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Synchrotron microbeam radiation therapy for rat brain tumor palliation—influence of the microbeam width at constant valley dose
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IOP PUBLISHING
PHYSICS IN MEDICINE AND BIOLOGY
Phys. Med. Biol. 54 (2009) 6711–6724
doi:10.1088/0031-9155/54/21/017
Synchrotron microbeam radiation therapy for rat brain tumor palliation—influence of the microbeam width at constant valley dose Rapha¨el Serduc1,2 , Audrey Bouchet3 , Elke Br¨auer-Krisch3 , Jean A Laissue4 , Jenny Spiga5 , Sukh´ena Sarun3 , Alberto Bravin3 , Caroline Fonta1,2 , Luc Renaud1,2 , Jean Boutonnat6 , Erik Albert Siegbahn7 , Fran¸cois Est`eve8 and G´eraldine Le Duc3 1
Universit´e de Toulouse, UPS, Centre de Recherche Cerveau et Cognition, France CNRS, CerCo, Toulouse, France 3 European Synchrotron Radiation Facility, F38043 Grenoble, France 4 Institute of Pathology, University of Bern, Switzerland 5 Department of Physics, University of Cagliari, s.p. Monserrato-Sestu, Monserrato (CA) 09042, Italy 6 TIMC lab, UMR CNRS 5525, Univ Joseph Fourier, CHU, Grenoble, France 7 Department of Medical Physics, Karolinska Universitetssjukhuset, 17176 Stockholm, Sweden 8 INSERM U836, Equipe 6, Institut des Neurosciences de Grenoble, 38043 Grenoble Cedex, France 2
E-mail:
[email protected]
Received 8 March 2009, in final form 15 September 2009 Published 20 October 2009 Online at stacks.iop.org/PMB/54/6711 Abstract To analyze the effects of the microbeam width (25, 50 and 75 μm) on the survival of 9L gliosarcoma tumor-bearing rats and on toxicity in normal tissues in normal rats after microbeam radiation therapy (MRT), 9L gliosarcomas implanted in rat brains, as well as in normal rat brains, were irradiated in the MRT mode. Three configurations (MRT25, MRT50, MRT75), each using two orthogonally intersecting arrays of either 25, 50 or 75 μm wide microbeams, all spaced 211 μm on center, were tested. For each configuration, peak entrance doses of 860, 480 and 320 Gy, respectively, were calculated to produce an identical valley dose of 18 Gy per individual array at the center of the tumor. Two, 7 and 14 days after radiation treatment, 42 rats were killed to evaluate histopathologically the extent of tumor necrosis, and the presence of proliferating tumors cells and tumor vessels. The median survival times of the normal rats were 4.5, 68 and 48 days for MRT25, 50 and 75, respectively. The combination of the highest entrance doses (860 Gy per array) with 25 μm wide beams (MRT25) resulted in a cumulative valley dose of 36 Gy and was excessively toxic, as it led to early death of all normal rats and of ∼50% of tumor-bearing rats. The short survival times, particularly of rats in the MRT25 group, restricted adequate observance of the therapeutic effect of the 0031-9155/09/216711+14$30.00
© 2009 Institute of Physics and Engineering in Medicine
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method on tumor-bearing rats. However, microbeams of 50 μm width led to the best median survival time after 9L gliosarcoma MRT treatment and appeared as the better compromise between tumor control and normal brain toxicity compared with 75 μm or 25 μm widths when used with a 211 μm on-center distance. Despite very high radiation doses, the tumors were not sterilized; viable proliferating tumor cells remained present at the tumor margin. This study shows that microbeam width and peak entrance doses strongly influence tumor responses and normal brain toxicity, even if valley doses are kept constant in all groups. The use of 50 μm wide microbeams combined with moderate peak doses resulted in a higher therapeutic ratio.
Introduction Microbeam radiation therapy (MRT) (Slatkin et al 1995) is a preclinical form of radiosurgery which was first dedicated to brain tumor treatment in rodents. It uses quasi-parallel, extremely high dose rate x-rays produced by electron bunches circulating in a synchrotron storage ring. MRT was initiated at the Brookhaven National Laboratory (New York, USA) (Laissue et al 1992, Slatkin et al 1995) and then developed at the European Synchrotron Radiation Facility (ESRF, Grenoble, France (Br¨auer-Krisch et al 2003, Siegbahn 2007, Smilowitz et al 2006, Laissue et al 2001)). Tumors are exposed to an array (unidirectional irradiation) or two arrays (bidirectional irradiation) of a few tens of microns thick, parallel beams separated by regular intervals of 50–400 μm. The high flux of synchrotron light allows very high rates of dose deposition (several hundreds Gy within 1 s). This unique irradiation geometry may prevent normal brain tissue necrosis (Laissue et al 2007, Serduc et al 2006, Slatkin et al 1995), mainly through normal brain vessel sparing (Serduc et al 2006, 2008a, 2008b). Previous studies showed that MRT might be a promising tool for palliation of brain tumors (table 1) (Dilmanian et al 2002, Laissue et al 1998, Regnard et al 2008, Serduc et al 2009, Smilowitz et al 2006). Laissue et al (1998) first reported the palliative and curative effects of MRT on 9L gliosarcoma-bearing rats. Dilmanian and co-workers (2002), using varying MRT configurations, reported the survival of 9LGS-bearing rats 2 years after irradiation. Due to the particular irradiation geometry used in MRT, one can describe two main regions of dose depositions. First, the peak region in which the microplanar beams deliver a high dose in their path. Second, the valley region, the tissue slices wedged between the peaks, exposed to a lower dose resulting from the diffusion of secondary electrons and photons derived from the adjacent peaks. The MRT-filtered spectrum is optimized to produce lateral microbeam profiles with a very sharp dose falloff. The detailed analysis of the contribution of photons and electrons to the dose deposition in MRT has been described by various authors (Siegbahn et al 2005, Spiga et al 2007, Stepanek et al 2000). All MRT parameters (i.e. peak dose, microbeam width and spacing, irradiation field, number of microbeams, photon spectrum, etc) are adjustable and influence strongly the dose deposition in the valley (De Felici et al 2005, Siegbahn et al 2005, 2006, Stepanek et al 2000). Previous studies have characterized the impact of some of these parameters (e.g. microbeam spacing, peak dose, uni- or bidirectional irradiations) on the survival of 9LGS-bearing rats (Dilmanian et al 2002, Laissue et al 1998, Regnard et al 2008). The effects of the microbeam width (MBW) have not been systematically studied. Some data on deuteron microbeams published in the 1960s have, for the first time,
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Table 1. Median survival time (MST, days) obtained by various authors for different MRT configurations used for the treatment of the 9LGS implanted in Fischer rats. ‘2×’ and ‘3×’ reflect crossfired and three-dimensional intersecting irradiations, respectively.
MST of nonirradiated 9LGSbearing rats (days)
MRT parameters: entrance dose (Gy)—MBW (μm)—on-center distance (μm)
No of rats per MRT subgroup
Array width x array height (mm)
Laissue et al 1998
20 (n = 9)
2 × 312–25–100 625–25–100 2 × 625–25–100
11 11 14
10 × 12
116 44 159
Dilmanian et al 2002
19 (n = 17)
23 (n = 6)
Serduc et al 2009 Smilowitz et al 2006
18 (n = 12)
5 5 5 5 6 6 3 11 10 9
8 to 10 × 11.4
Regnard et al 2008
150–27–50 250–27–50 300–27–50 250–27–75 300–27–75 500–27–75 500–27–100 625–25–100 625–25–200 3 × 400–50–200
10 × 12
98 136 55 171 62,5 31 170 67 37.5 57
21 (n = 14)
625–27–211
25
10.5 × 11
46
Studies
10 × 12
MST of irradiated rats (days)
highlighted the importance of MBW for normal brain tissue sparing (Zeman et al 1961). Empirically, MBWs of about 25 μm have mostly been used for experimental tumor treatment. The aim of this study was to observe the effects of the MBW on the median survival time (MST) of normal rats and of rats bearing implanted intracerebral 9LGS. MST was correlated with histological, immunohistochemical and in vitro studies. In this work, brain tumors have been irradiated with high radiation doses, using two orthogonal intersecting arrays of microbeams of 25, 50 or 75 μm widths. The valley dose has been kept identical between each irradiation configuration by modulating the peak entrance doses. Methods All operative procedures related to animal care strictly conformed to the Guidelines of the French Government with licenses 380324 and A3818510002. All experiments were performed under anesthesia with the following parameters: 5% isoflurane for induction and intraperitoneal injection of xylazine/ketamine (64.5/5.4 mg kg−1) for maintenance. Brain tumor inoculation The 9LGS cells were implanted in the brain of male Fisher 344 rats (Charles River, France) as previously described (Regnard et al 2008). Briefly, anesthetized animals were placed on a stereotactic frame, and 104 9LGS cells suspended in 1 μl culture medium with antibiotics were injected through a burr hole in the right caudate nucleus (3.5 mm lateral from the bregma, 5.5 mm below the skull surface).
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Radiation source and MRT setup MRT was performed at the ID17 biomedical beamline at the European Synchrotron Radiation Facility (Grenoble, France) (Thomlinson et al 2000). MRT uses x-rays emitted tangentially to the ring from relativistic electron bunches circulating in a storage ring. The wiggler source produces a white spectrum of photons which extends after filtration (Be (0.5 mm), C (1.5 mm), Al (1.5 mm) and Cu (1.0 mm)) from 50 to 350 keV (mean energy of 90 keV (Br¨auer-Krisch et al 2003)). The quasi-laminar beam was spatially fractionated into an array of microbeams by using an adjustable multislit collimator (TECOMET (Br¨auer-Krisch et al 2005)) positioned 33 m from the photon source and 80 cm upstream from the head of the animals. Downstream from the multislit collimator, the dose rate was approximatively 20 000 Gy s−1. Monte Carlo simulation and dose calculations The source used in the simulations reported here is represented by unpolarized planar microbeams with energies sampled from the x-ray spectrum measured at the ID 17 beamline of the ESRF. The x-ray microbeam impinges perpendicularly to the flat surface of the phantom which is a PMMA box with constant dimensions of 7 × 7 × 10 cm3. All computations of dose profiles have been done for a single microbeam. The dose distribution for an array of microbeams has been obtained subsequently by superposition. The height of the planar irradiation field is 14 mm, while three different microbeam widths have been considered (25, 50 and 75 μm). All relevant physical processes for photons (photoelectric, Rayleigh effects and Compton) as well as for electrons (elastic scattering and ionization) are considered for any of the calculations, and to increase the accuracy of the simulations, cut-off values have been kept as low as possible (1 eV). All the PVDR values for an irradiation field of 14 × 10 mm2 have been calculated in the middle of the square bundle of beams (5 mm). The doses were computed using the PENELOPE libraries (Salvat et al 2003) included in the GEANT4 (Geometry and Tracking) toolkit which has already been tested for MRT purposes (Spiga et al 2007). In vivo irradiation methods Rats bearing intracerebral 9LGS. Tumor cells were implanted in 59 rats. Fourteen days after tumor inoculation, the animals were positioned prone on a Kappa-type goniometer (Huber, Germany) in front of the x-rays source, on a home-made Plexiglas frame. Animals were divided into a MRT-treated group (n = 47), further subdivided into three subgroups which received three different configurations of irradiation, and a sham-irradiated control subgroup (SHAM) of 12 rats (table 2). Two orthogonal intersecting, 14 mm high arrays of microbeams were used. The on-center distance was fixed at 211 μm for all configurations. Twenty-five rats of the first subgroup were irradiated by 51 microbeams, 25 μm wide, with a skin entrance dose of 860 Gy in each array (MRT25). A second subgroup of 12 rats was exposed to 50 μm wide microbeams, with a skin entrance dose of 480 Gy in each array (MRT50). The last MRT-treated subgroup of 10 rats was irradiated by 75 μm wide microbeams, with a skin entrance dose of 320 Gy in each array (MRT75, table 2). Rats were first placed perpendicularly to the beam and received a lateral irradiation, from their anatomically right side to their left. Then, a 90◦ angle was applied to the motorized goniometer and the second irradiation was performed in the anatomically anteroposterior direction. The total procedure lasted about 2 min. Animal immobility during exposure was checked on three control video screens located in the control hutch. The spatial configuration of the microbeams was checked by radiochromic films (Gafchromic, HD-810) (Crosbie et al 2008).
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Table 2. Median survival times/increases in lifespan obtained for the different microbeam irradiations and detailed MRT parameters used in this study. Field size: two orthogonally intersecting, 14 mm high and ∼10.5 mm wide arrays of microbeams were used, with a fixed on-center distance of 211 μm.
Subgroup No of rats Peak entrance dose (Gy) Valley dose (Gy, at 1 cm depth) PVDR (at 1 cm depth) Peak/valley (cross-sectional area,%) MST (days after implantation) MST (days after irradiation) ILS (%)
No of rats MST (days after irradiation)
Intracerebral 9L gliosarcoma-bearing rats SHAM MRT 25 MRT 50 MRT 75 12 0 0 0 – 18 –
25 12 10 2 × 860 2 × 480 2 × 320 2 × 18 2 × 18 2 × 18 ∼48 ∼27 ∼18 12 24 35.3 18 53 40 4 39 26 100 194 122 MRT 25 6 4.5
Normal rats MRT 50 MRT 75 5 5 68 48
The 9LGS cell line used in our laboratory is highly radioresistant. High radiation doses— possibly excessive in terms of normal tissue damage—are required to control 9LGS growth in vitro and in vivo (Bencokova et al 2008). Kim et al (1999) reported that a 50% radiosurgical control of smaller (12 days old, versus 14 days old in the present study) 9LGS was only obtained after a single dose of 40 Gy of seamless photons in a small target volume (defined by a circular, 6 mm diameter collimator), i.e. a dose which clearly exceeds the normal tissue tolerance dose. The minimum radiation threshold dose for rat optic nerve damage in their study was ∼25 Gy (Kim et al 1999). In comparison, the MST of rats bearing 9LGS—comparable to those used in our study—and treated with whole head irradiation using 22.5 Gy of 250 kVp x-rays was 35 days, with only 20% long-term (i.e. 70 to approximately 365 days) survivors (Coderre et al 1994). Thus, we had to use a relatively high valley dose (18 Gy per array, 36 Gy in the volume of two intersecting arrays) to achieve a rapid and marked differential effect on 9LGS growth depending on the configuration of MRT parameters. Further, the brain tissue volume covered by the two intersecting arrays was relatively large, approximately 1.6 cm3, in relation to a rat brain weighing about 2 g. We would also like to stress that valley and peak doses delivered to 9L tumors in our study were in the range of and often lower than those described in other MRT studies of intracerebral 9LGS, such as those quoted in table 1. Normal rats without tumor. For each of the three configurations previously described, 16 normal rats, 5 or 6 in each subgroup, were exposed to the same irradiation procedure to test the toxicity of the radiation treatment. The time between implantation or irradiation and death was recorded as survival time (1 day was added in case of euthanasia as stated by endpoint guidelines of French Ethical Committees). MSTs were calculated and Kaplan–Meier survival data were plotted versus time after tumor implantation or irradiation. The increase in lifespan (ILS) in percentage is the difference between the MST for treated and untreated rats divided by the MST for untreated
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rats. The survival curves were compared using a log-rank test in GraphPad Prism (GraphPad Software, USA). Differences with a p value
