Br. J. Cancer (1978) 37,
Suppl. 1II, 220
INFLUENCE OF TUMOUR SIZE ON RADIOSENSITIZATION BY MISONIDAZOLE J. A. STANLEY, M. J. PECKHAMH AND G. G. STEEL
Froam the Radiotherapy Research Unit, Divisions of Radiotherapy and Biophysics, In?stitute of Cancer Research, Belmont, Sutton, Surrey SM2 5PX
Summary.-The administration of misonidazole shortly before irradiation increased the radiation response of the Lewis lung tumour growing either as approximately 500mm3 subcutaneous implants or as 15mm3 lung colonies. In the subcutaneous tumours, the dose enhancement ratio was approximately half the oxygen enhancement ratio. In the lung colonies the sensitization of hypoxic cells appeared to be complete. Similar studies on subcutaneous B16 melanoma showed that the drug increased radiosensitivity to a level comparable with that of cells in small pulmonary nodules, whose hypoxic fraction appears to be below 10-3. The results indicate complete 4ose modification of hypoxic cells where their natural proportion is small, with incomplete enhancement of radiosensitivity where the natural hypoxic fraction is larger.
CURRENT clinical interest in the treatment of occult metastatic disease by prophylactic cytotoxic therapy is supported experimentally by the observation that cells in small tumours are more sensitive to radiation and chemotherapy than are those in larger tumours (Fu, Phillips and Wharam, 1976; Steel, Adams and Stanley, 1976; Stanley, Shipley and Steel, 1977). However, our finding that even pulmonary tumours less than 20 mm3 in volume contain substantial numbers of hypoxic radioresistant cells may lead one to expect limited success from prophylactic radiotherapy alone. The potential advantage of hypoxic cell sensitizers in this situation is considerable. The purpose of the present study was to investigate the effect of the radiosensitizer misonidazole (1 -(2-nitro- 1 -imidazolyl)-3-methoxy-2-propanol) on the radiation response of the Lewis lung tumour treated as 15mm3 pulmonary nodules and 500mm3 subcutaneous tumours, whose hypoxic fractions have been found to be 4% and 36% respectively (Shipley, Stanley and Steel, 1975). The effect of the drug on the radiosensitivity of 500mm3 subcutaneous B16 melanomas, which have
a hypoxic fraction of 12% at this size, was
also studied to investigate whether there were any differences in dose-modification between tumours of similar volumes but containing different proportions of hypoxic cells. MATERIALS AND METHODS The Lewis lung tumour and B16 melanoma that were used in this study have been extensively studied in this laboratory, and descriptions of the tumours and the experimental methods for the production of cell
suspensions have been published elsewhere (Hill and Stanley, 1975a). Subcutaneous tumours were produced by the implantation of 105-106 cells. Lung nodules were produced by the intravenous injection of 104 viable cells mixed with 106 plastic microspheres. The growth rate of pulmonary nodules of the Lewis lung tumour was higher than for the B16 tumour and nodules were available for dissection between Days 10 and 20 for the Lewis tumour, compared with 15-25 days for the melanoma. The radiosensitizer, misonidazole, was obtained from Roche Products Ltd., Welwyn Garden City, Herts., and the normal dose was 1 mg/g in saline injected i.p. 30 min before irradiation. Some results on the effect of varying dose and time are reported.
221
TUMOUR-SIZE DEPENDENCE OF RADIOSENSITIZATION
Irradiation was given as whole-body doses to unanaesthetized mice, using a 60Co irradiator. The dose-rate was 250-300 rad/min at 30 cm. In vitro cloning assays for the two tumours have been described (Courtenay, 1976). These allow the measurement of surviving fractions down to 5 X 10-4 and they were supplemented for the data shown in Fig. 2 by measurements with the end-point dilution and lung-colony assays which have a somewhat higher sensitivity (Steel and Adams, 1975; 1977). RESULTS
The effect of misonidazole combined with radiation on pulmonary nodules of the Lewis lung tumour When irradiation treatment of 15mm3 pulmonary nodules of the Lewis lung tumour was preceded by misonidazole at a dose of 1 mg/g body weight, there was a marked increase in radiosensitivity. In Fig. 1 the results so obtained are compared with 15mm3 Lewis lung pulmonary nodules which had received radiation treatment alone, and which are known to contain a considerable proportion of hypoxic cells (Stanley et al., 1977). Also shown are the radiation survival data for 1mm3 pulmonary nodules of the same tumour whose response to radiation indicates no hypoxic cells at the levels of survival measured (Shipley et al., 1975). The points derived from 15mm3 tumours which had received misonidazole before radiation treatment fit very closely to the line derived from the radiation response of 1mm3 pulmonary tumours. A Do value of 106 rad for 1mm3 nodules was derived by linear regression analysis on the dose response data in the terminal region of the curve. The data on the terminal portion of the survival curve for 15mm3 nodules are consistent with a Do of 275 rad, the value derived from pulmonary tumours irradiated under hypoxic conditions (Stanley et al., 1977).
1000
2000
JUUU
DOSE (rad) FIG. 1.-60Co y-ray cell survival curves for Lewis lung tumour cells irradiated in situ in air-breathing unanaesthetized mice: 0 Cells frorn lmm3 pulmonary nodules; A Cells from 15mm3 pulmonary nodules; A Cells from 15mm3 pulmonary nodules treated 30 min before irradiation with 1 mg/g misonidazole. Colony-forming ability assayed in vitro in soft agar and the lines were fitted by linear regression analysis to the data points without sensitizer that are below 10-1
when the tumour-bearing mice were pretreated with 1 mg/g body weight of misonidazole administered 30 min before radiation treatment. The results shown in Fig. 2 indicate that the radiation response of pretreated subcutaneous tumours (terminal Do 225 rad) was intermediate between that of tumours of similar size that had not received the drug (terminal Do = 315 rad) and that of single cells irradiated under well oxygenated conditions (terminal Do- 110 rad). The results of the in vitro assay of cell survival in subcutaneous tumours treated The effect of misonidazole on subcutaneous with the sensitizer (Fig. 2 points down to Lewis lung tumours 5 X 10-4 survival) do not rule out the The radiosensitivity of 500mm3 sub- possibility that there was a resistant comcutaneous tumours was also enhanced ponent of a small proportion of cells that
222
J. A. STANLEY, M. J. PECKHAM AND G. G. STEEL
points fall below the radiation-alone points and that the difference in the dose range 400-1000 rad is marked. The effect of misonidazole on subcutaneous B 16 melanoma The results in Fig. 3 indicate that the radiosensitizer considerably reduced, and may have completely removed, the hypoxic fraction from 500mm3 subcutaneous B 16 melanoma when tumour-bearing animals each received a dose of 1 mg/g body weight of the drug 30 min before irradiation treatment. Large tumours which had not been pretreated showed a
i62
10 ILL
S:
a 10
1(57 gm
1000
3000 2000 DOSE (rad )
4000
FIa. 2. 60Co y-ray cell survival curves for Lewis lung tumour cells irradiated in vivo under well(-&, A, [) and in vitro (0) oxygenated conditions. Animals which were treated with 1 mg/g body weight of misonidazole (A) were paired during irradiation with saline-treated controls (A). Points numbered 1 and 2 were obtained by the colony assay and end-point dilution assay respectively. For all other points colony-forming ability was assayed in -vitro in soft agar.
retained the hypoxic Do. To investigate this, the experiments were repeated using the end-point dilution and lung-colony assays (Steel and Adams, 1975; 1977) and radiation dose levels that were expected to give a surviving fraction of 10-5. The results, shown in Fig. 2, are in reasonable agreement with our expectation and confirmed that in this system misonidazole is dose-modifying. We therefore have no reason to believe that any cells fail to be as fully sensitized as the majority of hypoxic cells. In the lower dose range, the experiments were conducted with paired points (with and without the sensitizer at the same radiation dose). It will be seen that with one exception the sensitizer-treated
0
I~
0 (I)
DOSE (rad) FIG. 3.-60Co y-ray cell survival curves for B16 melanoma cells within 500mm3 subcutaneous tumours (A, E1, A, *) and within 1mm3 pulmonary tumours (0) irradiated in air-breathing unanaesthetized mice. The lines were fitted by linear regression analysis to the non-sensitizer points below 10-1. Closed symbols (A, U) are for animals pretreated with 0-5 mg/g and 1-0 mg/g body weight of misonidazole respectively, the square points (-) being paired with saline-treated controls (WE )
during irradiation. Colony-forming ability
was assayed in vitro in soft agar.
TUMOUR-SIZE DEPENDENCE OF RADIOSENSITIZATION
223
Lewis lung tumour and 500mm3 subcutaneous B 16 melanomas received 1 mg/g body weight of misonidazole before irradiation. In both cases the radiation survival data lie on the curves derived from radiation treatment of 1mm3 pulmonary tumours. There was no evidence of a hypoxic "tail" and Do values in the terminal regions of the curves are close to those measured for fully oxygenated single-cell suspensions irradiated in vitro (Shipley et al., 1975; Hill and Stanley, 1975b). The response seen for 15mm3 pulmonary tumours is similar to that recently reported by Brown (1978) for in vivo treatment of pulmonary nodules of the EMT6 tumour with misonidazole given before irradiation. The radiosensitizer was not as effective in increasing the radiosensitivity of 500mm3 subcutaneous Lewis lung tumours. We have examined the possibility that this was a reflection of the failure of the drug to reach all cells in adequate concentration, and the in vivo assay points shown in Fig. 2 do not support this hypothesis. Similar findings have been reported by Brown (1975) using the EMT6 tumour, by Rauth and Kaufman (1975) using the KHT sarcoma, and by McNally (1975) using Sarcoma F. Since our data suggest that the radiosensitizer exerts a dose-modifying effect on the radiation response of hypoxic cells it is possible to calculate a dose-modifying factor from the ratio of the terminal Do values for treatment with and without the drug. These dose-modifying factors are found to be 2-6 (3 0) for 15mm3 Lewis lung tumours, 1-4 (2.8) for 500mm3 Lewis lung tumours, and 2-0 (2.7) for 500 mm3 B16 melanomas. The values given in brackets are the oxygen enhancement ratios (OER) found from the ratio of the Do values for irradiation in air and in well-oxygenated cell suspension (Shipley et al., 1975). Howin view of the radiobiological eviever, DISCUSSION dence that cells in small lung colonies are The most important result of this study fully oxygenated, it may be more approis the high degree of radiosensitization priate to calculate OER values by referseen when 15mm3 pulmonary nodules of ence to the Do for well-oxygenated tumour
characteristic radiation dose-response curve terminating in an exponential portion with a Do of 409 rad and earlier experiments (Hill and Stanley, 1975b) had demonstrated that 12% of cells in this size of tumour were hypoxic. The radiation response of 1mm3 B 16 melanoma pulmonary nodules appeared to follow a similar pattern to Lewis lung tumour nodules of the same size; the terminal exponential portion of the curve indicated a Do of 201 rad which is considerably lower than that obtained from large subcutaneous tumours. There was no evidence of a hypoxic "tail". The points obtained from animals which had received the radiosensitizer lay consistently along the curve derived from 1mm3 pulmonary nodules down to survival levels of 10-4. The effect of varying both the dose of radiosensitizer and the time of exposure was studied. Although the majority of experiments were conducted allowing 30 min between administration of the drug and commencement of the radiation treatment, time intervals ranging from 20-60 min were tested using the Lewis lung tumour and there was no variation of response during this time. In the studies on the B16 melanoma (Fig. 3) no effect due to increasing the time interval to 60 min was observed, although there was a slight indication that a reduction in sensitizer dose to 0 5 mg/g body weight may have reduced the sensitizing effect. In each experiment a control group treated only with the radiosensitizer at a dose of 1 mg/g body weight was included in order to investigate whether there was any cytotoxic effect due to the drug alone. The ratio of plating efficiencies of the radiosensitizer controls to the saline control varied between 0-6 and 1-4 and the data showed no consistent evidence of a cytotoxic effect.
224
J. A. STANLEY, M. J. PECKHAM AND G. G. STEEL
cells in situ than to the Do for a welloxygenated cell suspension. The Do values for cells in small Lewis lung or B 16 lung nodules are slightly greater than those for oxic cell suspensions (Shipley et al., 1975) perhaps due to phenomenon connected with cell contact. Since in Figs. 1 and 3 the data with misonidazole coincide with those for 1mm3 lung tumours, it is clear that the OER values calculated in this way would equal the dose-modifying factors given above. Other points of interest also emerged from the data for 500mm3 tumours. In the case of both B16 melanoma and Lewis lung tumour the data points derived from treatment with 0 5 mg/g body weight of misonidazole indicate that sensitization may be quite considerable at this reduced dose. Brown (1975) in his studies with the EMT6 tumour noticed that drug doses as low as 0 3 mg/g were effective, and observed that this result was promising with respect to applications to radiotherapy. Of additional interest to radiotherapy is the observation that, in experiments on 500mm3 subcutaneous Lewis lung tumours, the sensitizer was markedly effective in the dose range 400-1000 rad. This study was partly supported by National Cancer Institute Contract No. NCI-CM-23717. We acknowledge with gratitude the continuing support and helpful advice of Professor L. F. Lamerton and the help of Dr N. M. Blackett in analysing the data.
REFERENCES BROWN, J. M. (1975) Selective Radiosensitization of the Hypoxic Cells of Mouse Tumours with the Nitroimidazole and Ro-07-0582. Rad. Res., 64, 633. BROWN, J. M. (1978) Cytotoxic Effects of the Hypoxic Cell Radiosensitizer Ro-07-0582 on Tumour Cells in vivo. Rad. Res. (in press). COURTENAY, V. D. (1976) A Soft Agar Colony Assay for Lewis Lung Tumour and B 16 Melanoma Taken Directly from the Mouse. Br. J. Cancer, 34, 39. Fu, K. K., PHILLIPS, T. L. & WHARAM, M. D. (1976) Radiation Response of Artificial Pulmonary Metastases of the EMT6 Tumor. Int. J. Rad. Oncol. Biol. Phys., 1, 257. HILL, R. P. & STANLEY, J. A. (1975a) The Lung Colony Assay: Extension to the Lewis Lung Tumour and the B16 Melanoma: Radiosensitivity of B16 Melanoma Cells. Int. J. Radiat. Biol., 27, 377. HILL, R. P. & STANLEY. J. A. (1975b) The Response of Hypoxic B1 6 Melanoma Cells to In vivo Treatment with Chemotherapeutic Agents. Cancer Res., 35, 1147. McNALLY, N. J. (1975) The Effect of an Hypoxic Cell Sensitizer on Tumour Growth Delay and Cell Survival. Implications for Cell Survival in situ and in vitro. Br. J. Cancer, 32, 610. RAUTH, A. M. & KAUFMAN, K. (1975) In vivo Testing of Hypoxic Radiosensitizers usinlg the KHT Murine Tumour Assayed by the Lung Colony Technique. Br. J. Radiol., 48, 209. SHIPLEY, W. U., STANLEY, J. A. & STEEL, G. G. (1975) Tumour Size Dependency in the Radiation Response of the Lewis Lung Carcinoma. Cancer Res., 35, 2488. STANLEY, J. A., SHIPLEY, W. U. & STEEL, G. G. (1977) Influence of Tumour Size on Hypoxic Fraction and Therapeutic Sensitivity of Lewis Lung Tumour. Br. J. Cancer, 36, 105. STEEL, G. G. & ADAMS, K. (1975) Stern-cell Survival and Tumour Control in the Lewis Lung Carcinoma. Cancer Res., 35, 1530. STEEL, G. G. & ADAMS, K. (1977) Enhancement by Cytotoxic Agents of Artificial Pulmonary Metastases. Br. J. Cancer, 36, 653. STEEL, G. G., ADAMS, K. & STANLEY, J. A. (1976) Size-dependence of the Response of Lewis Lung Tumours to BCNU. Cancer Treat. Rep., 60, 1743.
Suppl. 1II, 220
INFLUENCE OF TUMOUR SIZE ON RADIOSENSITIZATION BY MISONIDAZOLE J. A. STANLEY, M. J. PECKHAMH AND G. G. STEEL
Froam the Radiotherapy Research Unit, Divisions of Radiotherapy and Biophysics, In?stitute of Cancer Research, Belmont, Sutton, Surrey SM2 5PX
Summary.-The administration of misonidazole shortly before irradiation increased the radiation response of the Lewis lung tumour growing either as approximately 500mm3 subcutaneous implants or as 15mm3 lung colonies. In the subcutaneous tumours, the dose enhancement ratio was approximately half the oxygen enhancement ratio. In the lung colonies the sensitization of hypoxic cells appeared to be complete. Similar studies on subcutaneous B16 melanoma showed that the drug increased radiosensitivity to a level comparable with that of cells in small pulmonary nodules, whose hypoxic fraction appears to be below 10-3. The results indicate complete 4ose modification of hypoxic cells where their natural proportion is small, with incomplete enhancement of radiosensitivity where the natural hypoxic fraction is larger.
CURRENT clinical interest in the treatment of occult metastatic disease by prophylactic cytotoxic therapy is supported experimentally by the observation that cells in small tumours are more sensitive to radiation and chemotherapy than are those in larger tumours (Fu, Phillips and Wharam, 1976; Steel, Adams and Stanley, 1976; Stanley, Shipley and Steel, 1977). However, our finding that even pulmonary tumours less than 20 mm3 in volume contain substantial numbers of hypoxic radioresistant cells may lead one to expect limited success from prophylactic radiotherapy alone. The potential advantage of hypoxic cell sensitizers in this situation is considerable. The purpose of the present study was to investigate the effect of the radiosensitizer misonidazole (1 -(2-nitro- 1 -imidazolyl)-3-methoxy-2-propanol) on the radiation response of the Lewis lung tumour treated as 15mm3 pulmonary nodules and 500mm3 subcutaneous tumours, whose hypoxic fractions have been found to be 4% and 36% respectively (Shipley, Stanley and Steel, 1975). The effect of the drug on the radiosensitivity of 500mm3 subcutaneous B16 melanomas, which have
a hypoxic fraction of 12% at this size, was
also studied to investigate whether there were any differences in dose-modification between tumours of similar volumes but containing different proportions of hypoxic cells. MATERIALS AND METHODS The Lewis lung tumour and B16 melanoma that were used in this study have been extensively studied in this laboratory, and descriptions of the tumours and the experimental methods for the production of cell
suspensions have been published elsewhere (Hill and Stanley, 1975a). Subcutaneous tumours were produced by the implantation of 105-106 cells. Lung nodules were produced by the intravenous injection of 104 viable cells mixed with 106 plastic microspheres. The growth rate of pulmonary nodules of the Lewis lung tumour was higher than for the B16 tumour and nodules were available for dissection between Days 10 and 20 for the Lewis tumour, compared with 15-25 days for the melanoma. The radiosensitizer, misonidazole, was obtained from Roche Products Ltd., Welwyn Garden City, Herts., and the normal dose was 1 mg/g in saline injected i.p. 30 min before irradiation. Some results on the effect of varying dose and time are reported.
221
TUMOUR-SIZE DEPENDENCE OF RADIOSENSITIZATION
Irradiation was given as whole-body doses to unanaesthetized mice, using a 60Co irradiator. The dose-rate was 250-300 rad/min at 30 cm. In vitro cloning assays for the two tumours have been described (Courtenay, 1976). These allow the measurement of surviving fractions down to 5 X 10-4 and they were supplemented for the data shown in Fig. 2 by measurements with the end-point dilution and lung-colony assays which have a somewhat higher sensitivity (Steel and Adams, 1975; 1977). RESULTS
The effect of misonidazole combined with radiation on pulmonary nodules of the Lewis lung tumour When irradiation treatment of 15mm3 pulmonary nodules of the Lewis lung tumour was preceded by misonidazole at a dose of 1 mg/g body weight, there was a marked increase in radiosensitivity. In Fig. 1 the results so obtained are compared with 15mm3 Lewis lung pulmonary nodules which had received radiation treatment alone, and which are known to contain a considerable proportion of hypoxic cells (Stanley et al., 1977). Also shown are the radiation survival data for 1mm3 pulmonary nodules of the same tumour whose response to radiation indicates no hypoxic cells at the levels of survival measured (Shipley et al., 1975). The points derived from 15mm3 tumours which had received misonidazole before radiation treatment fit very closely to the line derived from the radiation response of 1mm3 pulmonary tumours. A Do value of 106 rad for 1mm3 nodules was derived by linear regression analysis on the dose response data in the terminal region of the curve. The data on the terminal portion of the survival curve for 15mm3 nodules are consistent with a Do of 275 rad, the value derived from pulmonary tumours irradiated under hypoxic conditions (Stanley et al., 1977).
1000
2000
JUUU
DOSE (rad) FIG. 1.-60Co y-ray cell survival curves for Lewis lung tumour cells irradiated in situ in air-breathing unanaesthetized mice: 0 Cells frorn lmm3 pulmonary nodules; A Cells from 15mm3 pulmonary nodules; A Cells from 15mm3 pulmonary nodules treated 30 min before irradiation with 1 mg/g misonidazole. Colony-forming ability assayed in vitro in soft agar and the lines were fitted by linear regression analysis to the data points without sensitizer that are below 10-1
when the tumour-bearing mice were pretreated with 1 mg/g body weight of misonidazole administered 30 min before radiation treatment. The results shown in Fig. 2 indicate that the radiation response of pretreated subcutaneous tumours (terminal Do 225 rad) was intermediate between that of tumours of similar size that had not received the drug (terminal Do = 315 rad) and that of single cells irradiated under well oxygenated conditions (terminal Do- 110 rad). The results of the in vitro assay of cell survival in subcutaneous tumours treated The effect of misonidazole on subcutaneous with the sensitizer (Fig. 2 points down to Lewis lung tumours 5 X 10-4 survival) do not rule out the The radiosensitivity of 500mm3 sub- possibility that there was a resistant comcutaneous tumours was also enhanced ponent of a small proportion of cells that
222
J. A. STANLEY, M. J. PECKHAM AND G. G. STEEL
points fall below the radiation-alone points and that the difference in the dose range 400-1000 rad is marked. The effect of misonidazole on subcutaneous B 16 melanoma The results in Fig. 3 indicate that the radiosensitizer considerably reduced, and may have completely removed, the hypoxic fraction from 500mm3 subcutaneous B 16 melanoma when tumour-bearing animals each received a dose of 1 mg/g body weight of the drug 30 min before irradiation treatment. Large tumours which had not been pretreated showed a
i62
10 ILL
S:
a 10
1(57 gm
1000
3000 2000 DOSE (rad )
4000
FIa. 2. 60Co y-ray cell survival curves for Lewis lung tumour cells irradiated in vivo under well(-&, A, [) and in vitro (0) oxygenated conditions. Animals which were treated with 1 mg/g body weight of misonidazole (A) were paired during irradiation with saline-treated controls (A). Points numbered 1 and 2 were obtained by the colony assay and end-point dilution assay respectively. For all other points colony-forming ability was assayed in -vitro in soft agar.
retained the hypoxic Do. To investigate this, the experiments were repeated using the end-point dilution and lung-colony assays (Steel and Adams, 1975; 1977) and radiation dose levels that were expected to give a surviving fraction of 10-5. The results, shown in Fig. 2, are in reasonable agreement with our expectation and confirmed that in this system misonidazole is dose-modifying. We therefore have no reason to believe that any cells fail to be as fully sensitized as the majority of hypoxic cells. In the lower dose range, the experiments were conducted with paired points (with and without the sensitizer at the same radiation dose). It will be seen that with one exception the sensitizer-treated
0
I~
0 (I)
DOSE (rad) FIG. 3.-60Co y-ray cell survival curves for B16 melanoma cells within 500mm3 subcutaneous tumours (A, E1, A, *) and within 1mm3 pulmonary tumours (0) irradiated in air-breathing unanaesthetized mice. The lines were fitted by linear regression analysis to the non-sensitizer points below 10-1. Closed symbols (A, U) are for animals pretreated with 0-5 mg/g and 1-0 mg/g body weight of misonidazole respectively, the square points (-) being paired with saline-treated controls (WE )
during irradiation. Colony-forming ability
was assayed in vitro in soft agar.
TUMOUR-SIZE DEPENDENCE OF RADIOSENSITIZATION
223
Lewis lung tumour and 500mm3 subcutaneous B 16 melanomas received 1 mg/g body weight of misonidazole before irradiation. In both cases the radiation survival data lie on the curves derived from radiation treatment of 1mm3 pulmonary tumours. There was no evidence of a hypoxic "tail" and Do values in the terminal regions of the curves are close to those measured for fully oxygenated single-cell suspensions irradiated in vitro (Shipley et al., 1975; Hill and Stanley, 1975b). The response seen for 15mm3 pulmonary tumours is similar to that recently reported by Brown (1978) for in vivo treatment of pulmonary nodules of the EMT6 tumour with misonidazole given before irradiation. The radiosensitizer was not as effective in increasing the radiosensitivity of 500mm3 subcutaneous Lewis lung tumours. We have examined the possibility that this was a reflection of the failure of the drug to reach all cells in adequate concentration, and the in vivo assay points shown in Fig. 2 do not support this hypothesis. Similar findings have been reported by Brown (1975) using the EMT6 tumour, by Rauth and Kaufman (1975) using the KHT sarcoma, and by McNally (1975) using Sarcoma F. Since our data suggest that the radiosensitizer exerts a dose-modifying effect on the radiation response of hypoxic cells it is possible to calculate a dose-modifying factor from the ratio of the terminal Do values for treatment with and without the drug. These dose-modifying factors are found to be 2-6 (3 0) for 15mm3 Lewis lung tumours, 1-4 (2.8) for 500mm3 Lewis lung tumours, and 2-0 (2.7) for 500 mm3 B16 melanomas. The values given in brackets are the oxygen enhancement ratios (OER) found from the ratio of the Do values for irradiation in air and in well-oxygenated cell suspension (Shipley et al., 1975). Howin view of the radiobiological eviever, DISCUSSION dence that cells in small lung colonies are The most important result of this study fully oxygenated, it may be more approis the high degree of radiosensitization priate to calculate OER values by referseen when 15mm3 pulmonary nodules of ence to the Do for well-oxygenated tumour
characteristic radiation dose-response curve terminating in an exponential portion with a Do of 409 rad and earlier experiments (Hill and Stanley, 1975b) had demonstrated that 12% of cells in this size of tumour were hypoxic. The radiation response of 1mm3 B 16 melanoma pulmonary nodules appeared to follow a similar pattern to Lewis lung tumour nodules of the same size; the terminal exponential portion of the curve indicated a Do of 201 rad which is considerably lower than that obtained from large subcutaneous tumours. There was no evidence of a hypoxic "tail". The points obtained from animals which had received the radiosensitizer lay consistently along the curve derived from 1mm3 pulmonary nodules down to survival levels of 10-4. The effect of varying both the dose of radiosensitizer and the time of exposure was studied. Although the majority of experiments were conducted allowing 30 min between administration of the drug and commencement of the radiation treatment, time intervals ranging from 20-60 min were tested using the Lewis lung tumour and there was no variation of response during this time. In the studies on the B16 melanoma (Fig. 3) no effect due to increasing the time interval to 60 min was observed, although there was a slight indication that a reduction in sensitizer dose to 0 5 mg/g body weight may have reduced the sensitizing effect. In each experiment a control group treated only with the radiosensitizer at a dose of 1 mg/g body weight was included in order to investigate whether there was any cytotoxic effect due to the drug alone. The ratio of plating efficiencies of the radiosensitizer controls to the saline control varied between 0-6 and 1-4 and the data showed no consistent evidence of a cytotoxic effect.
224
J. A. STANLEY, M. J. PECKHAM AND G. G. STEEL
cells in situ than to the Do for a welloxygenated cell suspension. The Do values for cells in small Lewis lung or B 16 lung nodules are slightly greater than those for oxic cell suspensions (Shipley et al., 1975) perhaps due to phenomenon connected with cell contact. Since in Figs. 1 and 3 the data with misonidazole coincide with those for 1mm3 lung tumours, it is clear that the OER values calculated in this way would equal the dose-modifying factors given above. Other points of interest also emerged from the data for 500mm3 tumours. In the case of both B16 melanoma and Lewis lung tumour the data points derived from treatment with 0 5 mg/g body weight of misonidazole indicate that sensitization may be quite considerable at this reduced dose. Brown (1975) in his studies with the EMT6 tumour noticed that drug doses as low as 0 3 mg/g were effective, and observed that this result was promising with respect to applications to radiotherapy. Of additional interest to radiotherapy is the observation that, in experiments on 500mm3 subcutaneous Lewis lung tumours, the sensitizer was markedly effective in the dose range 400-1000 rad. This study was partly supported by National Cancer Institute Contract No. NCI-CM-23717. We acknowledge with gratitude the continuing support and helpful advice of Professor L. F. Lamerton and the help of Dr N. M. Blackett in analysing the data.
REFERENCES BROWN, J. M. (1975) Selective Radiosensitization of the Hypoxic Cells of Mouse Tumours with the Nitroimidazole and Ro-07-0582. Rad. Res., 64, 633. BROWN, J. M. (1978) Cytotoxic Effects of the Hypoxic Cell Radiosensitizer Ro-07-0582 on Tumour Cells in vivo. Rad. Res. (in press). COURTENAY, V. D. (1976) A Soft Agar Colony Assay for Lewis Lung Tumour and B 16 Melanoma Taken Directly from the Mouse. Br. J. Cancer, 34, 39. Fu, K. K., PHILLIPS, T. L. & WHARAM, M. D. (1976) Radiation Response of Artificial Pulmonary Metastases of the EMT6 Tumor. Int. J. Rad. Oncol. Biol. Phys., 1, 257. HILL, R. P. & STANLEY, J. A. (1975a) The Lung Colony Assay: Extension to the Lewis Lung Tumour and the B16 Melanoma: Radiosensitivity of B16 Melanoma Cells. Int. J. Radiat. Biol., 27, 377. HILL, R. P. & STANLEY. J. A. (1975b) The Response of Hypoxic B1 6 Melanoma Cells to In vivo Treatment with Chemotherapeutic Agents. Cancer Res., 35, 1147. McNALLY, N. J. (1975) The Effect of an Hypoxic Cell Sensitizer on Tumour Growth Delay and Cell Survival. Implications for Cell Survival in situ and in vitro. Br. J. Cancer, 32, 610. RAUTH, A. M. & KAUFMAN, K. (1975) In vivo Testing of Hypoxic Radiosensitizers usinlg the KHT Murine Tumour Assayed by the Lung Colony Technique. Br. J. Radiol., 48, 209. SHIPLEY, W. U., STANLEY, J. A. & STEEL, G. G. (1975) Tumour Size Dependency in the Radiation Response of the Lewis Lung Carcinoma. Cancer Res., 35, 2488. STANLEY, J. A., SHIPLEY, W. U. & STEEL, G. G. (1977) Influence of Tumour Size on Hypoxic Fraction and Therapeutic Sensitivity of Lewis Lung Tumour. Br. J. Cancer, 36, 105. STEEL, G. G. & ADAMS, K. (1975) Stern-cell Survival and Tumour Control in the Lewis Lung Carcinoma. Cancer Res., 35, 1530. STEEL, G. G. & ADAMS, K. (1977) Enhancement by Cytotoxic Agents of Artificial Pulmonary Metastases. Br. J. Cancer, 36, 653. STEEL, G. G., ADAMS, K. & STANLEY, J. A. (1976) Size-dependence of the Response of Lewis Lung Tumours to BCNU. Cancer Treat. Rep., 60, 1743.
