IN. J. Rudiufion Oncology Biol. Phys., Vol. 5, p. 1559-1563 p‘ Pergamon Press Ltd., 1979. Printed in the U. l .A.

0 Nitrosourea

COMBINATION THERAPY OF A SOLID MURINE TUMOR WITH 1, 3 BIS (2-CHLOROETHYL)-l-NITROSOUREA AND IRRADIATION-t

ADRIAN C. BEGG, Ph.D., KAREN K. Fu, M.D., JOANN VENNARI and THEODORE L. PHILLIPS, M.D.

Department of Radiation Oncology, University of California, San Francisco, CA 94143, U.S.A.

The EMT6/SF tumor growing subcutaneously in mice was used to study the combined effects of BCNU and radiation, using both growth delay and in vitro assays. BCNU (15 mg/kg) appeared to potent&e the effect of X-rays (800 rad) when assayed by growth delay, the largest effect occurring when drug was given 1 to 8 hours after irradiation. The regrowth rates of tumors after combined treatment were slower than untreated tumors, but not stower than the X-ray control, and therefore any attempt to correct for the different regrowth rates did not alter the conch&on that potentiation between drug and radiation occurred. Of the 9 drugs studied in our laboratory, BCNU caused the largest amount of cell killing for a given growth delay when assayed in vitro. Consequently a lower drug dose of 7.5 mg/kg, giving a surviving fraction of lO%, was used with the in vitn, assay. Cyclic variation in cell survival were seen as the interval between drug and radiation was varied between -48 and +48 hours. Maximum WBing occurred when drug was given 1 hr after X-rays but was only slightly greater than expected from additivity. Tumor control (TCD,,,) experiments on small tumors were also carried out to investigate the effti of high drug and radiition doses.

BCNU plus irradiation, Solid murine tumor, In vitro assay, Cyclophosphamide.

INTRODUCTION I,3 bis (Zchloroethyl)-1-nitrosourea (BCNU), an al- kylating agent, is the most widely used drug in the treatment of human brain tumors6*12 and also has significant activity against other human solid tumors, such as colorectal cancer and me1anoma.3 In combi- nation with X-rays, BCNU has been shown to re- duce the shoulder of the radiation dose response curve of brain tumor cells in culture” and also of EMT6/SF tumor cells treated in viva and assayed in vitro.1° The present work extends the latter study by investigating the effects of dose of each agent and time between treatments on the response of the EMT6/SF tumor assayed either in vitro or in situ by growth delay or tumor control.

METHODS AND MATERIALS The EMT6/SF mammary carcinoma grown sub-

cutaneously in the flank or leg of male BALB/c mice were used throughout. The tumor was maintained by passaging alternately in vivo and in vitro. Tumors for both maintenance and experiments were produced

by subcutaneous inoculation of between lo5 and lo6 cells in 0.05 mls.

For the growth delay experiments, tumors were inoculated in the left hind leg and treated when they reached 6-8 mm mean diameter. The three perpen- dicular diameters were measured with vernier calip- ers at intervals of 2 or 3 days beginning on the first day of treatment until the tumors exceeded 4 times the treatment volume (1.59 times the treatment di- ameter), the size chosen as the endpoint at which to calculate the growth delay. The growth delay is the difference in time to reach the endpoint size between the control and treated groups.

Tumor control experiments were carried out by treating subcutaneous leg tumors with X-rays and/or drug when the tumors reached approximately 2 mm mean diameter. The small tumor size was chosen because preliminary experiments with larger tumor (6-8 mm) showed extensive death of animals from lung metastases before the endpoint time, a problem avoided by using small tumors. Mice were observed twice per week following treatment and a recurrence

tSuppotted by the National Cancer Institute Research Grant CA-20529.

Acknowledgements-The drugs utilized in this experi-

ment were supplied by the National Cancer Institute, Di- vision of Cancer Treatment, Drug Development Branch.

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1560 Radiation Oncology 0 Biology 0 Physics September 1979, Volume 5, Number 9

was recorded only if a tumor increased in size on three successive measurement days.

To investigate the cause of slower tumor growth after treatment, retransplant experiments were car- ried out in which cells from treated tumors in the regrowth phase (usually around 10 mm) were im- planted into untreated recipient mice, and also where cells from untreated tumors were implanted into pre- treated mice. In all cases l(r tumor cells (treated or untreated) were implanted subcutaneously in ‘the left hind leg. Pretreatment was either with X-rays to the left hind leg only and/or BCNU given IP. The pre- treatment was always given 24 hours before the tumor cell implant. The subsequent growth rate of tumors was measured by using vernier caliper meas- urements of tumor diameters at regular intervals.

The in vitro assay experiments were carried out as previously described, and involved excising tumors after treatment in viva, preparing cell suspensions by mechanical chopping, incubation with 0.01% trypsin for 15 minutes, filtering through nylon mesh, cen- trifuging, and resuspending for cell counting. Known numbers of cells were plated in 25 cm* flasks and incubated at 37°C for 11 days to allow for clonal growth. The plating efficiency (number of clones/ number of cells plated) was calculated for each treatment group and the surviving fraction calculated by dividing the plating efficiency of the treated group by that of the control. Tumors for in vitro assay experiments were grown in the flank for comparison with previous experiments. Preliminary growth delay experiments showed that flank and leg tumors had similar growth rates and drug responses.

Mice were irradiated unanesthetized in lead boxes from which the left hind leg containing the tumor protruded.4 Irradiations were carried out using a Westinghouse Quadrocondex X-ray machine operat- ing at 230 kVp and 15 mA, employing 0.5 mm Cu + 1 mm Al filtration giving a HVL of 1.8 mm Cu. The mice were positioned 40 cm from the target where the dose rate was 195 t-ad per minute. BCNU was dissolved in absolute ethanol to a concentration of 20 mg/ml, with further dilutions made in saline to give an injection volume of 0.01 mls/g, given IP.

Cyclophosphamide (CY) was diluted in distilled water and injected IP at a concentration of 0.01 ml/g. Misonidazole (Ro-07-0582) was dissolved in saline and injected IP at a concentration of 0.03 ml/g.

RESULTS In vitro assay experiments. In three separate in

vitro assay experiments employing 7.5 mg/kg BCNU and 800 rad X-rays (Figure 1), only one showed cell killing after combined treatment which was in excess of that predicted from independent action of the two

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FIG. 1. In vitro assay experiments investigating the effects of sequence of time between treatments of 7.5 mg/kg BCNU and 800 rad X-rays. Each panel represents a sepa- rate experiment. In A and B, each point is data from 4 tumors combined into 2 pairs for plating; the errors repre- sent the range of surviving fractions of the pair. In C, each point is data from 8 tumors pooled before plating. The triangles and squares are the drug and radiation control data, respectively. The broken lines are the additive re- sponses calculated by multiplying the surviving fractions of the appropriate drug and X-ray controls. In D, the curves

from panels A-C are plotted together for comparison.

agents (Figure lc). Cyclic fluctuations in survival as a function of time between treatments were seen in all three experiments, with survivals at the best and worst intervals differing by a factor of 10 or greater. Plotting all three experiments together (Figure Id) demonstrates the variation between the different ex- perimental tumor batches, but also indicates good and bad treatment intervals, e.g., 48 hours between treatments with drug given either before or after X-rays was generally bad, while giving BCNU simul- taneously or 1 hour after X-rays was generally bet- ter.

Growth delay experiments. The two growth delay experiments (Figure 2), like the in vitro assay exper- iments, differ from one another in the pattern of response as a function of time between treatments. Both experiments, however, show potentiation (greater than additive delay) at some time intervals, and, in general, greater delays were seen when drug was given after X-rays. (There was excessive loss of tumors due to ulceration from the group where BCNU was given 8 hours before X-rays shown in Figure 3a. The data had to be extrapolated and are therefore unreliable at this point.)

Retransplant experiments. To test for residual damage in cells treated with BCNU, cells from tumors regrowing after 15 mg/kg BCNU treatment were implanted into normal mice (Figure 3a). There

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FIG. 2. Two growth delay experiments investigating the effects of sequence and time between 15 mgkg BCNU and 800 rad X-rays. There were 8 mice in each treatment group. Errors are +l SEM. The point for BCNU given 8 hours before X-rays in panel A was extrapolated (see text). The additive response was obtained by adding the delays of the drug and X-ray controls, and the error on the additive delay by calculating the root mean square of

the drug and X-ray control errors.

was a significant difference not dnly in the time of appearance but also in the growth rate of macro- scopic tumors of BCNU-treated cells compared with the controls. The doubling times from 8 to 10.1 mm for BCNU-treated and control tumors were 3.5 and 2.5 days, respectively.

To test for a host effect in slowing tumor growth cells, untreated tumors were implanted into mice which had been pretreated with either 15 mg/kg BCNU, or 800 rad X-ray, or both (Figure 3b). BCNU alone had a slight effect in slowing tumor growth while X-rays alone had a large effect. Adding BCNU to the X-ray lessened, if anything, the damag- ing effect of the pretreatment compared with X-ray alone. This is consistent with data from the experi-

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FIG. 3. Retransplant experiments. Growth curves of EMT/SF leg tumors. A, Cells from tumors either left un- treated or given 15 mgkg BCNU implanted into untreated recipients. B, Untreated tumor cells implanted into mice pretreated 24 hours earlier with the treatment indicated against each curve. Doses given were 15 mgkg BCNU,

800 rad X-rays, or 15 mgkg given 4 hours after 800 rad.

ments shown in Figure 2, where tumors after com- bined treatment were found to regrow slightly faster than the X-ray controls.

Tumor control experiments. Figure 4 shows the data from a preliminary experiment attempting to use tumor control as the endpoint to assay the effects of combined drug and X-ray treatment. BCNU and CY doses were chosen to give equivalent amounts of cell killing by the in vitro assay. The TCD,, was not reached in any of the three groups and so a conclu- sion as to which drug was superior could not be made. Of interest, however, is that for approx- imately equal tumor control rates, the X-ray/BCNU treated tumors had all recurred by 27 days after treatment (middle panel) while the X-ray/CY treated tumors had not all recurred until 47 days (right panel).

Two other points relevant to the use of small tumors in tumor control experiments are shown in Figure 5. Figure Sa shows results from an experi- ment with the hypoxic cell sensitizer misonidazole

1562 Radiation Oncology ??Biology 0 Physics September 1979, Volume 5, Number 9

FIG. 4. Tumor control experiments. Percent of tumors controlled as a function of days after treatment plotted. The doses of X-rays used are indicated against each curve. The tumors were subcutaneous in the leg irradiated when 2 mm mean diameter. Drug doses were 24 mgikg BCNU and 160 mgikg CY given 1 hour after the dose of X-rays. There

were 12 mice per group.

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FIG. 5. Growth delay experiments on 2 mm leg tumors. A, Effect of 0.67 mg/g misonidazole given 45 minutes before 1600 t-ad X-rays. B, Comparison of drug response in small and large tumors. Drug doses were 24 mg/kg BCNU and

160 mgkg CY. Errors are 2 1 SEM, 6-8 mice per group.

where the substantially increased response when misonidazole was given 45 minutes before X-rays indicated that 2 mm tumors contain a substantial proportion of hypoxic cells. Figure 5b shows results of treating small (2 mm) and large (7 mm) tumors with either 24 mg/kg BCNU or 160 mg/kg CY, equaling cell killing doses when assayed in vitro. The small

tumors were considerably more sensitive than large tumors to CY (14.9 versus 6.4 days delay, respec- tively) whereas the response to long delay induced by CY confirms previous results’ and is consistent with the slower recurrence time seen in Figure 4.

DISCUSSION Potentiation of damage as assayed in vitro was

only seen in one of three experiments, although potentiation was seen in both growth delay experi- ments. However, the BCNU dose chosen for the in vitro assay was half that used in the growth delay experiments. The reason for the choice of different doses was that there is too much cell killing for the amount of growth delay caused. If 15 mg/kg had been used in the in vitro assay, the lower limit of the assay’s response range may have been crossed. Conversely, 7.5 mg/kg would have produced very little growth delay. It is therefore unwise to conclude from the present experiments that the amount of potentiation seen, if any, was dose dependent, since a different assay was necessarily used with the two different doses.

In contrast to data with CY”, the potentiation ob- served in the growth delay experiments could not be explained by slower regrowth of tumors after com- bined treatment. In fact, tumors treated with BCNU plus X-rays regrew, on average, approximately 20% faster than the X-ray controls. This is consistent with the retransplant experiments showing faster growth in mice treated with both agents compared with X-ray pretreatment alone. The retransplant ex- periments also demonstrate that the major cause of slower growth after BCNU treatment is residual damage to tumor cells and not a host effect. Slower tumor growth after treatment is one of the main reasons why growth delay and in vitro assays would not necessarily be consistent, and retransplant exper- iments are of value in delineating the causes of the slower growth.

In the tumor control experiments, BCNU and CY were compared to test the hypothesis that if the difference in growth delay for the same amount of cell killing was purely due to CY survivors regrow- ing more slowly than BCNU survivors,8 then results from tumor control experiments should agree with cell survival measurements since regrowth rate is not a factor in either. Unfortunately, high enough doses of X-rays were not chosen in these preliminary ex- periments to answer the question. The initial exper- iments do reveal, however, that the endpoint time for the TCD,,, assay, if chosen from either the ir- radiation or irradiation plus BCNU data, may be too short when using a drug such as CY where regrowth is evidently slowed substantially. The choice should

be made of a long enough endpoint time to include an adequate safety margin for such drugs.

In addition, it is comforting from an experimental viewpoint that small tumors have a significant pro- portion of hypoxic cells (Figure 5a) partially validat- ing the use of such small tumors, i.e., from the standpoint of drugs having to diffuse to hypoxic areas, small tumors should be no more easy or dif- ficult to cure than large tumors, and mechanisms such as reoxygenation after drug treatment can be investigated. However, 2 mm tumors are growing approximately twice as fast as 7 mm tumors, which may significantly affect drug sensitivity, particularly for cell cycle specific drugs. Cyclophosphamide is not cell cycle specific but does appear to kill cycling cells more efficiently,’ probably largely explaining the dependence of growth delay on size for the

Combined effects of BCNU and X-rays 0 A. C. BEGG CI d. 1.563

EMT6/SF tumor. The response to BCNU, however, does not appear so size dependent, maybe because it kills noncyclic cells at least as efficiently as cycling cells.5.” Because of the kinetic differences between small and large tumors, any results of combination treatments with small tumors may not necessarily be applicable to large tumors, despite both having an apparently similar hypoxic cell problem.

In summary, potentiation was seen between 15 mg/kg BCNU and 800 tad X-rays when assayed by growth delay, whereas little potentiation was seen with the lower BCNU dose of 7.5 mg/kg when as- sayed in vitro. Further experiments are required to establish the dose dependence of potentiation and to clarify by tumor control experiments the discrepancy between assays when comparing CY with BCNU.

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Single agent chemotherapy of a solid murine tumor; comparison of growth delay and cell survival assays. Cancer Res. in press.

2. Begg, A. C., Fu, K. K., Shrieve, D. C., Phillips, T. L.: Combination therapy of a solid murine tumor with cyclophosphamide and radiation: The effects of time, dose, and assay method. Int. J. Radiat. One. Biol. Phys. 5: 1559-1563, 1979.

3. Carter, S. K., Bakowski, M. T., Hellman,K.: Chemotherapy of Cancer. New York: John Wiley Sons, Inc., 1977.

4. Douglas, B. G., Fowler, J. F.: The effect of small doses of X-rays on skin reactions in the mouse and a basic interpretation. Radiat. Res. 66: 401-426, 1976.

5. Hahn, G. M., Gordon, L. F., Kujian, S. P.: Re- sponses of cycling and noncycling cells to I-3-bis (2- chloroethyl)-1-nitrosourea and to bleomycin. Cancer Res. 34: 2373-2377, 1974.

6. Levin, V. A.: A pharmacologic basis for brain tumor chemotherapy. Sem. in. Oncol. 2: 57-61, 1975.

7. Steel, G. G., Adams, K.: Stem cell survival and tumor control in the Lewis lung carcinoma. Cancer Res. 35:

1530-1535, 1975. 8. Stephens, T. C., Peacock, J. H.: Tumour volume re-

sponse initial cell kill and cellular repopulation in B16 melanoma treated with cyclophosphamide and l- (2-chlorotheyl)-3-cyclohescyl-I-nitrosourea. Br. J. Cancer 35: 208-217, 1977.

9. Twentyman, P. R., Bleehen, N. M.: The sensitivity to cytotoxic agents of the EMT6 tumour in viva. Com- parative response of lung nodules in rapid exponential growth and of the solid flank tumour. Br. J. Cancer 33: 320-328, 1976.

10. Wharam, M. D., Phillips, T. L., Kane, L. J., Utley, J. F.: Response of a murine solid tumor to in viva com- bined chemotherapy and irradiation. Radiology 109: 451-455, 1973.

11. Wheeler, K. T., Deen, D. F., Wilson, C. B., Williams, M. E., Sheppard, S.: Modification of the in vitro radi- ation response of rat 9L brain tumor cells by BCNU. Int. J. Radiat. Oncol. Biol. Phys. 2: 7!UV3, 1977.

12. Wilson, C. B., Boldrey, E. B., Enot, K. J.: 1,3-bis (2-chloroethyl)-I-nitrosourea (NSC-409962) in the treatment of brain tumors. Cancer Chemother. Rep. 54: 273-281, 1970.