Radiobiological hypoxia of a transplanted rat tumour and the effect of treatment with cyclophosphamide

Europ. J. Cancer,V o l .

14. pp. F383 1387.

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Radiobiological Hypoxia of a Transplanted Rat Tumour and the Effect of Treatment with Cyclophosphamide* BRYAN DIXON,J" JAMES V. MOORE++§ and HELEN SPEAKMAN { "fRadiobiology Department, Regional Radiotherapy Centre, and {Radiotherapy Department, University of Leeds, Leeds LS16 6QB, United Kingdom

Abstract--Isolog, u~ tumoms, 8-10mm in diameter, of the 37th-40th generation spontaneous adenocarcinoma LMC, have been grown subcutaneous~ in 150-200g female Wistar rats and irradiated with 6°Co 7-rays beJore or 1, 4, 7 or 10days after intraperitoneal ir!jection of 150 mg/kg of cyclophosphamide (CP). Dose-response curves for delay in growth of the tumour given aerobic or hyperbaric (HBO) irradiation alone were biphasic and, above 15 Gy (AIR) and 20 Gy (HBO), their response was governed by a component of hypoxic cells. One day after CP the response of the tumour to aerobic irradiation was the same as when rendered hypoxic by clamping 15 min prior to treatment indicating that only hypoxic tumour cells survive in animals treated with drug. Three days later hypoxia is reduced by reoxygenation, but the hypoxic fraction is still significantly higher than before treatment with CP. Only by 7 and 10days had the hypoxic fraction of clonogenic cells been reduced to a level characteristic of the untreated tumour. Even though at 4days after giving drug the hypoxic component of cells was significantly larger than normal for rats breathing air, no resistant component was found in the tumour responsefor HBO irradiation. This suggests that processes leading to the reo:~ygenation of a turnout after an initial treatment with drug may themselves permit an enhanced effect of subsequent HBO" irradiation. rimental tumour whilst subjected to elevated, normal or reduced conditions of oxygenation, to irradiation alone or after pre-treatment by the cycle-active alkylating agent cyclophosphamide (CP). This tumour contains a clonogenic cell population relatively resistant to the action of the drug [2] and has a hypoxic fraction, measured by cell assay in vitro of approximately 18 °/o [5]. The present experiments employed the endpoint of delay in the macroscopic growth of the tumour [6].

INTRODUCTION 'Erie use of cytotoxic drugs as adjuvants to radiotherapy offers a potential therapeutic advantage in improved control of primary tumours. Thus doses of cytotoxic agents tolerated by the host may be used to kill a large proportion of cells in the tumour prior to irradiation [1, 2]. However, many such agents are less effective against non-proliferating than against cycling populations of cells [3]. Some at least of the non-proliferating cells in solid tumours may also be hypoxic [4], so that unless reoxygenation occurs in the conditions prevailing after drug treatmeflt, subsequent irradiation will have a less than optimal effect. We have examined the response of an expe-

MATERIALS AND METHODS

Accepted 30 May 1978. *Work supported by a TENOVUS Studentship 0VM). ]Address for reprints. §Present address: Paterson Laboratories, Christie th,~l)iml. Manchester M2()9BX, United Kingdom.

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Tumours of the 37th-40th transplant generations of the rat adenocarcinoma L M C z [7] were implanted subcutaneously in the flank of female 150-200g Wistar rats of John's strain, using the method of Thomlinson [8]. Animals were assigned randomly to treatment groups when their tumours first reached a mean diameter of 8 - 1 0 m m , as determined by ca-

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Bryan Dixon, J a m e s t\ Moore and Helen Speakman

liper measurements. Atker treatment they were caged in groups of 6, allowed tbod and water ad libitum and their tumours measured daily. Cyclophosphamide ('Endoxana'; Ward Blenkinsop, Bracknell) was injected intraperitoneally at a standard dose of 150mg/kg of body weight. This dose reduces the surviving ti'action of clonogenic ceils in the tumour to about 3 °,....o [2] and also results in a less than two-fold change in tumour volume over the first 10 days after treatment [5]. Rats were immobilised for irradiation by intraperitoneal injection of 65mg/kg of sodium amylobarbitone ('Amytal'; Lilly, Basingstoke) and immediately after treatment were given 0.8 mg/kg of 'Megimide' (Nicholas, Slough) to facilitate recovery from the hypnotic. Tumours were irradiated locally with 6°Co 7-rays at a dose-rate of approximately 1.90 Gy/min, the rats lying within a pressure chamber [6]. When treated with the rats breathing hyperbaric oxygen (HBO, 4 At abs) or air, the tumour lay unsupported in the irradiation beam. Tumours were made artiticially hypoxic by occluding the blood supply to the turnout with a 'U' shaped clamp applied 15rain prior to irradiation of rats breathing air. All irradiations were given as single doses. In rats treated first with CP, irradiation of the tumour in air or under hypoxia was carried out 1, 4, 7 or 10 days later. Irradiation in HBO was also used at 4 days after CP treatment. After treatment the time taken for each 810ram tumour to grow to an average diameter of 25 mm was measured. The difference in mean times taken by tumours in treated and untreated groups of rats constitutes the growth delay. The rates of regrowth of tumours treated by irradiation alone and those in rats given CP before irradiation has previously been shown to be the same [5].

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when this was displaced vertically upward, to an extent greater for irradiation in HBO (Curve C, Fig. 1) than in air (Curve D, Fig.

1). The delay in growth of L M C s tumours tbllowing injection of rats with 150mg/kg CP alone was 12.8_+0.3days. When this mean value was subtracted from the total delay obtained for tumours irradiated when clamped, but 1, 4, 7 or 10 days after treatment of the rats with drug, the delays due to the irradiation did not differ significantly from those fbr hypoxic tumours in rats not given CP (Fig. 2). After CP and with irradiation HBO

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After irradiation alone, the growth delay tbr tumours treated when clamped increased progressively with dose, while the growth delay data for tumours irradiated in rats breathing air or HBO were biphasic (Fig. 1). Data for doses up to and including 20 Gy (HBO) and 15 Gy (air) could be fitted by redrawing the dose response curve for irradiation under hypoxic conditions assuming oxygen enhancement ratios of 3.0 and 2.7 respectively (curves A and B, Fig. 1). With larger doses given in either condition, delay data were consistent with the response curve for hypoxic tumours

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DOSE (Gy) Fig. 2. Do,~e response ,fin L M C I irradiated under hypoxic condilion,~ q/Tee lreahnent ~ rat,i wilh CP, • 1 day; • 4 da~s: • 7 days; or • l0 days previou@. Five to si~ rats per trealmeu! group. Alean delax~ + 2 S.E.; dashed curves are ./)'ee-ha*ld,fit~ /o me(m.~ (;/ e~p~rimenlal data giT,e~l in 1@,~. I.

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given in re,tobit conditions at day 1, the growth delay of the tumom's duc to irradiation was also consistent with the radiation dose response curve for hypoxic tumours in rats not previously treated with drug (Fig. 3).

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oosE t~y) Fig. 3. Dose-response for LMCt irradiated in air after treatment oJrats with CP: @ I dax: • 4 days: • 7 days or • 10days previous~. Five to si.~ rats per treatment group. Mean delays + 2 S.E. Dashed curves as in Fig. 2.

However, 4 days after CP, a biphasic curve for irradiation in air was restored but the delays due to 30 and 40 Gy remained significantly less than the corresponding delays for aerobic irradiation alone (P>0.01). By 7 and 10 days after CP, the tumour response to all doses of aerobic irradiation was the same as for tumours in rats that had not been treated previously with drug.* When tumours were irradiated in untreated hosts breathing HBO, a residual resistant component was revealed at high dose (Fig. 1). Four days after CP when the resistant component in air breathing rats is even higher than normal, this residual component could not be shown in the radiation response of tumours when treated in HBO, none having yet regrown after 30 or 40 Gy more than 150 days after treatment (Fig. 4). DISCUSSION

The shapes and positions of curves for delay in tumour growth (Figs. 1-4) are believed to be determined mainly by the relative sensitivities of clonogenic cells within a population, *Radiation doses greater than 40 Gy were in general not given to C P treated animals since the combined delay in turnout growth permitted an increased incidence and more widespread development of metas-

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Fig. 4. Dose-response for LMC l irradiated in, • air or HBO, 4 days after treatment of rats with CP. Five to six rats per treatment group, mean delays __+2S.E. Dashed curves as in Figs. 2 and 3.

rather than the absolute size of that population. Thus Thomlinson [10] found that for the Fibrosarcoma R I B 5 40min after 35 Gy with the tumour clamped (for R I B s C reducing the clonogenic cell population by approximately 103 [11]) the delay data obtained for second doses given in air were changed little from that for aerobic irradiation of previously untreated tumours. We have found also that the delay curves for LMCI tumours irradiated at 5 m m diameter either clamped or in air, (Moore, unpublished) resemble closely those treated at 9 + 1 mm (Fig. 1). It seems valid then, to determine changes in the proportion of hypoxic cells by shifts in the growth delay curves for tumours even though the total number of cells may alter with time after an initial treatment, as occurs for LMC~ in the days following injection of CP [2]. Thus tbr rats treated with CP and where all the clonogenie cells within the tumour had been made uniformly resistant to radiation by clamping, little change was encountered in the position and shape of the dose-response curve for growth delay determined at any of the chosen intervals after treatment with drug. The delay data for tumours irradiated under aerobic conditions does however reveal differences dependent upon the time elapsed after giving drug. Notably, at 1 day after CP the response to radiation was almost indistinguishable from that for a tumour in which

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Br~an Did'on, James K Moore and Helen Speakman

all cells had been m a d e hypoxic by clamping (Fig. 3). A similar shift has been observed for the t u m o u r R I B 5 when second doses of radiation were given 4 0 m i n after a first dose sufficient to destroy all the well-oxygenated cells [10]. However for L M C l the ettkct persisted m u c h longer; the d a t a indicated that the proportion of hypoxic cells had still not been reduced by reoxygenation to pretreatment value even 4 days after CP. That alkylating agents m a y preferentially spare hypoxic clonogenic cells has been shown recently for the K H T sarcoma treated with nitrogen mustard [1]. T h e p h e n o m e n o n however, is not universal; the proportion of hypoxic cells in the B16 m e l a n o m a was unchanged when assayed 2 h r after CP [12]. In the cases where the proportion of hypoxic cells a m o n g survivors is raised, as for LMC1, the rate of reoxygenation m a y vary between t u m o u r types, as it does after a dose of radiation [13]. T h e L M C ! turnout has a relatively low Cell Loss Factor of 0.3 [7] and after 150mg/kg CP but before regrowth occurs, its volume never regresses below that on the day of treatment [2]. It is unlikely then, that the type of reoxygenation seen for the C3H m a m m a r y carcinoma which is rapid and complete [12], would occur in L M C b which in this respect more closely resembles the very slowly reoxygenating osteosarcoma described by van Putten [14]. In the light of these observations, the extent to which H B O enhanced the effect of the larger doses of radiation given 4 days after CP, is puzzling (Fig. 4). In rats not treated with CP, irradiation in H B O revealed a residual proportion of hypoxic cells (Fig. 1). T h a t reduced competition for oxygen from sterilized t u m o u r cells in rats trealed with CP would enable this hypoxic component to reoxygenate is reasonable but the same a r g u m e n t holds for tumours in rats breathing air where the hypoxic component is larger than normal

tour days alter CP (Fig. 4). However at this time reoxygenation is still in progress and normal levels of t u m o u r hypoxia are not attained until 7-10 days (Fig. 3). Thus, the situation prevailing after treatment with CP is in contrast to that before, when the oxygen concentration of critical cells is decreasing and hypoxia is a transient state leading to cell death and necrosis. On this basis, factors operating in favour of reoxygenation of the tumour alter CP treatment also m a y favour the more efficient oxygenation of erstwhile hypoxic cells during irradiation in HBO. Apart from this apparent enhanced effect of H B O irradiation 4 days after CP, which requires further studies at other time intervals, the results of these experiments clearly indicate that the systemic use of drug belore local radiotherapy under aerobic conditions m a y either prejudice local control of the t u m o u r (i.e. at 1 and 4 day intervals for L M C j ) or at best (7-10 days) have little or no synergistic effect. From the clinical standpoint the therapeutic optinnnn, i.e. a reduction in the hypoxic fraction below a lcvcl characteristic of the untreated tumour, was not achieved. If this depends ultimately upon a reduction of t u m o u r volume and a diminution of inter-capillary distances this is not achievable with L M C l using single doses of other chemotherapeutic agents e.g. Methotrexate, T h i o t e p a and Adriamycin (Dixon, unpublished). Doses of CP greater than 150mg/kg result in an unacceptable mortality, and though this m a y be avoided by the use of a split dose, actual regression of the L M C 1 t u m o u r does not occur [15].

Acknowledgements--We are gralethl Io Tenovus [iw their financial support (J.V.M.) and to Proli'ssor C..\. Joslin t-or advice and encouragement. We are espcciallx indebted to Dr. R. H. Thomlinson tbr the use of the experimental HBO chamber, Mr. J. Mouncev of the Yorkshire Regional Medical Physics Unit tot assistance with the irradiation dosimetry and Mrs. ,J. Haines tbr secretarial assistance.

REFERENCES 1.

R. P. HILL and R. S. BUSH, A new method of determining the fraction of hypoxic cells in a transplantable murine sarcoma. Radiat. Res. 70, 141 (1977). 2. J. V. MOORE and B. DixoN, The gross and cellular response of a rat mammary tumour to single doses of cyclophosphamide. Europ. j . Cancer 14, 91 (1978). 3. L. M. VAN PUTTEN, P. LELIEVELD and L. K. J. KRAM-IDSENGA, Cell cycle specificity and therapeutic effectiveness of cytostatic agents. Cancer Chemother. Rep. 56, 691 (1972). 4. I. TANNOCK, The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumour. Brit. J. Ccmcer22, 258 (1968).

Radiobiological Hypoxia of a Transplanted Rat Tumour 5. J . V . MooRE, The response of a rat mammary tumour to cyclophosphamide and to subsequent irradiation. Ph.D. Thesis, University of'Leeds (1976). 6. R . H . 'FnoMLINSON and E. A. CRADDOCK, Gross response of an experimental turnout to single doses of X-rays. Brit. J. Cancer 9, 539 (1967). 7. J. V. MOORE and B. DIXON, Serial transplantation, histology and cellular kinetics of a rat adenocarcinoma. Cell Tiss. Kinet. 10, 91 (1978). 8. R. H. TnO~LINSON, An experimental method for comparing treatments of intact malignant tumours in animals and its application to the use of oxygen in radiotherapy. Brit. J. Cancer 14, 555 (1960). 9. J . V . MooRE and B. DIXON, Metastasis of a transplantable mammary tumour in rats treated with cyclophosphamide and/or irradiation. Brit. J. Cancer 36, 221 (1977). 10. R. H. THOMLINSON, Changes of oxygenation in tumours in relation to irradiation. Front. Radiat. Ther. Oncol. 3, 109 (1968). 11. N . J . McNALLV, A comparison of the effects of radiation on tumour growth delay and cell survival. The effect of oxygen. Brit. J. Radiol. 4 ~ 450 (1973). 12. R . P . HILL and J. A. STAY~.EV, The response of B16 melanoma cells to in vivo treatment with chemotherapeutic agents. Cancer Res. 35, 1147 (1975). 13. R . F . KALL~aAN, The phenomenon of reoxygenation and its implications for fractionated radiotherapy. Radiology 1115, 136 (1972). 14. L. M. VAN PVTVEN, Oxygenation and cell kinetics after irradiation in a transplantable osteosarcoma. In Effects of Radiation in Cellular Proliferation and Differentiation. p. 493. I.A.E.A., Vienna (1968). 15. J. V. MooRE and B. Dixon, Host and tumour response to single and split doses of cyclophosphamide. Submitted tbr publication (1978),

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