications arising from reoxygenation, the latter dose was always given to tumors which had been rendered maximally hypoxic by nitrogen asphyxiation of the host animals. The survival of tumor cells exposed to this regimen varied over a 2or 3-fold range, and the curve fitted to these data showed statistically significant periodicity with a I
1
I
I
I
1.000
I 0
0
I
10
I
I
I
1
20
30
40
50
TIME
IN HOURS
Fig. 2. Survival of cells in solid EMT6 tumors after 2 doses of radiation. All tumors were irradiated locally with 300 rad of X-rays at time 0. At the specified times after this first treatment, tumors received a second treatment with 600 rad delivered in hypoxia. 0, A, Two independent experiments; 0, A, survivals of cells irradiated only with the first dose of irradiation and explanted at different times after treatment to test for PLD repair. From Kallman and Rockwell.z5
Facts and models applied
to tumor radiotherapy
sensitive-resistant period averaging IO-12 hr. The ageresponse function of EMT6 cells is characterized by 2 phases of radiosensitivity and 2 phases of radioresistance during each cell cycle, and the mean cell cycle time is 20-22 hr. There is no evidence for circadian rhythmicity in the radiosensitivity of these tumor cells. Because of the likelihood that this kind of parasynchronous redistribution is the same for normal cells,S.‘“,S3 the deliberate induction of redistribution by radiation is not a simple strategy for increasing the effectiveness of therapy. Nonetheless, the possibility remains that capitalizing upon induced redistribution in this way may be a potential means of increasing appreciably the therapeutic ratio. The importance of redistribution and the differential effect that could be generated in P cells, as contrasted with Q cells, is provided by the hypothetical situation were comsketched by Withers:S6 if redistribution plete between successive doses of, say, 100 rad, tissue/tumor response and a net survival curve would be governed largely by the more sensitive cells. If there were little or no redistribution, a condition that might be experienced by normal non-proliferating tissues, the response would soon become that of the more resistant cells, and those cells would dominate the net response to the second and all subsequent doses. In the case of an actively proliferating tumor which must be irradiated together with largely non-proliferating normal tissue cells, therefore, redistribution must be taken into account in any realistic modeling exercise. RECRUITMENT As stated above, recruitment is the term used to designate the movement of cells from the Q to the P compartment. It should be recognized that “Q cells” is not synonymous with “Go cells”; the latter2” are found in normal tissues and may be either quiescent stem cells or functional differentiated cells subject to specific regulatory processes that control their proliferation. It has been suggested’” that plateau phase cells in uitro are analogous with Q cells of tumors in viva; this is significant since it is to be expected that plateau phase cells might react differently to perturbations than -would Go cells in normal tissues. The need to incorporate Q cells and their purported recruitment into realistic models of tumors during the course of irradiation is obvious; it has been shown2.22 that non-cycling tumor cells are potentially clonogenie and therefore tumorigenic and net tumor radiosensitivity, or radiocurability, depends strongly on the number of clonogenic cells that must be sterilized. Although there is good experimental evidence’9,22 that Q cells are recruited into P in certain experimental tumors, it remains to be determined exactly when recruited Q cells start cycling,
0
R. F. KALLMAN
1107
the number of cells recruited per unit dose, and the timing of this phenomenon as related to dose. The limited evidence presently available suggests that, perhaps surprisingly, several days are required for the recruitment of significant numbers of Q cells after moderate doses of radiation (300-800 rad). In addition to the growth fraction3* which is a major determinant in the sensitivity of tumors to irradiation and other modalities, it is also essential to know the rates of cell 10~s.~~ The extent to which clonogenic tumor cells are lost, either from unperturbed tumors or from irradiated tumors, must be taken into account; and it is essential to know whether cell loss in unperturbed as well as in treated tumors involves P cells, Q cells, or both these major compartments. Recent evidence” suggests that cell loss is a random event in that both.P and Q cells may be lost with the same probability; furthermore, this does not appear to be changed in the few days following irradiation at moderate levels. REPOPULATION This refers to the replacement of cells which have been destroyed by irradiation and is synonymous with the older term, regeneration. It is perhaps easiest to appreciate the contribution of this R to tumor radiosensitivity, as the response to irradiation depends so strongly on the total number of cells which must be sterilized. Especially in the design of multifraction regimens, with daily or near-daily irradiations given over several weeks, repopulation of tumor cells between doses may be critical in determining the overall response of the tumor. This is demonstrated in the experiments of Suit et a1.4 Isotransplants of a spontaneous mammary carcinoma in C3H mice were irradiated locally either under hypoxic conditions, normal air-breathing conditions, or hyperbaric conditions-with mice breathing oxygen at 30 psi. For tumors irradiated hypoxically (with the blood supply occluded by a clamp placed temporarily at the base of the tumor), which rules out complicating effects of reoxygenation, The TCD50 for a specified number of fractions, u, appears to be independent of the time between treatments until some critical time is reached. After this, the TCDso increases rapidly; and the increasing steepness of the slope of the curve of TCDso vs overall treatment time suggests that repopulation becomes relatively more important in determining the TCD,, as the treatment time increases. It is suggested that this occurs because surviving tumor cells may repopulate more rapidly because of a radiation-induced shortening in cell cycle time.18 Despite the elegance of these latter experiments, however, it must be recognized that evidence has been presented by others’2.4’ that cells cycle less rapidly after irradiation.
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Although it is essential that post-irradiation kinetics be thoroughly understood in order that istic and predictive models of tumor radiation sitivity be developed, this understanding is simply available at present. The literature is replete contradictory conflicts
truly
and
evidence, remain
comprehensive
to
be
resolved.
models
several Until
of tumor
cell realsennot with
important this
is done,
July 1979, Volume 5, No. 7
cannot be formulated; in fact, models advanced on the basis of premature, inadequate, or incorrect experimental evidence may be dangerously misleading. This would have the result of inhibiting further necessary and constructive research and, more importantly,
depriving
improvements
the
in radiation
cancer
patient
of achievable
therapy.
radiosensitivity REFERENCES
I. Adams, G.E.: Hypoxic cell sensitizers for radiotherapy. In Cancer, A Comprehensive Treatise, Vol. 6, ed. by Becker, F.F., New York, Plenum Press, 1977, pp. 181223. 2. Barendsen, G.W., Roelse, H., Hermens, A.F., Madhuizen, H.T., Van Peperzeel, H.A.. Rutgers, D.H.: Clonogenic capacity of proliferating and non-proliferating cells of a transplantable rat rhabdomyosarcoma in relation to its radiosensitivity. J. Nut/ Cancer Instit. 51: 1521-1526, 1973. 3. Bush, R.S., Hill, R.P.: Biologic discussions augmenting radiation effects and model systems. Laryngoscope 85: I 119-l 133, 1975. 4. Bush, R.S., Jenkins, R.D.T., Allt, W.E.C., Beale, F.A., Bean, H., Dembo, A.J., Pringle, J.F.: Definitive evidence for hypoxic cells influencing cure in cancer therapy. Br. J. Cancer 37 (Suppl. III): 302-306, 1978. 5. Denekamp, J., Ball, M.M., Fowler, J.F.: Recovery and repopulation in mouse skin as a function of time after X-irradiation. Radiut. Res. 37: 361-370, 1969. 6. Elkind, M.M.: Sublethal X-ray damage and its repair in mammalian cells. In Radiation Research, ed. by Silini, G., North-Holland, Amsterdam, 1967, pp. 558-586. 7. Elkind, M.M., Sutton, H.: X-Ray damage and recovery in mammalian cells in culture. Nature (Land.) 184: 1293-1295, 1959. 8. Elkind, M.M., Swain, R.W., Alescio, T., Sutton, H., Moses, W.B.: Oxygen, nitrogen, recovery, and radiation therapy. In Cellular Radiation Biology, Baltimore, Williams & Wilkins, 1965, pp. 442-461. 9. Elkind, M.M., Whitmore, G.F.: The Radiobiology of Cultured Mammalian Cells, New York, Gordon & Breach Scientific Publishers, Inc., 1967, 615 pp. IO. Fowler, J.F., Denekamp, J., Sheldon, P.W., Smith, A.M., Begg, A.C., Harris, S.R., Page. A.L.: Optimum fractionation in the X-ray treatment of C3H mouse mammary tumors. Br. J. Radiol. 47: 781-789, 1974. I I. Franko, A.J., Kallman, R.F., Rapacchietta, D.: Evaluation of cell loss in EMT6 tumors using ‘*‘IUdR release. Rudiut. Res. 74: 526, 1978. 12. Frindel, E., Vassort, F., Tubiana, M.: Effects of irradiation on the cell cycle of an experimental ascites tumor of the mouse. Int. J. Radiut. Biol. 17: 329-337, 1970. 13. Grav. L.H.: Radiobiologic basis of oxvgen as a modifving factor in radiation therapy. Am. j.-Roentgenol. 85: 803-815, 1961. 14. Hahn, G.M.: Metabolic aspects of the role of hyperthermia in mammalian cell inactivation and their possible relevance to cancer treatment. Cancer Res. 34: 3117-3123, 1974. 15. Hahn. G.M., Bagshaw, M.A., Evans, R.G., Gordon, L.F.: Repair of potentially lethal lesions in X-irradiated, hamster cells: metabolic density-inhibited Chinese effects and hypoxia. Radiut. Res. 55: 280-290, 1973. 16. Hahn, G.M., Little, J.B.: Plateau phase cultures of
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18.
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27. 28. 29.
mammalian cells: An in vitro model for human cancer. Curr. Topics Rudiut. Res. 8: 39-83, 1972. Hahn, G.M., Rockwell, S., Kallman, R.F., Gordon, L.F., Frindel, E.: Repair of potentially lethal damage in Cancer viva in solid tumor cells after X-irradiation. Res. 34: 351-354, 1974. Hermens, A.F.: Variations in the cell kinetics and the growth rate in an experimental tumour during natural growth and after irradiation. Publ. No. 835, Radiobiological Institute TNO, Rijswijk (ZH), The Netherlands, 1973. Hermens, A.F., Barendsen, G.W.: Effects of ionizing radiation on the growth kinetics of tumors. In Growth Kinetics and Biochemical Regulation of Normul and Mulignant Cells. Proc. 29th Ann. M. D. Anderson Symp., Houston, Texas, lo-12 Mar. 1976, 1977, pp. 531-546. Hewitt. H.B., Wilson, C.W.: A survival curve for mammalian leukaemia cells: Irradiated in vivo (implications for the treatment of mouse leukaemia by whole-body irradiation). Br. J. Cancer 13: 69-75, 1959. Howes, A.E.: An estimation of changes in the proportions and absolute numbers of hypoxic cells after irradiation of transplanted C3H mouse mammary turnours. Br. J. Rudiol. 42: 441-447, 1969. Kallman, R.F.: On the recruitment of non-cycling tumor cells by irradiation. In Growth Kinetics und Biochemical Regulation of Normal and Malignant Cells. Proc. 29th Ann. M. D. Anderson Symp., Houston, Texas, IO-12 Mar. 1976, 1977, pp. 545-546. Kallman, R.F.: The oxygen effect and reoxygenation. In Excerptu Medica Internutional Congress Series No. 353, Vol. 5: Surgery, Radiotherapy, and Chemotherapy of Cancer. Proc. XI Intl. Cancer Congr., Florence, 1974, pp. 136-140. Kallman, R.F.: The phenomenon of reoxygenation and fractionated for radiotherapy. implications its Radiology 105: 135-142, 1972. Kallman, R.F., Rockwell, S.: Effects of radiation on animal tumor models. In Cancer, A Comprehensive Treatise, Vol. 6, ed. by Becker, F.F., New York, Plenum Press, 1977, pp. 225-279. Kallman, R.F., Silini, G., Taylor, H.M., III: Recuperation from lethal injury by whole-body irradiation--II. Kinetic aspects in radiosensitive BALB/c mice, and cyclic fine structure during the 4 days after conditioning irradiation. Rudiat. Res. 29: 362-394, 1966. Koch, C.J., Kruuv, J.: The effect of extreme hypoxia on recovery after radiation by synchronized mammalian cells. Rudiut. Res. 48: 74-85, 1971. Lajtha, L.C.: On the concept of the cell cycle. J. Cell. Camp. Physiol. 62: 143-144 (Suppl. I), 1963. Littbrand, B., RCvCsz. L.: The effect of oxygen on cellular survival and recovery after radiation. Br. J. Rudiol. 42: 914-924, 1969.
Facts
30.
31.
32.
33.
34.
35.
36.
37.
38.
39. 40.
41.
42.
and models applied
to tumor
Little, J.B.: Repair of sub-lethal and potentially lethal radiation damage in plateau phase cultures of human cells. Nature (London) 224: 804-806, 1969. Little, J.B., Hahn, G.M., Frindel, E., Tubiana, M.: Repair of potentially lethal radiation damage in vitro and in viva. Radiology 106: 689-694, 1973. Mendelsohn, M.L.: Autoradiographic analysis of cell proliferation in spontaneous breast cancer of the C3H mouse-III. The growth fraction. J. Nat1 Cancer Instit. 28: 1015-1029, 1962. Moulder, J.E., Fischer, J.J., Milardo, R.: Time-dose relationships for the cure of an experimental rat tumor with fractionated radiation. Int. J. Radiat. Oncol. Biol. Phys. 1: 431438, 1976. Phillips, R.A., Tolmach, L.J.: Repair of potentially lethal damage in X-irradiated HeLa cells. Radiat. Res. 29: 413432, 1966. Powers, W.E., Tolmach, L.J.: A multicomponent X-ray survival curve for mouse lymphosarcoma cells irradiated in in uiuo. Nature (London) 197: 710-711, 1963. Rockwell, S., Kallman, R.F.: Cyclic radiation-induced variations in cellular radiosensitivity in a mouse mammary tumor. Radiat. Res. 57: 132-147, 1974. Shipley, W.U., Stanley, J.A., Courtenay, V.D., Field, S.B.: Repair of radiation damage in Lewis lung carwith fast neucinoma cells following in situ treatment trons and y-rays. Cancer Res. 35: 932-938, 1975. Sinclair, W.K., Morton, R.A.: X-Ray and UV sensitivity of synchronized Chinese hamster cells at various stages of the cell cyc!e. Biophys. J. 5: l-25, 1965. Steel, G.G.: Cell loss from experimental turnours. Cell Tissue Kinet. 1: 193-207, 1968. Suit, H.D., Howes, A.E., Hunter, N.: Dependence of response of a C3H mammary carcinoma to fractionated irradiation on fractionation number and intertreatment interval. Radiat. Res. 72: 440-454, 1977. Szczepanski, L., Trott, K.R.: Post-irradiation proliferation kinetics of a serially transplanted murine adenocarcinoma. Br. J. Radiol. 48: 200-208, 1975. Tannock, I.F.: Oxygen diffusion and the distribution of cellular radiosensitivity in tumours. Br. J. Radiol. 45: 5 15-524. 1972.
radiotherapy
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43. Tannock, I.F.: The relation between cell proliferation and the vascular systems in a transplanted mouse mammary tumour. Br. J. Cancer 22: 258-273, 1968. 44. Tannock, I.F., Steel, G.G.: Tumor growth and cell kinetics in chronically hypoxic animals. J. Nat1 Cancer Instit. 45: 123-133, 1970. T., Tolmach, L.J.: Variation in several re45. Terasima, sponses of HeLa cells to X-irradiation during the division cycle. Biophys. J. 3: 11-33, 1963. 46. Thomlinson, R.H.: Reoxygenation as a function of tumor size and histopathologic type. In Conference in Time and Dose Relationships in Radiobiology as Applied to Radiotherapy BNL 50230 (C-57), Springfield, Virginia, Clearinghouse for Federal Scientific and Technical Information, 1970, pp. 242-247. R.H., Gray, L.H.: The histological struc47. Thomlinson, ture of some human lung cancers and the possible implications for radiotherapy. Br. J. Cancer 9: 539-549, 1955. 48. van Putten, L.M.: Oxygenation and cell kinetics after irradiation in a transplantable osteosarcoma. In E#ects of Radiation on Cellular Proliferation, Vienna, Intl Atomic Energy Association, 1968, pp. 493-505. 49. van Putten, L.M., Kallman, R.F.: Oxygenation status of a transplantable tumor during fractionated radiation therapy. J. Nat! Cancer Instit. 40: 441-451, 1968. H.R.: Cell renewal systems concept and the 50. Withers, radiation response. In Frontiers of Radiation Therapy and Oncology, Vol. 6, ed. by Vaeth, J.M., Baltimore, University Park Press, 1972, pp. 93-107. H.R.: Recovery and repopulation in uivo by 51. Withers, mouse skin epithelial cells during fractionated irradiation. Radiat. Res. 32: 227-239, 1967. 52. Withers, H.R.: The 4 R’s of radiotherapy. Adu. Radiat. Biol. 5: 241-271, 1975. H.R., Hunter, N., Barkley, Jr., H.T., Reid, 53. Withers, B.O.: Radiation survival and regeneration characteristics of spermatogenic stem cells of mouse testis. Radiat. Res. 57: 88-103, 1974. 54. Withers, H.R., Mason, K.A.: The kinetics of recovery in irradiated colonic mucosa of the mouse. Cancer 34: 896-903, 1974.
l Radiation
PhyJ Vol 5. pp. 1103.1109 Printed m the U.S.A
Sensitivity:
FACTS
0306.3016179/0701-,103/$02.CKWO
Facts and Models
AND MODELS
APPLIED
ROBERT Radiobiology
Research
Division,
TO TUMOR
RADIOTHERAPYt
F. KALLMAN, Ph.D.
Department of Radiology, Stanford Stanford, CA 94305, U.S.A.
University
School of Medicine,
The therapeutic effectiveness of radiation, i.e. the preferential elimination of tumor cells relative to normal cells, lies in the ability of radiation to alter tumor physiology during the course of treatment, not in inherent radiosensitivity differences between normal and tumor cells. The SRs, reoxygenation, repair, repopulation, redistribution, and recruitment are all initiated by irradiation, but only reoxygenation is tumor-specific. Comprehensive models of the response of tumors to irradiation must therefore take account of all these factors, but much more information is required before realistic and accurate models can be formulated. Radiation curability,
Reoxygenation,
Repair, Repopulation,
Redistribution,
Recruitment.
provements in radiotherapeutic effectiveness might be achieved. The post-irradiation events which must be taken into account in assembling facts and models relevant to tumor radiotherapy may conveniently be referred to and grouped under 5 R’s. Four of these have been discussed elsewhere25,52; they are reoxygenation, repair, repopulation, and redistribution. The fifth is recruitment; although perhaps it may be a special kind of redistribution, it is of sufficient importance to justify separate consideration. The first R listed, reoxygenation, is solely a property of tumors. The other 4 occur in normal as well as malignant tissues.
Although this symposium is designed to explore radiation sensitivity, this paper is concerned less with radiation sensitivity per se than with radiation curability, for it is the latter which is sought in the application of radiation to cancers in humans. It will be the thesis of this presentation that the effectiveness of radiation as a therapeutic modality lies not in any greater inherent sensitivity of cancer cells to radiation, but in the manner in which radiation is applied. It has been repeatedly demonstrated empirically that tumor cure or control may be brought about with high frequency when the radiation is administered by a number of fractionation or protraction schedules, all of which extend over intervals from several days to several weeks. Thus, it seems obvious that therapeutic effectiveness rests in the ability of radiation to alter the physiology of tumors, both within cells and between cells, during the course of treatment. In a typical fractionated regimen, each dose has 2 major kinds of effect: (a) to inflict damage that is lethal to cells without further extrinsic perturbation, and (b) to perturb individual cells or their organized “community” so they will be more efficiently destroyed by a subsequent treatment. The first class of events is the subject of most of the other papers in this symposium, and it is the latter class that is the principal concern of this paper. Indeed, it is through the understanding and, hopefully, successful manipulation of these post-irradiation events that useful models might be conceived and that im-
This term was introduced49 to denote the reacquisition of radiosensitivity by cells which have survived irradiation because they were hypoxic at the time of exposure. As it has been shown experimentally that hypoxic cells that survive irradiation in a tumor become better oxygenated afterwards, the term has proved to be appropriate. The oxygen concentration that is necessary to ensure maximum radiation effectiveness in mammalian cells is low relative to the degree of oxygenation that obtains throughout a typical mammal. Essentially complete sensitivity is obtained at intracellular oxygen concentrations of approximately 70 PM (corresponding to a partial pressure of 40 mm Hg), and less damage is produced
tThis research was supported by Public Health Service Research Grants Nos. CA-03353, CA-20527, and CA-10372,
awarded by the National Cancer Institute, Education and Welfare.
REOXYGENATION
1103
Depts of Health,
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Oncology
0 Biology
0 Physics
with lower oxygen concentrations.” When the extent of cell killing is measured for cells irradiated in the presence as compared with the absence of oxygen, oxygen enhancement ratios (OERs) are usually found to be 3.0 or slightly lower. This kind of OER has been demonstrated repeatedly both in vitro and in vivo, and because many tumors have either a limited or grossly inadequate blood supply, it is reasonable to suspect that radioresistant tumors contain significant numbers of clonogenic cells that are not killed by the usual therapeutic radiation regimens simply because these cells are hypoxic and therefore radioresistant. Many tumors have been found to consist of cords of healthy-looking tumor cells surrounding necrotic cells, or vice versa.42-44,47 The survival curves of many experimental solid tumors irradiated in viuo have a characteristic shape first described by Powers and Tolmach.75 There is a bi-phasic pattern with an initial portion similar to the survival curves of well oxygenated cells (in vitro), and a resistant tail portion with a slope similar to those of hypoxic cells in vitro or ‘of cells in tumors made hypoxic in vim. The vertical displacement of the resistant tail reflects the proportion of hypoxic cells in the tumor as well as the intrinsic radiosensitivity of these tumor cells; the proportion of hypoxic cells in the tumor can be estimated from such a survival curve by the procedure popularized by Hewitt and Wilson.*’ Because it is impossible to measure quantitative levels of tumor oxygenation and reoxygenation directly, virtually all data available on this subject are from indirect measurements: analyses of tumor-cell survival curves or 50% tumor control doses (TCD,,‘s). In the vast majority of experimental animal tumors, the proportion of clonogenic cells which have survival curves characteristic of hypoxic cells, i.e. the “hypoxic fraction”, is between 0.12 and 0.35.14 Well-oxygenated tumor cells are preferentially killed by radiation, as reflected by the hypoxic fraction of the surviving, clonogenic tumor cells, which has been shown to approach 1.0 when it is measured immediately after irradiation. At least 4 different patterns of reoxygenation occur in experimental tumors (Fig. 1). Although these different kinetic patterns may be influenced by the different methods used in tracing reoxygenation, it is more likely that these differences are characteristic of different kinds of tumors. Except reoxygenation is prompt and for the osteosarcoma,48 rapid. The effective proportion of hypoxic cells begins to decrease immediately after irradiation. In some cases, evidence suggests that the proportion of hypoxic cells does not simply decrease to its preirradiation value where it might remain indefinitely or until a subsequent radiation exposure; in some tumors, there is a suggestion of a second rise in the
July 1979, Volume 5, No. 7
Days after
rradmtion
Fig. 1. Reoxygenation kinetics of 4 experimental tumors following a conditioning irradiation (1000 or 1500 rad) at 0 time. The data are as follows: A, Thomlinson;46 0, Howes;*’
0,
van Putten;
*, Kallman.23
From
Kallman.z3
hypoxic fraction, possibly a consequence of radiation damage to the vasculature.6 In other tumors, the hypoxic fraction eventually falls to values significantly lower than those of the same tumors without irradiation, possibly because of extensive shrinkage or improved microcirculation.*’ We2’ have studied the speed of reoxygenation in 3 different mouse tumors, using the same technique to follow this phenomenon; our data demonstrate that the kinetics of reoxygenation in a tumor is a function of its radiation history. In contrast to the pattern that is seen after a single dose of IOOOrad, wherein reoxygenation is prompt and virtually completed by about 12 hr, it does not start until at least 12 hr after 2000rad. Although this is a provocative finding, it may not be directly applicable to human tumors undergoing fractionated radiotherapy by the customary daily irradiation regimens, because a single dose of 2000rad is not equivalent to the customary fractionated course to the same or even higher total doses. In fact, earlier evidence suggests that reoxygenation is not noticeably inhibited in tumors exposed to large total doses when these are accumulated by daily treatment with 200 rad fractions.49 Understanding the mechanism(s) responsible for reoxygenation is necessary for the formulation of realistic models that can account for the radiobiological findings and, hopefully, predict the effects of novel radiation treatments. Although possible mechanisms have been suggested,*’ the mechanism, or combination of mechanisms, responsible for reoxygenation remains to be established. Hypoxic cells limit the effectiveness of single doses of radiation in eradicating the cells of solid tumors in animals and probably in people as well. The greater therapeutic effectiveness of fractionated radiotherapy, relative to large single radiation treatments, probably results largely from reoxygenation,
Facts
and models applied to tumor radiotherapy
as the reoxygenation induced by each radiation treatment tends to ensure that more, formerly hypoxic, cells would be treated in an oxic environment in subsequent fractions, and therefore would be sterilized. However, not all fractionation schedules are equally successful in eliminating hypoxic cells in experimental rat and mouse tumors. The therapeutic ratios of different regimens vary significantly as a result of differences in reoxygenation and repopulation (see below) between fractions.‘0333 Similarly, there is evidence that the problem of hypoxic cells is not completely eliminated for all human tumors by reoxygenation occurring during the conventional courses of fractionated radiotherapy, and that residual, chronically hypoxic cells may limit the curability of certain human tumors by present therapeutic regimens.3 There are several means that might be employed to enhance the killing of hypoxic cells during radiotherapy; they include using an adjuvant to radiotherapy, such as a chemical agent such as the electron affinic nitroimidazole, misonidazole (Ro-07-0582), which will selectively radiosensitize hypoxic cells in a tumor;’ radiation therapy also could be combined with a second modality, such as hyperthermia,14 that is selectively toxic to hypoxic cells. These approaches are currently under intensive investigation and appear promising, but considerably more must be learned before data generated by such experiments can either be used reliably in the clinic or applied in more meaningful modeling studies. REPAIR For general purposes, the definition of repair includes all of the events which lead to the return or recovery of a biological system to its preirradiated state, after it has been injured by exposure to radiation or other therapeutic modalities. Because other papers in this symposium deal with specific aspects of repair, this discussion will include only those questions that are especially relevant to tumors. Two broad classes of phenomena are important here: the repair of sublethal damage (SLD) and of potentially lethal damage (PLD). SLD refers to injury which is insufficient to kill a cell and which may be repaired, usually in hours, if no further insult is received. As long as this damage is not completely repaired, additional injury may add to it and kill. Because of this, a dose of X-rays is less effective when it is subdivided into several fractions separated by time than when it is delivered in a single exposure. In the manner originally described by Elkind and Sutton,7.9 SLD is detected by comparing the survival of cells exposed to single treatments with survival of cells exposed to 2 or more treatments. The environment of a tumor, both extra- and in-
0
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tracellular, is of paramount importance in determining the fate of potentially lethal damage. Some of the injury experienced by irradiated cells may become lethal under certain conditions of growth or metabolism, but it may be repaired under other conditions and thus be non-lethal. Such PLD is detected not by examining the consequences of a second radiation exposure but by tracing cell survival when the postirradiation environment is altered.34 Both SLD and PLD repair occur in uico-in tumors’7,3’.37 and in normal tissuess’.54 as well. In addition to the importance of oxygenation status to the response of tumor cells to radiation, the condition of tumor cells with respect to cycling, or generative, activity is of major importance. In a typical established tumor, usually only a minority of the potentially clonogenic cells is actively proliferating; many if not most of the remaining cells are viable, look normal, and are either temporarily or permanently non-proliferating.‘* Do all tumor cells share the same capacities to repair radiation damage, whether they are oxygenated or non-oxygenated, or proliferating or non-proliferating? It is common to use, as a model for proliferating tumor cells, exponentially growing cultures of mammalian cells of various types. The laboratory model generally used to simulate non-proliferating tumor cells is the plateau phase characteristic of cells maintained in vitro.16 Well-oxygenated, exponentially growing mammalian cells in vitro have repeatedly been shown to accumulate SLD and be capable of SLD repair. At moderate levels of hypoxia, both accumulation and repair of SLD occur, while it has been reported*’ that severely hypoxic cells are less able to accumulate SLD and totally anoxic cells are unable to accumulate SLD. This, however, is not universally accepted, as others’.*’ have found conflicting results. These discrepancies relate to the repair of SLD as well. If it were possible to confirm that all severely hypoxic tumor cells are incapable of normal SLD repair, this would have obvious relevance to a comprehensive model of tumor radiosensitivity. It would constitute another tumor-specific phenomenon similar to reoxygenation, as normal tissue cells are reasonably adequately oxygenated. On the other hand, tumor cells in exponential growth would be incapable of PLD repair if indeed they resemble exponentially growing mammalian cells in culture. When these cells cease proliferating, however, they should be able to repair PLD if they resemble their in uitro counterparts.‘s.34 Unlike SLD repair, PLD repair involves a change in the slope of the survival curve and is essentially independent of the shoulder. Several experimental observations in vitro suggest that non-cycling cells in solid tumors should be especially capable of PLD
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Radiation Oncology 0 Biology 0 Physics
repair: (I) intercellular contact appears to increase the capacity for PLD repair; cells in dense monolayers exhibit 2-5 times as much PLD repair as the same cells irradiated after exposure in suspension. (2) Repair of PLD is enhanced when the irradiated cells are held in a nonoptimum nutrient medium which supports proliferation only poorly, and is greater still in a medium which does not support proliferation at all.‘5,70 (3) Cultured plateau-phase cells are capable of extensive PLD repair under conditions of moderate hypoxia.lh It is clear then that these repair properties must be incorporated into any model which would accurately take account of important determinants of tumor radiation sensitivity. All subpopulations of cells within a tumor, uniquely determined by oxygenation status, proliferation status, nutrient status, or other important physiological conditions, must be characterized by their abilities to repair PLD and SLD. REDISTRIBUTION The clonogenic cells of a tumor may be classified into either of 2 compartments: actively proliferating, P, or non-proliferating (or, perhaps, extremely slowly proliferating), Q. By redistribution, we refer to changes within the P compartment in the distribution and numbers of cells among the phases of the cell generation cycle. Movement of cells from the Q to the P compartment is a special case of redistribution, termed recruitment, which will be discussed below. It was both understood and expected that the P cell compartment would be altered by radiation, or by any other kind of experimental perturbation, after the classical findings of Terasima and Tolmach4’ and later, Sinclair and Morton:38 cellular radiosensitivity undergoes pronounced and regular changes as cells progress through the generation cycle, the wellknown “age-response function”. Terasima and Tolmach4’ also reported the important observation that the duration of the division delay experienced by irradiated cells depends not only on radiation dose but also on cell age. This, coupled with the differential killing of cells at more sensitive stages, supports the expectation that an exponentially growing and therefore asynchronous population of cells will be effectively, albeit temporarily, synchronized by moderate radiation exposure, as cells tend to progress beyond the transition point as a cohort when they recover from the reversible division delay lesion. This kind of expected redistribution was suggested by the experiments of Elkind” in which the survival of Chinese hamster cells in vitro was examined at 37°C and 24°C as a function of time between 2 doses of X-rays. If cells were held at 24°C between treatthrough the cell cycle was ments, progression effectively inhibited; and although SLD repair pro-
July 1979, Volume 5, NO. 7
ceeded normally, the subsequent fall and rise in survival observed at 37°C did not occur, presumably because redistribution could not be expressed at this temperature. Thus, redistribution leads to a state of partial growth synchrony among surviving cycling cells, and this should appear shortly after irradiation. It should be evidenced by changes in net cellular radiosensitivity as the surviving cohort continues to progress through the cell cycle. Because of the great heterogeneity which is characteristic of virtually all cell populations and the possibly stochastic nature of the control of growth and the transition from one phase of the cycle to the next, synchrony would not be expected to persist more than a short time-approximately one average cycle time for the cells in question. Evidence that these expectations are most reasonable and that indeed redistribution occurs in the cells of tumors and normal tissues has been provided.24.‘6 The data in Fig. 2 illustrate the cyclic fluctuation in radiosensitivity of the clonogenic cells of the EMT6 mouse tumor. Tumors were irradiated in situ with a first dose of 300 rad, and at 2-hr intervals thereafter they were exposed to a second dose of 600rad. To avoid complications arising from reoxygenation, the latter dose was always given to tumors which had been rendered maximally hypoxic by nitrogen asphyxiation of the host animals. The survival of tumor cells exposed to this regimen varied over a 2or 3-fold range, and the curve fitted to these data showed statistically significant periodicity with a I
1
I
I
I
1.000
I 0
0
I
10
I
I
I
1
20
30
40
50
TIME
IN HOURS
Fig. 2. Survival of cells in solid EMT6 tumors after 2 doses of radiation. All tumors were irradiated locally with 300 rad of X-rays at time 0. At the specified times after this first treatment, tumors received a second treatment with 600 rad delivered in hypoxia. 0, A, Two independent experiments; 0, A, survivals of cells irradiated only with the first dose of irradiation and explanted at different times after treatment to test for PLD repair. From Kallman and Rockwell.z5
Facts and models applied
to tumor radiotherapy
sensitive-resistant period averaging IO-12 hr. The ageresponse function of EMT6 cells is characterized by 2 phases of radiosensitivity and 2 phases of radioresistance during each cell cycle, and the mean cell cycle time is 20-22 hr. There is no evidence for circadian rhythmicity in the radiosensitivity of these tumor cells. Because of the likelihood that this kind of parasynchronous redistribution is the same for normal cells,S.‘“,S3 the deliberate induction of redistribution by radiation is not a simple strategy for increasing the effectiveness of therapy. Nonetheless, the possibility remains that capitalizing upon induced redistribution in this way may be a potential means of increasing appreciably the therapeutic ratio. The importance of redistribution and the differential effect that could be generated in P cells, as contrasted with Q cells, is provided by the hypothetical situation were comsketched by Withers:S6 if redistribution plete between successive doses of, say, 100 rad, tissue/tumor response and a net survival curve would be governed largely by the more sensitive cells. If there were little or no redistribution, a condition that might be experienced by normal non-proliferating tissues, the response would soon become that of the more resistant cells, and those cells would dominate the net response to the second and all subsequent doses. In the case of an actively proliferating tumor which must be irradiated together with largely non-proliferating normal tissue cells, therefore, redistribution must be taken into account in any realistic modeling exercise. RECRUITMENT As stated above, recruitment is the term used to designate the movement of cells from the Q to the P compartment. It should be recognized that “Q cells” is not synonymous with “Go cells”; the latter2” are found in normal tissues and may be either quiescent stem cells or functional differentiated cells subject to specific regulatory processes that control their proliferation. It has been suggested’” that plateau phase cells in uitro are analogous with Q cells of tumors in viva; this is significant since it is to be expected that plateau phase cells might react differently to perturbations than -would Go cells in normal tissues. The need to incorporate Q cells and their purported recruitment into realistic models of tumors during the course of irradiation is obvious; it has been shown2.22 that non-cycling tumor cells are potentially clonogenie and therefore tumorigenic and net tumor radiosensitivity, or radiocurability, depends strongly on the number of clonogenic cells that must be sterilized. Although there is good experimental evidence’9,22 that Q cells are recruited into P in certain experimental tumors, it remains to be determined exactly when recruited Q cells start cycling,
0
R. F. KALLMAN
1107
the number of cells recruited per unit dose, and the timing of this phenomenon as related to dose. The limited evidence presently available suggests that, perhaps surprisingly, several days are required for the recruitment of significant numbers of Q cells after moderate doses of radiation (300-800 rad). In addition to the growth fraction3* which is a major determinant in the sensitivity of tumors to irradiation and other modalities, it is also essential to know the rates of cell 10~s.~~ The extent to which clonogenic tumor cells are lost, either from unperturbed tumors or from irradiated tumors, must be taken into account; and it is essential to know whether cell loss in unperturbed as well as in treated tumors involves P cells, Q cells, or both these major compartments. Recent evidence” suggests that cell loss is a random event in that both.P and Q cells may be lost with the same probability; furthermore, this does not appear to be changed in the few days following irradiation at moderate levels. REPOPULATION This refers to the replacement of cells which have been destroyed by irradiation and is synonymous with the older term, regeneration. It is perhaps easiest to appreciate the contribution of this R to tumor radiosensitivity, as the response to irradiation depends so strongly on the total number of cells which must be sterilized. Especially in the design of multifraction regimens, with daily or near-daily irradiations given over several weeks, repopulation of tumor cells between doses may be critical in determining the overall response of the tumor. This is demonstrated in the experiments of Suit et a1.4 Isotransplants of a spontaneous mammary carcinoma in C3H mice were irradiated locally either under hypoxic conditions, normal air-breathing conditions, or hyperbaric conditions-with mice breathing oxygen at 30 psi. For tumors irradiated hypoxically (with the blood supply occluded by a clamp placed temporarily at the base of the tumor), which rules out complicating effects of reoxygenation, The TCD50 for a specified number of fractions, u, appears to be independent of the time between treatments until some critical time is reached. After this, the TCDso increases rapidly; and the increasing steepness of the slope of the curve of TCDso vs overall treatment time suggests that repopulation becomes relatively more important in determining the TCD,, as the treatment time increases. It is suggested that this occurs because surviving tumor cells may repopulate more rapidly because of a radiation-induced shortening in cell cycle time.18 Despite the elegance of these latter experiments, however, it must be recognized that evidence has been presented by others’2.4’ that cells cycle less rapidly after irradiation.
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Although it is essential that post-irradiation kinetics be thoroughly understood in order that istic and predictive models of tumor radiation sitivity be developed, this understanding is simply available at present. The literature is replete contradictory conflicts
truly
and
evidence, remain
comprehensive
to
be
resolved.
models
several Until
of tumor
cell realsennot with
important this
is done,
July 1979, Volume 5, No. 7
cannot be formulated; in fact, models advanced on the basis of premature, inadequate, or incorrect experimental evidence may be dangerously misleading. This would have the result of inhibiting further necessary and constructive research and, more importantly,
depriving
improvements
the
in radiation
cancer
patient
of achievable
therapy.
radiosensitivity REFERENCES
I. Adams, G.E.: Hypoxic cell sensitizers for radiotherapy. In Cancer, A Comprehensive Treatise, Vol. 6, ed. by Becker, F.F., New York, Plenum Press, 1977, pp. 181223. 2. Barendsen, G.W., Roelse, H., Hermens, A.F., Madhuizen, H.T., Van Peperzeel, H.A.. Rutgers, D.H.: Clonogenic capacity of proliferating and non-proliferating cells of a transplantable rat rhabdomyosarcoma in relation to its radiosensitivity. J. Nut/ Cancer Instit. 51: 1521-1526, 1973. 3. Bush, R.S., Hill, R.P.: Biologic discussions augmenting radiation effects and model systems. Laryngoscope 85: I 119-l 133, 1975. 4. Bush, R.S., Jenkins, R.D.T., Allt, W.E.C., Beale, F.A., Bean, H., Dembo, A.J., Pringle, J.F.: Definitive evidence for hypoxic cells influencing cure in cancer therapy. Br. J. Cancer 37 (Suppl. III): 302-306, 1978. 5. Denekamp, J., Ball, M.M., Fowler, J.F.: Recovery and repopulation in mouse skin as a function of time after X-irradiation. Radiut. Res. 37: 361-370, 1969. 6. Elkind, M.M.: Sublethal X-ray damage and its repair in mammalian cells. In Radiation Research, ed. by Silini, G., North-Holland, Amsterdam, 1967, pp. 558-586. 7. Elkind, M.M., Sutton, H.: X-Ray damage and recovery in mammalian cells in culture. Nature (Land.) 184: 1293-1295, 1959. 8. Elkind, M.M., Swain, R.W., Alescio, T., Sutton, H., Moses, W.B.: Oxygen, nitrogen, recovery, and radiation therapy. In Cellular Radiation Biology, Baltimore, Williams & Wilkins, 1965, pp. 442-461. 9. Elkind, M.M., Whitmore, G.F.: The Radiobiology of Cultured Mammalian Cells, New York, Gordon & Breach Scientific Publishers, Inc., 1967, 615 pp. IO. Fowler, J.F., Denekamp, J., Sheldon, P.W., Smith, A.M., Begg, A.C., Harris, S.R., Page. A.L.: Optimum fractionation in the X-ray treatment of C3H mouse mammary tumors. Br. J. Radiol. 47: 781-789, 1974. I I. Franko, A.J., Kallman, R.F., Rapacchietta, D.: Evaluation of cell loss in EMT6 tumors using ‘*‘IUdR release. Rudiut. Res. 74: 526, 1978. 12. Frindel, E., Vassort, F., Tubiana, M.: Effects of irradiation on the cell cycle of an experimental ascites tumor of the mouse. Int. J. Radiut. Biol. 17: 329-337, 1970. 13. Grav. L.H.: Radiobiologic basis of oxvgen as a modifving factor in radiation therapy. Am. j.-Roentgenol. 85: 803-815, 1961. 14. Hahn, G.M.: Metabolic aspects of the role of hyperthermia in mammalian cell inactivation and their possible relevance to cancer treatment. Cancer Res. 34: 3117-3123, 1974. 15. Hahn. G.M., Bagshaw, M.A., Evans, R.G., Gordon, L.F.: Repair of potentially lethal lesions in X-irradiated, hamster cells: metabolic density-inhibited Chinese effects and hypoxia. Radiut. Res. 55: 280-290, 1973. 16. Hahn, G.M., Little, J.B.: Plateau phase cultures of
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mammalian cells: An in vitro model for human cancer. Curr. Topics Rudiut. Res. 8: 39-83, 1972. Hahn, G.M., Rockwell, S., Kallman, R.F., Gordon, L.F., Frindel, E.: Repair of potentially lethal damage in Cancer viva in solid tumor cells after X-irradiation. Res. 34: 351-354, 1974. Hermens, A.F.: Variations in the cell kinetics and the growth rate in an experimental tumour during natural growth and after irradiation. Publ. No. 835, Radiobiological Institute TNO, Rijswijk (ZH), The Netherlands, 1973. Hermens, A.F., Barendsen, G.W.: Effects of ionizing radiation on the growth kinetics of tumors. In Growth Kinetics and Biochemical Regulation of Normul and Mulignant Cells. Proc. 29th Ann. M. D. Anderson Symp., Houston, Texas, lo-12 Mar. 1976, 1977, pp. 531-546. Hewitt. H.B., Wilson, C.W.: A survival curve for mammalian leukaemia cells: Irradiated in vivo (implications for the treatment of mouse leukaemia by whole-body irradiation). Br. J. Cancer 13: 69-75, 1959. Howes, A.E.: An estimation of changes in the proportions and absolute numbers of hypoxic cells after irradiation of transplanted C3H mouse mammary turnours. Br. J. Rudiol. 42: 441-447, 1969. Kallman, R.F.: On the recruitment of non-cycling tumor cells by irradiation. In Growth Kinetics und Biochemical Regulation of Normal and Malignant Cells. Proc. 29th Ann. M. D. Anderson Symp., Houston, Texas, IO-12 Mar. 1976, 1977, pp. 545-546. Kallman, R.F.: The oxygen effect and reoxygenation. In Excerptu Medica Internutional Congress Series No. 353, Vol. 5: Surgery, Radiotherapy, and Chemotherapy of Cancer. Proc. XI Intl. Cancer Congr., Florence, 1974, pp. 136-140. Kallman, R.F.: The phenomenon of reoxygenation and fractionated for radiotherapy. implications its Radiology 105: 135-142, 1972. Kallman, R.F., Rockwell, S.: Effects of radiation on animal tumor models. In Cancer, A Comprehensive Treatise, Vol. 6, ed. by Becker, F.F., New York, Plenum Press, 1977, pp. 225-279. Kallman, R.F., Silini, G., Taylor, H.M., III: Recuperation from lethal injury by whole-body irradiation--II. Kinetic aspects in radiosensitive BALB/c mice, and cyclic fine structure during the 4 days after conditioning irradiation. Rudiat. Res. 29: 362-394, 1966. Koch, C.J., Kruuv, J.: The effect of extreme hypoxia on recovery after radiation by synchronized mammalian cells. Rudiut. Res. 48: 74-85, 1971. Lajtha, L.C.: On the concept of the cell cycle. J. Cell. Camp. Physiol. 62: 143-144 (Suppl. I), 1963. Littbrand, B., RCvCsz. L.: The effect of oxygen on cellular survival and recovery after radiation. Br. J. Rudiol. 42: 914-924, 1969.
Facts
30.
31.
32.
33.
34.
35.
36.
37.
38.
39. 40.
41.
42.
and models applied
to tumor
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radiotherapy
0
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