First published online August 17, 2006
British Journal of Radiology (2006) 79, 991-998
© 2006 British Institute of Radiology
doi: 10.1259/bjr/10893584
The usefulness of a continuous administration of tirapazamine combined with reduced dose-rate irradiation using 
-rays or reactor thermal neutrons
S Masunaga, MD, PhD1,
Y Sakurai, PhD4,
K Nagata, MD, PhD1,
M Suzuki, MD, PhD1,
A Maruhashi, PhD2,
Y Kinashi, MD, PhD3,
H Nagasawa, PhD5,
Y UTO, PhD6,
H Hori, PhD6 and
K Ono, MD, PhD1
1 Radiation Oncology Research Laboratory, 2 Division of Radiation Life Science, 3 Division of Radiation Safety, Research Reactor Institute, Kyoto University, Osaka, 4 Department of Physics, Faculty of Medicine, Sapporo Medical University, Sapporo, 5 Laboratory of Pharmaceutical Chemistry, Gifu Pharmaceutical University, Gifu, 6 Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, Tokushima, Japan
Correspondence: Shin-ichiro Masunaga, Radiation Oncology Research Laboratory, Research Reactor Institute, Kyoto University, 2-1010 Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494, Japan. E-mail: smasuna{at}rri.kyoto-u.ac.jp
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Abstract
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We clarified the usefulness of the continuous administration of tirapazamine (TPZ) in combination with reduced dose-rate irradiation (RDRI) using 
-rays or reactor thermal neutrons. Squamous cell carcinoma (SCC) VII tumour-bearing mice received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. Then, they received a single intraperitoneal injection or 24 h continuous subcutaneous infusion of TPZ in combination with conventional dose-rate irradiation (CDRI) or RDRI using -rays or thermal neutrons. After irradiation, the tumour cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labelling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total tumour cells was determined using tumours that were not pre-treated with BrdU. The sensitivity of both total and Q cells, especially of Q cells, was significantly reduced with RDRI compared with CDRI. Combination of TPZ increased the sensitivity of both populations, with a slightly more remarkable increase in Q cells. Furthermore, the continuous administration of TPZ raised the sensitivity of both total and Q cell populations, especially the former, more markedly than the single administration, whether combined with CDRI or RDRI using -rays or thermal neutrons. From the viewpoint of solid tumour control as a whole, including intratumour Q-cell control, the use of TPZ, especially when administered continuously, combined with RDRI, is useful for suppressing the reduction in the sensitivity of tumour cells caused by the decrease in irradiation dose rate in vivo.
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Introduction
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Many cells in solid tumours are quiescent in situ, but are still clonogenic [1]. These quiescent (Q) tumour cells have been thought to be more resistant to irradiation because of their much higher hypoxic fractions and greater potentially lethal damage repair (PLDR) capacities than proliferating (P) tumour cells, mainly based on the characteristics of plateau-phase cultured cells in vitro [1]. Employing our newly developed method for selectively detecting the response of intratumour Q cell populations in vivo, we have already shown that all these characteristics could be applied to Q state cells in solid tumours in vivo under conventional high dose-rate irradiation conditions [2, 3]. On the other hand, clinically, reduced dose-rate irradiation has also been found to spare normal tissue and to yield greater therapeutic gains [4].
Meanwhile, the development of bioreductive agents that are particularly toxic to hypoxic cells is considered a promising approach to solving the problem of radio-resistant tumour hypoxia in cancer radiotherapy [5]. Tirapazamine (TPZ), a lead compound as a bioreductive hypoxic cytotoxin, in combination with radiation has been shown to be very useful for controlling solid tumours as a whole, especially for controlling hypoxia-rich Q tumour cell populations [2]. Tumour hypoxia results from either limited oxygen diffusion (chronic hypoxia) or limited perfusion (acute hypoxia, transient hypoxia or ischaemic hypoxia). Chronically hypoxic tumour cells existing on the rim of the oxygen diffusion distance are supposed to be killed by just a single administration of the cytotoxin [2]. Acutely hypoxic tumour cells occurring sporadically throughout solid tumours are thought to be able to be killed by the cytotoxin during the long-term continuous administration. Recently, we have shown that the continuous administration of hypoxic cytotoxin was very useful for sensitizing tumour cells in vivo [6].
In the current study, the usefulness of combining TPZ administered singly or continuously with conventional or reduced dose-rate irradiation with low linear energy transfer (LET) -rays was evaluated in terms of the responses of the total ( = P+Q) and Q tumour cell populations [2]. In the same manner, in combination with conventional or reduced dose-rate irradiation using high LET reactor thermal neutron beams, which are available at our institute, the usefulness of TPZ was also evaluated.
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Methods and materials
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Mice and tumours
SCC VII squamous cell carcinoma (Department of Radiology, Kyoto University) derived from C3H/He mice was maintained in vitro in Eagle's minimum essential medium supplemented with 12.5% fetal bovine serum. Cells were collected from exponentially growing cultures, and 1.0x105 cells were inoculated subcutaneously into the left hind legs of 8-week-old to 11-week-old syngeneic female C3H/He mice (Japan Animal Co., Ltd., Osaka, Japan). 14 days after inoculation, each tumour had reached approximately 1 cm in diameter. At treatment, the body weight of the tumour-bearing mice was 22.1 g (19.8–24.4 g) (mean (95% confidence limit)). Mice were handled according to the Recommendations for Handling of Laboratory Animals for Biomedical Research, compiled by the Committee on Safety and Ethical Handling Regulations for Laboratory Animal Experiments, Kyoto University. All experimental procedures mentioned here were in accordance with institutional guidelines for the care and use of laboratory animals in research.
Incidentally, the p53 status of the SCC VII tumour cells is the wild type [7].
Labelling with 5-bromo-2'-deoxyuridine (BrdU)
9 days after the tumour cell inoculation, mini-osmotic pumps (Durect Corporation, Cupertino, CA) containing BrdU dissolved in physiological saline (250 mg ml–1) were implanted subcutaneously to label all P cells for 5 days. Administration of BrdU did not affect the tumour growth rate. The tumours were 1 cm in diameter on treatment. The labelling index after continuous labelling with BrdU was 55.3% (50.8–59.8%) (mean (95% confidence limit)), and reached a plateau level at this stage. Therefore, we regarded tumour cells not incorporating BrdU after continuous labelling as Q cells.
Treatment
After the labelling with BrdU, TPZ dissolved in physiological saline was administered at a dose of 224 mmol kg–1 (40 mg kg–1) singly by intraperitoneal injection or continuously for 24 h by subcutaneously implanting mini-osmotic pumps (Durect Corporation, Cupertino, CA) containing TPZ dissolved in physiological saline. Directly following the intraperitoneal injection of TPZ, during the 24 h of continuous subcutaneous infusion of TPZ, or without TPZ administration, whole body irradiation to the tumour-bearing mice was carried out using -rays or reactor thermal neutron beams available at our reactor institute.
-ray irradiation was performed with a cobalt-60 -ray irradiator at a dose rate of 2.75 Gy min–1 as conventionally employed high dose-rate irradiation (CDRI) with tumour-bearing mice held in a specially constructed device with the tail firmly fixed with an adhesive tape. Reduced dose-rate irradiation (RDRI) was performed at a dose rate of 0.039 Gy min–1 by keeping an appropriate distance between the cobalt-60 radiation source and the irradiated tumour-bearing mouse fixed within the specially constructed device.
Thermal neutron irradiation was performed using a reactor neutron beam with a cadmium (Cd) ratio of 160. The neutron fluence was measured from the radioactivation of gold foil at both the front and back of the tumours. The tumours were small and located just beneath the surface. The neutron fluence was assumed to decrease linearly from the front to back of the tumours. Thus, we used the average neutron fluence determined from the values measured at the front and back. Contaminating -ray, including secondary -ray, doses were measured with a thermoluminescence dosemeter powder at the back of the tumours. For the estimation of neutron energy spectra, eight types of activation foil and 14 types of nuclear reaction were used. The absorbed dose was calculated using the flux-to-dose conversion factor [8]. The tumours contained, in weight percentage, H (10.7%), C (12.1%), N (2.0%), O (71.4%) and others (3.8%) [9].
For thermal neutron beam irradiation at a conventionally employed dose rate, a device made of acrylic resin and capable of holding 16 mice was used, and the tumour-bearing mice fixed in position with adhesive tape were irradiated with a reactor thermal neutron beam at a maximum neutron flux just in front of the neutron-radiating bismuth layer of the heavy water facility at our reactor. The neutron flux (n cm–2 s–1) and kerma rate (cGy h–1) of the obtained beam were 2.0x109 and 96 for the thermal neutron range (less than 0.6 keV), 2.8x107 and 1.03 for the epithermal neutron range (0.6–10 keV), and 6.6x106 and 28.4 for the fast neutron range (more than 10 keV), respectively. The contaminating -ray dose rate was 120 cGy h–1. Overall, the irradiation dose rate was 0.041 Gy min–1. This value is almost the maximum dose rate available at our reactor.
Thermal neutron beam irradiation at a reduced dose rate was carried out by placing the tumour-bearing mouse 15 cm from the aperture of the collimator installed in front of the neutron-radiating bismuth layer. Each tumour-bearing mouse was fixed within the specially constructed device. The neutron flux (n cm–2 s–1) and kerma rate (cGy h–1) of the obtained beam were 3.3x108 and 14.0 for the thermal neutron range (less than 0.6 keV), 3.1x106 and 0.10 for the epithermal neutron range (0.6–10 keV), and 1.4x105 and 0.60 for the fast neutron range (more than 10 keV), respectively. The contaminating -ray dose rate was 32.2 cGy h–1. As a whole, the irradiation dose rate was 0.0078 Gy min–1.
Each irradiation group also included mice that were not pre-treated with BrdU.
Immunofluorescence staining of BrdU-labelled cells and micronucleus (MN) assay
Following irradiation, tumours were excised from the mice given BrdU, minced and trypsinized. Tumour cell suspensions thus obtained were incubated for 72 h in tissue culture dishes containing complete medium and 1.0 µg ml–1 of cytochalasin-B to inhibit cytokinesis while allowing nuclear division, and the cultures were then trypsinized and cell suspensions were fixed. After the centrifugation of fixed cell suspensions, the cell pellet was re-suspended with cold Carnoy's fixative (ethanol:acetic acid = 3:1 in volume). The suspension was then placed on a glass microscope slide and the sample was dried at room temperature. The slides were treated with 2 M hydrochloric acid for 60 min at room temperature to dissociate the histones and partially denature the DNA. The slides were then immersed in borax-borate buffer (pH 8.5) to neutralize the acid. BrdU-labelled tumour cells were detected by indirect immunofluorescence staining using monoclonal anti-BrdU antibody (Becton Dickinson, San Jose, CA) and fluorescein isothiocyanate (FITC)-conjugated antimouse IgG antibody (Sigma, St. Louis, MO). To observe the double staining of tumour cells with green-emitting FITC and red-emitting propidium iodide (PI), cells on the slides were treated with PI [2 µg ml–1 in phosphate-buffered saline (PBS)] and monitored under a fluorescence microscope.
The MN frequency in cells not labelled with BrdU could be examined by counting the micronuclei in the binuclear cells that showed only red fluorescence. The MN frequency was defined as the ratio of the number of micronuclei in the binuclear cells to the total number of binuclear cells observed [2].
The ratios obtained in tumours not pre-treated with BrdU indicated the MN frequency at all phases in the total (P+Q) tumour cell population. More than 400 binuclear cells were counted to determine the MN frequency.
Clonogenic cell survival assay
The clonogenic cell survival assay was also performed on the mice given no BrdU using an in vivo–in vitro assay method. Tumours were disaggregated by stirring for 20 min at 37°C in PBS containing 0.05% trypsin and 0.02% ethylenediamine tetra-acetic acid. The cell yield was 4.5 (3.4–5.6)x107 g–1 tumour weight. Appropriate numbers of viable tumour cells from the single cell suspension were plated on 60 mm or 100 mm tissue culture dishes and, 12 days later, colonies were fixed with ethanol, stained with Giemsa and counted. For the tumours that received no irradiation, the plating efficiencies for the total tumour cell populations and the MN frequencies for the total and Q cell populations are shown in Table 1
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Three mice were used to assess each set of conditions and each experiment was repeated twice. To examine the differences between pairs of values, Student's t-test was used when variances of the two groups could be assumed to be equal; otherwise, the Welch t-test was used. p-values are from two-sided tests.
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Results
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Overall, the MN frequencies for Q cells were significantly larger than those for total cells (p<0.05) (Table 1
). Singly or continuously, TPZ administration induced a significantly lower plating efficiency and significantly higher MN frequency in both the total and Q cell populations than no drug treatment (p<0.05). Continuous administration caused a lower plating efficiency and higher MN frequency in both the populations than did a single intraperitoneal administration (p<0.05).
Figure 1 shows the clonogenic cell survival curves after -ray irradiation (left panel) or reactor thermal neutron beam irradiation (right panel) with CDRI or RDRI in combination with TPZ or without TPZ. On -ray or neutron irradiation, RDRI yielded significantly larger surviving fractions (SFs) than CDRI under all conditions. However, the degree of the increase in the SF was more marked under -ray irradiation than under neutron irradiation. Furthermore, this increase was effectively suppressed by TPZ, especially continuous TPZ administration, whether under -ray or neutron irradiation.
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Figure 1. The clonogenic cell survival curves after-ray irradiation (left panel) or reactor thermal neutron beam irradiation (right panel) with conventional dose-rate irradiation (CDRI) or reduced dose-rate irradiation (RDRI) in combination with tirapazamine (TPZ) or without TPZ. TPZ was given as a single intraperitoneal injection (i.p.) or by continuous subcutaneously administration (cont.). Bars represent standard deviations.
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Figures 2 and 3 show the normalized MN frequencies after -ray irradiation (Figure 2) and reactor thermal neutron beam irradiation (Figure 3), respectively, with CDRI or RDRI in combination with or without TPZ administration in the total (left panel) and Q (right panel) tumour cell populations. When TPZ was employed, even if no radiation was given, MN frequencies were higher than when no TPZ was administered, because of the slight genotoxicity of the drug (Table 1). Therefore, for background correction, we used the normalized MN frequency to exclude the effects of the genotoxicity of TPZ. The normalized frequency is the frequency in the irradiated tumours minus the frequency in the non-irradiated tumours. All MN frequencies were actual values induced under each set of conditions. Namely, on neutron irradiation, they included the values due to contaminating fast neutrons and -rays in the neutron beam.
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Figure 2. The normalized micronucleus frequencies after-ray irradiation with conventional dose-rate irradiation (CDRI) or reduced dose-rate irradiation (RDRI) in combination with tirapazamine (TPZ) or without TPZ in the total (left panel) and Q (right panel) tumour cell populations. TPZ was given as a single intraperitoneal injection (i.p.) or by continuous subcutaneously administration (cont.). Bars represent standard deviations.
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Figure 3. The normalized micronucleus frequencies after reactor thermal neutron beam irradiation with conventional dose-rate irradiation (CDRI) or reduced dose-rate irradiation (RDRI) in combination with or without tirapazamine (TPZ) administration in the total (left panel) and Q (right panel) tumour cell populations. TPZ was given as a single intraperitoneal injection (i.p.) or by continuous subcutaneously administration (cont.). Bars represent standard deviations.
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On -ray or neutron irradiation, RDRI induced significantly lower normalized MN frequencies than CDRI under all conditions, especially in Q cells. The degree of the decrease in the normalized MN frequency was more marked under -ray irradiation than under neutron irradiation in both cell populations. Furthermore, this decrease in the normalized MN frequency was effectively suppressed by TPZ, especially continuous TPZ administration, whether under -ray or neutron irradiation.
To assess the relative biological effectiveness (RBE) of the reactor thermal neutron beams in both total and Q cell populations under CDRI and RDRI compared with -rays, the RBE was calculated using the data for irradiation only conditions without TPZ treatment given in Figures 1 to 3 (Table 2). In both total and Q cells, the values of RBE under RDRI were significantly larger than those under CDRI, reflecting that the degree of the change in the SF and the normalized MN frequency caused by reducing the irradiation dose rate was more marked under -ray irradiation than under neutron irradiation. In addition, the values in Q cells were significantly larger than those in the total cell populations under both CDRI and RDRI. This means that the degree of the change in the normalized MN frequency caused by reducing the dose rate was more marked in Q cells than in total cells.
To evaluate the effect of TPZ treatment on the surviving fractions in the total cell populations and the normalized MN frequencies in the total and Q cell populations, the enhancement ratio (ER) was calculated using the data given in Figures 1 to 3 (Table 3). The values under -ray irradiation, in Q cells, and under RDRI were larger than those under neutron irradiation, in total cells and under CDRI, respectively. In both cell populations, under both CDRI and RDRI, the values for the continuous subcutaneous administration were higher than those for the single intraperitoneal injection. In addition, the extent of the increase in the value caused by the continuous administration compared with the single injection in the total cells was slightly larger than that in the Q cells.
To assess the effects of TPZ treatment on the change in the SFs and the normalized MN frequency caused by reducing the dose rate, the effects of TPZ on the repair in the SF and the normalized MN frequency by RDRI compared with CDRI was calculated using the data given in Figures 1 to 3 (Table 4). With or without TPZ treatment, the values in Q cells were significantly higher than those in total cells (p<0.05). Whether under -ray or neutron irradiation, in both the total and Q cells, the values were reduced by TPZ treatment, especially continuous administration. Furthermore, the extent of this reduction with TPZ was slightly more remarkable in the Q cells than in the total cell populations.
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Discussion
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In contrast to normal tissues, most solid tumours have regions of low oxygen (hypoxia), low pH and low levels of glucose [5]. Therefore, the hypoxic microenvironment in solid tumours represents an attractive target for tumour-selective cancer therapy [10]. So far, we have shown the usefulness of TPZ in combination with -ray (low LET) irradiation or reactor thermal neutron (high LET) beam exposure with a conventionally employed dose rate, in terms of the cytocidal effects on both total and Q cell populations in solid tumours by employing our newly developed method for selectively detecting the response of intratumour Q cell populations in vivo [2]. Through the current study, we tried to reveal the effectiveness of the combined use of TPZ and -ray or thermal neutron irradiation with a reduced dose rate, compared with a conventionally employed dose rate.
On thermal neutron irradiation only, the RBE values obtained with a conventionally employed dose rate were similar to those reported previously in both total and Q cells (Table 2) [11]. But, the reduced dose rate induced significantly larger RBE values than the conventionally employed dose rate in both cell populations (Table 2), probably because with a reduced dose rate, significantly more radiation-induced DNA damage was repaired under -ray irradiation than under neutron irradiation in both cell populations. Meanwhile, whether using -rays or thermal neutrons, Q cells showed significantly larger RBE values than the total cells (Table 2), indicating that under a reduced dose rate, significantly more radiation-induced DNA damage is repaired in Q cells than in total cells. Namely, under a reduced dose rate, -ray irradiation induced significantly greater repair of radiation-induced DNA damage than did neutron irradiation, especially in the Q cell populations. This was compatible with the results in our previous report concerning PLDR in the total and Q cell populations after a conventionally employed dose-rate irradiation using -rays or reactor thermal neutron beams [12].
When low LET radiation is employed, it has been thought that decreasing the dose rate reduces late effects much more than it decreases tumour control. Thus, the "therapeutic ratio" (ratio of tumour control to complications) increases as the dose rate decreases. Furthermore, the difference between early and late effects for low dose rate radiotherapy, as well as itself improving the therapeutic ratio, allows the delivery of a complete treatment in a short time, allowing the effects of tumour repopulation to be minimized. In other words, decreasing the dose rate increases the therapeutic ratio, limited only by tumour cell repopulation [4]. This is the primary rationale for low dose rate radiotherapy using low LET radiation. However, this rationale does not take into account the response of Q cell populations in solid tumours at all. The current study showed that lowering the dose rate decreases the effect on Q cells much more than it reduces the effect on the total cells (Table 4). Therefore, considering the Q-cell response, it follows that the therapeutic ratio does not always increase when the dose rate is reduced.
In this study, the enhancing effect of TPZ was more remarkable under -ray irradiation, in Q cells, and with a reduced dose rate than under neutron irradiation, in total cells, and with a conventional dose rate, respectively (Table 3). This enhancement when TPZ is combined with radiation is thought to be advantageous for increasing the sensitivity of intratumour cells, especially radioresistant Q-cells, to low dose rate -ray irradiation. Actually, TPZ suppressed the decrease in the sensitivity of tumour cells by reducing the dose rate to a considerable extent, whether under -ray or neutron irradiation, in both the total and Q cells (Table 4). On the other hand, large intercapillary distances resulting from rapid tumour cell proliferation lead to chronically hypoxic cells existing at the rim of the oxygen diffusion distance [13]. Factors such as vessel plugging by blood cells or circulating tumour cells, the collapse of vessels in regions of high tumour interstitial pressure, or spontaneous vasomotor activity in normal tissue vessels incorporated into the tumour which subsequently affects flow in downstream tumour microvessels cause intermittent blood flow in tumours, which results in acute hypoxia [14]. Thus, acute hypoxic areas can develop throughout a tumour depending on the causative factors and can occur sporadically in large areas of a solid tumour. Thus, when administered continuously, TPZ can produce DNA breaks in larger areas of solid tumours where acute hypoxia has occurred during the administration period than a single intraperitoneal administration [6]. Consequently, a continuous subcutaneous administration was more effective at enhancing the effects on both total and Q cell populations than a single intraperitoneal injection (Tables 3 and 4). Incidentally, according to our previous report [3], in the SCC VII tumour the hypoxic fraction (HF) of total cells includes a large acutely HF and small chronically HF. In contrast, the HF of Q cells is made up of a large chronically HF and a small acutely HF. Therefore, the enhancing effect of continuous administration compared with a single administration might be larger in total cells than in Q cells (Table 3). Anyway, the use of TPZ, especially when administered continuously, combined with low dose-rate irradiation, is very useful for suppressing the decrease in sensitivity of tumour cells by decreasing the irradiation dose rate.
Solid tumours, especially human tumours, are thought to contain a high proportion of Q cells [15]. The presence of these cells is probably due, in part, to hypoxia and the depletion of nutrition in the tumour core, and this is another consequence of poor vascular supply [15]. This might promote the formation of micronuclei in Q tumour cells (Table 1). We have reported that Q cell populations have less sensitivity, greater PLDR capacities, and a larger HF than P cell populations in solid tumours in vivo [2]. Actually, also in this study, Q cells always showed significantly less sensitivity than total cells under all irradiation conditions, although the difference in the sensitivity between total and Q cells could be slightly reduced in combination with TPZ treatment, especially with continuous administration (Table 5). This means that more Q cells survive after radiation therapy than P cells. Consequently, the control of Q cells has a great impact on the outcome of radiation therapy. Meanwhile, in pre-clinical and clinical studies, severe neuropathy (both peripheral and central) such as muscle cramping and hearing loss was the dose-limiting toxicity and precluded safe and efficacious use in combination with radiotherapy [16]. From the viewpoint of the tumour cell-killing effect as a whole, including intratumour Q cell control, following elucidation of normal tissue toxicity, the use of TPZ that is more toxic to hypoxia-rich Q cells than total cells, especially with continuous administration, can be regarded as a promising modality as a combined treatment with conventional low LET radiation and high LET neutron therapy.
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Acknowledgments
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This study was supported, in part, by a Grant-in-aid for Scientific Research (C) (18591380) from the Japan Society for the Promotion of Science.
Received for publication May 10, 2006.
Revision received June 15, 2006.
Accepted for publication June 21, 2006.
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Abstract
Introduction
