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The combination of methyl-CCNU and irradiation: Cell survival studies on a human tumor xenograft
036&3Olfi79/091545-04$02.00/O
Int. J. Radiation
Oncology Biol. Phys.,, Vol. 5, p. 1541-1548 Q Pergamon Press Ltd., 1979. Printed m the U. d .A.
??Nitrosourea
THE COMBINATION OF METHYL-CCNU AND IRRADIATION: CELL SURVIVAL STUDIES ON A HUMAN TUMOR XENOGRAFT ANGELA E.BATEMAN,D. Phil., KAREN K. Fu,M.D.t G. D. W.Tows~,B.Tech. Radiotherapy Research Department, Sutton,
Biophysics Division, Institute Surrey. SM2 SPX, England
and
of Cancer Research
Cell survival studies on a human tumor xenogreft grown in mice have demonstrated that the chemotherapeutic drug methyl-CCNU can increase the cytotoxic effect of y-irradiation. Injection of 25 mgikg of methyl-CCNU three hours before irradiation was the most effective time schedule of those tested. This pre-treatment reduced the DOof the radiation survival curve from 231 rad to 135 rad without showing any evidence of a reduced shoulder. Human tumor xenograft, Methyl-CCNU, y-Irradiation.
INTRODUCTION
suppressed mice. Tumors from passages 8 to 20were used in this study. Methyl-CCNU was used from capsules intended for oral administration (10 mg NSC 95441, containing mannitol and magnesium stearate). The contents of one capsule were homogenized in 0.4 ml dimethyl sulphoxide then 3.6 ml 5% Tween 80 in saline was added, and 0.1 ml per 10 g of body weight was injected intraperitoneally. For tumor irradiation, unanaesthetized air-breathing mice were given wholebody exposure of two equal doses directed to right and left sides of the mouse from a 6oCo source (dose rate approximately 200 rad per min). Tumors were excised 16 to 20 hours after irradiation and were finely chopped and cell suspensions prepared by colIagenase treatment (30 min at 37°C in 2 mg/ml collagenase type 1 in Ham’s F12 medium) followed by five minutes incubation in 0.05% trypsin in calcium- and magnesium-free balanced salts solution. The resultant cell suspension was poured through a sterile polyester mesh of pore size 25 pm and mixed with calf serum (10% of total volume). The refractile tumor cells were counted on a hemocytometer. Appropriate cell dilutions were made and cells were plated in 0.3% agar medium containing rat erythrocytes and 20% special Bobby calf serum in Ham’s F12 medium.4 One ml agar
A combined treatment of chemotherapy and irradiation is currently being investigated as an adjuvant therapy for resectable cancers and has been demonstrated to prolong survival in patients with locally unresectable disease. ls6 Methyl-CCNU (Me-CCNU) has given a response-rate in patients with cancer of the large bowel comparable with that of the more frequently used 5-fluorouracil treatment and the combination of methyl-CCNU with irradiation in treatment of gutassociated cancers may therefore prove clinically useful. The availability of human tumors growing as xenografts in immune-suppressed mice has enabled us to test the combination of Me-CCNU and irradiation on an anaplastic carcinoma of human origin.
METHODSANDMATERIALS Immune-suppressed CBA/lac mice were prepared according to Steel et al.‘, by thymectomy at four weeks of age, followed by intraperitoneal injection of 200 mg/kg arabinosyl cytosine (Ara-C) two days before 900 rad whole-body s°Co irradiation. The tumor, an anaplastic adenocarcinoma obtained from a patient with a suspected tumor of the pancreas, was established as a xenograft (HX32) in 1973 and maintained by passaging in leg muscles of immune-
supported by an American Cancer Society Clinical Junior Faculty Fellowship. Reprint request to: A. E. Bateman, D. Phil.
TDivision of Radiation Oncology, University of Califorma, San Francisco, CA., U.S.A. Acknowledgements-Funded by National Institutes of Health grant no. R01 CA 20519. Dr. Fu was partially 1545
1546
Radiation Oncology 0 Biology 0 Physics
September
1979, Volume 5, Number 9
cultures, containing either 300 control cells or up to 3x lo4 treated cells were gassed with a 5% 02, 5% COP, 90% N, mixture and were fed after 7 and 14 days with 1.5 ml fresh medium. Cell colonies were scored after three weeks. The plating efficiency (PE) of the untreated tumor cells was approximately 30%. The ratio of PE of treated cells to PE of control cells was used to calculate the fraction of clonogenic cells surviving treatment (SF). Control PE was determined in each experiment by plating cells from each of at least two untreated mice; from two to six mice were given each test treatment and the SF of tumor clonogenic cells was determined for each individual mouse. All liquids which had come into contact with the human tumor material were autoclaved before disposal, .all plastics and glassware were either incinerated or bleached in hypochlorite solution before reuse. RESULTS The LD,, (30 days) for methyl-CCNU in our CBA/Iac mice is approximately 40 mg/kg. Twentyfive mg/kg of this drug was known to kill about 90% of HX32 clonogenic cells, and this dose was chosen for combination with irradiation. We investigated the change in SF with time of assay after drug injection; Fig. 1 shows a decrease in SF between two and 24 hours after treatment, as reported earlier for B16 mouse melanoma.5 This implies that the toxic products of this drug are active in vivo for longer than two hours. In the following experiments tumors were excised 1624 hours after Me-CCNU injection, by which time the maximum toxic effect of the drug should have occurred. The time between irradiation and tumor excision varied in these experiments between 16 and 20 hours, to allow the repair of “potentially lethal” radiation damage which is known to occur in this tumor.3 Radiation doses from 200-800 rad were given three hours after an injection of Me-CCNU or of the vehicle alone. The SF data are shown in Fig. 2. The lines drawn through the data were derived by linear regression of In SF with radiation dose. The expected addition curve is drawn parallel to the irradiation alone curve but intersecting the ordinate at the SF due to Me-CCNU alone (0.109, the mean of 16 assays). Calculated Do values are 231 rad for irradiation alone and 135 rad for irradiation three hours after Me-CCNU and the slope of the radiation plus Me-CCNU curve is significantly greater than that of the curve for radiation alone (one-tailed r-test p
‘ij g z & 0
0.01
I
I
2L
LO
Time
FIG. 1. The cytotoxic
I
I
15
2
of methyl-CCNU injection before assay) (hours effect
of 25 mg/kg Me-CCNU
meas-
ured at various times after injection. Me-CCNU was injected into two tumor-bearing mice at each time-point. The total cell yield per tumor (N) and the plating efficiency (PE) were measured. The proportion of clonogenic cells per tumor which survive Me-CCNU=N,,,,,,,,, x PE Itreated flLcuntrull x PElrontrull. Data from one representative experiment. The times referred to are those between drug administration and tumor removal for in vitro culture.
1.36 (67% confidence limits 1.13-1.65). The extrapolation number for irradiation after Me-CCNU is 0.285, which is 2.6 times the surviving fraction due to Me-CCNU alone (67% confidence limits 1.5-4.6 times SFlh,e_CCNUI). There is thus no evidence that prior exposure to Me-CCNU reduces the shoulder of the irradiation dose/response curve. Methyl-CCNU was injected at times between seven hours prior to and 30 min after 600 rad and the results are shown in Fig. 3 where the range of expected additive effect is shown as the shaded area. This range was calculated as the maximum and minimum values of the product of SFfradiationl and
Irradiation
and methyl-CCNU
1547
E. BATEMAN cftrl.
0 A.
-.
1.0 vi = 0, ” 2 2
0
0
0.1
g T;
i 0
5
a
N ” z
c
0
0.2
0
0.01
I
0
I
-7
z ._ :: 2
I
-5 Time
O-001
? ._ > ._ 2 l? 0
0~0001
0
I
1
I
200
LOO
Radiation
dose
I
I
800
between
I
-1
-3 methyl-CCNU
’
and
?i
irradiation
+l
’ (hours)
FIG. 3. Potentiation of radiation cytotoxicity by MeCCNU as a function of time between drug injection and irradiation. Data from several experiments are shown, in which the time of injection of 25 mgkg Me-CCNU was varied with respect to irradiation with 600 rad. A relative survival of 1.0 is the mean expected additive SF (SFlirradiafionl X SF ,a,,,,). The shaded area shows the range of additive survival (see text). The data points (0) are the values of SF observed for the combined treatments. Each open circle represents the results of an individual mouse tumor survival evaluation using in V~VOtreatment and in vitro culture.
in rad
FIG. 2. Radiation dose/response curves with or without the injection of 25 mgkg Me-CCNU three hours before irradiation. Each data point represents the SF of tumor cells from an individual mouse. 0 without pretreatment. DC,=231 rad. 0 three hours after IP injection of 25 mg/kg Me-CCNU D,,=135 rad. The dash line indicates survival expected of the effect if Me-CCNU were strictly additive to that of irradiation.
SF Ldrupl.The SF values obtained when Me-CCNU preceded irradiation by three hours appear to be lower than for other schedules, and this may be the optimum timing for potentiation. We conclude that Me-CCNU potentiated radiation damage to tumor cells after intraperitoneal injection into mice three hours before irradiation. DISCUSSION Earlier studies on the combined treatment of experimental tumors with drugs and irradiation include the detailed study of the effects of 5-fluorouracil and X-rays on the AKR lymphomas in which the time between treatments of this rapidly-proliferating diffuse tumor was varied. Cell killing much greater than additive was observed when 5-fluorouracil was administered about 20 hours before irradiation, and
also from immediately after to eight hours after irradiation. However, in the B16 murine solid tumor, no time-dependent potentiation of radiation damage by 5-fluorouracil was detected8 and in studies on the EMT6 murine solid tumor only one drug out of four tested gave a radiation dose/response curve which was steeper two hours after drug administration than the control radiation dose/response curve.‘O The effective drug in the latter study was the nitrosourea BCNU, and our results suggest that the cytotoxic activities of irradiation and Me-CCNU are not independent, so the potentiation of radiation cytotoxicity by nitrosoureas injected two or three hours before irradiation may occur in several tumor systems. Nitrosoureas may be interacting with DNA, as previously demonstrated for CCNU? and therefore increasing the effectiveness of radiation damage to DNA or inhibiting the repair of sub-lethal damage. The potential benefit of any treatment depends on the effect on the tumor relative to the toxicity to normal tissues. We have calculated a therapeutic ratio for Me-CCNU given to mice three hours before irradiation. The drug reduces the radiation dose necessary for a fixed surviving fraction by a factor of 2.83 for tumor cell survival (from Fig. 2 Me-CCNU plus 460 rad is equivalent to 1300 rad) and by a
1548
Radiation Oncology 0 Biology 0 Physics
factor
of 1.67 for gut microcolony survival (Meplus 780 rad is equivalent to 1300 rad, unpublished data of Dr. T. Phelps). The therapeutic
CCNU
September
1979, Volume 5, Number 9
ratio of this combination is therefore 1.69 at that radiation dose, and may indicate a beneficial treatment.
REFERENCES S.K.: Large-bowel cancer-the current status of treatment. J. Nat. Cancer Inst. 56: 3-10, 1976.
1. Carter,
2. Cheng, C.J., Fujimara, S., Grunberger, D., Weinstein, I. B.: Interaction of l-(2-chloroethyI)-3-cyclohexylI-nitrosourea (NSC-79037) with nucleic acids and proteins in vivo and in vitro. Cancer Res. 32: 22-27, 1972. 3. Courtenay, V. D., Smith, I. E., Peckham, J. J., Steel, G. G.: In vitro and in vivo radiosensitivity of human tumor cells obtained from a pancreatic carcinoma xenograft. Nature 263: 771-772, 1976. 4. Courtenay, V. D., Mills, J.: An in vitro colony assay for human tumors grown in immune-suppressed mice and treated in vivo with cytotoxic agents. Br. J. Cancer 37: 261-268, 1978. 5. Hill, R. P., Stanley, J. A.: The response of hypoxic B16 melanoma cells to in vivo treatment with chemotherapeutic agents. Cancer Res. 35: 1147-l 153, 1975. 6. Moertel, C. G., Childs, D. S., Jr., Reitemeier, R. J., Colby, M. Y., Holbrook, M. A.: Combined
7.
8.
9.
10.
5fluorouracil and supervoltage radiation therapy of locally unresectable gastrointestinal cancer. Lancet II: 865-867, 1%9. Steel, G. G., Courtenay, V. D., Rostom, A. Y.: Improved immune-suppression techniques for the xenografting of human tumors. Br. J. Cancer 37: 224230, 1978. Stephens, T. C., Peacock, J. H., Steel, G. G.: Cell survival in B16 melanoma after treatment with combinations of cytotoxic agents: lack of potentiation. Br. J. Cancer 36: 84-93, 1977. Vietti, T., Eggerding, F., Valeriote, F.: Combined effect of X radiation and 5-fluorouracil on survival of transplanted leukemic cells. J. Nat. Cancer Inst. 47: 865-870, 197 1. Wharam, M. D., Phillips, T. L., Kane, L., Utley, J. F.: Response of a murine solid tumor to in vivo combined chemotherapy and irradiation. Radiology 109: 451-455, 1973.
Int. J. Radiation
Oncology Biol. Phys.,, Vol. 5, p. 1541-1548 Q Pergamon Press Ltd., 1979. Printed m the U. d .A.
??Nitrosourea
THE COMBINATION OF METHYL-CCNU AND IRRADIATION: CELL SURVIVAL STUDIES ON A HUMAN TUMOR XENOGRAFT ANGELA E.BATEMAN,D. Phil., KAREN K. Fu,M.D.t G. D. W.Tows~,B.Tech. Radiotherapy Research Department, Sutton,
Biophysics Division, Institute Surrey. SM2 SPX, England
and
of Cancer Research
Cell survival studies on a human tumor xenogreft grown in mice have demonstrated that the chemotherapeutic drug methyl-CCNU can increase the cytotoxic effect of y-irradiation. Injection of 25 mgikg of methyl-CCNU three hours before irradiation was the most effective time schedule of those tested. This pre-treatment reduced the DOof the radiation survival curve from 231 rad to 135 rad without showing any evidence of a reduced shoulder. Human tumor xenograft, Methyl-CCNU, y-Irradiation.
INTRODUCTION
suppressed mice. Tumors from passages 8 to 20were used in this study. Methyl-CCNU was used from capsules intended for oral administration (10 mg NSC 95441, containing mannitol and magnesium stearate). The contents of one capsule were homogenized in 0.4 ml dimethyl sulphoxide then 3.6 ml 5% Tween 80 in saline was added, and 0.1 ml per 10 g of body weight was injected intraperitoneally. For tumor irradiation, unanaesthetized air-breathing mice were given wholebody exposure of two equal doses directed to right and left sides of the mouse from a 6oCo source (dose rate approximately 200 rad per min). Tumors were excised 16 to 20 hours after irradiation and were finely chopped and cell suspensions prepared by colIagenase treatment (30 min at 37°C in 2 mg/ml collagenase type 1 in Ham’s F12 medium) followed by five minutes incubation in 0.05% trypsin in calcium- and magnesium-free balanced salts solution. The resultant cell suspension was poured through a sterile polyester mesh of pore size 25 pm and mixed with calf serum (10% of total volume). The refractile tumor cells were counted on a hemocytometer. Appropriate cell dilutions were made and cells were plated in 0.3% agar medium containing rat erythrocytes and 20% special Bobby calf serum in Ham’s F12 medium.4 One ml agar
A combined treatment of chemotherapy and irradiation is currently being investigated as an adjuvant therapy for resectable cancers and has been demonstrated to prolong survival in patients with locally unresectable disease. ls6 Methyl-CCNU (Me-CCNU) has given a response-rate in patients with cancer of the large bowel comparable with that of the more frequently used 5-fluorouracil treatment and the combination of methyl-CCNU with irradiation in treatment of gutassociated cancers may therefore prove clinically useful. The availability of human tumors growing as xenografts in immune-suppressed mice has enabled us to test the combination of Me-CCNU and irradiation on an anaplastic carcinoma of human origin.
METHODSANDMATERIALS Immune-suppressed CBA/lac mice were prepared according to Steel et al.‘, by thymectomy at four weeks of age, followed by intraperitoneal injection of 200 mg/kg arabinosyl cytosine (Ara-C) two days before 900 rad whole-body s°Co irradiation. The tumor, an anaplastic adenocarcinoma obtained from a patient with a suspected tumor of the pancreas, was established as a xenograft (HX32) in 1973 and maintained by passaging in leg muscles of immune-
supported by an American Cancer Society Clinical Junior Faculty Fellowship. Reprint request to: A. E. Bateman, D. Phil.
TDivision of Radiation Oncology, University of Califorma, San Francisco, CA., U.S.A. Acknowledgements-Funded by National Institutes of Health grant no. R01 CA 20519. Dr. Fu was partially 1545
1546
Radiation Oncology 0 Biology 0 Physics
September
1979, Volume 5, Number 9
cultures, containing either 300 control cells or up to 3x lo4 treated cells were gassed with a 5% 02, 5% COP, 90% N, mixture and were fed after 7 and 14 days with 1.5 ml fresh medium. Cell colonies were scored after three weeks. The plating efficiency (PE) of the untreated tumor cells was approximately 30%. The ratio of PE of treated cells to PE of control cells was used to calculate the fraction of clonogenic cells surviving treatment (SF). Control PE was determined in each experiment by plating cells from each of at least two untreated mice; from two to six mice were given each test treatment and the SF of tumor clonogenic cells was determined for each individual mouse. All liquids which had come into contact with the human tumor material were autoclaved before disposal, .all plastics and glassware were either incinerated or bleached in hypochlorite solution before reuse. RESULTS The LD,, (30 days) for methyl-CCNU in our CBA/Iac mice is approximately 40 mg/kg. Twentyfive mg/kg of this drug was known to kill about 90% of HX32 clonogenic cells, and this dose was chosen for combination with irradiation. We investigated the change in SF with time of assay after drug injection; Fig. 1 shows a decrease in SF between two and 24 hours after treatment, as reported earlier for B16 mouse melanoma.5 This implies that the toxic products of this drug are active in vivo for longer than two hours. In the following experiments tumors were excised 1624 hours after Me-CCNU injection, by which time the maximum toxic effect of the drug should have occurred. The time between irradiation and tumor excision varied in these experiments between 16 and 20 hours, to allow the repair of “potentially lethal” radiation damage which is known to occur in this tumor.3 Radiation doses from 200-800 rad were given three hours after an injection of Me-CCNU or of the vehicle alone. The SF data are shown in Fig. 2. The lines drawn through the data were derived by linear regression of In SF with radiation dose. The expected addition curve is drawn parallel to the irradiation alone curve but intersecting the ordinate at the SF due to Me-CCNU alone (0.109, the mean of 16 assays). Calculated Do values are 231 rad for irradiation alone and 135 rad for irradiation three hours after Me-CCNU and the slope of the radiation plus Me-CCNU curve is significantly greater than that of the curve for radiation alone (one-tailed r-test p
‘ij g z & 0
0.01
I
I
2L
LO
Time
FIG. 1. The cytotoxic
I
I
15
2
of methyl-CCNU injection before assay) (hours effect
of 25 mg/kg Me-CCNU
meas-
ured at various times after injection. Me-CCNU was injected into two tumor-bearing mice at each time-point. The total cell yield per tumor (N) and the plating efficiency (PE) were measured. The proportion of clonogenic cells per tumor which survive Me-CCNU=N,,,,,,,,, x PE Itreated flLcuntrull x PElrontrull. Data from one representative experiment. The times referred to are those between drug administration and tumor removal for in vitro culture.
1.36 (67% confidence limits 1.13-1.65). The extrapolation number for irradiation after Me-CCNU is 0.285, which is 2.6 times the surviving fraction due to Me-CCNU alone (67% confidence limits 1.5-4.6 times SFlh,e_CCNUI). There is thus no evidence that prior exposure to Me-CCNU reduces the shoulder of the irradiation dose/response curve. Methyl-CCNU was injected at times between seven hours prior to and 30 min after 600 rad and the results are shown in Fig. 3 where the range of expected additive effect is shown as the shaded area. This range was calculated as the maximum and minimum values of the product of SFfradiationl and
Irradiation
and methyl-CCNU
1547
E. BATEMAN cftrl.
0 A.
-.
1.0 vi = 0, ” 2 2
0
0
0.1
g T;
i 0
5
a
N ” z
c
0
0.2
0
0.01
I
0
I
-7
z ._ :: 2
I
-5 Time
O-001
? ._ > ._ 2 l? 0
0~0001
0
I
1
I
200
LOO
Radiation
dose
I
I
800
between
I
-1
-3 methyl-CCNU
’
and
?i
irradiation
+l
’ (hours)
FIG. 3. Potentiation of radiation cytotoxicity by MeCCNU as a function of time between drug injection and irradiation. Data from several experiments are shown, in which the time of injection of 25 mgkg Me-CCNU was varied with respect to irradiation with 600 rad. A relative survival of 1.0 is the mean expected additive SF (SFlirradiafionl X SF ,a,,,,). The shaded area shows the range of additive survival (see text). The data points (0) are the values of SF observed for the combined treatments. Each open circle represents the results of an individual mouse tumor survival evaluation using in V~VOtreatment and in vitro culture.
in rad
FIG. 2. Radiation dose/response curves with or without the injection of 25 mgkg Me-CCNU three hours before irradiation. Each data point represents the SF of tumor cells from an individual mouse. 0 without pretreatment. DC,=231 rad. 0 three hours after IP injection of 25 mg/kg Me-CCNU D,,=135 rad. The dash line indicates survival expected of the effect if Me-CCNU were strictly additive to that of irradiation.
SF Ldrupl.The SF values obtained when Me-CCNU preceded irradiation by three hours appear to be lower than for other schedules, and this may be the optimum timing for potentiation. We conclude that Me-CCNU potentiated radiation damage to tumor cells after intraperitoneal injection into mice three hours before irradiation. DISCUSSION Earlier studies on the combined treatment of experimental tumors with drugs and irradiation include the detailed study of the effects of 5-fluorouracil and X-rays on the AKR lymphomas in which the time between treatments of this rapidly-proliferating diffuse tumor was varied. Cell killing much greater than additive was observed when 5-fluorouracil was administered about 20 hours before irradiation, and
also from immediately after to eight hours after irradiation. However, in the B16 murine solid tumor, no time-dependent potentiation of radiation damage by 5-fluorouracil was detected8 and in studies on the EMT6 murine solid tumor only one drug out of four tested gave a radiation dose/response curve which was steeper two hours after drug administration than the control radiation dose/response curve.‘O The effective drug in the latter study was the nitrosourea BCNU, and our results suggest that the cytotoxic activities of irradiation and Me-CCNU are not independent, so the potentiation of radiation cytotoxicity by nitrosoureas injected two or three hours before irradiation may occur in several tumor systems. Nitrosoureas may be interacting with DNA, as previously demonstrated for CCNU? and therefore increasing the effectiveness of radiation damage to DNA or inhibiting the repair of sub-lethal damage. The potential benefit of any treatment depends on the effect on the tumor relative to the toxicity to normal tissues. We have calculated a therapeutic ratio for Me-CCNU given to mice three hours before irradiation. The drug reduces the radiation dose necessary for a fixed surviving fraction by a factor of 2.83 for tumor cell survival (from Fig. 2 Me-CCNU plus 460 rad is equivalent to 1300 rad) and by a
1548
Radiation Oncology 0 Biology 0 Physics
factor
of 1.67 for gut microcolony survival (Meplus 780 rad is equivalent to 1300 rad, unpublished data of Dr. T. Phelps). The therapeutic
CCNU
September
1979, Volume 5, Number 9
ratio of this combination is therefore 1.69 at that radiation dose, and may indicate a beneficial treatment.
REFERENCES S.K.: Large-bowel cancer-the current status of treatment. J. Nat. Cancer Inst. 56: 3-10, 1976.
1. Carter,
2. Cheng, C.J., Fujimara, S., Grunberger, D., Weinstein, I. B.: Interaction of l-(2-chloroethyI)-3-cyclohexylI-nitrosourea (NSC-79037) with nucleic acids and proteins in vivo and in vitro. Cancer Res. 32: 22-27, 1972. 3. Courtenay, V. D., Smith, I. E., Peckham, J. J., Steel, G. G.: In vitro and in vivo radiosensitivity of human tumor cells obtained from a pancreatic carcinoma xenograft. Nature 263: 771-772, 1976. 4. Courtenay, V. D., Mills, J.: An in vitro colony assay for human tumors grown in immune-suppressed mice and treated in vivo with cytotoxic agents. Br. J. Cancer 37: 261-268, 1978. 5. Hill, R. P., Stanley, J. A.: The response of hypoxic B16 melanoma cells to in vivo treatment with chemotherapeutic agents. Cancer Res. 35: 1147-l 153, 1975. 6. Moertel, C. G., Childs, D. S., Jr., Reitemeier, R. J., Colby, M. Y., Holbrook, M. A.: Combined
7.
8.
9.
10.
5fluorouracil and supervoltage radiation therapy of locally unresectable gastrointestinal cancer. Lancet II: 865-867, 1%9. Steel, G. G., Courtenay, V. D., Rostom, A. Y.: Improved immune-suppression techniques for the xenografting of human tumors. Br. J. Cancer 37: 224230, 1978. Stephens, T. C., Peacock, J. H., Steel, G. G.: Cell survival in B16 melanoma after treatment with combinations of cytotoxic agents: lack of potentiation. Br. J. Cancer 36: 84-93, 1977. Vietti, T., Eggerding, F., Valeriote, F.: Combined effect of X radiation and 5-fluorouracil on survival of transplanted leukemic cells. J. Nat. Cancer Inst. 47: 865-870, 197 1. Wharam, M. D., Phillips, T. L., Kane, L., Utley, J. F.: Response of a murine solid tumor to in vivo combined chemotherapy and irradiation. Radiology 109: 451-455, 1973.