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Radiation enhancement of lung nodule formation in mice is not potentiated by treatment with a perfluorochemical emulsion and carbogen
Radiotherapy and Oncology, 14 (1989)49-53 Elsevier
49
RTO 00524
Radiation enhancement of lung nodule formation in mice is not potentiated by treatment with a perfluorochemical emulsion and carbogen Sara R o c k w e l l a n d M a r i a n n e Kelley Department of Therapeutic Radiology, Yale University School of Medicine, New Haven. CT, U.S.A.
(Received23 March 1988, revisionreceived27 June 1988, accepted 5 July 1988)
Key words: Perfluorochemicalemulsion; Fluosol-DA, 20%; Lung metastases; Radiation damage, lung
Summary The ability of intraveneously-injected mouse mammary tumor cells to form lung tumors is increased by irradiation of the thorax 24 h previously. We examined the effects of treatment with a perfluorochemical emulsion (Fluosol-DA, 20%) plus carbogen before and during irradiation on the radiation-induced enhancement of lung nodule formation. We found no evidence that treatment with Fluosol plus carbogen altered the development of tumor nodules in irradiated mouse lungs.
Introduction Perfluorochemical emulsions (PFC-E) were originally developed as O2-transport agents, to replace transfused blood [15,19]. The observation that PFC-E could ameliorate ischemic injury after experimental vascular occlusion lead to the idea that these agents might be used to improve oxygenation Addressfor correspondence: Sara Rockwell,Department of Therapeutic Radiology,Yale UniversitySchoolof Medicine, 333 Cedar Street, New Haven, CT 06510-8040, U.S.A.
in the hypoxic areas of solid tumors and therefore improve the results of radiotherapy [2,8,17]. This has been tested using several solid tumor systems; treatment with PFC-E and 02 or carbogen has been found to increase the efficacy of radiation in killing tumor cells, perturbing tumor growth, and producing local tumor control [3,8,9,11,13,17]. As a result, one PFC-E, Fluosoi-DA, 20%, has entered clinical trials as an adjunct to radiotherapy for carcinoma of the head and neck, glioma, and carcinoma of the lung. Extensive studies of the physiologic effects and
0167-8140/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)
50 toxicities of Fluosol have been performed in laboratory animals, human volunteers, and patients treated for trauma or anemia [15,19]. However, the use of this agent in cancer therapy raises questions not encountered previously. One must ask, for example, whether the PFC-E/O2 treatments alter the acute or chronic toxicities of radiation to normal tissues. One must also ask whether treatments with a PFC-E and oxygen have physiologic or toxic effects which alter progression of neoplastic disease. The experiments reported here are part of a series of studies addressing these questions [2,3,8-11]. Here we extend these studies to ask whether treatment with a PFC-E and carbogen during thoracic irradiation alters the radiation-induced augmentation of lung colony formation; such augmentation might occur if this treatment sensitized the lung to radiation injury. This question has recently acquired clinical significance, because of the initiation of clinical trials requiring irradiation of lung tissue in patients treated with a PFC-E and oxygen.
Methods and materials Mice and tumors. Female BALB/c Rw mice 2.5 months of age were used throughout these studies. Mice were bred and maintained under SPF conditions [8,10]. The characteristics of the EMT6 Rw mouse mammary tumor cells used in these studies are detailed elsewhere [8-12].
The ability of EMT6 cells to form lung tumors was assayed as detailed previously [7,10,12]. Cell suspensions prepared from exponentially-growing cultures were counted and diluted to obtain a series of single cell suspensions containing 500-5000 cells per 0.2 ml aliquot; no heavily irradiated cells or microspheres were admixed. Two dilutions were tested for most irradiated groups; the group(s) developing optimal numbers of colonies for counting were used for analysis. All mice to be inoculated with a given dilution were ear tagged, randomized, and inoculated in random order. Animals were sacrificed 14 days after injection' and the lungs were removed, cleaned, fixed in Bouin's and Lung colonies.
washed with ethanol. Lungs were coded and counted blind. Tumor nodules on the lung surfaces were counted using a dissecting microscope. Fluosol-DA, 20% (Fluosol), was manufactured by Green Cross Corporation, Japan, and provided without cost by Alpha Therapeutic Corporation, Los Angeles. The composition and characteristics of this emulsion are detailed elsewhere [2,8,15,19]. Fiuosol formulated without hydroxyethyl starch was used for these studies. Fluosol was administered i.v. at a dose of 15 ml/kg. Immediately afterward, mice were placed in an atmosphere of 95% 02/5% CO2 (carbogen) for 30 min before irradiation. During irradiation, carbogen was administered through a nose cone. This Fluosol/carbogen treatment has been shown to improve the radiation response of EMT6 tumors in BALB/c mice [8,9,11]; similar regimens enhance the radiation responses of other rodent tumors [3,13,17]. Because we found previously [7,12] that the stress associated with experimental manipulations and repeated injections can alter the incidence of lung nodules, control mice received i.v. injections of 15 ml/kg sterile, pyrogenfree, physiologic saline. Fluosol.
Mice were anesthetized with chloral hydrate, then positioned on their backs, with lead shields covering the lower body (up to the diaphragm) and the head and neck (down to the apex of the lung); legs and tails were under the shields. The lungs were irradiated with 250 kV X-rays (15 mA, 2 mm A1 filtration, 1.48 Gy/min). Doses to tissues under the shields were < 5% of the lung doses. Tumor cells were injected 24 h after irradiation. Irradiation.
Results
Data from four experiments examining tumor formation in irradiated lungs are shown in Table I. To facilitate comparisons between groups injected with different cell numbers, values are shown as colonyforming efficiencies (lung nodules/100 cells). There was considerable experiment-to-experiment variability in the colony-forming efficiencies in control
51 TABLE I Effects of thoracic irradiation on the development of lung nodules from intraveneously-injectedEMT6 tumor cells. Radiation dose
0 Gy
5 Gy
S/A
S/A
F/C
S/A
F/C
S/A
F/C
1.9 4- 0.3 3.2 + 0.3 -
1.7 + 0.3 3.5 + 0.6 -
1.7 + 0.4 4.6 + 0.4 4.1 + 0.5
1.6 + 0.3 4.5 4- 0.6 5.7 + 0.8
8.7 + 0.4 5.4 + 0.9
4.9 4- 0.9 5.7 + 0.3
0.15 0.31 1.6 0.66
! + + +
0.02 0.06 0.1 0.11
10 Gy
15 Gy
Values shown are the colony-forming efficiencies(nodules formed/100 cells injected). ValuEs are means _+ S.E.M.s for 8-12 mice. S/A = mice injected with saline and breathing air; F/C = mice injected with Fluosol and breathing carbogen. Data from four independent experiments are shown.
mice. However, thoracic i r r a d i a t i o n always increased the n u m b e r o f l u n g colonies over that in u n i r r a d i a t e d a n i m a l s from the same experiment (Table I). L u n g - c o l o n y n u m b e r s increased linearly with increasing r a d i a t i o n dose (Fig. 1). The effect of F l u o s o l / c a r b o g e n t r e a t m e n t o n lung
n o d u l e f o r m a t i o n in irradiated mice was e x a m i n e d in the same experiments. In only one o f the seven direct paired c o m p a r i s o n s o f perfluorochemicaltreated a n d c o n t r o l mice was there a statistically significant difference in l u n g - c o l o n y n u m b e r ; in this case, mice treated with r a d i a t i o n alone had more t u m o r s t h a n mice also treated with F l u o s o l a n d carbogen. The overall dose-responce curve for the enh a n c e m e n t o f lung n o d u l e f o r m a t i o n was similar in perfluorochemical-treated a n d c o n t r o l mice. The experiments reported here did n o t include studies of u n i r r a d i a t e d mice treated with P F C - E plus carbogen, because previous studies [10] revealed n o effects for single F l u o s o l / c a r b o g e n treatm e n t s given one or 7 days before i n o c u l a t i o n o f tum o r cells or for a m u l t i - t r e a t m e n t regimen with Fluosol/carbogen t r e a t m e n t s given 8, 6, 4 a n d one day before i n o c u l a t i o n of t u m o r cells.
,_.1 LU 6 0 o o L.U .._1 0 0 z
3
Discussion
2
0
I
I
I
5
I0
15
R A D I A T I O N D O S E (Gy)
Fig. I. Lung colonies developing in Fluosol/carbogen-treated (O) and saline-injected/air-breathing (O) mice as a function of the dose of radiation. Values are means from 2 to 4 independent experiments; S.E.M.s are shown for n >/ 3.
The use of P F C - E as adjuncts to r a d i o t h e r a p y raises questions n o t e n c o u n t e r e d in the prior uses o f these agents in t r a u m a , surgery, or anemia. Past studies in o u r own a n d other laboratories ask whether t r e a t m e n t with a P F C - E plus oxygen could alter the progression of the m a l i g n a n t disease, either t h r o u g h direct effects o n the viability or growth o f the malign a n t cells or via effects on the host, such as physio-
52 logic changes or immune perturbations. Treatment with Fluosol did not alter the growth or viability of either EMT6 cells in vitro or cells in solid EMT6 tumors [8,9,11]. Moreover, treatment with Fluosol plus carbogen did not alter the growth of solid intradermal EMT6 tumors [11]. Fluosol therefore appears to have no direct effect on tumor cell viability or the growth of established tumors in this tumor/ host system. Lung metastases might still be altered if the PFCE/carbogen treatment injured the lung or produced other effects (e.g. hemodynamic changes, immune effects) which altered the entrapment, implantation, or growth of the tumor cells in the lung. Our previous studies [10], however, revealed no effects of single or multiple PFC-E/carbogen treatments on the development of lung tumors from i.v.-injected tumor cells. Any effects noted in the present studies of tumors in irradiated lungs would therefore reflect a change in the radiosensitivity of the lung tissue, rather than toxic effects of the Fluosol/carbogen treatment on the host or direct effects of the treatment on the malignant cells. Fluosol/carbogen treatments do not produce significant changes in the radiation response of the hematopoietic stem cells, gut, or testes [2,9], but can alter radiation reactions in mouse skin [9,13]. The effects of local thoracic irradiation on the development of lung nodules from intraveneouslyinjected tumor cells have been studied extensively [1,5,6,14,16,18,20,21]. Irradiation of the lungs enhances the development of tumors; the number of lung nodules increases roughly linearly with the radiation dose. The effect of irradiation varies with time; it is maximal ~ 1 day after irradiation, but may show a second peak several months later [1,5,16,18]. Augmentation of lung nodule formation has also been reported after treatment with chemotherapeutic agents [5,14,21], hypoxic cell sensitizers [5,12], and manipulations producing stress [5,7,20]. Injury to the lung appears to be the major factor promoting metastasis [5]. This does not appear to alter the initial entrapment of tumor cells in the lung, but rather to increase the ability of the trapped cells to form tumors [5,6,16]. Damage to
the capillary walls, which facilitates crossing of tumor cells from the lumen of the capillary to the extravascular spaces, has been hypothesized to be critical [5]. Immunologic factors may also p!ay a limited role, especially after treatment with certain drugs [5,20]. Treatment of mice with radiosensitizers and radioprotectors before irradiation alters the number of lung nodules, but this does not reflect sensitization/protection. Misonidazole augments lung nodule formation in mice treated with X-rays or cyclophosphamide; this appears to reflect the additional injury from the misonidazole [5]. WR2721 increases the number of colonies developing in irradiated lungs, rather than decreasing lung nodule formation as might be expected from its protective effects against radiation pneumonitis [5]. As WR2721 increases tumor formation in unirradiated lungs, this "enhancement" probably reflects a summation of damage from WR2721 and radiation, which overrides the radioprotective effects of the drug [5]. The relatively low RBE for augmentation of lung metastases with neutron irradiation and the fact that fractionation of the radiation into two equal fractions separated by 4 h increases the number of lung nodules [5] show that alteration of radiation regimens need not produce the changes in lung nodule formation predicted from simple considerations. Because of these data, we felt that we could not predict a priori the effect of Fluosol/carbogen treatment on radiation-enhanced lung nodule formation. In the experiments reported here, localized thoracic irradiation increased the number of lung nodules developing from tumor cells injected i.v. 24 h later. The number of tumors increased linearly with the radiation dose over the range from 5 to 15 Gy. This increase was consistent with those in other tumor/host systems [1,5,6,16,18,22]. The incidence of lung nodules was not altered when mice were injected with Fluosol 30 min before irradiation and treated with carbogen before and during irradiation. Therefore, treatment with Fluosol plus carbogen during irradiation did not sensitize the lungs to the production of the radiation-induced lesions which enhance lung nodule formation.
53
Acknowledgements This research was supported by Grant CA35215 from the NCI. We thank Dr. Norma Lowe of Alpha Therapeutic Corporation for providing the Fluosol-DA, 20%, for these studies and Ms. Jacqueline Mendes for her assistance with the experiments.
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 Fischer, J.J., Rockwell, S. and Martin, D.F. Perfluorochemicals and hyperbaric oxygen in radiation therapy. Int. J. Radiat. Oncol. Biol. Phys. 12: 95-102, 1986. 3 Martin, D.F., Porter, E.A., Fischer, J.J. and Rockwell, S. Effect of a perfluorochemical emulsion on the radiation response of BAI 112 rhabdomyosarcomas. Radiat. Res. 112: 45-53, 1987. 4 Mason, K.A., Withers, H.R. and Steckel, R.J. Acute effects of a perfluorochemical oxygen carrier on the normal tissues of the mouse. Radiat. Res. 104: 387-394, 1985. 5 Milas, L. and Peters, L.J. Conditioning of tissues for metastasis formation by radiation and cytotoxic drugs. In: Cancer Invasion and Metastasis: Biologic and Therapeutic Aspects, pp. 321-336. Editors: G.L. Nicolson and L. Milas. Raven Press, New York, 1984. 6 Peters, L.J., Mason, K., McBride, W.H. and Patt, Y.Z. Enhancement of lung colony-forming efficiency by local thoracic irradiation: interpretation of labelled cell studies. Radiology 126: 499-505, 1978. 7 Rockwell, S. Maintenance of tumor systems and appropriate treatment techniques for experimental tumors. In: Rodent Tumors in Experimental Cancer Therapy, pp. 29-36. Editor: R.F. Kallman. Plenum Press, New York, 1987. 8 Rockwell, S. Use of a perfluorochemical emulsion to improve oxygenation in a solid tumor. Int. J. Radiat. Oncol. Biol. Phys. 11: 97-103, 1985. 9 Rockwell, S., Mate, T.P., lrvin, C.G. and Nierenburg, M. Reactions of tumors and normal tissues in mice to irradia-
I0
11
12
13
14
15
16
17
18 19
20
21
22
tion in the presence and absence of a perfluorochemical emulsion. Int. J. Radiat. Oncol. Biol. Phys. 12:1311-1313, 1986. Rockwell, S., Irvin, C.G., and Nierenburg, M. Effect of a perfluorochemical emulsion on the development of artificial lung metastases in mice. Clin. Expl. Metast. 4: 45-50, 1986. Rockwell, S., lrvin, C.G. and Nierenburg, M. Preclinical studies of a perfluorochemical emulsion as an adjunct to radiotherapy. Int. J. Radiat. Oncol. Biol. Phys., in press, 1988. Rockwell, S., Nierenburg, M. and Irvin, C.G. Enhancement of artificial lung metastases by misonidazole. Int. J. Radiat. Oncol. Biol. Phys. 10: 1395-1398, 1984. Song, C.W., Lee, I., Hasegawa, T., Rhee, J.G. and Levitt, S.H. Increase in pO 2 and radiosensitivity of tumors by Fluosol-DA (20%) and carbogen. Cancer Res. 47: 442-446, 1987. Steel, G.G. and Adams, K. Enhancement by cytotoxic agents of artificial pulmonary metastasis. Br. J. Cancer 36: 653~58, 1977. Suyama, T., Yokoyama, K. and Naito, R. Development of a perfluorochemical whole blood substitute (Fluosol-DA, 20%) - an overview of clinical studies with 185 patients. In: The Red Cell, pp. 609-626. Alan R. Liss, New York, 1981. Tanaka, Y. Effect of lung irradiation on the incidence of pulmonary metastases and its mechanism. Acta Radiol. Ther. Phys. Biol. (Jpn) 15: 141-148, 1976. Teicher, B.A. and Rose, C.M. Perfluorochemical emulsions can increase tumor radiosensitivity. Science 223: 934-936, 1984. Thompson, S.C. Tumour colony growth in the irradiated mouse lung. Br. J. Cancer 30: 337-341, 1974. Tremper, K.K., Friedman, A.E., Levine, E.M., Lapin, R. and Camarillo, D. The preoperative treatment of severely anemic patients with a perfluorochemical oxygen-transport fluid, Fluosol-DA. N. Engl. J. Med. 307: 277-283, 1982. Van den Brenk, H.A.S., Stone, M.G., Kelly, H. and Sharpington, G. Lowering of innate resistance of the lungs to the growth of blood-borne cancer cells in states of topical and systemic stress. Br. J. Cancer 33: 60-78, 1976. van Putten, L.M., Kram, L.K.J. van Dierendonck, H.H.C., Smink, T. and Fuzy, M. Enhancement by drugs of metastatic lung nodule formation after intraveneous tumour cell injection. Int. J. Cancer 15: 588-595, 1975. Withers, H.R. and Milas, L. Influence of preirradiation of lung on development of artificial pulmonary metastases of fibrosarcoma in mice. Cancer Res. 33: 1931-1936, 1973.
49
RTO 00524
Radiation enhancement of lung nodule formation in mice is not potentiated by treatment with a perfluorochemical emulsion and carbogen Sara R o c k w e l l a n d M a r i a n n e Kelley Department of Therapeutic Radiology, Yale University School of Medicine, New Haven. CT, U.S.A.
(Received23 March 1988, revisionreceived27 June 1988, accepted 5 July 1988)
Key words: Perfluorochemicalemulsion; Fluosol-DA, 20%; Lung metastases; Radiation damage, lung
Summary The ability of intraveneously-injected mouse mammary tumor cells to form lung tumors is increased by irradiation of the thorax 24 h previously. We examined the effects of treatment with a perfluorochemical emulsion (Fluosol-DA, 20%) plus carbogen before and during irradiation on the radiation-induced enhancement of lung nodule formation. We found no evidence that treatment with Fluosol plus carbogen altered the development of tumor nodules in irradiated mouse lungs.
Introduction Perfluorochemical emulsions (PFC-E) were originally developed as O2-transport agents, to replace transfused blood [15,19]. The observation that PFC-E could ameliorate ischemic injury after experimental vascular occlusion lead to the idea that these agents might be used to improve oxygenation Addressfor correspondence: Sara Rockwell,Department of Therapeutic Radiology,Yale UniversitySchoolof Medicine, 333 Cedar Street, New Haven, CT 06510-8040, U.S.A.
in the hypoxic areas of solid tumors and therefore improve the results of radiotherapy [2,8,17]. This has been tested using several solid tumor systems; treatment with PFC-E and 02 or carbogen has been found to increase the efficacy of radiation in killing tumor cells, perturbing tumor growth, and producing local tumor control [3,8,9,11,13,17]. As a result, one PFC-E, Fluosoi-DA, 20%, has entered clinical trials as an adjunct to radiotherapy for carcinoma of the head and neck, glioma, and carcinoma of the lung. Extensive studies of the physiologic effects and
0167-8140/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)
50 toxicities of Fluosol have been performed in laboratory animals, human volunteers, and patients treated for trauma or anemia [15,19]. However, the use of this agent in cancer therapy raises questions not encountered previously. One must ask, for example, whether the PFC-E/O2 treatments alter the acute or chronic toxicities of radiation to normal tissues. One must also ask whether treatments with a PFC-E and oxygen have physiologic or toxic effects which alter progression of neoplastic disease. The experiments reported here are part of a series of studies addressing these questions [2,3,8-11]. Here we extend these studies to ask whether treatment with a PFC-E and carbogen during thoracic irradiation alters the radiation-induced augmentation of lung colony formation; such augmentation might occur if this treatment sensitized the lung to radiation injury. This question has recently acquired clinical significance, because of the initiation of clinical trials requiring irradiation of lung tissue in patients treated with a PFC-E and oxygen.
Methods and materials Mice and tumors. Female BALB/c Rw mice 2.5 months of age were used throughout these studies. Mice were bred and maintained under SPF conditions [8,10]. The characteristics of the EMT6 Rw mouse mammary tumor cells used in these studies are detailed elsewhere [8-12].
The ability of EMT6 cells to form lung tumors was assayed as detailed previously [7,10,12]. Cell suspensions prepared from exponentially-growing cultures were counted and diluted to obtain a series of single cell suspensions containing 500-5000 cells per 0.2 ml aliquot; no heavily irradiated cells or microspheres were admixed. Two dilutions were tested for most irradiated groups; the group(s) developing optimal numbers of colonies for counting were used for analysis. All mice to be inoculated with a given dilution were ear tagged, randomized, and inoculated in random order. Animals were sacrificed 14 days after injection' and the lungs were removed, cleaned, fixed in Bouin's and Lung colonies.
washed with ethanol. Lungs were coded and counted blind. Tumor nodules on the lung surfaces were counted using a dissecting microscope. Fluosol-DA, 20% (Fluosol), was manufactured by Green Cross Corporation, Japan, and provided without cost by Alpha Therapeutic Corporation, Los Angeles. The composition and characteristics of this emulsion are detailed elsewhere [2,8,15,19]. Fiuosol formulated without hydroxyethyl starch was used for these studies. Fluosol was administered i.v. at a dose of 15 ml/kg. Immediately afterward, mice were placed in an atmosphere of 95% 02/5% CO2 (carbogen) for 30 min before irradiation. During irradiation, carbogen was administered through a nose cone. This Fluosol/carbogen treatment has been shown to improve the radiation response of EMT6 tumors in BALB/c mice [8,9,11]; similar regimens enhance the radiation responses of other rodent tumors [3,13,17]. Because we found previously [7,12] that the stress associated with experimental manipulations and repeated injections can alter the incidence of lung nodules, control mice received i.v. injections of 15 ml/kg sterile, pyrogenfree, physiologic saline. Fluosol.
Mice were anesthetized with chloral hydrate, then positioned on their backs, with lead shields covering the lower body (up to the diaphragm) and the head and neck (down to the apex of the lung); legs and tails were under the shields. The lungs were irradiated with 250 kV X-rays (15 mA, 2 mm A1 filtration, 1.48 Gy/min). Doses to tissues under the shields were < 5% of the lung doses. Tumor cells were injected 24 h after irradiation. Irradiation.
Results
Data from four experiments examining tumor formation in irradiated lungs are shown in Table I. To facilitate comparisons between groups injected with different cell numbers, values are shown as colonyforming efficiencies (lung nodules/100 cells). There was considerable experiment-to-experiment variability in the colony-forming efficiencies in control
51 TABLE I Effects of thoracic irradiation on the development of lung nodules from intraveneously-injectedEMT6 tumor cells. Radiation dose
0 Gy
5 Gy
S/A
S/A
F/C
S/A
F/C
S/A
F/C
1.9 4- 0.3 3.2 + 0.3 -
1.7 + 0.3 3.5 + 0.6 -
1.7 + 0.4 4.6 + 0.4 4.1 + 0.5
1.6 + 0.3 4.5 4- 0.6 5.7 + 0.8
8.7 + 0.4 5.4 + 0.9
4.9 4- 0.9 5.7 + 0.3
0.15 0.31 1.6 0.66
! + + +
0.02 0.06 0.1 0.11
10 Gy
15 Gy
Values shown are the colony-forming efficiencies(nodules formed/100 cells injected). ValuEs are means _+ S.E.M.s for 8-12 mice. S/A = mice injected with saline and breathing air; F/C = mice injected with Fluosol and breathing carbogen. Data from four independent experiments are shown.
mice. However, thoracic i r r a d i a t i o n always increased the n u m b e r o f l u n g colonies over that in u n i r r a d i a t e d a n i m a l s from the same experiment (Table I). L u n g - c o l o n y n u m b e r s increased linearly with increasing r a d i a t i o n dose (Fig. 1). The effect of F l u o s o l / c a r b o g e n t r e a t m e n t o n lung
n o d u l e f o r m a t i o n in irradiated mice was e x a m i n e d in the same experiments. In only one o f the seven direct paired c o m p a r i s o n s o f perfluorochemicaltreated a n d c o n t r o l mice was there a statistically significant difference in l u n g - c o l o n y n u m b e r ; in this case, mice treated with r a d i a t i o n alone had more t u m o r s t h a n mice also treated with F l u o s o l a n d carbogen. The overall dose-responce curve for the enh a n c e m e n t o f lung n o d u l e f o r m a t i o n was similar in perfluorochemical-treated a n d c o n t r o l mice. The experiments reported here did n o t include studies of u n i r r a d i a t e d mice treated with P F C - E plus carbogen, because previous studies [10] revealed n o effects for single F l u o s o l / c a r b o g e n treatm e n t s given one or 7 days before i n o c u l a t i o n o f tum o r cells or for a m u l t i - t r e a t m e n t regimen with Fluosol/carbogen t r e a t m e n t s given 8, 6, 4 a n d one day before i n o c u l a t i o n of t u m o r cells.
,_.1 LU 6 0 o o L.U .._1 0 0 z
3
Discussion
2
0
I
I
I
5
I0
15
R A D I A T I O N D O S E (Gy)
Fig. I. Lung colonies developing in Fluosol/carbogen-treated (O) and saline-injected/air-breathing (O) mice as a function of the dose of radiation. Values are means from 2 to 4 independent experiments; S.E.M.s are shown for n >/ 3.
The use of P F C - E as adjuncts to r a d i o t h e r a p y raises questions n o t e n c o u n t e r e d in the prior uses o f these agents in t r a u m a , surgery, or anemia. Past studies in o u r own a n d other laboratories ask whether t r e a t m e n t with a P F C - E plus oxygen could alter the progression of the m a l i g n a n t disease, either t h r o u g h direct effects o n the viability or growth o f the malign a n t cells or via effects on the host, such as physio-
52 logic changes or immune perturbations. Treatment with Fluosol did not alter the growth or viability of either EMT6 cells in vitro or cells in solid EMT6 tumors [8,9,11]. Moreover, treatment with Fluosol plus carbogen did not alter the growth of solid intradermal EMT6 tumors [11]. Fluosol therefore appears to have no direct effect on tumor cell viability or the growth of established tumors in this tumor/ host system. Lung metastases might still be altered if the PFCE/carbogen treatment injured the lung or produced other effects (e.g. hemodynamic changes, immune effects) which altered the entrapment, implantation, or growth of the tumor cells in the lung. Our previous studies [10], however, revealed no effects of single or multiple PFC-E/carbogen treatments on the development of lung tumors from i.v.-injected tumor cells. Any effects noted in the present studies of tumors in irradiated lungs would therefore reflect a change in the radiosensitivity of the lung tissue, rather than toxic effects of the Fluosol/carbogen treatment on the host or direct effects of the treatment on the malignant cells. Fluosol/carbogen treatments do not produce significant changes in the radiation response of the hematopoietic stem cells, gut, or testes [2,9], but can alter radiation reactions in mouse skin [9,13]. The effects of local thoracic irradiation on the development of lung nodules from intraveneouslyinjected tumor cells have been studied extensively [1,5,6,14,16,18,20,21]. Irradiation of the lungs enhances the development of tumors; the number of lung nodules increases roughly linearly with the radiation dose. The effect of irradiation varies with time; it is maximal ~ 1 day after irradiation, but may show a second peak several months later [1,5,16,18]. Augmentation of lung nodule formation has also been reported after treatment with chemotherapeutic agents [5,14,21], hypoxic cell sensitizers [5,12], and manipulations producing stress [5,7,20]. Injury to the lung appears to be the major factor promoting metastasis [5]. This does not appear to alter the initial entrapment of tumor cells in the lung, but rather to increase the ability of the trapped cells to form tumors [5,6,16]. Damage to
the capillary walls, which facilitates crossing of tumor cells from the lumen of the capillary to the extravascular spaces, has been hypothesized to be critical [5]. Immunologic factors may also p!ay a limited role, especially after treatment with certain drugs [5,20]. Treatment of mice with radiosensitizers and radioprotectors before irradiation alters the number of lung nodules, but this does not reflect sensitization/protection. Misonidazole augments lung nodule formation in mice treated with X-rays or cyclophosphamide; this appears to reflect the additional injury from the misonidazole [5]. WR2721 increases the number of colonies developing in irradiated lungs, rather than decreasing lung nodule formation as might be expected from its protective effects against radiation pneumonitis [5]. As WR2721 increases tumor formation in unirradiated lungs, this "enhancement" probably reflects a summation of damage from WR2721 and radiation, which overrides the radioprotective effects of the drug [5]. The relatively low RBE for augmentation of lung metastases with neutron irradiation and the fact that fractionation of the radiation into two equal fractions separated by 4 h increases the number of lung nodules [5] show that alteration of radiation regimens need not produce the changes in lung nodule formation predicted from simple considerations. Because of these data, we felt that we could not predict a priori the effect of Fluosol/carbogen treatment on radiation-enhanced lung nodule formation. In the experiments reported here, localized thoracic irradiation increased the number of lung nodules developing from tumor cells injected i.v. 24 h later. The number of tumors increased linearly with the radiation dose over the range from 5 to 15 Gy. This increase was consistent with those in other tumor/host systems [1,5,6,16,18,22]. The incidence of lung nodules was not altered when mice were injected with Fluosol 30 min before irradiation and treated with carbogen before and during irradiation. Therefore, treatment with Fluosol plus carbogen during irradiation did not sensitize the lungs to the production of the radiation-induced lesions which enhance lung nodule formation.
53
Acknowledgements This research was supported by Grant CA35215 from the NCI. We thank Dr. Norma Lowe of Alpha Therapeutic Corporation for providing the Fluosol-DA, 20%, for these studies and Ms. Jacqueline Mendes for her assistance with the experiments.
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