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Temporal interactions in the Lewis lung tumour between cytotoxic drugs and acute or fractionated radiotherapy
Radiotherapy and Oncology, 5 (1986) 137-146 Elsevier
137
RTO 00177
Temporal interactions in the Lewis lung tumour between cytotoxic drugs and acute or fractionated radiotherapy T. C. Stephens, K. A da m s , J. H. P e a c o c k and G. G. Steel Radiotherapy Research Unit, Institute of Cancer Research, Clifton Ave., Sutton, Surrey SM2 5PX, U.K.
(Received 11 February 1985,revision received24 June 1985, accepted 31 July 1985)
Key words: Cyclophosphamide;c/s-Platinum; MeCCNU; Radiation; Fractionation; Interactions
Summary The suggestion was examined, that acute irradiation might be combined with some cytotoxic drugs to produce a supra-additive antitumour effect by timing the second treatment to coincide with the period of repopulation by tumour cells that survived the first treatment. The possible interaction of fractionated irradiation with drugs was also studied. Lewis lung tumours were treated in vivo with acute radiation (20 Gy) and 1-(2chloroethyl)-3-(4-methyl cyclohexyl)-1-nitrosourea (MeCCNU, 10 mg 9 kg-2), cyclophosphamide (CY, 120 mg 9 kg-2) or cis-dichlorodiammine platinum (cis-Pt, 10 mg 9 kg-1) with time intervals ranging from simultaneous to 9 days in either sequence. Tumour response was measured by regrowth delay and combination responses were evaluated relative to calculated "additivity envelopes". With CY and cis-Pt, tumour volume response was approximately additive, whether the drugs were administered simultaneously, or up to 7 days before or after radiation. However, with simultaneous administration, MeCCNU and radiation were supra-additive in terms of volume response, although only additive using a clonogenic cell survival assay. A transplantation experiment suggested that the supra-additive volume response may be due to retarded growth rate in regrowing tumours. In the fractionation studies, drugs were given either at the beginning or at the end of a regime of 5 daily 6 Gy doses. With CY and MeCCNU the drugs were more effective when given with the first radiation dose, but with cis-Pt either regime was equally effective. There was no evidence that repopulation could be exploited to improve therapeutic effect with any of the combination treatments used in this study.
Introduction Combinations of cytotoxic drugs with radiotherapy have been examined in a variety of animal and human tumours in the hope of improving thera-
peutic ratios. One of the ways in which this might be achieved is by exploiting time-dependent synergistic interactions between radiation and drugs
0167-8140/86/$03.50 9 1986ElsevierSciencePublishers B.V. (BiomedicalDivision)
138 against tumours, in so far as they do not occur in limiting normal tissues [24]. Many workers have approached this problem first by attempting to demonstrate that certain combinations of drugs and radiation exhibit time-dependent fluctuations in effectiveness against experimental tumours [29,35]. In such studies, the time scales over which interactions have been sought have most often ranged between simultaneous treatment and separation of treatments by 1 or 2 days, with the radiation administered as an acute treatment, although a few longer term studies have been reported [11,23]. Drugs such as CY, eis-Pt and nitrosoureas (MeCCNU, C C N U and BCNU) have been extensively examined [1,3,4,11,12,23,31,32], but in most cases there has been little evidence of significant synergistic interactions with radiation. In this paper we have re-examined these agents in combination with acute irradiation of the Lewis lung tumour, covering periods up to 9 days between treatments. In addition to simultaneous administration, we have timed our second treatment to coincide with the beginning, the middle and the end of repopulation by tumour cells surviving the first treatment, on the premise that rapidly repopulating tumour cells may have an altered sensitivity to insult by a second agent. The idea of attempting to use cell cycle specific drugs to exploit the rapid cell proliferation that may occur during repopulation of tumours, has been alluded to by several workers [6,12,13,16,33,34], along with the suggestion that tumours that shrink after chemotherapy, might become more radiosensitive due to reoxygenation [19]. The drugs used in this study are not thought of as classical cell cycle phase specific agents, and such agents were not chosen since they are generally ineffective against the Lewis lung tumour. Although CY kills non-cycling cells, it may kill cycling cells more efficiently [34], the nitrosoureas appear equally effective against non-cycling and cycling cells [34], while cis-Pt kills at all phases of the cell cycle but preferentially in G1 phase [18], and has radiosensitising properties [7]. We have also begun to examine the clinically more interesting combination of drugs given at the
beginning or the end of a course of fractionated radiotherapy.
Methods
Lewis lung (LL) tumours were routinely maintained by intra-muscular (i.m.) transplantation of tumour brei (containing about 106 viable cells per implant) into the gastrocnemius muscles of 25 to 30 g male C57B1/cbi mice supplied by the Institute of Cancer Research breeding centre. In all experiments mice had only one tumour sited in the left hind leg. Groups of mice were used for experiments when their tumours reached a mean weight of 0.2 g ( + 0.05 g). Tumour sizes were evaluated by passing tumour bearing legs through a series of calibrated holes in a perspex disc. This measure had previously been related to tumour weight by a calibration curve technique [21]. In most experiments a growth delay assay was used to assess the antitumour effects of single and combination treatments. Each treatment group consisted of 8 mice. Treated and control tumours were measured regularly until they exceeded 4 x the pretreatment volume and volume response was expressed as median /'4 • (i.e. time to reach 4 x pretreatment volume). Error bars on the T4 • values represent 25 and 75 percentiles. Growth delay (GD) may be calculated as: median T4 • for treated group - median T4 • for untreated control group. Some experiments were also performed using an excision cell survival assay. Tumours were disaggregated using trypsin and the cells plated into soft-agar for colony formation as described previously [5,26]. Of relevance to the transplantation experiments described below is that fact that the Lewis lung tumour has very little immunogenicity in our strain of C57B1 mice [21]. The TDs0 (cell dose to give 50% takes) for intramuscular implantation was below 3 cells in untreated mice and only 10 to 30 time higher in pre-immunised recipients. CY was obtained in 100 mg vials from Farmitalia Carlo Erba Ltd, Barnet, Hertfordshire, England, and dissolved in 0.15 M NaC1 for intraperitoneal
139 (i.p.) injection. It was used within 1 h of preparation [27]. M e C C N U was obtained from the National Cancer Institute, Bethesda, M D , USA. A stock solution at 20 mg 9 m l - 1 in D M S O was prepared and stored in 0.5 ml aliquots at - 2 0 ~ F o r i.p. injection, M e C C N U at 1 mg 9m l - 1 was prepared by diluting an aliquot of frozen stock 1 in 20 with 5% Tween 80 in PBSA. It was used immediatly [25]. cis~Pt was obtained from Johnson-Mathe Chemicals Ltd, London. It was dissolved at a concentration o f 1 m g 9 ml -~ in PBSA for i.p. injection [15]. 6~ radiation was administered locally to t u m o u r bearing legs of non-anaesthetised mice using a jig and shield arrangement described previously [26]. Dosimetry was performed using a Baldwin-Farmer substandard dosimeter.
Results
Single-treatment response curves Volume response versus dose curves for the single drugs and acute irradiation, are shown in Fig. 1. The tumours were much more sensitive to M e C C N U and CY, c o m p a r e d to cis-Pt. G r o w t h delays approaching 20 days could be achieved near the L D 10 (dose to kill 10% of mice) of C Y (300 mg
CY DOSE(mg.kg"I )
MeCCNU [X3s (mg.kg-1) IO
3o
i'
'
20
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DAYS AFTER MeCCNU O
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Fig. 2. Regrowth curves for the combination of MeCCNU (10 rag. kg- 1) with radiation (20 Gy). In the upper panel MeCCNU was administered first, while in the lower panel irradiation was the first treatment. The symbols represent controls (Q), and combinations administered either simultaneously (O), or with intervals of 1 day (A), 5 days (D) or 8 days (W). The upper panel also shows the effect of MeCCNU alone (-Q-) and the lower, radiation alone (-Q-).
9 kg -1) or M e C C N U (40 mg 9 kg-1), but only 5 days with c&-Pt (12 mg 9 kg-1). The dose-response curves from Fig. 1 were used to select doses for combination treatments. D r u g doses were chosen which by themselves produced a median 7"4 • of about 10 days. Thus, for radiation 20 Gy was chosen, for M e C C N U 10 mg 9 kg -1 and for C Y 120 mg - kg l. However, cis-Pt produced unacceptable mortality at 12 mg 9 k g - x (when the T4 x was 9 days), so a lower dose of 10 m g - k g - 1 , giving a T4 • of 7 days, was used.
Combinations of acute radiation with drugs I ol
o
,
, 10
,
2{}
,
, 30
RADIATION DOSE (Oy)
,
I
,
CISPLATIN DOSE(mg-kg-I)
Fig. 1. Dose-response curves for growth delay of LL turnouts treated with single doses of MeCCNU (E]), radiation ( ~ ) , CY (O), and cis-Pt (~7). Error bars are the 25th and 75th percentiles of the median T4 •
0
DAYS AFTER IRRADIATION
Fig. 2 shows t u m o u r regrowth curves for the combination of acute radiation with M e C C N U . Each acute-radiation/drug combination was administered in both sequences, drug first or radiation first. In order that turnout sizes were c o m p a r a b l e for each regime, the first treatment was always given at
140 60
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MeCCNU
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20 [
cis-Pt
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IRRADIATION (days)
Fig. 3. Summary plots showing "time-lines" for the interaction of 20 Gy radiation with 10 mg 9 kg-1 M e C C N U , 120 mg 9 k g - 1 CY and 10 mg 9 kg-1 cis-Pt. Full symbols ( I , O , V ) indicate the regrowth responses for combinations and the effect of radiation alone (A) and single drugs ([~, O, V ) are shown for comparison. The shaded areas represent envelopes of additivity for combination treatments (see text for method of calculation), and untreated control 2"4 • for each set of experiments are also shown.
a mean tumour weight of 0.2 g and in the experiment shown in Fig. 2, the alternate treatment was given either simultaneously, 1, 5 or 8 days later. It was clear from this experiment that tumours responded best when irradiation coincided with M e C C N U administration, and that there was progressively less effect as the treatments were separated by 1 to 5 days. This result is summarised in Fig. 3, as a "time-line" plot. In order to assess the significance of the combination responses we have in this study, calculated "envelopes o f additivity" (shown as shaded regions in Fig. 3) which include all those responses that could conceivably result from addition of the single agent responses. We have discussed elsewhere the dubious significance of a simple expected "additive" response calculated as the sum of the single agent responses [24], in situations where (as here) the individual dose-response curves are non-linear. The use of an "additivity envelope" circumvents this problem to some extent by taking into account the non-linearity of response curves. The precise methods of calculating the limits of the "envelope
of additivity", that are known as Mode I (where the addition is performed by taking increments in dose of both agents starting from zero) and Mode II (where the increment in dose of the first agent is taken from zero, but the increment in dose of the second agent is taken from the steepest part of its response curve), have been discussed in detail by Steel and Peckham [20,24]. In performing Mode II calculations we have also taken into account the sequence of treatment [14]. It can be seen that the combination was supraadditive when M e C C N U was given simultaneously with radiation, but at other time intervals the combination was within the envelope of additivity. Also summarised in Fig. 3 are the results with CY and cis-Pt (note difference in 2"4 • scale compared to MeCCNU). For these agents combinations were generally close to additive, with no marked supra-additive effect similar to that seen with radiation plus M e C C N U . However, CY given 9 days before radiation appeared least effective, and marginally better responses were seen when cis-Pt was administered after radiation than before.
141 5-0
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Fig. 4. Regrowth curves for the combination o f CY (120 mg 9 kg- 1), with a course o f fractionated radiotherapy comprising 5 daily doses o f 6 Gy. The drug was given with the first (V), or the last radiation dose (Tq) and drug alone (O), radiation alone (A) and untreated controls ( 0 ) are also shown.
~'
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RADIATION
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Fig. 5. Summary plot showing the effects o f combining 10 mg k g - 1 M e C C N U , 120 mg 9 k g - 1 CY or I0 mg 9 k g - 1 cis-Pt with the first or last doses of a fractionated radiotherapy protocol o f 5 x 6 Gy daily. Full symbols indicate the response to drugs plus radiation ( I I , O , V ) ; open symbols indicate the response to fractionated radiotherapy alone (A) and to the single drugs (Fq,O,V) given at the time when tumours would normally receive the first radiation dose. The simple (Mode I) additive combination response is also shown (---). The error bars indicate the 25th and 75th precentiles of treated groups and the shaded regions indicate the variation in :/4 • of untreated controls.
peared equally effective (and approximately additive) with either regime tested.
Combinations of fractionated radiation with drugs The MeCCNU~radiation interaction Cytotoxic drugs were given either with the first or the last dose of a fractionated radiotherapy regime consisting of 5 daily doses, each of 6 Gy. Fig. 4 shows the tumour regrowth curves obtained for the combination of CY with fractionated radiotherapy. A greater tumour volume response was obtained when CY was administered with the first dose of radiation than with the last. This is shown in Fig. 5, which also summarizes the combination results obtained with MeCCNU and eis-Pt. Because full dose response curves for fractionated irradiation were not available, only Mode I additive is shown in this Fig. 5. The combination of MeCCNU with fractionated radiotherapy was significantly better when the drug was given with the first fraction of radiotherapy than with the last, while the cis-Pt combination ap-
Two additional types of experiment were performed in order to explore more fully the interaction between MeCCNU and simultaneous singledose irradiation. We looked for synergistic tumour cell killing by the combination and for retardation in the growth rate of tumour cells surviving the simultaneous combination. LL tumours were treated in vivo with MeCCNU either alone, or in simultaneous combination with radiation. The tumours were excised 24 h later, cell suspensions prepared, and tumour cell survival measured by colony formation in soft-agar. Due to the limitations of the cell survival assay, it was necessary to use lower drug and radiation doses in this experiment compared to Fig. 3, thus regrowth delay was also measured for both the simultaneous
142 TABLE I
combination
and combinations
with a 3 day inter-
Cell survival response of LL tumours treated with MeCCNU and radiation alone or in combination.
val between
treatments.
only 4 mg
MeCCNU
and
With
10 G y
radiation
9 kg-1
a substantially
g r e a t e r g r o w t h d e l a y w a s still o b s e r v e d f o r s i m u l TreatmenP
S.F. per tumour b
taneous
treatments
with a 3 day Expt 1. 4 mg 9 kg -1 MeCCNU alone 2.3 x 10 -3 10 Gy radiation alone 3.2 x 10 -2 MeCCNU plus radiation 1.2 • 10 -~ Predicted additive (Mode I) 7.1 • 10 -5
Expt 2. 3.6 4.2 3.8 1.5
x x • x
10 -a 10 -2 10 -4 10 -4
a Mice bearing i.m. LL tumours were treated simultaneously with MeCCNU and radiation, and an excision cell survival assay was performed 24 h later. The control plating efficiency (PE) was 60%. b S.F. per tumour was calculated as: number of colony-forming tumour cells per treated tumour number of colony-forming tumour cells per control tumour"
(/'4•
=
9.2 d a y s ) c o m p a r e d
interval between
treatments
(with
e i t h e r s e q u e n c e , T4 • = 4.9 d a y s ) . T h e cell s u r v i v a l results for simultaneous treatment, obtained in two separate though
experiments,
are shown
in Table
I. A l -
the growth delay data suggest that simul-
t a n e o u s t r e a t m e n t w a s s u p r a - a d d i t i v e , t h e cell s u r vival response was not apparently greater than additive. Unfortunately, be performed,
only Mode I addition could
s i n c e cell s u r v i v a l w i t h s i n g l e a g e n t s
could not be measured
at high enough doses, but
because the radiation
s u r v i v a l c u r v e is b i - p h a s i c ,
Mode
II addition
would
be expected
to predict
h i g h e r s u r v i v a l t h a n M o d e I. T h u s , s u p r a - a d d i t i v e cell k i l l i n g d o e s n o t a p p e a r
to explain the supra-
additive regrowth delay results. However,
we can-
TABLE II Transplantation of untreated, or drug and radiation treated LL ceils into pretreated or non-pretreated recipient mice. Cells a
Recipients b
Time to 0.5 g (days)~
Td (days) r
Untreated Untreated Untreated
untreated radiation d MeCCNU ~
10.8 (10.2-11.2) 11.0 (10.8-11.8) 9.0 ( 8.9- 9.9)
1.6 (1.2-1.8) 1.9 (1.3-2.0) 2.0 (1.2-2.6)
Radiation Radiation Radiation
untreated radiation MeCCNU
12.8 (12.2-13.2) 13.8 (12.9-14.2) 12.4 (I 1.9-12.8)
1.8 (l.l-1.9) 1.7 (1.1-2.3) 1.7 (1.2-1.8)
MeCCNU MeCCNU MeCCNU
untreated radiation MeCCNU
14.1 (13.3-14.5) 16.9 (16.4-18.3) 13.0 (12.8-14.0)
1.7 (1.1-3.6) 2.7 (2.0-2.9) 1.6 (1.3-2.3)
Rad + MeCCNU f Rad + MeCCNU Rad + MeCCNU
untreated radiation MeCCNU
29.8 (26.0-48.9) 45.0 (40.0-49.0) 31.3 (28.6-33.6)
2.5 (2.2-2.7) 4.8 (3.5-6.8) 2.0 (1.9-3.1)
a Tumours were treated in vivo with MeCCNU and/or radiation and disaggregated 24 h later. Recipients received subcutaneous implants of 5 • 104 tumour cells. Recipients were treated with MeCCNU or radiation 6 h before tumour cells were implanted. c Values are median, 25th and 75th percentiles. d Radiation dose = 10 Gy. ~ MeCCNU dose = 4 mg 9 kg-1. f Radiation-MeCCNU combination given simultaneously.
143 not rule out the possibility of supra-additive cell killing at higher drug and radiation doses. The possibility that the synergistic effect of simultaneous treatment seen with the growth delay end-point might reflect a retardation in the growth rate of cells surviving the combined treatment is suggested by the less steep regrowth curve following simultaneous treatment in Fig. 2. Table II shows the results of a transplantation experiment designed specifically to explore this question. Three batches of mice were prepared: untreated controls, mice whose left hind leg had been locally treated with l0 Gy of gamma-rays and mice which had been injected i.p. with 4 mg 9kg- 1 MeCCNU. 6 h later the batches of mice were sub-divided into four groups (each of 6 mice), and injected i.m. into the left hind leg with 5 x 104 tumour cells that had been prepared from untreated LL tumours or those treated 24 h earlier in three ways: irradiated to 10 Gy, treated with 4 mg 9 kg-1 MeCCNU or treated simultaneously with radiation and MeCCNU. The time for each implant to grow to a target size of 0.5 g and the growth rate (tumour volume doubling time, Td) estimated from the slope of the growth curve of each implant at that size, were measured. Although the same numbers of structurally intact tumour cells receiving the different pretreatments were implanted, it is important to remember that radiation and MeCCNU either alone or in combination killed many tumour cells. Therefore, the time for tumours to grow from each type of pretreated cell implant, regardless of which recipient is used, will depend very much on the extent of cell killing by the pretreatment. Thus, we should only compare the growth time for a given cell treatment group in the three different recipients. However, the tumour growth rate at 0.5 g, for each type of cell in different recipients can be compared, since it should not depend on the number of viable cells initially implanted. The results (Table II) show clearly that tumour cells that had been pretreated with MeCCNU alone or with MeCCNU plus radiation took longer to attain the target size and grew significantly more slowly (greater Td), if they were implanted i.m. into recipients with irradiated legs, compared to all other combinations.
Discussion
In this paper we have examined the suggestion that ionising radiation may be combined with some cytotoxic drugs (e.g. MeCCNU, CY and cis-Pt) to produce a beneficial antitumour effect by timing the second treatment to coincide with the period of repopulation by tumour cells that survive the first treatment. Single large doses of radiation and a daily five fraction regime were both studied. The experiments were performed using the murine Lewis lung tumour where we have previously described the pattern of repopulation which occurs after single large radiation doses [26], and during a course of fractionated radiotherapy [2]. Sequential excision assays were used to monitor repopulation. Following acute doses of 15 or 25 Gy, repopulation had begun within 2 days of treatment and gradually accelerated to a maximum rate (Td = 1 day) between 5 and 8 days after treatment. The rate of repopulation then slowed down as the surviving fraction approached 1. During fractionated regimens of 10 daily doses of 3.2 to 6.5 Gy or twice daily doses of 2.3 to 4.7 Gy, we were unable to detect significant repopulation, although it began promptly after the last fraction. When the cytotoxic drugs were combined with single large radiation doses and regrowth delay measured, two distinct patterns of response were observed: (i) MeCCNU was clearly much more effective when given simultaneously or within 1 day of irradiation than given between 1 and 9 days before or after irradiation. Using the classification of Steel [15] the simultaneous treatment response was supra-additive, while at other times the response was within the envelope of additivity. (ii) CY and cis-Pt appeared to be marginally more effective when administered between 2 and 6 days before or after irradiation than when given simultaneously with irradiation. However, at all times of drug administration from 7 days before to 7 days after radiation the response was within the additivity envelope. When combined with fractionated irradiation, MeCCNU and CY appeared to be slightly more
144 effective when given simultaneously with the first radiation dose than with the last. The other combinations of these drugs and cis-Pt with radiation, were all close to additive. More detailed studies to explore the mechanism of the apparent M e C C N U interaction with simultaneous acute irradiation suggested that this is not due to enhanced tumour cell killing, since cell survival measured with an excision clonogenic assay was additive. Instead, it would seem that the supra-additive regrowth delay response results from retardation of the growth of tumour cells that had been pre-treated with either M e C C N U or M e C C N U plus radiation, when they attempted to grow in an irradiated site. This phenomenon has been widely described for radiation alone, (where it is known as the " t u m o u r bed effect" [8]). However, it should be stressed that a classical tumour bed effect was not seen; the growth of untreated or irradiated cells was not retarded when they were implanted into irradiated recipients. It is also interesting to note that the growth of C C N U treated B 16 melanoma cells may be retarded if they are implanted into animals that have been pre-treated with CY, although there was no retardation if cells were implanted into C C N U pretreated mice [28]. Additional support for the above mechanism is provided by the fact that simultaneous treatment of tumours with radiation and M e C C N U reduced the regrowth rate by a factor of 2 to 3 (See growth curves in Fig. 2), which compares well with the retardation in growth rate of radiation-MeCCNU treated cells implanted into irradiated recipients (Table II). Further, M e C C N U treated cells in M e C C N U treated mice, and irradiated cells in irradiated mice appeared to have no effect on growth rate in either tumour, or transplantation experiments. Although we set out to determine whether the increased tumour cell proliferation that occurs during repopulation, either after a large acute treatment with radiation or drugs, or during fractionated irradiation, may be an exploitable mechanism to obtain greater tumour response in combined modality treatments, we have found little indication that this is the case with the agents chosen for this
study. We conclude that with CY and cis-Pt the response is essentially the same whether the agents are give together or separated by a few days when repopulation is maximal, thus repopulation seems not to be an important determinant of response. On the other hand, with M e C C N U , the response is substantially better if the treatments are given simultaneously, and any attempt to exploit repopulation is detrimental. In the fractionated radiation studies, the poorer response seen when the drugs were administered with the last dose o f radiation may reflect the development of chemo-resistance due to the increase in size of tumours compared to their size at the start of treatment. Changes in drug and radiation response that are associated with tumour size have been widely reported [9,10,17,19,22]. Alternatively, our previous radiosensitivity studies with the Lewis lung tumour [2] show that during fractionated regimes tumours become progressively more hypoxic even though they shrink slightly in volume during treatment, and this may reduce chemo-responsiveness [9,30]. However, this might not be true for cis-Pt, since it apparently kills oxic and hypoxic cells equally well in vitro [30], and has radiosensitising properties [7]. Our results generally confirm those previous reports in which longer time intervals between drugs and radiation have been examined using the growth delay endpoint. There were no marked supra-additive interactions between either CY or B C N U and radiation in the E M T 6 tumour when treatments were separated by up to 3 days [3,4], and responses to CY-radiation were approximately additive in the hepatoma 3924A, for treatment intervals between - 7 and + 4 days between the two agents [11]. Using a tumour cure endpoint to assess the combination o f CY-radiation in the Lewis lung tumour, Steel et al. [23] found that it was best to administer the agents within a day of one another. A fixed dose of CY was unable to sterilise the increasing number of cells present in tumours as they repopulated after irradiation. If our findings applied to human tumours in situ, then simultaneous administration of M e C C N U and radiation might be good for palliation (i.e. prolonging survival time), but are unlikely to influence
145 survival rates, compared to regimens in which longer intervals are left between the treatments.
Acknowledgements We are grateful for the support of Prof. M. J. Peckham; the work was partially supported by NCI Grant No. RO1 CA 26059.
References 1 Bateman, A. E., Fu, K. K. and Towse, G. D. W. The combination of methyl-CCNU and radiation: Cell survival studies on a human tumour xenograft. Int. J. Rad. Oncol. Biol. Phys. 5: 1545-1548, 1979. 2 Beck-Bornholdt, H-. P., Peacock, J. H. and Stephens, T. C. Kinetics of cellular inactivation by fractionated irradiation in Lewis lung tumour. Int. J. Rad. Oncol. Biol. Phys. 11: 1171-1177, 1985. 3 Begg, A. C., Fu, K. K., Shrieve, D. C. and Phillips, T. L. Combination therapy of a solid murine tumour with cyclophosphamide and radiation. Int. J. Rad. Oncol. Biol. Phys. 5: 1433-1439, 1979. 4 Begg, A. C., Fu, K. K., Vennari, J. and Phillips, T. L. Combination therapy of a solid murine tumour with 1,3-bis (2chloroethyl)-1-nitrosourea and radiation. Int. J. Rad. Oncol. Biol. Phys. 5: 1559-1563, 1979. 5 Courtenay, V. D. A soft agar colony assay for Lewis lung tumour and B16 melanoma taken directly from the mouse. Br. J. Cancer 34: 39-45, 1976. 6 Dethlefsen, L. A. Cellular recovery kinetic studies relevant to combined-modality research and therapy. Int. J. Rad. Oncol. Biol. Phys. 5:1197-1203, 1979. 7 Douple, E. B. and Richmond, R. C. Radiosensitisation of hypoxic tumor cells by cis- and trans-dichlorodiammine platinum (II). Int. J. Rad. Oncol. Biol. Phys. 5: 1369-1372, 1979. 8 Hewitt, H. B. and Blake, E. R. The growth of transplanted tumours in pre-irradiated sites. Br. J. Cancer 22: 808-824, 1968. 9 Hill, R. P. and Stanley, J. A. The response of hypoxic B16 melanoma cells to in vitro treatment with chemotherapeutic agents. Cancer Res. 35: 1147-1153, 1975. 10 Hill, R. P. and Stanley, J. A. Pulmonary metastases of the Lewis lung tumour - Cell kinetics and response to cyclophosphamide at different sizes. Cancer Treatment Rep. 6: 29-36, 1977. 11 Hopkins, H. A., Ritenour, E. R., MacLeod, M. S. and Looney, W. B. Theeffect ofvarying the time between irradiation and cyclophosphamide on growth response of hepatoma 3924A. Int. J. Oncol. Biol. Phys. 5: 1455-1459, 1979.
12 Lelieveld, P. R., Twentyman, P. R., Kallman, R. F. and Brown, J. M. The effect of time between X-irradiation and chemotherapy on the growth of three solid mouse tumours. IV. BCNU. Int. J. Rad. Oncol. Biol. Phys. 5:1549 1552, 1979. 13 Mulder, J. H., Smink, T. and VanPutten, L. M. Schedule dependent effectiveness of CCNU and 5-fluorouracil in experimental chemotherapy. Eur. J. Cancer 13: 1123-1131, 1977. 14 Redpath, J. L. Mechanisms in combination therapy: isobologram analysis and sequencing. Int. J. Rad. Biol. 38: 355 356, 1980. 15 Rose, C. M., Millar, J. L., Peacock, J. H., Phelps, T. A. and Stephens, T. C. Differential enhancement of melphalan cytotoxicity in tumour and normal tissue by misonidazole. In: Radiation Sensitizers: Their Use in the Clinical Management of Cancer. Editor L. W. Brady; Masson Publishers, Paris, 1980. 16 Schenken, L. L. and Hagemann, R . F . Recruitment oncotherapy schedules for enhanced efficacy of cycle active agents. Proc. Am. Assoc. Cancer Res. 17: 88, 1976. 17 Shipley, W. U., Stanley, J. A. and Steel, G. G. Tumour size dependency in the radiation response of the Lewis lung carcinoma. Cancer Res. 35: 2488-2493, 1975. 18 Sigdestad, C. P. and Grdina, D. J. In vivo cell cycle phase-preferential killing of murine fibrosarcoma cells by cisplatin. Cancer Treat. Rep. 65: 845-851, 1981. 19 Stanley, J. A., Shipley, W. U. and Steel, G. G. Influence of tumour size on hypoxic fraction and therapeutic sensitivity of Lewis lung tumour. Br. J. Cancer 36: 105-113, 1977. 20 Steel, G. G. Terminology in the description of drug-radiation interactions. Int. J. Rad. Oncol. Biol. Phys. 5:11451150, 1979. 21 Steel, G. G. and Adams, K. Stem-cell survival and turnout control. Cancer Res. 35: 1530-1535, 1975. 22 Steel, G. G., Adams, K. and Stanley, J. Size dependence of the response of Lewis lung tumour to BCNU. Cancer Treatment Rep. 60:1743 1748, 1976. 23 Steel, G. G., Hill, R. P. and Peckham, M. J. Combined radiotherapy-chemotherapy of Lewis lung carcinoma. Int. J. Rad. Oncol. Biol. Phys. 4: 49-52, 1978. 24 Steel, G. G. and Peckham, M. J. Exploitable mechanisms in combined radiotherapy-chemotherapy: the concept of additivity. Int. J. Rad. Oncol. Biol. Phys. 5: 85-91, 1979. 25 Stephens, T. C., Adams, K. and Peacock, J. H. Identification of a subpopulation of MeCCNU resistant cells in previously untreated Lewis lung tumours. Br. J. Cancer 50: 77-83, 1984. 26 Stephens, T. C., Currie, G. A. and Peacock, J. H. Repopulation of gamma-irradiated Lewis lung carcinoma by malignant cells and host macrophage progenitors. Br. J. Cancer 38: 573-582, 1978. 27 Stephens, T. C. and Peacock, J. H. Tumour volume response, initial cell kill and cellular repopulation in B 16 melanoma treated with cyclophosphamide and l-(2-chloroe-
146
28
29
30
31
thyl)-3-cyclohexyl-l-nitrosourea. Br. J. Cancer 36: 313-321, 1977. Stephens, T. C. and Peacock, J. H. Comparison of growth delay and cell survival as end-points of tumour response following treatment with combinations of cytotoxic agents. Br. J. Cancer 41, Suppl. IV, 288-293, 1980. Stephens, T. C. and Peacock, J. H. and Steel, G. G. Cell survival in B 16 melanoma after treatment with combinations of cytotoxic agents: lack of potentiation. Br. J. Cancer 36: 84-93, 1977. Teicher, B. A., Lazo, J. S. and Sartorelli, A. C. Classification of antineoplastic agents by their selective toxicities toward oxygenated and hypoxic tumour cells. Cancer Res. 41: 7381, 1981. Twentyman, P. R., Kallman, R. F. and Brown, J. M. The effect of time between X-irradiation and chemotherapy on
32
33
34
35
the growth of three solid mouse tumours. III. cis-Diamminedichloroplatinum. Int. J. Rad. Oncol. Biol. Phys. 5: 1365-1367, 1979. Twentyman, P. R., Kallman, R. F. and Brown, J. M. The effect of time between X-irradiation and chemotherapy on the growth of three solid mouse tumours. II. cyclophosphamide. Int. J. Rad. Oncol. Biol. Phys. 5: 1425-1427, 1979. Valeriote, F. and VanPutten, L. M. Proliferation-dependent cytotoxicity of anticancer agents: A review Cancer Res. 35: 2619-2630, 1975. Van Putten, L. M. Are cell kinetic data relevant for the design of tumour chemotherapy schedules? Cell Tissue Kinet. 7: 493-504, 1974. Vietti, T. J., Eggerding, F. and Valeriote, F. Combined effect of X-radiation and 5-Fluorouracil on survival of transplanted leukemic cells. J. Natl. Cancer Inst. 47: 865-870, 1971.
137
RTO 00177
Temporal interactions in the Lewis lung tumour between cytotoxic drugs and acute or fractionated radiotherapy T. C. Stephens, K. A da m s , J. H. P e a c o c k and G. G. Steel Radiotherapy Research Unit, Institute of Cancer Research, Clifton Ave., Sutton, Surrey SM2 5PX, U.K.
(Received 11 February 1985,revision received24 June 1985, accepted 31 July 1985)
Key words: Cyclophosphamide;c/s-Platinum; MeCCNU; Radiation; Fractionation; Interactions
Summary The suggestion was examined, that acute irradiation might be combined with some cytotoxic drugs to produce a supra-additive antitumour effect by timing the second treatment to coincide with the period of repopulation by tumour cells that survived the first treatment. The possible interaction of fractionated irradiation with drugs was also studied. Lewis lung tumours were treated in vivo with acute radiation (20 Gy) and 1-(2chloroethyl)-3-(4-methyl cyclohexyl)-1-nitrosourea (MeCCNU, 10 mg 9 kg-2), cyclophosphamide (CY, 120 mg 9 kg-2) or cis-dichlorodiammine platinum (cis-Pt, 10 mg 9 kg-1) with time intervals ranging from simultaneous to 9 days in either sequence. Tumour response was measured by regrowth delay and combination responses were evaluated relative to calculated "additivity envelopes". With CY and cis-Pt, tumour volume response was approximately additive, whether the drugs were administered simultaneously, or up to 7 days before or after radiation. However, with simultaneous administration, MeCCNU and radiation were supra-additive in terms of volume response, although only additive using a clonogenic cell survival assay. A transplantation experiment suggested that the supra-additive volume response may be due to retarded growth rate in regrowing tumours. In the fractionation studies, drugs were given either at the beginning or at the end of a regime of 5 daily 6 Gy doses. With CY and MeCCNU the drugs were more effective when given with the first radiation dose, but with cis-Pt either regime was equally effective. There was no evidence that repopulation could be exploited to improve therapeutic effect with any of the combination treatments used in this study.
Introduction Combinations of cytotoxic drugs with radiotherapy have been examined in a variety of animal and human tumours in the hope of improving thera-
peutic ratios. One of the ways in which this might be achieved is by exploiting time-dependent synergistic interactions between radiation and drugs
0167-8140/86/$03.50 9 1986ElsevierSciencePublishers B.V. (BiomedicalDivision)
138 against tumours, in so far as they do not occur in limiting normal tissues [24]. Many workers have approached this problem first by attempting to demonstrate that certain combinations of drugs and radiation exhibit time-dependent fluctuations in effectiveness against experimental tumours [29,35]. In such studies, the time scales over which interactions have been sought have most often ranged between simultaneous treatment and separation of treatments by 1 or 2 days, with the radiation administered as an acute treatment, although a few longer term studies have been reported [11,23]. Drugs such as CY, eis-Pt and nitrosoureas (MeCCNU, C C N U and BCNU) have been extensively examined [1,3,4,11,12,23,31,32], but in most cases there has been little evidence of significant synergistic interactions with radiation. In this paper we have re-examined these agents in combination with acute irradiation of the Lewis lung tumour, covering periods up to 9 days between treatments. In addition to simultaneous administration, we have timed our second treatment to coincide with the beginning, the middle and the end of repopulation by tumour cells surviving the first treatment, on the premise that rapidly repopulating tumour cells may have an altered sensitivity to insult by a second agent. The idea of attempting to use cell cycle specific drugs to exploit the rapid cell proliferation that may occur during repopulation of tumours, has been alluded to by several workers [6,12,13,16,33,34], along with the suggestion that tumours that shrink after chemotherapy, might become more radiosensitive due to reoxygenation [19]. The drugs used in this study are not thought of as classical cell cycle phase specific agents, and such agents were not chosen since they are generally ineffective against the Lewis lung tumour. Although CY kills non-cycling cells, it may kill cycling cells more efficiently [34], the nitrosoureas appear equally effective against non-cycling and cycling cells [34], while cis-Pt kills at all phases of the cell cycle but preferentially in G1 phase [18], and has radiosensitising properties [7]. We have also begun to examine the clinically more interesting combination of drugs given at the
beginning or the end of a course of fractionated radiotherapy.
Methods
Lewis lung (LL) tumours were routinely maintained by intra-muscular (i.m.) transplantation of tumour brei (containing about 106 viable cells per implant) into the gastrocnemius muscles of 25 to 30 g male C57B1/cbi mice supplied by the Institute of Cancer Research breeding centre. In all experiments mice had only one tumour sited in the left hind leg. Groups of mice were used for experiments when their tumours reached a mean weight of 0.2 g ( + 0.05 g). Tumour sizes were evaluated by passing tumour bearing legs through a series of calibrated holes in a perspex disc. This measure had previously been related to tumour weight by a calibration curve technique [21]. In most experiments a growth delay assay was used to assess the antitumour effects of single and combination treatments. Each treatment group consisted of 8 mice. Treated and control tumours were measured regularly until they exceeded 4 x the pretreatment volume and volume response was expressed as median /'4 • (i.e. time to reach 4 x pretreatment volume). Error bars on the T4 • values represent 25 and 75 percentiles. Growth delay (GD) may be calculated as: median T4 • for treated group - median T4 • for untreated control group. Some experiments were also performed using an excision cell survival assay. Tumours were disaggregated using trypsin and the cells plated into soft-agar for colony formation as described previously [5,26]. Of relevance to the transplantation experiments described below is that fact that the Lewis lung tumour has very little immunogenicity in our strain of C57B1 mice [21]. The TDs0 (cell dose to give 50% takes) for intramuscular implantation was below 3 cells in untreated mice and only 10 to 30 time higher in pre-immunised recipients. CY was obtained in 100 mg vials from Farmitalia Carlo Erba Ltd, Barnet, Hertfordshire, England, and dissolved in 0.15 M NaC1 for intraperitoneal
139 (i.p.) injection. It was used within 1 h of preparation [27]. M e C C N U was obtained from the National Cancer Institute, Bethesda, M D , USA. A stock solution at 20 mg 9 m l - 1 in D M S O was prepared and stored in 0.5 ml aliquots at - 2 0 ~ F o r i.p. injection, M e C C N U at 1 mg 9m l - 1 was prepared by diluting an aliquot of frozen stock 1 in 20 with 5% Tween 80 in PBSA. It was used immediatly [25]. cis~Pt was obtained from Johnson-Mathe Chemicals Ltd, London. It was dissolved at a concentration o f 1 m g 9 ml -~ in PBSA for i.p. injection [15]. 6~ radiation was administered locally to t u m o u r bearing legs of non-anaesthetised mice using a jig and shield arrangement described previously [26]. Dosimetry was performed using a Baldwin-Farmer substandard dosimeter.
Results
Single-treatment response curves Volume response versus dose curves for the single drugs and acute irradiation, are shown in Fig. 1. The tumours were much more sensitive to M e C C N U and CY, c o m p a r e d to cis-Pt. G r o w t h delays approaching 20 days could be achieved near the L D 10 (dose to kill 10% of mice) of C Y (300 mg
CY DOSE(mg.kg"I )
MeCCNU [X3s (mg.kg-1) IO
3o
i'
'
20
1(30 ,
3C
,
2~
300
-Tit
2,
/t /
DAYS AFTER MeCCNU O
10 i
5.0
20 ,
30 i
!
t
l
i
20
30
i
SO
/10 ,
i
I
,
1"0
0-5 .-/-
t.O
O-1 ~_ 5-0
7"
< 0"5
0.1
I
0
l
10
I
40
Fig. 2. Regrowth curves for the combination of MeCCNU (10 rag. kg- 1) with radiation (20 Gy). In the upper panel MeCCNU was administered first, while in the lower panel irradiation was the first treatment. The symbols represent controls (Q), and combinations administered either simultaneously (O), or with intervals of 1 day (A), 5 days (D) or 8 days (W). The upper panel also shows the effect of MeCCNU alone (-Q-) and the lower, radiation alone (-Q-).
9 kg -1) or M e C C N U (40 mg 9 kg-1), but only 5 days with c&-Pt (12 mg 9 kg-1). The dose-response curves from Fig. 1 were used to select doses for combination treatments. D r u g doses were chosen which by themselves produced a median 7"4 • of about 10 days. Thus, for radiation 20 Gy was chosen, for M e C C N U 10 mg 9 kg -1 and for C Y 120 mg - kg l. However, cis-Pt produced unacceptable mortality at 12 mg 9 k g - x (when the T4 x was 9 days), so a lower dose of 10 m g - k g - 1 , giving a T4 • of 7 days, was used.
Combinations of acute radiation with drugs I ol
o
,
, 10
,
2{}
,
, 30
RADIATION DOSE (Oy)
,
I
,
CISPLATIN DOSE(mg-kg-I)
Fig. 1. Dose-response curves for growth delay of LL turnouts treated with single doses of MeCCNU (E]), radiation ( ~ ) , CY (O), and cis-Pt (~7). Error bars are the 25th and 75th percentiles of the median T4 •
0
DAYS AFTER IRRADIATION
Fig. 2 shows t u m o u r regrowth curves for the combination of acute radiation with M e C C N U . Each acute-radiation/drug combination was administered in both sequences, drug first or radiation first. In order that turnout sizes were c o m p a r a b l e for each regime, the first treatment was always given at
140 60
30 F
MeCCNU
3o-
CY
20 [
cis-Pt
20
iiIiJ
X
z <~ --20 uJ Z
T
10
IC
.
.
0 -I0
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-5
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0
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TIME BEFORE OR AFTER
+5
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.
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.
.
.
.
.
.
J
I
0
+5
1
+10
IRRADIATION (days)
Fig. 3. Summary plots showing "time-lines" for the interaction of 20 Gy radiation with 10 mg 9 kg-1 M e C C N U , 120 mg 9 k g - 1 CY and 10 mg 9 kg-1 cis-Pt. Full symbols ( I , O , V ) indicate the regrowth responses for combinations and the effect of radiation alone (A) and single drugs ([~, O, V ) are shown for comparison. The shaded areas represent envelopes of additivity for combination treatments (see text for method of calculation), and untreated control 2"4 • for each set of experiments are also shown.
a mean tumour weight of 0.2 g and in the experiment shown in Fig. 2, the alternate treatment was given either simultaneously, 1, 5 or 8 days later. It was clear from this experiment that tumours responded best when irradiation coincided with M e C C N U administration, and that there was progressively less effect as the treatments were separated by 1 to 5 days. This result is summarised in Fig. 3, as a "time-line" plot. In order to assess the significance of the combination responses we have in this study, calculated "envelopes o f additivity" (shown as shaded regions in Fig. 3) which include all those responses that could conceivably result from addition of the single agent responses. We have discussed elsewhere the dubious significance of a simple expected "additive" response calculated as the sum of the single agent responses [24], in situations where (as here) the individual dose-response curves are non-linear. The use of an "additivity envelope" circumvents this problem to some extent by taking into account the non-linearity of response curves. The precise methods of calculating the limits of the "envelope
of additivity", that are known as Mode I (where the addition is performed by taking increments in dose of both agents starting from zero) and Mode II (where the increment in dose of the first agent is taken from zero, but the increment in dose of the second agent is taken from the steepest part of its response curve), have been discussed in detail by Steel and Peckham [20,24]. In performing Mode II calculations we have also taken into account the sequence of treatment [14]. It can be seen that the combination was supraadditive when M e C C N U was given simultaneously with radiation, but at other time intervals the combination was within the envelope of additivity. Also summarised in Fig. 3 are the results with CY and cis-Pt (note difference in 2"4 • scale compared to MeCCNU). For these agents combinations were generally close to additive, with no marked supra-additive effect similar to that seen with radiation plus M e C C N U . However, CY given 9 days before radiation appeared least effective, and marginally better responses were seen when cis-Pt was administered after radiation than before.
141 5-0
NeCCNU
cis-Pt
cY
30
A c~ "o
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x2O
ILl
Z
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....
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~o
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S~ 6GyDAILY
0"1
I
0
I
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1
10
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20
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DAYS
Fig. 4. Regrowth curves for the combination o f CY (120 mg 9 kg- 1), with a course o f fractionated radiotherapy comprising 5 daily doses o f 6 Gy. The drug was given with the first (V), or the last radiation dose (Tq) and drug alone (O), radiation alone (A) and untreated controls ( 0 ) are also shown.
~'
4'
r
4'
RADIATION
,~
r
~,
tt
,I,
{5x 6Gy)
Fig. 5. Summary plot showing the effects o f combining 10 mg k g - 1 M e C C N U , 120 mg 9 k g - 1 CY or I0 mg 9 k g - 1 cis-Pt with the first or last doses of a fractionated radiotherapy protocol o f 5 x 6 Gy daily. Full symbols indicate the response to drugs plus radiation ( I I , O , V ) ; open symbols indicate the response to fractionated radiotherapy alone (A) and to the single drugs (Fq,O,V) given at the time when tumours would normally receive the first radiation dose. The simple (Mode I) additive combination response is also shown (---). The error bars indicate the 25th and 75th precentiles of treated groups and the shaded regions indicate the variation in :/4 • of untreated controls.
peared equally effective (and approximately additive) with either regime tested.
Combinations of fractionated radiation with drugs The MeCCNU~radiation interaction Cytotoxic drugs were given either with the first or the last dose of a fractionated radiotherapy regime consisting of 5 daily doses, each of 6 Gy. Fig. 4 shows the tumour regrowth curves obtained for the combination of CY with fractionated radiotherapy. A greater tumour volume response was obtained when CY was administered with the first dose of radiation than with the last. This is shown in Fig. 5, which also summarizes the combination results obtained with MeCCNU and eis-Pt. Because full dose response curves for fractionated irradiation were not available, only Mode I additive is shown in this Fig. 5. The combination of MeCCNU with fractionated radiotherapy was significantly better when the drug was given with the first fraction of radiotherapy than with the last, while the cis-Pt combination ap-
Two additional types of experiment were performed in order to explore more fully the interaction between MeCCNU and simultaneous singledose irradiation. We looked for synergistic tumour cell killing by the combination and for retardation in the growth rate of tumour cells surviving the simultaneous combination. LL tumours were treated in vivo with MeCCNU either alone, or in simultaneous combination with radiation. The tumours were excised 24 h later, cell suspensions prepared, and tumour cell survival measured by colony formation in soft-agar. Due to the limitations of the cell survival assay, it was necessary to use lower drug and radiation doses in this experiment compared to Fig. 3, thus regrowth delay was also measured for both the simultaneous
142 TABLE I
combination
and combinations
with a 3 day inter-
Cell survival response of LL tumours treated with MeCCNU and radiation alone or in combination.
val between
treatments.
only 4 mg
MeCCNU
and
With
10 G y
radiation
9 kg-1
a substantially
g r e a t e r g r o w t h d e l a y w a s still o b s e r v e d f o r s i m u l TreatmenP
S.F. per tumour b
taneous
treatments
with a 3 day Expt 1. 4 mg 9 kg -1 MeCCNU alone 2.3 x 10 -3 10 Gy radiation alone 3.2 x 10 -2 MeCCNU plus radiation 1.2 • 10 -~ Predicted additive (Mode I) 7.1 • 10 -5
Expt 2. 3.6 4.2 3.8 1.5
x x • x
10 -a 10 -2 10 -4 10 -4
a Mice bearing i.m. LL tumours were treated simultaneously with MeCCNU and radiation, and an excision cell survival assay was performed 24 h later. The control plating efficiency (PE) was 60%. b S.F. per tumour was calculated as: number of colony-forming tumour cells per treated tumour number of colony-forming tumour cells per control tumour"
(/'4•
=
9.2 d a y s ) c o m p a r e d
interval between
treatments
(with
e i t h e r s e q u e n c e , T4 • = 4.9 d a y s ) . T h e cell s u r v i v a l results for simultaneous treatment, obtained in two separate though
experiments,
are shown
in Table
I. A l -
the growth delay data suggest that simul-
t a n e o u s t r e a t m e n t w a s s u p r a - a d d i t i v e , t h e cell s u r vival response was not apparently greater than additive. Unfortunately, be performed,
only Mode I addition could
s i n c e cell s u r v i v a l w i t h s i n g l e a g e n t s
could not be measured
at high enough doses, but
because the radiation
s u r v i v a l c u r v e is b i - p h a s i c ,
Mode
II addition
would
be expected
to predict
h i g h e r s u r v i v a l t h a n M o d e I. T h u s , s u p r a - a d d i t i v e cell k i l l i n g d o e s n o t a p p e a r
to explain the supra-
additive regrowth delay results. However,
we can-
TABLE II Transplantation of untreated, or drug and radiation treated LL ceils into pretreated or non-pretreated recipient mice. Cells a
Recipients b
Time to 0.5 g (days)~
Td (days) r
Untreated Untreated Untreated
untreated radiation d MeCCNU ~
10.8 (10.2-11.2) 11.0 (10.8-11.8) 9.0 ( 8.9- 9.9)
1.6 (1.2-1.8) 1.9 (1.3-2.0) 2.0 (1.2-2.6)
Radiation Radiation Radiation
untreated radiation MeCCNU
12.8 (12.2-13.2) 13.8 (12.9-14.2) 12.4 (I 1.9-12.8)
1.8 (l.l-1.9) 1.7 (1.1-2.3) 1.7 (1.2-1.8)
MeCCNU MeCCNU MeCCNU
untreated radiation MeCCNU
14.1 (13.3-14.5) 16.9 (16.4-18.3) 13.0 (12.8-14.0)
1.7 (1.1-3.6) 2.7 (2.0-2.9) 1.6 (1.3-2.3)
Rad + MeCCNU f Rad + MeCCNU Rad + MeCCNU
untreated radiation MeCCNU
29.8 (26.0-48.9) 45.0 (40.0-49.0) 31.3 (28.6-33.6)
2.5 (2.2-2.7) 4.8 (3.5-6.8) 2.0 (1.9-3.1)
a Tumours were treated in vivo with MeCCNU and/or radiation and disaggregated 24 h later. Recipients received subcutaneous implants of 5 • 104 tumour cells. Recipients were treated with MeCCNU or radiation 6 h before tumour cells were implanted. c Values are median, 25th and 75th percentiles. d Radiation dose = 10 Gy. ~ MeCCNU dose = 4 mg 9 kg-1. f Radiation-MeCCNU combination given simultaneously.
143 not rule out the possibility of supra-additive cell killing at higher drug and radiation doses. The possibility that the synergistic effect of simultaneous treatment seen with the growth delay end-point might reflect a retardation in the growth rate of cells surviving the combined treatment is suggested by the less steep regrowth curve following simultaneous treatment in Fig. 2. Table II shows the results of a transplantation experiment designed specifically to explore this question. Three batches of mice were prepared: untreated controls, mice whose left hind leg had been locally treated with l0 Gy of gamma-rays and mice which had been injected i.p. with 4 mg 9kg- 1 MeCCNU. 6 h later the batches of mice were sub-divided into four groups (each of 6 mice), and injected i.m. into the left hind leg with 5 x 104 tumour cells that had been prepared from untreated LL tumours or those treated 24 h earlier in three ways: irradiated to 10 Gy, treated with 4 mg 9 kg-1 MeCCNU or treated simultaneously with radiation and MeCCNU. The time for each implant to grow to a target size of 0.5 g and the growth rate (tumour volume doubling time, Td) estimated from the slope of the growth curve of each implant at that size, were measured. Although the same numbers of structurally intact tumour cells receiving the different pretreatments were implanted, it is important to remember that radiation and MeCCNU either alone or in combination killed many tumour cells. Therefore, the time for tumours to grow from each type of pretreated cell implant, regardless of which recipient is used, will depend very much on the extent of cell killing by the pretreatment. Thus, we should only compare the growth time for a given cell treatment group in the three different recipients. However, the tumour growth rate at 0.5 g, for each type of cell in different recipients can be compared, since it should not depend on the number of viable cells initially implanted. The results (Table II) show clearly that tumour cells that had been pretreated with MeCCNU alone or with MeCCNU plus radiation took longer to attain the target size and grew significantly more slowly (greater Td), if they were implanted i.m. into recipients with irradiated legs, compared to all other combinations.
Discussion
In this paper we have examined the suggestion that ionising radiation may be combined with some cytotoxic drugs (e.g. MeCCNU, CY and cis-Pt) to produce a beneficial antitumour effect by timing the second treatment to coincide with the period of repopulation by tumour cells that survive the first treatment. Single large doses of radiation and a daily five fraction regime were both studied. The experiments were performed using the murine Lewis lung tumour where we have previously described the pattern of repopulation which occurs after single large radiation doses [26], and during a course of fractionated radiotherapy [2]. Sequential excision assays were used to monitor repopulation. Following acute doses of 15 or 25 Gy, repopulation had begun within 2 days of treatment and gradually accelerated to a maximum rate (Td = 1 day) between 5 and 8 days after treatment. The rate of repopulation then slowed down as the surviving fraction approached 1. During fractionated regimens of 10 daily doses of 3.2 to 6.5 Gy or twice daily doses of 2.3 to 4.7 Gy, we were unable to detect significant repopulation, although it began promptly after the last fraction. When the cytotoxic drugs were combined with single large radiation doses and regrowth delay measured, two distinct patterns of response were observed: (i) MeCCNU was clearly much more effective when given simultaneously or within 1 day of irradiation than given between 1 and 9 days before or after irradiation. Using the classification of Steel [15] the simultaneous treatment response was supra-additive, while at other times the response was within the envelope of additivity. (ii) CY and cis-Pt appeared to be marginally more effective when administered between 2 and 6 days before or after irradiation than when given simultaneously with irradiation. However, at all times of drug administration from 7 days before to 7 days after radiation the response was within the additivity envelope. When combined with fractionated irradiation, MeCCNU and CY appeared to be slightly more
144 effective when given simultaneously with the first radiation dose than with the last. The other combinations of these drugs and cis-Pt with radiation, were all close to additive. More detailed studies to explore the mechanism of the apparent M e C C N U interaction with simultaneous acute irradiation suggested that this is not due to enhanced tumour cell killing, since cell survival measured with an excision clonogenic assay was additive. Instead, it would seem that the supra-additive regrowth delay response results from retardation of the growth of tumour cells that had been pre-treated with either M e C C N U or M e C C N U plus radiation, when they attempted to grow in an irradiated site. This phenomenon has been widely described for radiation alone, (where it is known as the " t u m o u r bed effect" [8]). However, it should be stressed that a classical tumour bed effect was not seen; the growth of untreated or irradiated cells was not retarded when they were implanted into irradiated recipients. It is also interesting to note that the growth of C C N U treated B 16 melanoma cells may be retarded if they are implanted into animals that have been pre-treated with CY, although there was no retardation if cells were implanted into C C N U pretreated mice [28]. Additional support for the above mechanism is provided by the fact that simultaneous treatment of tumours with radiation and M e C C N U reduced the regrowth rate by a factor of 2 to 3 (See growth curves in Fig. 2), which compares well with the retardation in growth rate of radiation-MeCCNU treated cells implanted into irradiated recipients (Table II). Further, M e C C N U treated cells in M e C C N U treated mice, and irradiated cells in irradiated mice appeared to have no effect on growth rate in either tumour, or transplantation experiments. Although we set out to determine whether the increased tumour cell proliferation that occurs during repopulation, either after a large acute treatment with radiation or drugs, or during fractionated irradiation, may be an exploitable mechanism to obtain greater tumour response in combined modality treatments, we have found little indication that this is the case with the agents chosen for this
study. We conclude that with CY and cis-Pt the response is essentially the same whether the agents are give together or separated by a few days when repopulation is maximal, thus repopulation seems not to be an important determinant of response. On the other hand, with M e C C N U , the response is substantially better if the treatments are given simultaneously, and any attempt to exploit repopulation is detrimental. In the fractionated radiation studies, the poorer response seen when the drugs were administered with the last dose o f radiation may reflect the development of chemo-resistance due to the increase in size of tumours compared to their size at the start of treatment. Changes in drug and radiation response that are associated with tumour size have been widely reported [9,10,17,19,22]. Alternatively, our previous radiosensitivity studies with the Lewis lung tumour [2] show that during fractionated regimes tumours become progressively more hypoxic even though they shrink slightly in volume during treatment, and this may reduce chemo-responsiveness [9,30]. However, this might not be true for cis-Pt, since it apparently kills oxic and hypoxic cells equally well in vitro [30], and has radiosensitising properties [7]. Our results generally confirm those previous reports in which longer time intervals between drugs and radiation have been examined using the growth delay endpoint. There were no marked supra-additive interactions between either CY or B C N U and radiation in the E M T 6 tumour when treatments were separated by up to 3 days [3,4], and responses to CY-radiation were approximately additive in the hepatoma 3924A, for treatment intervals between - 7 and + 4 days between the two agents [11]. Using a tumour cure endpoint to assess the combination o f CY-radiation in the Lewis lung tumour, Steel et al. [23] found that it was best to administer the agents within a day of one another. A fixed dose of CY was unable to sterilise the increasing number of cells present in tumours as they repopulated after irradiation. If our findings applied to human tumours in situ, then simultaneous administration of M e C C N U and radiation might be good for palliation (i.e. prolonging survival time), but are unlikely to influence
145 survival rates, compared to regimens in which longer intervals are left between the treatments.
Acknowledgements We are grateful for the support of Prof. M. J. Peckham; the work was partially supported by NCI Grant No. RO1 CA 26059.
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