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Reoxygenation in a rat rhabdomyosarcoma tumor following X-irradiation
In! J Rodrolmn Onroh~y RIO/. Phys Vol. Printed ,n the U.S.A. All nghts reserved.
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413-471 Copyright
03603016/91 $3.00 + .OO 0 199 Pergamon Press plc
I
0 Original Contribution REOXYGENATION IN A RAT RHABDOMYOSARCOMA FOLLOWING X-IRRADIATION S. M. JAVED
AFZAL,
PH.D.,’
T. S. TENFORDE,
AND S. B. CURTIS, ‘Cell and Molecular Biology Division, and ‘Life Sciences Center,
PH.D.,~
TUMOR
K. S. KAVANAU,
B.S.’
PH.D.’
Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720; Pacific Northwest Laboratory. P.O. Box 999, Richland. WA 99352
The paired survival curve technique was used to characterize the rate at which the fraction of hypoxic cells in rat rhabdomyosarcoma R-l tumors returns to the preirradiation value of 37% following a single dose of 225kVp X rays. Tumors were administered a conditioning x-ray dose of 15Gy, followed at 0, 3, 6, 12, 24, or 48 hr by a 5 Gy, IO-Gy, or IS-Gy dose of X rays under air-breathing conditions or under hypoxic conditions produced by nitrogen-gas asphyxiation 5 min prior to irradiation. Cellular surviving fractions were determined by the tumor excision assay following in viva irradiation. From the ratio of the survival fractions measured for tumor cells from air-breathing and hypoxic animals, the fraction of hypoxic ceils was determined as a function of time postirradiation. These results indicated that immediately following a 15Gy dose of X rays, essentially 180% of the viable cells remaining were hypoxic. The tumors reoxygenated rapidly, returning to the preirradiation level of 37% during the first 6 hr postirradiation. Rat rhabdomyosarcoma tumors, Reoxygenation, Hypoxic fraction.
The existence of clonogenic hypoxic cells has been demonstrated in a variety of animal tumors (2, 1 1, 22. 26,29, 37, 40, 41). and their importance in determining radiosensitivity is widely accepted. Even though evidence for the existence of hypoxic cells in human tumors is indirect (3, 9, 10, 38, 39), the importance of killing radioresistant hypoxic cells to optimize the radiocurability of human tumors has often been emphasized (3, 5, 17, 19, 20, 25, 27, 3 1). Because of the inhomogeneous supply of oxygen within the tumor, cells are in varying state of oxygenation. After X-irradiation, the oxygenated cells are killed preferentially, thereby leading to a preponderance of surviving cells that are mainly hypoxic. After treatment with either a single or fractionated doses of X rays, a proportion of surviving cells that were initially hypoxic becomes reoxygenated, thereby changing the oxygenation status of the tumor during the course of radiation treatment (12, 13, 15, 17, 20, 2 1, 30). It is therefore of importance to determine the kinetics and extent of these changes in order to assess the relative effectiveness of fractionated treatments
with low- and high-LET radiation. Because of the reduced dependence on oxygen for cell killing (4, 6), high-LET heavy charged particles may offer an advantage in the radiocurability of tumors exhibiting reduced or no reoxygenation. Therefore, in continuation of our studies on the radiobiological response of rat rhabdomyosarcoma tumors to X rays and BEVALAC charged particle beams ( 1, 6-8, 32-34, 36), measurements have been made of the kinetics of reoxygenation following a single conditioning dose of X-irradiation. Furthermore, in our recent studies (35) on the kinetics of repopulation following single (20 Gy) or fractionated doses of X-irradiation, a IO-fold decrease in the fraction of clonogenic cells has been observed between the third and fourth day after irradiation. Based on our earlier cell survival studies (32), it could be calculated that all of the cells surviving the large doses in the repopulation studies were hypoxic at the time of irradiation. One of the arguments to explain this decrease could be the in vitro “rescue” of hypoxic cells from tumors that were excised and plated in cell culture medium during the first 2 days after irradiation. This argument is based on the assump-
Reprint requests to: S. M. Javed Afzal, Ph.D., Bldg. 74, I Cyclotron Road, Cell and Molecular Biology Division, Lawrence Berkeley Laboratory. University of California, Berkeley, CA 94720. Acknowledgments-The skillful assistance of Tennessee Gock in preparation of this manuscript is gratefully acknowledged.
Research support was received from Public Health Service Grant CA RO I - 174 11 awarded by the National Cancer Institute, and from the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC03-76SFOOO98 with the Lawrence Berkeley Laboratory. Accepted for publication 10 October 1990.
INTRODUCTION
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474
1. J. Radiation Oncology 0 Biology 0 Physics
tion that hypoxic cells left in situ may have reduced survival potential, but when removed from the tumor during the first 2 days postirradiation and cultured in vitro, these cells are “rescued” by the presence of a growth medium containing feeder cells, fetal calf serum, and other nutrients (23,24). For this hypothesis to hold, the cells would have to remain hypoxic for at least up to 2 days postirradiation. The present study was therefore conducted to measure the rate of reoxygenation in rat rhabdomyosarcoma tumors in order to test this hypothesis. METHODS
AND MATERIALS
Experimental tumor line The tumor used for the reoxygenation studies is a subline, designated R2C5, derived from the rat rhabdomyosarcoma R- 1 tumor. The characteristics and radiation response of the R2C5 tumor subline have been described previously (32). The fraction of chronically hypoxic cells in R2C5 tumors is 37%, as determined from the ratio of the surviving fraction of cells in the high-dose range (> 15 Gy) under air-breathing vs. hypoxic conditions (32). The R2C5 tumor has been used in our laboratory for all in vivo cell survival studies subsequent to 1982 because it produces nonnecrotic tumors in syngeneic WAG/Rij rats and has a plating efficiency in the tumor excision assay of -50%.
Irradiation Tumors were irradiated with X rays from a therapy unit* operated at 225 kVp with a total filtration of 0.35 mm Cu (mean photon energy = 75 keV; HVL = 1.08 mm Cu). The x-ray dose rate measured with a 250 R ionization chamber+ was 6 Gy/min at the tumor location. For tumor irradiations under air-breathing conditions, the animals were lightly anesthetized with metofane.* Cell survival assay Tumors were excised following irradiation, minced with scissors, and placed in 25 ml of 37°C tissue culture medium containing 1 mg/ml dispase.” The culture medium consisted of Hanks MEM with 50 units/ml penicillin and supplemented with 10% fetal 50 pg/ml streptomycin,** calf serum and 10% newborn calf serum.++ The tumor mince was stirred magnetically in the enzyme solution for 3 min, at which time the supernatant was decanted to remove erythrocytes and lysed tumor cells. Twentyfive ml of fresh enzyme solution was added and the dissociation procedure was continued at 37°C with magnetic stirring for 1 hr. At the end of this period, the cell suspension was chilled for 5 min in an ice bath to stop enzyme activity, and was then passed through a bone marrow fil-
* Model RT200/250, Philips, Eindhoven, The Netherlands. + Victoreen, Cleveland, OH. * Pitman-Moore, Washington Crossing, NJ. 5 Sigma Chemical Co., St. Louis, MO.
March 1991. Volume 20. Number 3
terS’ to remove clumps of cells. The cells were centrifuged (400 g, 10 min. 4°C). resuspended in tissue culture medium, and counted with a hemacytometer. After appropriate dilution, the single-cell suspension was seeded into 25-cm2 flasks”” for assay of colony-forming ability. The total number of cells per flask was adjusted to lo4 by the addition of lethally irradiated (42 Gy of 225-kVp X rays) feeder cells prepared from the cultured R2C5 cell line. Colony development was allowed to proceed for 12 days in a 37°C incubator continuously flushed with a 97.5% air -2.5% CO* humidified gas mixture. The culture medium was changed on the fourth day after the cells were plated. At the end of the 12-day growth period, colonies were fixed with Bouins and stained with hematoxylin. Colonies containing more than 50 cells were scored as radiation survivors. The average plating efficiency for nonirradiated control tumors was 52.9 + 7.5 (SE) %. The paired survival curve method (26) was used to determine the hypoxic fraction. The dose response curves were obtained by administering single doses to groups of tumors either in hosts under normal air-breathing conditions or in hosts that were asphyxiated with a stream of nitrogen gas 5 min prior to irradiation and tumor excision. Survival curves were fitted to the data using linear regression analysis. From the ratio of the survival fractions measured for air-breathing and hypoxic tumor cells, the fraction of hypoxic cells was determined. To determine the kinetics of reoxygenation, tumors were irradiated with a conditioning dose of 15 Gy in animals under normal air-breathing conditions, followed at 0, 3, 6, 12, or 48 hr by a 5-, lo-, or 15-Gy second dose of X rays under either normal air-breathing or hypoxic conditions. From the ratio of the survival fractions measured for air-breathing and hypoxic tumor cells at various intervals, the fraction of hypoxic cells was determined as a function of time. RESULTS Earlier studies of rhabdomyosarcoma
tumors irradiated and hypoxic conditions have shown that the survival curve for tumors irradiated in air-breathing hosts exhibit a hypoxic break over the dose range 5-10 Gy, and at high doses (> 15 Gy) the slopes of the air and nitrogen curves are parallel (32). Therefore, in the present study, doses greater than 15 Gy were used to determine the hypoxic fraction and the rate of reoxygenation. Survival curves for rat rhabdomyosarcoma tumors irradiated in situ with single 15-, 20-, 25-, and 30-Gy doses of X rays in air-breathing or nitrogen-asphyxiated hosts are presented in Figure 1. As reported in a previous study (32), the hypoxic fraction was calculated to be 37%.
in situ with 225-kVp X rays under air-breathing
** Gibco, Grand Island, NY. ” Flow Laboratories, Inglewood, CA. t* C. R. Bard, Inc., Fitzwilliam, NH. BBFalcon Plastics, Oxnard, CA.
Reoxygenation of R-l tumor 0 S. M. J. AFZAL efal.
0
IO
5
15
20
30
2.5
35
DOSE (Gy)
radiation, the hypoxic fraction was close to 100% since there was no change in the survival of tumor cells irradiated under air-breathing or hypoxic conditions (Fig. 2A). Therefore, it was assumed that all the cells surviving the 15 Gy of priming dose responded to additional radiation as being hypoxic. As the interval between the conditioning dose and the second dose increased, the hypoxic curves shifted gradually in the upward direction. We interepret the upward shift observed in the hypoxic curve as being due to the repair occurring in the tumor cell population during the interval before the cells were made hypoxic for the second dose of radiation. An upward shift in survival is not evident in the tumors irradiated in the air-breathing hosts because of the counteractive effect of the presence of reoxygenated cells at the time of administration of the second dose of radiation. Following a time interval of 3 hr (Fig. 2B) between the conditioning and the second dose, the two survival curves began to separate from one another, indicating that the hypoxic fraction was beginning to drop. During the following additional 3 hr (6 hr postirradiation), the hypoxic fraction decreased rapidly (Fig. 2C), returning to the preirradiation value of 37%. There was no difference between the hypoxic fractions measured at 6, 12, 24, or 48 hr postirradiation (Figs. 2D and 3). Data for 12 hr and 24 hr are not shown.
40
XBL 8915185
Fig. I. Surviving fraction of rat rhabdomyosarcoma tumor as a function of single dose given under air-breathing (0) hypoxic (0) conditions. Three to four tumors were pooled each data point. Standard errors from colony counts of tissue culture dishes are indicated.
cells and for four
Paired survival curves from tumors irradiated with various doses in air-breathing or nitrogen asphyxiated hosts after 0, 3, 6, or 48 hr following a single conditioning dose of 15 Gy under normal air-breathing conditions are shown in Figure
2. Immediately
after
the conditioning
dose
of
1 : : A
,001 .Ol :
15 Gy=b
0
5
IO
15 Total
‘!
2nd Dose
, , . , . ,‘“*,
, 20
25
Dose
30
35
B
15 Gy ‘-’ hr’b2nd
Total
Dose
. , . . , . . 35 40 30
001 I . . , . . , . 1 , . . , . . , 15 20 25 0 5 10
40
(Gy)
‘=’ hr’w2nd
47s
(Gy)
Dose
Dose
.l
.Ol
.OOI I-l 0
5
10
15 Total
20
25
Dose
30 (Gy)
35
40
,001 II 0
5
10
15 Total
20 Dose
25
30
35
40
(Gy)
Fig. 2. Surviving fraction of rat rhabdomyosarcoma tumor cells as a function of total dose, with the secondary dose given under air-breathing (0) or hypoxic (0) conditions at O(A), 3(B), 6(C), or 48(D) hr after a 15Gy conditioning dose. Three to four tumors were pooled for each data point. Standard errors from colony counts of four tissue culture dishes are indicated.
476
I. J. Radiation Oncology 0 Biology 0 Physics
=:
80
E
70
t
P 60 c
0 ._
Z
50
E LL
40
01 0
6
12
18
24
30
Time (Hours)
36
42
48
XBL L19151ed
Fig. 3. Percentage of hypoxic cells in rat rhabdomyosarcoma tumors as a function of time after a conditioning dose of 15 Gy. Error bars represent the standard error of the mean hypoxic fraction measured from the 3 dose points.
DISCUSSION The rate of reoxygenation following irradiation has been measured for several rodent tumor models (12-Z 1e 37. 40, 41). Most of the tumors except osteosarcoma have been shown to reoxygenate rapidly. with their hypoxic fraction returning to the preirradiation values within the first 6- 12 hrs. More recent and extensive studies of Rofstad (30) with human melanoma tumors grown as xenografts in nude mice have also indicated rapid and extensive reoxygenation. with the hypoxic fraction returning to the preirradiation value within 12 to 24 hr. These results are in agreement with the present study that indicated rapid and complete reoxygenation of rhabdomyosarcoma tumors within 6 hr after irradiation. Although the mechanism(s) of reoxygenation are not
March 199 I, Volume 20. Number ?
well understood, the rapid rate of reoxygenation observed in the present study is compatible with the assumption that in the rat rhabdomyosarcoma tumors it could be caused by a reduced rate of oxygen use by radiation-damaged (“doomed”) cells and/or resumption of normal blood flow in transiently non-functioning capillaries (30). Although we have no direct evidence that either mechanism is operative, recent studies have demonstrated a rapid increase in the blood flow rate following irradiation. Using the radiolabeled microsphere (RM) and paramagnetic assisted magnetic resonance imaging (PA-MRI) techniques, Ngo c~ful. (28) have measured tumor blood flow in irradiated rhabdomyosarcoma tumors. Preliminary data indicate that following local irradiation with lo- I5 Gy ‘j’Cs gamma rays, a significant increase in the overall tumor blood flow occurs within 30 min postirradiation. The rapid rate of reoxygenation observed in the present study contradicts the assumption that reoxygenation is caused by increased capillary density caused by tumor shrinkage (2. 17). which is manifested several days after irradiation. The data presented here also do not support the argument that a IO-fold decrease in the clonogenic cell survival at 3-4 days postirradiation observed in our tumor repopulation studies with 20 Gy of X rays (35) could be caused by the “rescue” of the hypoxic cells at O-2 days postirradiation. Given the fact that the decrease in the clonogenic cell survival occurred at 3-4 days postirradiation, it is unlikely to be caused by the “rescue” of hypoxic cells since they become reoxygenated within the first 6 hr after irradiation. Our recent studies (35) have attributed the decrease in cell survival at 3-4 days postirradiation to the influx of host diploid cells that are cytotoxic to the irradiated tumor cells. The present study supports our earlier research (34) on the combined effects of the radiosensitizer, misonidazole. and fractionated doses of low- and high-LET radiation. When misonidazole and X-irradiation were given in four daily fractions, the drug enhancement ratio decreased significantly. Based on the present study. this effect would be expected because reoxygenation of tumor cells is complete well within the 24-hr fractionation interval.
REFERENCES I. Afzal. S. M. J.: Tenforde,
T. S.; Parr, S. S.: Curtis, S. B. PLD repair in rat rhabdomyosarcoma tumor cells irradiated in vivo and in vitro with high-LET and low-LET radiation. Radiat. Res. 107:354-366; 1986. 2. Brown, J. M. Evidence for acutely hypoxic cells in mouse tumours, and a possible mechanism of reoxygenation. Br. J. Radiol. 52:650-656: 1979. 3. Bush, R. S.: Jenkin, R. D. T.: Allt, W. E. C.; Beale, F. A.: Bean, H.: Dembo, A. J.: Pringle. J. F. Definitive evidence for hypoxic cells influencing cure in cancer therapy. Br. J. Cancer 37 (Suppl. 111):302-306; 1978. 4. Castro, J. R.; Quivey, J. M.; Lyman, J. T.; Chen. G. T. Y.; Phillips, T. L.; Tobias, C. A.; Alpen, E. L. Current status of clinical particle radiotherapy at the Lawrence Berkeley Laboratory. Cancer 46:633-641: 1980.
5. Chapman, J. D.; Franko, A. J.; Sharplin. J. A marker for hypoxic cells in tumours with potential clinical applicability. Br. J. Cancer 43546-550; 198 I. 6. Curtis, S. B.; Schilling, W. A.: Tenforde. T. S.; Crabtree, K. E.; Tenforde. S. D.; Howard, J.; Lyman. J. T. Survival of oxygenated and hypoxic tumor cells in the extendedpeak regions of heavy charged-particle beams. Radiat. Res. 90:292-309; 1982. 7. Curtis. S. B.: Tenforde, T. S.: Afzal. S. M. J. Hypoxic cell sensitizers and heavy charged particle beams may play complementary roles in killing hypoxic tumor cells. Int. J. Radiat. Oncol. Biol. Phys. IO: 1203-1205; 1984. 8. Curtis, S. B.: Tenforde. T. S.; Parks D.: Schilling, W. A.; Lyman, J. T. Response of a rat rhabdomyosarcoma to neonand helium-ion irradiation. Radiat. Res. 74:274-288: 1978.
Reoxygenation of R-l tumor 0 S. M. J. AFZAL etal. 9. Dische, S. Hyperbaric oxygen: The Medical Research Council trials and their clinical significance. Br. J. Radio]. 5 1:888-894; 1979. IO. Dische, S.; Anderson, P. J.; Sealy. R.; Watson, E. R. Carcinoma of the cervix-anaemia, radiotherapy and hyperbaric oxygen. Br. J. Radiol. 56:251-255; 1983. 1I. Dixon, B.; Moore, J. V.; Speakman, H. Radiobiological hypoxia of a transplanted rat tumour and the effect of treatment with cyclophosphamide. Europ. J. Cancer 14: I3831387; 1978. 12. Dorie, M. J.; Kallman, R. F. Reoxygenation in the RIF-I tumor. Int. J. Radiat. Oncol. Biol. Phys. 10:687-694: 1984. 13. Dorie, M. J.: Kallman, R. F. Reoxygenation of the RIF-I tumor after fractionated radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 12:1853-1859; 1986. 14. Howes. A. E. An estimation of changes in the proportion and absolute numbers of hypoxic cells after irradiation of transplanted CjH mouse mammary tumours. Br. J. Radiol. 42144 l-447; 1969. 15. Howes. A. E.; Page, A.; Fowler, J. F. The effect of single and fractionated doses of X rays on the effective proportion of hypoxic cells in CJH mouse mammary turnouts. Br. J. Radio]. 45:250-256: 1972. 16. Kallman. R. F. Oxygenation and reoxygenation of a mouse mammary carcinoma. In: Duplan. J. F., Chapiro, A., Eds. Advances in radiation research, Vol. III, Proc. IV, International Congress of Radiation Research. New York: Gordon and Breach Science Publishers; 1970: I 195-l 204. 17. Kallman, R. F. The phenomenon of reoxygenation and its implications for fractionated radiotherapy. Radiology 105: 135-142; 1972. 18. Kallman. R. F. The oxygen effect and reoxygenation. In: Surgery, radiotherapy and chemotherapy of cancer, Vol. 5. Proc. XI International Cancer Congress. Amsterdam: Excerpta Medica; 1974: 136- 140. 19. Kallman. R. F. Hypoxia and reoxygenation in the radioresistance of solid tumors. In: Fletcher, G. H., Nervi, C., Withers. H. R., Eds. Biological basis and clinical implications of tumor radioresistance. New York: Masson Publishing; 1983:55-60. 20. Kallman, R. F.; Dorie. M. J. Tumor oxygenation and reoxygenation during radiation therapy: their importance in predicting tumor response. Int. J. Radiat. Oncol. Biol. Phys. I2:68 l-685; 1986. 2 I. Kallman, R. F.; Jardine. L. J.; Johnson, C. W. Effects of different schedules of dose fractionation on the oxygenation status of a transplantable mouse sarcoma. JNCI 44:369377; 1970. 22. Kummermehr, J.; Fink, R.; Preuss-Bayer, G.; Trott, K.-R. Reoxygenierung in einem transplantablen Fibrosarkom der Maus. Strahlenther. 157:272-276; I98 I. 23. McNally, N. J. The effect of an hypoxic cell sensitizer on tumor growth delay and cell survival. Implications for cell survival in sif~r and in vitro. Br. J. Cancer 32:6 10-6 18; 1975. 24. McNally. N. J. A comparison of the effects of radiation on tumor growth delay and cell survival. The effects of radiation quality. Br. J. Cancer 48: I4 I - 145: 1975. 25. Moulder, J. E.; Dutreix. J.: Rockwell. S.; Siemann, D. W. Applicability of animal tumor data to cancer therapy in humans. Int. J. Radiat. Oncol. Biol. Phys. l4:9 13-927; 1988. 26. Moulder. J. E.; Rockwell, S. Hypoxic fractions of solid tumors. Int. J. Radiat. Oncol. Biol. Phys. 10:695-712; 1984.
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27. Moulder, J. E.; Rockwell. S. Tumor hypoxia: its impact on cancer therapy. Cancer Metas. Rev. 5:3 13-342: 1987. 28. Ngo, F. Q. H.; Karam, R.; Belfi, C. A.; Knots, L.; Thomas, D.; Sibata, C.: Tefft, M.; Afzal, J. A study of heterogeneities in tumor blood flow and capillary permeability following local irradiation. Presented at the 38th Annual Meeting of The Radiation Research Society, New Orleans, April 7- 12, 1990. 29. Powers, W. E.; Tolmach, L. J. Demonstration of an anoxic component in a mouse tumor-cell population by in vivo assay of survival following irradiation. Radio]. 83:328-336: 1964. 30. Rofstad. E. K. Hypoxia and reoxygenation in human melanoma xenografts. Int. J. Radiat. Oncol. Biol. Phys. I7:8 I89; 1989. 31. Stanley, J. A.; Shipley, W. U.; Steel. G. G. Influence of tumor size on hypoxic fraction and therapeutic sensitivity of Lewis lung tumour. Br. J. Cancer 36: 105-I 13; 1977. 32. Tenforde, T. S.; Afzal, S. M. J.; Parr, S. S.: Howard, J.; Lyman. J. T.; Curtis, S. B. Cell survival in rat rhabdomyosarcoma tumors irradiated in rive with extended-peak silicon ions. Radiat. Res. 92:208-2 16; 1982. 33. Tenforde, T. S.; Curtis, S. B.; Crabtree, K. E.: Tenforde, S. D.; Schilling, W. A.; Howard. J.: Lyman, J. T. In viva cell survival and volume response characteristics of rat rhabdomyosarcoma tumors irradiated in the extended peak region of carbon- and neon-ion beams. Radiat. Res. 83:4256: 1980. 34. Tenforde, T. S.; Curtis, S. B.; Tenforde. S. D.; Parr, S. S.; Crabtree, K. E.; Flynn, M. J.: Howard, J.: Lyman, J. T. Misonidazole enhancement of radiation-induced growth delay in rat rhabdomyosarcoma tumors exposed to accelerated carbon and neon ions. Int. J. Radiat. Biol. 40:1 l7129: 1981. 35. Tenforde, T. S.; Kavanau. K. S.; Afzal. S. M. J.; Curtis, S. B. Host cell cytotoxicity, cellular repopulation dynamics, and phase-specific cell survival in X-irradiated rat rhabdomyosarcoma tumors. Radiat. Res. I23:32-43: 1990. 36. Tenforde, T. S.; Tenforde. S. D.; Crabtree, K. E.: Parks, D. L.; Schilling, W. A.; Parr, S. S.; Flynn. M. J.: Howard. J.; Lyman, J. T.; Curtis, S. B. RBE values for radiationinduced growth delay in rat rhabdomyosarcoma tumors exposed to plateau and peak carbon, neon and argon ions. Int. J. Radiat. Oncol. Biol. Phys. 72 17-22 I ; I98 I. 37. Thomlinson, R. H. Reoxygenation as a function of tumor size and histopathologic type. In: Conference on time and dose relationships in radiobiology as applied to radiotherapy BNL 50230 (C-57). Springfield, Va: Clearinghouse for Federal Scientific and Technical Information; 1970:242-247. 38. Thomlinson, R. H.; Gray. L. H. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br. J. Cancer 9:539-549: 1955. 39. Urtasun, R.: Band, P.; Chapman. J. D.; Feldstein, M. L.; Mielke, B.; Fryer, C. Radiation and high-dose metronidazole in supratentorial glioblastomas. N. Eng. J. Med. 294: l3641367; 1976. 40. van Putten, L. M. Tumour reoxygenation during fractionated radiotherapy; Studies with a transplantable mouse osteosarcoma. Europ. J. Cancer 4: 173- 182; 1968. 41. van Putten. L. M.; Kallman, R. F. Oxygenation status of a transplantable tumor during fractionated radiation therapy. JNCI 40:441-451: 1968.
20. PP
413-471 Copyright
03603016/91 $3.00 + .OO 0 199 Pergamon Press plc
I
0 Original Contribution REOXYGENATION IN A RAT RHABDOMYOSARCOMA FOLLOWING X-IRRADIATION S. M. JAVED
AFZAL,
PH.D.,’
T. S. TENFORDE,
AND S. B. CURTIS, ‘Cell and Molecular Biology Division, and ‘Life Sciences Center,
PH.D.,~
TUMOR
K. S. KAVANAU,
B.S.’
PH.D.’
Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720; Pacific Northwest Laboratory. P.O. Box 999, Richland. WA 99352
The paired survival curve technique was used to characterize the rate at which the fraction of hypoxic cells in rat rhabdomyosarcoma R-l tumors returns to the preirradiation value of 37% following a single dose of 225kVp X rays. Tumors were administered a conditioning x-ray dose of 15Gy, followed at 0, 3, 6, 12, 24, or 48 hr by a 5 Gy, IO-Gy, or IS-Gy dose of X rays under air-breathing conditions or under hypoxic conditions produced by nitrogen-gas asphyxiation 5 min prior to irradiation. Cellular surviving fractions were determined by the tumor excision assay following in viva irradiation. From the ratio of the survival fractions measured for tumor cells from air-breathing and hypoxic animals, the fraction of hypoxic ceils was determined as a function of time postirradiation. These results indicated that immediately following a 15Gy dose of X rays, essentially 180% of the viable cells remaining were hypoxic. The tumors reoxygenated rapidly, returning to the preirradiation level of 37% during the first 6 hr postirradiation. Rat rhabdomyosarcoma tumors, Reoxygenation, Hypoxic fraction.
The existence of clonogenic hypoxic cells has been demonstrated in a variety of animal tumors (2, 1 1, 22. 26,29, 37, 40, 41). and their importance in determining radiosensitivity is widely accepted. Even though evidence for the existence of hypoxic cells in human tumors is indirect (3, 9, 10, 38, 39), the importance of killing radioresistant hypoxic cells to optimize the radiocurability of human tumors has often been emphasized (3, 5, 17, 19, 20, 25, 27, 3 1). Because of the inhomogeneous supply of oxygen within the tumor, cells are in varying state of oxygenation. After X-irradiation, the oxygenated cells are killed preferentially, thereby leading to a preponderance of surviving cells that are mainly hypoxic. After treatment with either a single or fractionated doses of X rays, a proportion of surviving cells that were initially hypoxic becomes reoxygenated, thereby changing the oxygenation status of the tumor during the course of radiation treatment (12, 13, 15, 17, 20, 2 1, 30). It is therefore of importance to determine the kinetics and extent of these changes in order to assess the relative effectiveness of fractionated treatments
with low- and high-LET radiation. Because of the reduced dependence on oxygen for cell killing (4, 6), high-LET heavy charged particles may offer an advantage in the radiocurability of tumors exhibiting reduced or no reoxygenation. Therefore, in continuation of our studies on the radiobiological response of rat rhabdomyosarcoma tumors to X rays and BEVALAC charged particle beams ( 1, 6-8, 32-34, 36), measurements have been made of the kinetics of reoxygenation following a single conditioning dose of X-irradiation. Furthermore, in our recent studies (35) on the kinetics of repopulation following single (20 Gy) or fractionated doses of X-irradiation, a IO-fold decrease in the fraction of clonogenic cells has been observed between the third and fourth day after irradiation. Based on our earlier cell survival studies (32), it could be calculated that all of the cells surviving the large doses in the repopulation studies were hypoxic at the time of irradiation. One of the arguments to explain this decrease could be the in vitro “rescue” of hypoxic cells from tumors that were excised and plated in cell culture medium during the first 2 days after irradiation. This argument is based on the assump-
Reprint requests to: S. M. Javed Afzal, Ph.D., Bldg. 74, I Cyclotron Road, Cell and Molecular Biology Division, Lawrence Berkeley Laboratory. University of California, Berkeley, CA 94720. Acknowledgments-The skillful assistance of Tennessee Gock in preparation of this manuscript is gratefully acknowledged.
Research support was received from Public Health Service Grant CA RO I - 174 11 awarded by the National Cancer Institute, and from the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC03-76SFOOO98 with the Lawrence Berkeley Laboratory. Accepted for publication 10 October 1990.
INTRODUCTION
473
474
1. J. Radiation Oncology 0 Biology 0 Physics
tion that hypoxic cells left in situ may have reduced survival potential, but when removed from the tumor during the first 2 days postirradiation and cultured in vitro, these cells are “rescued” by the presence of a growth medium containing feeder cells, fetal calf serum, and other nutrients (23,24). For this hypothesis to hold, the cells would have to remain hypoxic for at least up to 2 days postirradiation. The present study was therefore conducted to measure the rate of reoxygenation in rat rhabdomyosarcoma tumors in order to test this hypothesis. METHODS
AND MATERIALS
Experimental tumor line The tumor used for the reoxygenation studies is a subline, designated R2C5, derived from the rat rhabdomyosarcoma R- 1 tumor. The characteristics and radiation response of the R2C5 tumor subline have been described previously (32). The fraction of chronically hypoxic cells in R2C5 tumors is 37%, as determined from the ratio of the surviving fraction of cells in the high-dose range (> 15 Gy) under air-breathing vs. hypoxic conditions (32). The R2C5 tumor has been used in our laboratory for all in vivo cell survival studies subsequent to 1982 because it produces nonnecrotic tumors in syngeneic WAG/Rij rats and has a plating efficiency in the tumor excision assay of -50%.
Irradiation Tumors were irradiated with X rays from a therapy unit* operated at 225 kVp with a total filtration of 0.35 mm Cu (mean photon energy = 75 keV; HVL = 1.08 mm Cu). The x-ray dose rate measured with a 250 R ionization chamber+ was 6 Gy/min at the tumor location. For tumor irradiations under air-breathing conditions, the animals were lightly anesthetized with metofane.* Cell survival assay Tumors were excised following irradiation, minced with scissors, and placed in 25 ml of 37°C tissue culture medium containing 1 mg/ml dispase.” The culture medium consisted of Hanks MEM with 50 units/ml penicillin and supplemented with 10% fetal 50 pg/ml streptomycin,** calf serum and 10% newborn calf serum.++ The tumor mince was stirred magnetically in the enzyme solution for 3 min, at which time the supernatant was decanted to remove erythrocytes and lysed tumor cells. Twentyfive ml of fresh enzyme solution was added and the dissociation procedure was continued at 37°C with magnetic stirring for 1 hr. At the end of this period, the cell suspension was chilled for 5 min in an ice bath to stop enzyme activity, and was then passed through a bone marrow fil-
* Model RT200/250, Philips, Eindhoven, The Netherlands. + Victoreen, Cleveland, OH. * Pitman-Moore, Washington Crossing, NJ. 5 Sigma Chemical Co., St. Louis, MO.
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terS’ to remove clumps of cells. The cells were centrifuged (400 g, 10 min. 4°C). resuspended in tissue culture medium, and counted with a hemacytometer. After appropriate dilution, the single-cell suspension was seeded into 25-cm2 flasks”” for assay of colony-forming ability. The total number of cells per flask was adjusted to lo4 by the addition of lethally irradiated (42 Gy of 225-kVp X rays) feeder cells prepared from the cultured R2C5 cell line. Colony development was allowed to proceed for 12 days in a 37°C incubator continuously flushed with a 97.5% air -2.5% CO* humidified gas mixture. The culture medium was changed on the fourth day after the cells were plated. At the end of the 12-day growth period, colonies were fixed with Bouins and stained with hematoxylin. Colonies containing more than 50 cells were scored as radiation survivors. The average plating efficiency for nonirradiated control tumors was 52.9 + 7.5 (SE) %. The paired survival curve method (26) was used to determine the hypoxic fraction. The dose response curves were obtained by administering single doses to groups of tumors either in hosts under normal air-breathing conditions or in hosts that were asphyxiated with a stream of nitrogen gas 5 min prior to irradiation and tumor excision. Survival curves were fitted to the data using linear regression analysis. From the ratio of the survival fractions measured for air-breathing and hypoxic tumor cells, the fraction of hypoxic cells was determined. To determine the kinetics of reoxygenation, tumors were irradiated with a conditioning dose of 15 Gy in animals under normal air-breathing conditions, followed at 0, 3, 6, 12, or 48 hr by a 5-, lo-, or 15-Gy second dose of X rays under either normal air-breathing or hypoxic conditions. From the ratio of the survival fractions measured for air-breathing and hypoxic tumor cells at various intervals, the fraction of hypoxic cells was determined as a function of time. RESULTS Earlier studies of rhabdomyosarcoma
tumors irradiated and hypoxic conditions have shown that the survival curve for tumors irradiated in air-breathing hosts exhibit a hypoxic break over the dose range 5-10 Gy, and at high doses (> 15 Gy) the slopes of the air and nitrogen curves are parallel (32). Therefore, in the present study, doses greater than 15 Gy were used to determine the hypoxic fraction and the rate of reoxygenation. Survival curves for rat rhabdomyosarcoma tumors irradiated in situ with single 15-, 20-, 25-, and 30-Gy doses of X rays in air-breathing or nitrogen-asphyxiated hosts are presented in Figure 1. As reported in a previous study (32), the hypoxic fraction was calculated to be 37%.
in situ with 225-kVp X rays under air-breathing
** Gibco, Grand Island, NY. ” Flow Laboratories, Inglewood, CA. t* C. R. Bard, Inc., Fitzwilliam, NH. BBFalcon Plastics, Oxnard, CA.
Reoxygenation of R-l tumor 0 S. M. J. AFZAL efal.
0
IO
5
15
20
30
2.5
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DOSE (Gy)
radiation, the hypoxic fraction was close to 100% since there was no change in the survival of tumor cells irradiated under air-breathing or hypoxic conditions (Fig. 2A). Therefore, it was assumed that all the cells surviving the 15 Gy of priming dose responded to additional radiation as being hypoxic. As the interval between the conditioning dose and the second dose increased, the hypoxic curves shifted gradually in the upward direction. We interepret the upward shift observed in the hypoxic curve as being due to the repair occurring in the tumor cell population during the interval before the cells were made hypoxic for the second dose of radiation. An upward shift in survival is not evident in the tumors irradiated in the air-breathing hosts because of the counteractive effect of the presence of reoxygenated cells at the time of administration of the second dose of radiation. Following a time interval of 3 hr (Fig. 2B) between the conditioning and the second dose, the two survival curves began to separate from one another, indicating that the hypoxic fraction was beginning to drop. During the following additional 3 hr (6 hr postirradiation), the hypoxic fraction decreased rapidly (Fig. 2C), returning to the preirradiation value of 37%. There was no difference between the hypoxic fractions measured at 6, 12, 24, or 48 hr postirradiation (Figs. 2D and 3). Data for 12 hr and 24 hr are not shown.
40
XBL 8915185
Fig. I. Surviving fraction of rat rhabdomyosarcoma tumor as a function of single dose given under air-breathing (0) hypoxic (0) conditions. Three to four tumors were pooled each data point. Standard errors from colony counts of tissue culture dishes are indicated.
cells and for four
Paired survival curves from tumors irradiated with various doses in air-breathing or nitrogen asphyxiated hosts after 0, 3, 6, or 48 hr following a single conditioning dose of 15 Gy under normal air-breathing conditions are shown in Figure
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20
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30 (Gy)
35
40
,001 II 0
5
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20 Dose
25
30
35
40
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Fig. 2. Surviving fraction of rat rhabdomyosarcoma tumor cells as a function of total dose, with the secondary dose given under air-breathing (0) or hypoxic (0) conditions at O(A), 3(B), 6(C), or 48(D) hr after a 15Gy conditioning dose. Three to four tumors were pooled for each data point. Standard errors from colony counts of four tissue culture dishes are indicated.
476
I. J. Radiation Oncology 0 Biology 0 Physics
=:
80
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Fig. 3. Percentage of hypoxic cells in rat rhabdomyosarcoma tumors as a function of time after a conditioning dose of 15 Gy. Error bars represent the standard error of the mean hypoxic fraction measured from the 3 dose points.
DISCUSSION The rate of reoxygenation following irradiation has been measured for several rodent tumor models (12-Z 1e 37. 40, 41). Most of the tumors except osteosarcoma have been shown to reoxygenate rapidly. with their hypoxic fraction returning to the preirradiation values within the first 6- 12 hrs. More recent and extensive studies of Rofstad (30) with human melanoma tumors grown as xenografts in nude mice have also indicated rapid and extensive reoxygenation. with the hypoxic fraction returning to the preirradiation value within 12 to 24 hr. These results are in agreement with the present study that indicated rapid and complete reoxygenation of rhabdomyosarcoma tumors within 6 hr after irradiation. Although the mechanism(s) of reoxygenation are not
March 199 I, Volume 20. Number ?
well understood, the rapid rate of reoxygenation observed in the present study is compatible with the assumption that in the rat rhabdomyosarcoma tumors it could be caused by a reduced rate of oxygen use by radiation-damaged (“doomed”) cells and/or resumption of normal blood flow in transiently non-functioning capillaries (30). Although we have no direct evidence that either mechanism is operative, recent studies have demonstrated a rapid increase in the blood flow rate following irradiation. Using the radiolabeled microsphere (RM) and paramagnetic assisted magnetic resonance imaging (PA-MRI) techniques, Ngo c~ful. (28) have measured tumor blood flow in irradiated rhabdomyosarcoma tumors. Preliminary data indicate that following local irradiation with lo- I5 Gy ‘j’Cs gamma rays, a significant increase in the overall tumor blood flow occurs within 30 min postirradiation. The rapid rate of reoxygenation observed in the present study contradicts the assumption that reoxygenation is caused by increased capillary density caused by tumor shrinkage (2. 17). which is manifested several days after irradiation. The data presented here also do not support the argument that a IO-fold decrease in the clonogenic cell survival at 3-4 days postirradiation observed in our tumor repopulation studies with 20 Gy of X rays (35) could be caused by the “rescue” of the hypoxic cells at O-2 days postirradiation. Given the fact that the decrease in the clonogenic cell survival occurred at 3-4 days postirradiation, it is unlikely to be caused by the “rescue” of hypoxic cells since they become reoxygenated within the first 6 hr after irradiation. Our recent studies (35) have attributed the decrease in cell survival at 3-4 days postirradiation to the influx of host diploid cells that are cytotoxic to the irradiated tumor cells. The present study supports our earlier research (34) on the combined effects of the radiosensitizer, misonidazole. and fractionated doses of low- and high-LET radiation. When misonidazole and X-irradiation were given in four daily fractions, the drug enhancement ratio decreased significantly. Based on the present study. this effect would be expected because reoxygenation of tumor cells is complete well within the 24-hr fractionation interval.
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