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Response of a poorly reoxygenating mouse osteosarcoma to X-rays and fast neutrons
Europ. 07. Cancer Vol. 7, pp. 153-160. Pergamon Press 1971. Printed in Great Britain
Response of a Poorly Reoxygenating Mouse Osteosarcoma to X-Rays and Fast Neutrons L. M. VAN PUTTEN, P. LELIEVELD and J. J. BROERSE
Radiobiological Institute TNO, 151 Lange Kleiweg, Rijswijk Z.H., The Netherlands A b s t r a c t - A transplantable osteosarcoma, which had previously been shown to reoxygenate its anoxic cells very slowly after a single large dose of X-rays, was studied after X- and neutron-irradiation. It was shown to be relatively resistant to fractionated X-ray therapy, probably due to anoxia. Nevertheless it could be shown that a small degree of reoxygenation must also occur in this tumour. Its response to treatment with 15 Me V neutrons was unusual; RBE valuesfor cell survival of 10-s werefor fractionated irradiation 2" 5,for single dose in vivo irradiation 1.8, andfor in vitro irradiation 1.6. The small difference between the RBE values for the well oxygenated osteosarcoma cells in vitro and for the partially hypoxic cells in vivo is associated with an unexpectedly high OER of 2" 0 for neutron irradiation of these cells in vitro as compared with the OER for X-rays of 2.4. The gain factor for neutrons, that is the ratio of OER values for X-rays and neutrons, is only 1.2, indicating that although hypoxia may be a limiting factor for the cure of this tumour byfractionated X-ray theory, the use of neutrons does not offer a major advantage. The searchfor other models of radiotherapy-resistant tumours should be continued.
INTRODUCTION
The present report is concerned with further studies, to learn whether reoxygenation occurs in this tumour during fractionated irradiation with daily doses of similar magnitude as used in clinical radiotherapy. In addition this report describes the effects of 1 5 M e V neutron irradiation of the osteosarcoma.
REOXYGENATION o f tumours in the course of
fractionated radiotherapy is a well known phenomenon. It has been found in all of the tumours tested for its occurrence with one exception; a transplantable mouse osteosarcoma (see Table 1). Since a tumour which reoxygenates slowly or not at all is of great interest for the application of fast neutrons, a number of further studies were devoted to this osteosarcoma. As described earlier [14], this tumour, which contains on the average between 10 and 20% anoxic cells, was found to reoxygenate slowly after a single dose of 1000 rads. After fractionated irradiation (4 dally doses of 250 rads) the mean anoxic cell fraction was o/ a figure which permitted no conclusion on 32/o, the occurrence of reoxygenation after these doses.
MATERIAL AND METHODS Tumours Osteosarcoma C22LR was used in serial transplantation passages 76-85 in (CBA/Rij × C57BL/Rij)FI hybrid mice, in which the tumour was originally induced [14]. T u m o u r cell survival was estimated by an endpoint dilution assay after suspending the tumour cells by the method of Reinhold [15]. In this estimate survival refers to reproductive 153
154
L. M. van Putten, P. Lelievdd and 07. o7. Broerse Table 1. Evidencefor tumour reoxygenation Host animal of original tumour
Tumour*
References
Evidence for tumour reoxygenation Mou, ge
C3Hkm
DBA C3H/J C3H/He C3H/(Barts) ]
(C3H× O)Ft ) R . H . C3H (Hammersmith) C3H/He
John's strain WAG/Rij
Sarcoma KHT
(L.T.) van Putten and Kallman [1, 2] Kallman et al. [3] (" (E.T.) Suit et al. [4] ] (E.T.) Suit and Maeda [5] [ (E.T.) Suit and Sehiavone [6] Mammary carcinoma ~ (Prim.) Hawkes et al. [7] and (E.T.) Cheshire and Lindop [8] ((E.T.) Howes [9] Fibrosarcoma (E.T.) Suit and Suchato [10] Squamous cell carcinoma (E.T.) Rat Sarcoma RIB5 Rhabdomyosarcoma
(L.T.) Thomlinson [11, 12] (L.T.) Barendsen and Broerse [13]
No evidence for reoxygenation (CBA/Rij × C57BL/Rij)F10steosarcoma C22LR mice
(L.T.) van Putten [14] i
*L.T. =Late transplant passages. E.T. =Early transplants. Prim. =Primary tumours.
integrity of the cells as assessed by their capacity to give rise to a tumour within 100 days after inoculation in an isologous recipient. The quantitative suspension technique used for estimating the total number of reproductively intact cells per tumour was described earlier [16]. This technique was used for evaluating cell survival after fractionated radiotherapy. T u m o u r volumes were estimated by caliper measurements of the tumour diameter in three dimensions; multiplication of these values and correction by a factor of 0" 525 gave a dependable measure for tumour volume as verified by excision and volume measurements by submersion. For all volume measurements 5 to 10 tumours per treatment group were followed simultaneously in order to obtain a reproducible average.
Irradiations X-rays were administered by a PhilipsMtiller machine operated under the following conditions: 300 kVp; 10 mA; H V L 3.0 m m Cu; dose rate 60rads per rain for in vivo irradiation and 130 rads per rain for in vitro irradiation. The 15 MeV neutrons were produced through the D - T reaction using a Van de Graaff generator working at 400 kV and 250 pA. Due to the limited output of the neutron generator, the irradiations were carried
out at small distances from the target. For the in vitro experiments the tumour cell suspensions were irradiated in culture dishes at a dose rate of 20 rads/min. The in vivo neutron irradiations were carried out at a dose rate of 15 rads/min. To compensate for the inhomogeneities in the neutron flux density distribution and to obtain multi-lateral exposure, the mice in centrifuge tubes were mounted on a styrofoam irradiation cylinder which was rotated in front of the target during the irradiations. The neutron dosimetry was carried out with a tissue equivalent ionization chamber and sulphur activation detectors [17, 18].
Cell oxygenation in vitro The oxygenation status of the cells during in vitro irradiation was controlled by the following methods: tumour cells suspended in Hanks' balanced salt solution with 5% calf serum were irradiated in a volume of 4-7 ml in a shallow culture dish with a surface of 15 cm 2. Before and during aerobic irradiation the cooled cell suspensions were agitated by a shaker and by a flow of humidified air of 600 ml per min, which was led over the surface of the suspension. For hypoxic irradiation the cell suspensions kept at room temperature were similarly agitated but gassed with pure nitrogen. A Hersch cell measured the oxygen content of
Response of a Poorly Reoxygenating Mouse Osteosarcomato X-rays and Fast Neutrons
less affected than the rat rhabdomyosarcoma described by Barendsen and Broerse, which regresses after 20 × 300 rads to a v o l u m e of less than 10% of its volume at the start of treatment [13]. Although the radiosensitivity in vitro for the cells of the two turnouts is rather similar [14, 19], the osteosarcoma does not decrease in size below the pre-irradiation volume, even after four weeks of treatment. This might be ascribed to persistent anoxia in the tumour associated with the poor reoxygenation observed after single high doses [14], but an alternative explanation for the poor response, might be found in rapid repopulation, associated with the high growth rate of the osteosarcoma. Its volume doubling time is approximately 1 "8 days. In an attempt to differentiate between these two possibilities a study was made of the comparative effect of 4 vs. 5 doses per week. If repopulation was important, the longer radiation free interval would cause a marked difference between these two regimens• I f however, anoxia is an important factor in the radiation resistance, the last dose of each week would in this slowly reoxygenating tumour certainly be less effective than the earlier doses, which after the radiation-free weekend might find a frac-
the outflow gas and after a 10 min equilibration period prior to irradiation an oxygen content below 20 ppm was reached. The culture dishes contained no materials which dissolve large quantities of oxygen; the walls were made of glass and the bottom of Melinex (polyethylene terephthalate).
High pressure oxygen treatment Mice anaesthetized with pentobarbital were irradiated in a stainless steel container covered with a 1 cm thick glass plate. After flushing the container with oxygen the pressure was slowly raised to 3 atm gauge pressure (4 atm absolute). After irradiation the pressure was lowered over a period of 20 min. Control irradiations were performed in the same container on similarly anaesthetized mice breathing air.
RESULTS Daily local X-irradiation of tumours located subcutaneously in the flank of anaesthetized mice was performed for 1, 2, 3 or 4 weeks with doses of 300 rads five times weekly. The tumour volume changes under these treatments are presented in Fig. I. It is evident that this tumour is very resistant, it is for example much 500 5 x 300 tad i::
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156
L. M. van Putten, P. Lelieveld and 07. 07. Broerse
tion of well-oxygenated cells. The results of these studies are presented in Fig. 2; for comparison the results of chemotherapy treatment with 4 and 5 doses per week are also presented. In these studies the mice were anaesthetized for irradiation, but not for chemotherapy; there was no significant effect on body weight of the treatments. The difference between the two irradiation regimens is much smaller than between the chemotherapy groups, suggesting that indeed anoxia may be very important in the radiation resistance of this tumour. Attempts to obtain more complete oxygenation during fractionated treatment of the tumour were performed by irradiation of tumours under high pressure oxygen. This w a s carried out as local irradiation of tumours located on the leg of mice under nembutal anaesthesia, both in mice breathing 4 atmospheres of oxygen and in control mice breathing air at ambient pressure. The results are presented in Fig. 3 (lines A and B). The tumours appear more sensitive under high pressure oxygen, but the most interesting results are seen in the different types of control mice. A comparison of the results in anaesthetized controls, line A, with line C, which shows the cell survival in tumours of total-body irradiated, non-anaesthetized control mice,
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indicates that anaesthesia caused a marked decrease in radiosensitivity, probably through anoxia. Some constriction of the mouse leg which carried the tumour, by the lead which shielded the rest of the body may have contributed to this anoxia, although such constriction was avoided as much as possible. Although the high pressure oxygen treatment was therefore not successful, this experiment demonstrated the complications caused by anaesthesia and this led us to study the effect of neutrons in non-anaesthetized mice. In Fig. 3 the effect of fractionated total body irradiation with fast neutrons is represented (line D). The RBE for 0-01% survival is 2"5, a disappointingly low value for an anoxic tumour. In order to analyse the reasons for this finding additional studies with fast neutrons were performed: the effect of single doses in vivo are presented in Fig. 4 and the results of in vitro irradiation of osteosarcoma cells with 15 MeV neutrons under different conditions of oxygenation are given in Fig. 5. Table 2 presents the parameters of the dose-effect curves for X-rays and neutrons and the derived values for RBE, OERs and gain factor. The most striking result is the finding of an O E R for fast neutrons of 2"0, which is very high, especially in comparison with the O E R of 2" 4 for X-rays. This may explain the relatively
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Response of a Poorly Reoxygenating Mouse Osteosarcoma to X-rays and Fast Neutrons
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Fig. 3. The effect of 5 daily doses of irradiation on tumour-cell survival. Survival is plotted as the ratio of the numberof reprvductively intact turnoutcells in treatedturnouts immediately after the last dose to the number in control animals at the time of thefirst treatment; in the treatmentperiod this cell number increases in the control tumours by a factor of 5.4. Line A: Localized turnout irradiation (X-rays) in anaesthetized mice. Line B: Localized turnout irradiation (X-rays) in anaesthetizedmice under hyperbaric oxygen. Line G: Total body X-irradiation without anaesthesia. Line D: Total body irradiation with fast neutrons without anaesthesia. The points * represent the effect of 5 daily doses of X-ray treatment on the rhabdomyosarcoma, described by Barendsen and Brverse [13].
low RBE for fast neutrons on this poorly oxygenated tumour.
DISCUSSION Three aspects of these studies deserve further discussion; the radioresistance of the tumour; its degree of oxygenation under fractionated radiotherapy and its response to fast neutrons. The radioresistance of this tumour to fractionated X-irradiation was demonstrated for anaesthetized mice by a study of the tumourvolume response. The later finding that the anaesthesia may affect the tumour sensitivity seems to diminish the value of these observations. Nevertheless there remains a marked resistance to X-ray treatment even without anaesthesia; this is clear from a comparison in Fig. 3 of line C which represents osteosarcoma cell survival after five daily doses of total body irradiation, with the data from Barendsen and Broerse [13] on rhabdomyosarcoma cell survival after five daily doses of local irradiation which are also presented in this figure.
An approximately tenfold-higher cell survival in the osteosarcoma after five days treatment is observed even when the mice are not anaesthetized. It would seem likely that this resistance is due to hypoxia. The study on the use of hyperbaric oxygen was not successful but we have obtained some information from the hypoxia caused by anaesthesia in this tumour. Many other transplanted tumours do not show this effect (e.g. [3, 9, 13] ) and this may be a peculiar property of the osteosarcoma, possibly related to its poor reoxygenation after single large doses of treatment. An analysis of the effect of anaesthesia (compare lines A and C of Fig. 3) indicates that it is responsible for an approximately fiftyfold difference in cell survival, presumably through hypoxia. If no reoxygenation took place in this tumour, at most a sixfold difference in survival could be ascribed to hypoxia, since initially about one-sixth of the cells are already anoxic. This suggests that at least an additional eight-
158
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fold ratio of survival must be explained by reoxygenation, and more if we assume that there are initially some oxygenated ceils in the tumour in anaesthetized mice. It is thus for example possible during four of the five treatments, there were 60% (or a fraction of 1/1.7) welloxygenated cells due to reoxygenation. In comparison with a completely anoxic tumour the survival ratios during the five treatment doses could thus have been 6 x 1.7 x 1- 7 x 1.7 x 1.7 =50. This example indicates that an approximately fiftyfold difference in survival would still be compatible with a degree of reoxygenation that is sub-optimal in the sense that the effectiveness of the fracionated treatment might still be limited by the presence of anoxic cells. A tumour with this property fulfills our search for a model of radiation resistance in which hypoxia is limiting the probability of cure. Such a tumour in man would provide the rationale for treatment with hyperbaric oxygen or other treatment modalities which are less affected by the oxygenation status of the cells. Unfortunately, the osteosarcoma did not
respond to neutrons in the expected way; an OER as high as 2" 0 implies that the neutrons are hardly more effective than X-rays in killing the anoxic cells of this tumour. The agreement between the results with regard to slope and RBE for anoxic cells in vitro, and terminal slope and RBE in vivo support the reality of this finding. For most well reoxygenating tumours one would not expect much difference in RBE on comparing single and fractionated doses in vivo since the higher effectiveness of neutrons on the anoxic cells, which is important in the single dose studies, is compensated for by the higher effectiveness of neutrons in fractionated exposure, associated with the smaller shoulder. For the osteosarcoma there is little higher effectiveness on anoxic cells, so the absence of repair of sub-lethal damage, giving a higher effectiveness of neutrons in fractionated irradiation, dominates and is thus responsible for the increased RBE with fractionation. It will be clear that the small gain factor does not permit an evaluation of the degree of reoxygenation from these results and our high expectations of
Response of a Poorly Reoxygenating Mouse Osteosarcoma to X-rays and Fast Neutrons finding tumour quence system. This
pected behaviour in a number of ways: it reoxygenates poorly, it is anoxic under anaesthesia and it shows a high O E R for neutron irradiation. Although the first two properties may well be
an elevated R B E for this resistant are not fulfilled, most likely as a conseof the high O E R for neutrons in this tumour thus demonstrates an unex1o
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Table 2. RBE
X-rays Do (racks)
for surviving fraction
Neutrons
n
Do (racks)
n
I0 -I
10-3
slope ratio
10-3
A. RBE of 15 MeV neutrons for survival of osteosarcoma cells
.In vitro Air Nitrogen
124 282
6"5 9.3
85 171
2"9 3.0
1-8 2"2
1.7 2.0
1 "6 1.9
1.5 1.7
246 287
3.2 45-6
151 137
1.85 8.8
1 "9 2.9
1.8 2.6
1.8 2.5
1.7 2" 1
In vivo Single doses Five daily doses
159
B. OER values for osteosarcoma cells in vitro
X-rays
Neutrons
Oxygen enhancement gain factor
For survival 10-t 10-2 10-3
2" 5 2.4 2.4
2" 0 2-0 2.0
1.2 1.2 1.2
Slope ratio
2.3
2.0
1.1
160
L. M . van Putten, P. Lelieveld and 07. 07. Broerse
related, we cannot see how the third one could be more than coincidental. A search among other experimental tumours for a suitable model for the radiotherapy-resistant clinical turnout should be continued in order to see whether other models show different patterns
of response, for which neutron therapy might indeed provide a therapeutic gain. Aclmowl~lg~entswThe capable technical assistance of Mrs. H. M. G. Uilenreef, Mrs. L. van der Burg, Miss L. K. J. Idsenga and Mr. A. C. Engels is gratefully acknowledged.
REFFJ~NClgS 1. L . M . VAN PUTTEN and R. F. KALLMAN,Effect of pre-irradiation on the ratio of oxygenated and anoxic cells in a transplanted mouse tumor. Abstract no. 910, Third Int. Congress of Radiation Research, Cortina d'Ampezzo (1966). 2. L . M . VAN PUTTENand R. F. KALLMAN,Oxygenation status of a transplantable tumor during fracfionated radiation therapy. 07. nat. Cancer Inst. 40~ 441 (1968). 3. R . F . KALLMAN,L. J. JAR.DINEand C. W. JOHNSON, The effects of different schedules of dose fractionation on the oxygenation status of a transplantable mouse sarcoma. 07. nat. Cancer Inst. 44~ 369 (1970). 4. H . D . SUIT, R. LINDBERO, C. SUCHATOand A. OZ~.NNE,Radiation dose fractionation and high pressure oxygen in radiotherapy of the DBA mouse mammary carcinoma. Am. 07. Roentgenol. 99, 895 (1967). 5. H . D . SUIT and M. MAEDA, Hyperbaric oxygen and radiobiology of a C3H mouse mammary carcinoma. 07. nat. Cancer Inst. 39, 639 (1967). 6. H . D . SUIT and J. V. SC~IAVONE,Effect of a single dose of radiation on proportion of hypoxic cells in a C3H mouse mammary carcinoma. Radiology 90~ 325 (1968). 7. M . J . HAwks, R. P. HILL and P. J. LINDOP, The response of C3H mammary tumours to irradiation in single and fractionated doses. Brit. 07. Radiol. 41, 134 (1968). 8. P . J . CHESm~ and P. J. LINDOP, The influence of intracellular recovery and hypoxic cells on the radiation response of mammary tumours and skin in C3H mice. Brit. 07. Radiol. 4~2, 215 (1969). 9. A.E. How~.s, An estimation of changes in the proportions and absolute numbers ofhypoxic cells after irradiation of transplanted C3H mouse mammary tumours. Brit. 07. Radiol. 42, 441 (1969). 10. H . D . SmT and C. SUCHATO,Hyperbaric oxygen and radiotherapy of a fibrosarcoma and of a squamous-cell carcinoma of C3H mice. Radiology 89, 713 (1967). 11. R . H . THOMLINSON,The effects of irradiation on the proportion of anoxic cells in tumours. Third Int. Congress of Radiation Research, Abstract no. 878, Cortina d'Ampezzo (1966). 12. R . H . THOMLINSON,Changes of oxygenation in tumours in relation to irradiation. Front. Radiat. Ther. Oncol. 3, 109 (1968). 13. G. W. BARENDSEN and J. J. BROERSE,Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X-rays. II. Effects of fracfionated treatments, applied five times a week for several weeks. Europ. 07. Cancer 6, 89 (1970). 14. L . M . VAN PUTTEN, Tumour reoxygenation during fractionated radiotherapy; studies with a transplantable mouse osteosarcoma. Europ. 07. Cancer 4, 173 (1968). 15. H. S. R~.INHOLD, A cell dispersion technique for use in quantitative transplantation studies with solid tumours. Europ. 07. Cancer 1, 67 (1965). 16. L . M . VaN PurrF.N, Oxygenation and cell kinetics after irradiation in a transplantable osteosarcoma. In Effects of Radiation on Cellular Proliferation and Differentiation, p. 493. IAEA, Vienna (1968). 17. J . J . BRO~RSE and H. VaN AMMERS,Dosimetry for fast neutron irradiations of cultured cells and intact animals. I. Characteristics of tissue-equivalent ionization chambers. Int. 07. Radiat. Biol. 10, 417 (1966). 18. J . J . BROERSE, Dosimetry for fast neutron irradiations of cultured ceils and intact animals. II. Comparison between activiation and ionization methods. Int. 07. Radiat. Biol. 10, 429 (1966). 19. H.S. REINHOLD, Quantitative evaluation of the radiosensitivity of cells of a transplantable rhabdomyosarcoma in the rat. Europ. 07. Cancer 2, 33 (1966).
Response of a Poorly Reoxygenating Mouse Osteosarcoma to X-Rays and Fast Neutrons L. M. VAN PUTTEN, P. LELIEVELD and J. J. BROERSE
Radiobiological Institute TNO, 151 Lange Kleiweg, Rijswijk Z.H., The Netherlands A b s t r a c t - A transplantable osteosarcoma, which had previously been shown to reoxygenate its anoxic cells very slowly after a single large dose of X-rays, was studied after X- and neutron-irradiation. It was shown to be relatively resistant to fractionated X-ray therapy, probably due to anoxia. Nevertheless it could be shown that a small degree of reoxygenation must also occur in this tumour. Its response to treatment with 15 Me V neutrons was unusual; RBE valuesfor cell survival of 10-s werefor fractionated irradiation 2" 5,for single dose in vivo irradiation 1.8, andfor in vitro irradiation 1.6. The small difference between the RBE values for the well oxygenated osteosarcoma cells in vitro and for the partially hypoxic cells in vivo is associated with an unexpectedly high OER of 2" 0 for neutron irradiation of these cells in vitro as compared with the OER for X-rays of 2.4. The gain factor for neutrons, that is the ratio of OER values for X-rays and neutrons, is only 1.2, indicating that although hypoxia may be a limiting factor for the cure of this tumour byfractionated X-ray theory, the use of neutrons does not offer a major advantage. The searchfor other models of radiotherapy-resistant tumours should be continued.
INTRODUCTION
The present report is concerned with further studies, to learn whether reoxygenation occurs in this tumour during fractionated irradiation with daily doses of similar magnitude as used in clinical radiotherapy. In addition this report describes the effects of 1 5 M e V neutron irradiation of the osteosarcoma.
REOXYGENATION o f tumours in the course of
fractionated radiotherapy is a well known phenomenon. It has been found in all of the tumours tested for its occurrence with one exception; a transplantable mouse osteosarcoma (see Table 1). Since a tumour which reoxygenates slowly or not at all is of great interest for the application of fast neutrons, a number of further studies were devoted to this osteosarcoma. As described earlier [14], this tumour, which contains on the average between 10 and 20% anoxic cells, was found to reoxygenate slowly after a single dose of 1000 rads. After fractionated irradiation (4 dally doses of 250 rads) the mean anoxic cell fraction was o/ a figure which permitted no conclusion on 32/o, the occurrence of reoxygenation after these doses.
MATERIAL AND METHODS Tumours Osteosarcoma C22LR was used in serial transplantation passages 76-85 in (CBA/Rij × C57BL/Rij)FI hybrid mice, in which the tumour was originally induced [14]. T u m o u r cell survival was estimated by an endpoint dilution assay after suspending the tumour cells by the method of Reinhold [15]. In this estimate survival refers to reproductive 153
154
L. M. van Putten, P. Lelievdd and 07. o7. Broerse Table 1. Evidencefor tumour reoxygenation Host animal of original tumour
Tumour*
References
Evidence for tumour reoxygenation Mou, ge
C3Hkm
DBA C3H/J C3H/He C3H/(Barts) ]
(C3H× O)Ft ) R . H . C3H (Hammersmith) C3H/He
John's strain WAG/Rij
Sarcoma KHT
(L.T.) van Putten and Kallman [1, 2] Kallman et al. [3] (" (E.T.) Suit et al. [4] ] (E.T.) Suit and Maeda [5] [ (E.T.) Suit and Sehiavone [6] Mammary carcinoma ~ (Prim.) Hawkes et al. [7] and (E.T.) Cheshire and Lindop [8] ((E.T.) Howes [9] Fibrosarcoma (E.T.) Suit and Suchato [10] Squamous cell carcinoma (E.T.) Rat Sarcoma RIB5 Rhabdomyosarcoma
(L.T.) Thomlinson [11, 12] (L.T.) Barendsen and Broerse [13]
No evidence for reoxygenation (CBA/Rij × C57BL/Rij)F10steosarcoma C22LR mice
(L.T.) van Putten [14] i
*L.T. =Late transplant passages. E.T. =Early transplants. Prim. =Primary tumours.
integrity of the cells as assessed by their capacity to give rise to a tumour within 100 days after inoculation in an isologous recipient. The quantitative suspension technique used for estimating the total number of reproductively intact cells per tumour was described earlier [16]. This technique was used for evaluating cell survival after fractionated radiotherapy. T u m o u r volumes were estimated by caliper measurements of the tumour diameter in three dimensions; multiplication of these values and correction by a factor of 0" 525 gave a dependable measure for tumour volume as verified by excision and volume measurements by submersion. For all volume measurements 5 to 10 tumours per treatment group were followed simultaneously in order to obtain a reproducible average.
Irradiations X-rays were administered by a PhilipsMtiller machine operated under the following conditions: 300 kVp; 10 mA; H V L 3.0 m m Cu; dose rate 60rads per rain for in vivo irradiation and 130 rads per rain for in vitro irradiation. The 15 MeV neutrons were produced through the D - T reaction using a Van de Graaff generator working at 400 kV and 250 pA. Due to the limited output of the neutron generator, the irradiations were carried
out at small distances from the target. For the in vitro experiments the tumour cell suspensions were irradiated in culture dishes at a dose rate of 20 rads/min. The in vivo neutron irradiations were carried out at a dose rate of 15 rads/min. To compensate for the inhomogeneities in the neutron flux density distribution and to obtain multi-lateral exposure, the mice in centrifuge tubes were mounted on a styrofoam irradiation cylinder which was rotated in front of the target during the irradiations. The neutron dosimetry was carried out with a tissue equivalent ionization chamber and sulphur activation detectors [17, 18].
Cell oxygenation in vitro The oxygenation status of the cells during in vitro irradiation was controlled by the following methods: tumour cells suspended in Hanks' balanced salt solution with 5% calf serum were irradiated in a volume of 4-7 ml in a shallow culture dish with a surface of 15 cm 2. Before and during aerobic irradiation the cooled cell suspensions were agitated by a shaker and by a flow of humidified air of 600 ml per min, which was led over the surface of the suspension. For hypoxic irradiation the cell suspensions kept at room temperature were similarly agitated but gassed with pure nitrogen. A Hersch cell measured the oxygen content of
Response of a Poorly Reoxygenating Mouse Osteosarcomato X-rays and Fast Neutrons
less affected than the rat rhabdomyosarcoma described by Barendsen and Broerse, which regresses after 20 × 300 rads to a v o l u m e of less than 10% of its volume at the start of treatment [13]. Although the radiosensitivity in vitro for the cells of the two turnouts is rather similar [14, 19], the osteosarcoma does not decrease in size below the pre-irradiation volume, even after four weeks of treatment. This might be ascribed to persistent anoxia in the tumour associated with the poor reoxygenation observed after single high doses [14], but an alternative explanation for the poor response, might be found in rapid repopulation, associated with the high growth rate of the osteosarcoma. Its volume doubling time is approximately 1 "8 days. In an attempt to differentiate between these two possibilities a study was made of the comparative effect of 4 vs. 5 doses per week. If repopulation was important, the longer radiation free interval would cause a marked difference between these two regimens• I f however, anoxia is an important factor in the radiation resistance, the last dose of each week would in this slowly reoxygenating tumour certainly be less effective than the earlier doses, which after the radiation-free weekend might find a frac-
the outflow gas and after a 10 min equilibration period prior to irradiation an oxygen content below 20 ppm was reached. The culture dishes contained no materials which dissolve large quantities of oxygen; the walls were made of glass and the bottom of Melinex (polyethylene terephthalate).
High pressure oxygen treatment Mice anaesthetized with pentobarbital were irradiated in a stainless steel container covered with a 1 cm thick glass plate. After flushing the container with oxygen the pressure was slowly raised to 3 atm gauge pressure (4 atm absolute). After irradiation the pressure was lowered over a period of 20 min. Control irradiations were performed in the same container on similarly anaesthetized mice breathing air.
RESULTS Daily local X-irradiation of tumours located subcutaneously in the flank of anaesthetized mice was performed for 1, 2, 3 or 4 weeks with doses of 300 rads five times weekly. The tumour volume changes under these treatments are presented in Fig. I. It is evident that this tumour is very resistant, it is for example much 500 5 x 300 tad i::
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156
L. M. van Putten, P. Lelieveld and 07. 07. Broerse
tion of well-oxygenated cells. The results of these studies are presented in Fig. 2; for comparison the results of chemotherapy treatment with 4 and 5 doses per week are also presented. In these studies the mice were anaesthetized for irradiation, but not for chemotherapy; there was no significant effect on body weight of the treatments. The difference between the two irradiation regimens is much smaller than between the chemotherapy groups, suggesting that indeed anoxia may be very important in the radiation resistance of this tumour. Attempts to obtain more complete oxygenation during fractionated treatment of the tumour were performed by irradiation of tumours under high pressure oxygen. This w a s carried out as local irradiation of tumours located on the leg of mice under nembutal anaesthesia, both in mice breathing 4 atmospheres of oxygen and in control mice breathing air at ambient pressure. The results are presented in Fig. 3 (lines A and B). The tumours appear more sensitive under high pressure oxygen, but the most interesting results are seen in the different types of control mice. A comparison of the results in anaesthetized controls, line A, with line C, which shows the cell survival in tumours of total-body irradiated, non-anaesthetized control mice,
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indicates that anaesthesia caused a marked decrease in radiosensitivity, probably through anoxia. Some constriction of the mouse leg which carried the tumour, by the lead which shielded the rest of the body may have contributed to this anoxia, although such constriction was avoided as much as possible. Although the high pressure oxygen treatment was therefore not successful, this experiment demonstrated the complications caused by anaesthesia and this led us to study the effect of neutrons in non-anaesthetized mice. In Fig. 3 the effect of fractionated total body irradiation with fast neutrons is represented (line D). The RBE for 0-01% survival is 2"5, a disappointingly low value for an anoxic tumour. In order to analyse the reasons for this finding additional studies with fast neutrons were performed: the effect of single doses in vivo are presented in Fig. 4 and the results of in vitro irradiation of osteosarcoma cells with 15 MeV neutrons under different conditions of oxygenation are given in Fig. 5. Table 2 presents the parameters of the dose-effect curves for X-rays and neutrons and the derived values for RBE, OERs and gain factor. The most striking result is the finding of an O E R for fast neutrons of 2"0, which is very high, especially in comparison with the O E R of 2" 4 for X-rays. This may explain the relatively
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Response of a Poorly Reoxygenating Mouse Osteosarcoma to X-rays and Fast Neutrons
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low RBE for fast neutrons on this poorly oxygenated tumour.
DISCUSSION Three aspects of these studies deserve further discussion; the radioresistance of the tumour; its degree of oxygenation under fractionated radiotherapy and its response to fast neutrons. The radioresistance of this tumour to fractionated X-irradiation was demonstrated for anaesthetized mice by a study of the tumourvolume response. The later finding that the anaesthesia may affect the tumour sensitivity seems to diminish the value of these observations. Nevertheless there remains a marked resistance to X-ray treatment even without anaesthesia; this is clear from a comparison in Fig. 3 of line C which represents osteosarcoma cell survival after five daily doses of total body irradiation, with the data from Barendsen and Broerse [13] on rhabdomyosarcoma cell survival after five daily doses of local irradiation which are also presented in this figure.
An approximately tenfold-higher cell survival in the osteosarcoma after five days treatment is observed even when the mice are not anaesthetized. It would seem likely that this resistance is due to hypoxia. The study on the use of hyperbaric oxygen was not successful but we have obtained some information from the hypoxia caused by anaesthesia in this tumour. Many other transplanted tumours do not show this effect (e.g. [3, 9, 13] ) and this may be a peculiar property of the osteosarcoma, possibly related to its poor reoxygenation after single large doses of treatment. An analysis of the effect of anaesthesia (compare lines A and C of Fig. 3) indicates that it is responsible for an approximately fiftyfold difference in cell survival, presumably through hypoxia. If no reoxygenation took place in this tumour, at most a sixfold difference in survival could be ascribed to hypoxia, since initially about one-sixth of the cells are already anoxic. This suggests that at least an additional eight-
158
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fold ratio of survival must be explained by reoxygenation, and more if we assume that there are initially some oxygenated ceils in the tumour in anaesthetized mice. It is thus for example possible during four of the five treatments, there were 60% (or a fraction of 1/1.7) welloxygenated cells due to reoxygenation. In comparison with a completely anoxic tumour the survival ratios during the five treatment doses could thus have been 6 x 1.7 x 1- 7 x 1.7 x 1.7 =50. This example indicates that an approximately fiftyfold difference in survival would still be compatible with a degree of reoxygenation that is sub-optimal in the sense that the effectiveness of the fracionated treatment might still be limited by the presence of anoxic cells. A tumour with this property fulfills our search for a model of radiation resistance in which hypoxia is limiting the probability of cure. Such a tumour in man would provide the rationale for treatment with hyperbaric oxygen or other treatment modalities which are less affected by the oxygenation status of the cells. Unfortunately, the osteosarcoma did not
respond to neutrons in the expected way; an OER as high as 2" 0 implies that the neutrons are hardly more effective than X-rays in killing the anoxic cells of this tumour. The agreement between the results with regard to slope and RBE for anoxic cells in vitro, and terminal slope and RBE in vivo support the reality of this finding. For most well reoxygenating tumours one would not expect much difference in RBE on comparing single and fractionated doses in vivo since the higher effectiveness of neutrons on the anoxic cells, which is important in the single dose studies, is compensated for by the higher effectiveness of neutrons in fractionated exposure, associated with the smaller shoulder. For the osteosarcoma there is little higher effectiveness on anoxic cells, so the absence of repair of sub-lethal damage, giving a higher effectiveness of neutrons in fractionated irradiation, dominates and is thus responsible for the increased RBE with fractionation. It will be clear that the small gain factor does not permit an evaluation of the degree of reoxygenation from these results and our high expectations of
Response of a Poorly Reoxygenating Mouse Osteosarcoma to X-rays and Fast Neutrons finding tumour quence system. This
pected behaviour in a number of ways: it reoxygenates poorly, it is anoxic under anaesthesia and it shows a high O E R for neutron irradiation. Although the first two properties may well be
an elevated R B E for this resistant are not fulfilled, most likely as a conseof the high O E R for neutrons in this tumour thus demonstrates an unex1o
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Table 2. RBE
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for surviving fraction
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.In vitro Air Nitrogen
124 282
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159
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160
L. M . van Putten, P. Lelieveld and 07. 07. Broerse
related, we cannot see how the third one could be more than coincidental. A search among other experimental tumours for a suitable model for the radiotherapy-resistant clinical turnout should be continued in order to see whether other models show different patterns
of response, for which neutron therapy might indeed provide a therapeutic gain. Aclmowl~lg~entswThe capable technical assistance of Mrs. H. M. G. Uilenreef, Mrs. L. van der Burg, Miss L. K. J. Idsenga and Mr. A. C. Engels is gratefully acknowledged.
REFFJ~NClgS 1. L . M . VAN PUTTEN and R. F. KALLMAN,Effect of pre-irradiation on the ratio of oxygenated and anoxic cells in a transplanted mouse tumor. Abstract no. 910, Third Int. Congress of Radiation Research, Cortina d'Ampezzo (1966). 2. L . M . VAN PUTTENand R. F. KALLMAN,Oxygenation status of a transplantable tumor during fracfionated radiation therapy. 07. nat. Cancer Inst. 40~ 441 (1968). 3. R . F . KALLMAN,L. J. JAR.DINEand C. W. JOHNSON, The effects of different schedules of dose fractionation on the oxygenation status of a transplantable mouse sarcoma. 07. nat. Cancer Inst. 44~ 369 (1970). 4. H . D . SUIT, R. LINDBERO, C. SUCHATOand A. OZ~.NNE,Radiation dose fractionation and high pressure oxygen in radiotherapy of the DBA mouse mammary carcinoma. Am. 07. Roentgenol. 99, 895 (1967). 5. H . D . SUIT and M. MAEDA, Hyperbaric oxygen and radiobiology of a C3H mouse mammary carcinoma. 07. nat. Cancer Inst. 39, 639 (1967). 6. H . D . SUIT and J. V. SC~IAVONE,Effect of a single dose of radiation on proportion of hypoxic cells in a C3H mouse mammary carcinoma. Radiology 90~ 325 (1968). 7. M . J . HAwks, R. P. HILL and P. J. LINDOP, The response of C3H mammary tumours to irradiation in single and fractionated doses. Brit. 07. Radiol. 41, 134 (1968). 8. P . J . CHESm~ and P. J. LINDOP, The influence of intracellular recovery and hypoxic cells on the radiation response of mammary tumours and skin in C3H mice. Brit. 07. Radiol. 4~2, 215 (1969). 9. A.E. How~.s, An estimation of changes in the proportions and absolute numbers ofhypoxic cells after irradiation of transplanted C3H mouse mammary tumours. Brit. 07. Radiol. 42, 441 (1969). 10. H . D . SmT and C. SUCHATO,Hyperbaric oxygen and radiotherapy of a fibrosarcoma and of a squamous-cell carcinoma of C3H mice. Radiology 89, 713 (1967). 11. R . H . THOMLINSON,The effects of irradiation on the proportion of anoxic cells in tumours. Third Int. Congress of Radiation Research, Abstract no. 878, Cortina d'Ampezzo (1966). 12. R . H . THOMLINSON,Changes of oxygenation in tumours in relation to irradiation. Front. Radiat. Ther. Oncol. 3, 109 (1968). 13. G. W. BARENDSEN and J. J. BROERSE,Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X-rays. II. Effects of fracfionated treatments, applied five times a week for several weeks. Europ. 07. Cancer 6, 89 (1970). 14. L . M . VAN PUTTEN, Tumour reoxygenation during fractionated radiotherapy; studies with a transplantable mouse osteosarcoma. Europ. 07. Cancer 4, 173 (1968). 15. H. S. R~.INHOLD, A cell dispersion technique for use in quantitative transplantation studies with solid tumours. Europ. 07. Cancer 1, 67 (1965). 16. L . M . VaN PurrF.N, Oxygenation and cell kinetics after irradiation in a transplantable osteosarcoma. In Effects of Radiation on Cellular Proliferation and Differentiation, p. 493. IAEA, Vienna (1968). 17. J . J . BRO~RSE and H. VaN AMMERS,Dosimetry for fast neutron irradiations of cultured cells and intact animals. I. Characteristics of tissue-equivalent ionization chambers. Int. 07. Radiat. Biol. 10, 417 (1966). 18. J . J . BROERSE, Dosimetry for fast neutron irradiations of cultured ceils and intact animals. II. Comparison between activiation and ionization methods. Int. 07. Radiat. Biol. 10, 429 (1966). 19. H.S. REINHOLD, Quantitative evaluation of the radiosensitivity of cells of a transplantable rhabdomyosarcoma in the rat. Europ. 07. Cancer 2, 33 (1966).