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Radiation response of artificial pulmonary metastases of the EMT6 tumor
Inl. 1. Radiation
Oncology Bill.
Phys., 1976, Vol.
1, pp. 257-260.
Pergamon Press.
Printed in the U.S.A.
RADIATION RESPONSE OF ARTIFICIAL PULMONARY METASTASES OF THE EMT6 TUMOR-t
KAREN K. Fu, M.D.,
THEODORE
and MOODY D. WHARAM, Division of Radiation
L.
PHILLIPS,
M.D.
M.D.+
Oncology and Laboratory of Radiobiology, San Francisco, CA 94143, U.S.A.
University
of California.
A method for studying the response of artfficiaUy produced pulmonary met&am of a mouse mammary tumor to treatment in situ is described. Cells grown and had&ted in small lung metmtam appeared more radfosensftive in that tbefr dose respome curve bad smaller ehoaklcrandD~valuesthnnthoseof~grownin~BnnktamorsPadhm~tcdinsituor in vitro.
fih=rY
mehstaw,
Radiinsftfvity,
Irradiation, Chemotherapy, Immmotherapy.
INTRODUCTION An assay
EMT6 tumor cells in small artificial metastatic pulmonary nodules to treatment in situ with radiation. The characteristics of the EMT6 tumor and its tissue-culture-adapted derivative have been described by Rockwell et aI.”
system that measures the cellular response of small metastatic pulmonary nodules to irradiation, chemotherapy, or immunotherapy in situ would provide an optimal model for determining improvement in the therapeutic ratio since it would have the unique feature of a small tumor mass residing in a very sensitive, dose-limiting normal tissue. Several investigators have worked on methods of developing lung assay systems. Hill and Bush’ developed a lung colony assay analogous to the spleen colony method of McCulloch and Till8 Fidler’ found that the incidence of experimental pulmonary metastases of the B16 melanoma varies with embolic homogeneity and size, cell number and tumor viability. Prior local irradiation of the lungs has been reported to increase the incidence of artificial pulmonary metastases from various tumors.‘~‘2~13~‘5 It was the purpose of this study to develop a method for investigating the response of
METHODS AND MATRRIALS Eight to ten week old BALB/c female mice obtained from Cumberland Laboratories (Clinton, Tennessee) were used in all experiments. The EMT6 tumor cells were grown as flank tumors in BALB/c mice after subcutaneous inoculation and as cell cultures in 32 oz. glass culture bottles in modified Eagle’s medium plus 15% fetal calf serum. The method for preparing single cell suspensions from solid tumors has been described elsewhere.” Briefly, after excision the tumor was chopped with a mechanical chopper, trypsinized, and resuspended in Ca++-free medium. Viable cells were counted in a hemacytometer using a phase-contrast microscope. Cells grown in culture bottles were
tWork performed under the auspices of the U.S. Energy Research and Development Administration and supported in part by Training Grant No. CA-05177 and Clinical Cancer Center Grant No. CA-11067 from the National Cancer Institute. *Present address: Department of Radiation
Oncology, Johns Hopkins Hospital, Baltimore, MD 21205, U.S.A. Acknowledgements-The authors wish to thank Ms. Mimi Zeiger for editorial assistance and Mr. Lawrence J. Kane and Mr. Gallo Velasco-Jackson for technical help. 257
258
Radiation Oncology 0 Biology 0 Physics
harvested 3 days after inoculation of lo5 cells in 35 ml of medium. After treatment with 0.05% trypsin for 15 min, they were resuspended in Cd’-free medium. For lung nodule production, single-cell suspensions of viable cells from flank tumors or cells from cultures were injected in O-2ml volumes through the tail vein into recipient mice that had received no radiation or 400 rad whole body radiation (230 kVp X-rays, H.V.L. l-6 mm Cu, 100 rad/min) 24 hr before injection. Lung nodules 0.5-2 mm in diameter appeared 14 days after injection and were counted under a dissecting microscope. Whole body irradiation of air-breathing, unanesthetized mice bearing EMT6 tumor lung nodules or flank tumors (approximately 10 mm in diameter) and in vitro irradiation of single cell suspensions prepared from flank tumors in open 2cm3 glass ampules were carried out with a self-contained, 2OOOCi, ‘“Cs irradiator at a dose rate of 275 radlmin. For in oitro assay of lung nodules, 2-8 mice were irradiated for each radiation dose in individual experiments. Two to four doses were used in each experiment. Multiple experiments were done and the surviving fraction for each radiation dose represents the mean of 2-4 experiments, in most cases four. Immediately after irradiation, the mice were sacrificed and the lungs were removed. The lung nodules were dissected out individually under a dissecting microscope, pooled, minced with iris scissors, treated with 0.05% buBered trypsin in a plastic Petri dish (35 x 10 mm, Falcon), and stirred for 15 min with a miniature magnetic stirring rod. Tumor cells were then resuspended in Ca*+-free medium. A known number of viable cells was plated in sealed plastic tissue-culture flasks and incubated at 37°C in an atmosphere consisting of 5% CO* and 95% filtered air. Nine days later the clones were fixed, stained and counted. Cell survivals were calculated from the ratios of the plating efficiencies of the irradiated cells and unirradiated controls. The plating efficiency for unirradiated control lung nodules was 14&4.7%. Survival of cells irradiated in situ as flank tumors or in vitro as single cell suspensions in glass ampules was assayed in vitro in the same
Vol. I, No. 34
manner as above. The plating efficiency of unirradiated control flank tumors was 35? 10%. RESULTS The number of lung nodules produced by single-cell suspensions prepared from flank tumors varied from mouse to mouse and from experiment to. experiment for the same number of viable cells injected. This was probably due to variation in the proportion of viable and dead cells and the presence of stromal elements in the flank tumors. A higher and more constant lung cloning efficiency (number of lung nodules per number of viable cells injected x 100%) was obtained by injecting single-cell suspensions prepared from cell cultures into preirradiated mice (Table 1). The average cloning efficiency was approximately 3.7%. To limit the number of lung nodules per mouse to 10-20 for the purpose of studying the radiation response of lung nodules in situ, the combination of 400rad of whole body irradiation of recipient mice and 350 cultured cells in O-2ml i.v. was adopted for all subsequent experiments. The dose response curve for cells grown and irradiated as small lung nodules (Fig. 1) clearly consisted of two components, the first portion having an extrapolation number (fi) of 5-3 and a Do of 98 rad and the second portion having a Do of 270rad. The dose response curve for cells grown and irradiated in situ as flank tumors had a broad shoulder with an extrapolation number of about 18 and a D,, of 167 rad in the region between 600 and 1000tad and a Do of 351 t-ad in the region between 1000 and 2400 rad. For cells prepared from flank tumors and irradiated in vitro in air, the dose response curve had an extrapolation number of 13.5 and a Do of 139rad. DISCUSSION Although the shape of the dose response curve of EMT6 tumor cells irradiated in situ as “metastatic” lung nodules was similar to that of cells irradiated as flank tumors, the extrapolation number and the Do were smaller in both the initial and the terminal portions of the dose response curve. The ii and Do of the initial portion of the dose response curve of
Radiation response of lung metastases 0 K. K. FU et al.
Table I. Cloning efficiency of intravenously
259
injected EMT6 tumor cells
Recipient mice [mean (% ‘_ SE.)] Experiment A :
Flank tumor Flank tumor Cell culture
D E
Cell culture Flank tumor
Cell Cell Cell Cell Cell Cell
CF H I J
K L
Cell
M N 0
Cell Cell Cell Cell Cell
4lN
culture culture culture culture culture culture culture culture culture culture culture culture
1200
with 400 rad
Numerous to confluent nodules after 200-600 cells i.v.
2.6 + 0.9 1.7-co.3 1.4a0.2 6.0 ” 0.5 2.3 + 0.3 5.420.6 2.7 f 0.3 3.120.3 1.820.1 2.7 2 0.2 12.9 + 1.4 l-8+0-3
k
J-+---
preirradiated
0.420.2 0.08 2 0.02 Numerous to contluent nodules after (l-3-5.2) x 10’cells i.v. 0.9 2 0.4
?? fUmTumRNsmJ
I
.I01
not preirradiated
Source of cells
Ido0
2wo
2400
Dc5EwJsl
Fig. 1. Dose response curve of EMT6 tumor cells irradiated as lung nodules (0.5-2 mm in diameter) in situ (m), as flank tumors (approximately 10 mm in diameter) in situ (0). and as single cell suspensions from flank tumors (A). Symbols represent the mean survival determined in 2-6 experiments. Bars represent standard errors of the mean.
the lung nodules were also smaller than those of the in vitro irradiated cells prepared from flank tumors. The larger Do of the terminal portion of the in situ dose response curves of the lung nodules and flank tumors are at least partly due to the presence of hypoxic cells. Cells from flank tumors irradiated in vitro were well oxygenated and therefore did not exhibit a change of radiosensitivity in the high dose region of the dose response curve. These results suggest greater radiosensitivity for cells in small lung nodules. This may be related to the smaller size (0.5-2 mm in diameter) and cell number of lung nodules. This finding is consistent both with the demonstration that metastatic occult tumor foci of C3HBA mouse adenocarcinoma are more radioresponsive than small tumors in the lung when assayed for nodule survival regardless of the source of radiation3 and with the finding of enhanced radiosensitivity of artificial pulmonary metastases of the Lewis Another factor affecting lung carcinoma.” radiosensitivity may be the difference in the proportion of proliferating and nonproliferating cells and distributions of cell ages. In addition, the radiation response of tumor cells may be influenced by other environmental
260
Vol. 1, No. 3-4
Radiation Oncology 0 Biology 0 Physics
factors: cells of lung nodules were grown in well oxygenated and vascularized tissue. The greater radiosensitivity of cells irradiated as metastatic lung nodules may be one of the possible explanations for the clinical observation that radiation in doses
considered suboptimal for control of certain primary tumors is capable of eradicating small pulmonary metastases and that low-dose elective lung irradiation is successful in decreasing the incidence of pulmonary metastasis.2*6q7*9
REFERENCES 1. Brown, J.M.: The effect of lung irradiation on the incidence of pulmonary metastases in mice. Br. J. Radiol. 46: 613-618, 1973. 2. Caldwell, W.: Elective whole lung irradiation. Cancer to be published. 3. El-Mahdi, A.M., Shaeffer, J., Aceto, H. Jr., Constable, W.C.: A comparison of radiation control of pulmonary metastases in C3H mice by helium ions or cobalt-60 photons. Cancer 34: 130-135, 1974. 4. Fidler, I.J.: The relationship of embolic homogeneity, number, size and viability to the incidence of experimental metastasis. Eur. J. Cancer 9: 223-227, 1973.
5. Hill, R.P., Bush, R.S.: A lung-colony assay to determine the radio-sensitivity of the cells of a solid tumour. lnt. J. Radiat. Biol. 15: 435-444, 1%9. 6. Jenkin, R.D.T.: Ewing’s sarcoma: A study of treatment methods. Clin. Radiol. 17: 97-106, 1966. 7. Margolis, L.W., Phillips, T.L.: Whole-lung irradiation for metastatic tumor. Radiology 93: 1173-1179, 1%9. 8. McCulloch, E.A., Till, J.E.: The sensitivity of cells from normal mouse bone marrow to gamma radiation in vitro and in vivo. Radiat. Res. 16: 822-832, 1962. 9. Newton, K.A., Spittle, M.F.: An analysis of 40
cases treated by total thoracic irradiation. Clin. Radial. 20: 19-22, 1969. 10. Rockwell, S.C., Kallman, R.F., Fajardo, L.F.: Characteristics of a serially transplanted mouse mammary tumor and its tissue-culture-adapted derivative. J. Narl Cancer. Inst. 49: 735-749, 1972. 11. Shipley, W.U., Stanley, J.A.: Enhanced tumor cell radiosensitivity in artificial pulmonary metastases of the Lewis lung carcinoma. Ink J. Radiat. Oncol. Biol. Phys. 1: 261-265, 1976. 12. Van den Brenk, H.A.S., Burch, W.M.. Orton, C., Sharpington, C.: Stimulation of clonogenic growth of tumour cells and metastases in the lungs by local X-radiation. Br. J. Cancer 27: 291-306, 1973. 13. Van den Brenk, H.A.S., Kelly, H.: Potentiating effect of prior local irradiation of the lungs on pulmonary metastases. Br. J. Radiol. 47: 332-336, 1974.
14. Wharam. M.D., Phillips, T.L., Kane, L., Utley, J.F.: Response of a murine solid tumor to in vioo combined chemotherapy and irradiation. Radiology IeS: 451-455, 1g3. 15. Withers, H-R., Milas, L.: In!?uence of preirradiation of lung on development of artiticial pulmonary metastases of fibrosarcoma in mice. Cancer Res. 33: 1931-1936, 1973.
Oncology Bill.
Phys., 1976, Vol.
1, pp. 257-260.
Pergamon Press.
Printed in the U.S.A.
RADIATION RESPONSE OF ARTIFICIAL PULMONARY METASTASES OF THE EMT6 TUMOR-t
KAREN K. Fu, M.D.,
THEODORE
and MOODY D. WHARAM, Division of Radiation
L.
PHILLIPS,
M.D.
M.D.+
Oncology and Laboratory of Radiobiology, San Francisco, CA 94143, U.S.A.
University
of California.
A method for studying the response of artfficiaUy produced pulmonary met&am of a mouse mammary tumor to treatment in situ is described. Cells grown and had&ted in small lung metmtam appeared more radfosensftive in that tbefr dose respome curve bad smaller ehoaklcrandD~valuesthnnthoseof~grownin~BnnktamorsPadhm~tcdinsituor in vitro.
fih=rY
mehstaw,
Radiinsftfvity,
Irradiation, Chemotherapy, Immmotherapy.
INTRODUCTION An assay
EMT6 tumor cells in small artificial metastatic pulmonary nodules to treatment in situ with radiation. The characteristics of the EMT6 tumor and its tissue-culture-adapted derivative have been described by Rockwell et aI.”
system that measures the cellular response of small metastatic pulmonary nodules to irradiation, chemotherapy, or immunotherapy in situ would provide an optimal model for determining improvement in the therapeutic ratio since it would have the unique feature of a small tumor mass residing in a very sensitive, dose-limiting normal tissue. Several investigators have worked on methods of developing lung assay systems. Hill and Bush’ developed a lung colony assay analogous to the spleen colony method of McCulloch and Till8 Fidler’ found that the incidence of experimental pulmonary metastases of the B16 melanoma varies with embolic homogeneity and size, cell number and tumor viability. Prior local irradiation of the lungs has been reported to increase the incidence of artificial pulmonary metastases from various tumors.‘~‘2~13~‘5 It was the purpose of this study to develop a method for investigating the response of
METHODS AND MATRRIALS Eight to ten week old BALB/c female mice obtained from Cumberland Laboratories (Clinton, Tennessee) were used in all experiments. The EMT6 tumor cells were grown as flank tumors in BALB/c mice after subcutaneous inoculation and as cell cultures in 32 oz. glass culture bottles in modified Eagle’s medium plus 15% fetal calf serum. The method for preparing single cell suspensions from solid tumors has been described elsewhere.” Briefly, after excision the tumor was chopped with a mechanical chopper, trypsinized, and resuspended in Ca++-free medium. Viable cells were counted in a hemacytometer using a phase-contrast microscope. Cells grown in culture bottles were
tWork performed under the auspices of the U.S. Energy Research and Development Administration and supported in part by Training Grant No. CA-05177 and Clinical Cancer Center Grant No. CA-11067 from the National Cancer Institute. *Present address: Department of Radiation
Oncology, Johns Hopkins Hospital, Baltimore, MD 21205, U.S.A. Acknowledgements-The authors wish to thank Ms. Mimi Zeiger for editorial assistance and Mr. Lawrence J. Kane and Mr. Gallo Velasco-Jackson for technical help. 257
258
Radiation Oncology 0 Biology 0 Physics
harvested 3 days after inoculation of lo5 cells in 35 ml of medium. After treatment with 0.05% trypsin for 15 min, they were resuspended in Cd’-free medium. For lung nodule production, single-cell suspensions of viable cells from flank tumors or cells from cultures were injected in O-2ml volumes through the tail vein into recipient mice that had received no radiation or 400 rad whole body radiation (230 kVp X-rays, H.V.L. l-6 mm Cu, 100 rad/min) 24 hr before injection. Lung nodules 0.5-2 mm in diameter appeared 14 days after injection and were counted under a dissecting microscope. Whole body irradiation of air-breathing, unanesthetized mice bearing EMT6 tumor lung nodules or flank tumors (approximately 10 mm in diameter) and in vitro irradiation of single cell suspensions prepared from flank tumors in open 2cm3 glass ampules were carried out with a self-contained, 2OOOCi, ‘“Cs irradiator at a dose rate of 275 radlmin. For in oitro assay of lung nodules, 2-8 mice were irradiated for each radiation dose in individual experiments. Two to four doses were used in each experiment. Multiple experiments were done and the surviving fraction for each radiation dose represents the mean of 2-4 experiments, in most cases four. Immediately after irradiation, the mice were sacrificed and the lungs were removed. The lung nodules were dissected out individually under a dissecting microscope, pooled, minced with iris scissors, treated with 0.05% buBered trypsin in a plastic Petri dish (35 x 10 mm, Falcon), and stirred for 15 min with a miniature magnetic stirring rod. Tumor cells were then resuspended in Ca*+-free medium. A known number of viable cells was plated in sealed plastic tissue-culture flasks and incubated at 37°C in an atmosphere consisting of 5% CO* and 95% filtered air. Nine days later the clones were fixed, stained and counted. Cell survivals were calculated from the ratios of the plating efficiencies of the irradiated cells and unirradiated controls. The plating efficiency for unirradiated control lung nodules was 14&4.7%. Survival of cells irradiated in situ as flank tumors or in vitro as single cell suspensions in glass ampules was assayed in vitro in the same
Vol. I, No. 34
manner as above. The plating efficiency of unirradiated control flank tumors was 35? 10%. RESULTS The number of lung nodules produced by single-cell suspensions prepared from flank tumors varied from mouse to mouse and from experiment to. experiment for the same number of viable cells injected. This was probably due to variation in the proportion of viable and dead cells and the presence of stromal elements in the flank tumors. A higher and more constant lung cloning efficiency (number of lung nodules per number of viable cells injected x 100%) was obtained by injecting single-cell suspensions prepared from cell cultures into preirradiated mice (Table 1). The average cloning efficiency was approximately 3.7%. To limit the number of lung nodules per mouse to 10-20 for the purpose of studying the radiation response of lung nodules in situ, the combination of 400rad of whole body irradiation of recipient mice and 350 cultured cells in O-2ml i.v. was adopted for all subsequent experiments. The dose response curve for cells grown and irradiated as small lung nodules (Fig. 1) clearly consisted of two components, the first portion having an extrapolation number (fi) of 5-3 and a Do of 98 rad and the second portion having a Do of 270rad. The dose response curve for cells grown and irradiated in situ as flank tumors had a broad shoulder with an extrapolation number of about 18 and a D,, of 167 rad in the region between 600 and 1000tad and a Do of 351 t-ad in the region between 1000 and 2400 rad. For cells prepared from flank tumors and irradiated in vitro in air, the dose response curve had an extrapolation number of 13.5 and a Do of 139rad. DISCUSSION Although the shape of the dose response curve of EMT6 tumor cells irradiated in situ as “metastatic” lung nodules was similar to that of cells irradiated as flank tumors, the extrapolation number and the Do were smaller in both the initial and the terminal portions of the dose response curve. The ii and Do of the initial portion of the dose response curve of
Radiation response of lung metastases 0 K. K. FU et al.
Table I. Cloning efficiency of intravenously
259
injected EMT6 tumor cells
Recipient mice [mean (% ‘_ SE.)] Experiment A :
Flank tumor Flank tumor Cell culture
D E
Cell culture Flank tumor
Cell Cell Cell Cell Cell Cell
CF H I J
K L
Cell
M N 0
Cell Cell Cell Cell Cell
4lN
culture culture culture culture culture culture culture culture culture culture culture culture
1200
with 400 rad
Numerous to confluent nodules after 200-600 cells i.v.
2.6 + 0.9 1.7-co.3 1.4a0.2 6.0 ” 0.5 2.3 + 0.3 5.420.6 2.7 f 0.3 3.120.3 1.820.1 2.7 2 0.2 12.9 + 1.4 l-8+0-3
k
J-+---
preirradiated
0.420.2 0.08 2 0.02 Numerous to contluent nodules after (l-3-5.2) x 10’cells i.v. 0.9 2 0.4
?? fUmTumRNsmJ
I
.I01
not preirradiated
Source of cells
Ido0
2wo
2400
Dc5EwJsl
Fig. 1. Dose response curve of EMT6 tumor cells irradiated as lung nodules (0.5-2 mm in diameter) in situ (m), as flank tumors (approximately 10 mm in diameter) in situ (0). and as single cell suspensions from flank tumors (A). Symbols represent the mean survival determined in 2-6 experiments. Bars represent standard errors of the mean.
the lung nodules were also smaller than those of the in vitro irradiated cells prepared from flank tumors. The larger Do of the terminal portion of the in situ dose response curves of the lung nodules and flank tumors are at least partly due to the presence of hypoxic cells. Cells from flank tumors irradiated in vitro were well oxygenated and therefore did not exhibit a change of radiosensitivity in the high dose region of the dose response curve. These results suggest greater radiosensitivity for cells in small lung nodules. This may be related to the smaller size (0.5-2 mm in diameter) and cell number of lung nodules. This finding is consistent both with the demonstration that metastatic occult tumor foci of C3HBA mouse adenocarcinoma are more radioresponsive than small tumors in the lung when assayed for nodule survival regardless of the source of radiation3 and with the finding of enhanced radiosensitivity of artificial pulmonary metastases of the Lewis Another factor affecting lung carcinoma.” radiosensitivity may be the difference in the proportion of proliferating and nonproliferating cells and distributions of cell ages. In addition, the radiation response of tumor cells may be influenced by other environmental
260
Vol. 1, No. 3-4
Radiation Oncology 0 Biology 0 Physics
factors: cells of lung nodules were grown in well oxygenated and vascularized tissue. The greater radiosensitivity of cells irradiated as metastatic lung nodules may be one of the possible explanations for the clinical observation that radiation in doses
considered suboptimal for control of certain primary tumors is capable of eradicating small pulmonary metastases and that low-dose elective lung irradiation is successful in decreasing the incidence of pulmonary metastasis.2*6q7*9
REFERENCES 1. Brown, J.M.: The effect of lung irradiation on the incidence of pulmonary metastases in mice. Br. J. Radiol. 46: 613-618, 1973. 2. Caldwell, W.: Elective whole lung irradiation. Cancer to be published. 3. El-Mahdi, A.M., Shaeffer, J., Aceto, H. Jr., Constable, W.C.: A comparison of radiation control of pulmonary metastases in C3H mice by helium ions or cobalt-60 photons. Cancer 34: 130-135, 1974. 4. Fidler, I.J.: The relationship of embolic homogeneity, number, size and viability to the incidence of experimental metastasis. Eur. J. Cancer 9: 223-227, 1973.
5. Hill, R.P., Bush, R.S.: A lung-colony assay to determine the radio-sensitivity of the cells of a solid tumour. lnt. J. Radiat. Biol. 15: 435-444, 1%9. 6. Jenkin, R.D.T.: Ewing’s sarcoma: A study of treatment methods. Clin. Radiol. 17: 97-106, 1966. 7. Margolis, L.W., Phillips, T.L.: Whole-lung irradiation for metastatic tumor. Radiology 93: 1173-1179, 1%9. 8. McCulloch, E.A., Till, J.E.: The sensitivity of cells from normal mouse bone marrow to gamma radiation in vitro and in vivo. Radiat. Res. 16: 822-832, 1962. 9. Newton, K.A., Spittle, M.F.: An analysis of 40
cases treated by total thoracic irradiation. Clin. Radial. 20: 19-22, 1969. 10. Rockwell, S.C., Kallman, R.F., Fajardo, L.F.: Characteristics of a serially transplanted mouse mammary tumor and its tissue-culture-adapted derivative. J. Narl Cancer. Inst. 49: 735-749, 1972. 11. Shipley, W.U., Stanley, J.A.: Enhanced tumor cell radiosensitivity in artificial pulmonary metastases of the Lewis lung carcinoma. Ink J. Radiat. Oncol. Biol. Phys. 1: 261-265, 1976. 12. Van den Brenk, H.A.S., Burch, W.M.. Orton, C., Sharpington, C.: Stimulation of clonogenic growth of tumour cells and metastases in the lungs by local X-radiation. Br. J. Cancer 27: 291-306, 1973. 13. Van den Brenk, H.A.S., Kelly, H.: Potentiating effect of prior local irradiation of the lungs on pulmonary metastases. Br. J. Radiol. 47: 332-336, 1974.
14. Wharam. M.D., Phillips, T.L., Kane, L., Utley, J.F.: Response of a murine solid tumor to in vioo combined chemotherapy and irradiation. Radiology IeS: 451-455, 1g3. 15. Withers, H-R., Milas, L.: In!?uence of preirradiation of lung on development of artiticial pulmonary metastases of fibrosarcoma in mice. Cancer Res. 33: 1931-1936, 1973.