This thesis presents interdisciplinary, collaborative research in the field of synchrotron microbeam radiation therapy (MRT). Synchrotron MRT is an experimental radiotherapy technique under consideration for clinical use, following demonstration of efficacy in tumour-bearing rodent models with remarkable sparing of normal tissue. A high flux, X-ray beam from a synchrotron is segmented into micro-planar arrays of narrow beams, typically 25 μm wide and with peak-to-peak separations of 200 μm. The radiobiological effect of MRT and the underlying cellular mechanisms are poorly understood. The ratio between dose in the ‘peaks’of the microbeams to the dose in the ‘valleys’, between the microbeams, has strong biological significance. However, there are difficulties in accurately measuring the dose distribution for MRT. The aim of this thesis is to address elements of both the dosimetric and radiobiological gaps that exist in the field of synchrotron MRT. A method of film dosimetry and microdensitometry was adapted in order to measure the peak-to-valley dose ratios for synchrotron MRT. Two types of radiochromic film were irradiated in a phantom and also flush against a microbeam collimator on beamline BL28B2 at the SPring-8 synchrotron. The HD-810 and EBT varieties of radiochromic film were used to record peak dose and valley dose respectively. In other experiments, a dose build-up effect was investigated and the half value layer of the beam with and without the microbeam collimator was measured to investigate the effect of the collimator on the beam quality. The valley dose obtained for films placed flush against the collimator was approximately 0.25% of the peak dose. Within the water phantom, the valley dose had increased to between 0.7–1.8% of the peak dose, depending on the depth in the phantom. We also demonstrated, experimentally and by Monte Carlo simulation, that the dose is not maximal on the surface and that there is a dose build-up effect. The microbeam collimator did not make an appreciable difference to the beam quality. The measured values of peak-to-valley dose ratio were higher than those predicted by previously published Monte Carlo simulation papers. For the radiobiological studies, planar (560 Gy) or cross-planar (2 x 280 Gy or 2 x 560 Gy) irradiations were delivered to mice inoculated with mammary tumours in their leg, on beamline BL28B2 at the SPring-8 synchrotron. Immunohistochemical staining for DNA double strand breaks, proliferation and apoptosis was performed on irradiated tissue sections. The MRT response was compared to conventional radiotherapy at 11, 22 or 44 Gy. The results of the study provides the first evidence for a differential tissue response at a cellular level between normal and tumour tissues following synchrotron MRT. Within 24 hours of MRT to tumour, obvious cell migration had occurred into and out of irradiated zones. MRT-irradiated tumours showed significantly less proliferative capacity by 24 hours post-irradiation (P = 0.002). Median survival times for EMT-6.5 and 67NR tumour-bearing mice following MRT (2 x 560 Gy) and conventional radiotherapy (22 Gy) increased significantly compared to unirradiated controls (P < 0.0005). However, there was markedly less normal tissue damage from MRT than from conventional radiotherapy. MRT-treated normal skin mounts a more coordinated repair response than tumours. Cell-cell communication of death signals from directly irradiated, migrating cells, may explain why tumours are less resistant to high dose MRT than normal tissue.

Microbeam radiation therapy (MRT) is a potential cancer therapy technique that uses an intense X-ray beam produced by a synchrotron. In MRT, an array of microplanar beams, called a microbeam, is delivered to a tumour. The dose at each centre of planar beams is extremely large (several hundred grays) while dose level in the valley between the peaks is below several tens of gray. Moreover, the width of each planar beam is typically 20 - 50 µm, and the distance from a centre of planar-beam to that of adjacent beam is 200 - 400 µm. For the latter reasons, the fundamental requirements for the dosimetry technique in MRT are (1) a micrometer-scale spatial resolution and (2) detection sensitivity at large doses (5 - 1000 Gy). No existing detectors can satisfy those two requirements together. The objective of the Ph.D. research is to develop a prototype dosimetry technique which fulfils the requirements for measuring the dose profile in the microbeam. The currently used approach relies on the indirect detection of X-rays; in which the X-ray dose is recorded on a detector plate, and then the recorded signals are digitized using a reader. Our proposed approach utilizes Sm3+-doped polycrystallites, glasses, and/or suitable glass-ceramics (though our approach is not limited to the use of Sm ion) for the detector plate, in which a valence reduction of Sm3+, that is the conversion of Sm3+ to Sm2+, takes place upon irradiation of X-rays. The extent of reduction is further read out using confocal fluorescence microscopy via the photoluminescence (PL) signals of Sm3+ and Sm2+. The work carried out throughout the course of the research includes the construction of confocal fluorescence microscopy, synthesis and characterizations of dosimeter materials, as well as application tests of our approach for measuring the dose profile of a microbeam used at synchrotron facilities -- Canadian Light Source (CLS), Saskatoon, Canada, European Synchrotron Radiation Facility (ESRF), Grenoble, France, and SPring-8, Hyogo, Japan. Further, the research has shown that 1 % Sm-doped fluoroaluminate glass is one of the best candidates for the type of dosimetric application. It has the dynamic range of ~1 to over 1000 Gy which covers the dose range used in MRT, excellent signal-to-noise ratio (large extent of Sm3+ → Sm2+ change), and excellent stability of recorded signal over time. The recorded signal in the detector is erasable by heating or exposing to light such as UV. Furthermore, with a use of confocal microscope, it has ability to measure the distribution pattern of dose over the cross-section of microbeam. Therefore, we believe that our approach is one of the most promising techniques available.

La radiothérapie par microfaisceaux (MRT) synchrotron est une méthode de radiothérapie alternative pour les tumeurs cérébrales, qui présente l'avantage unique de pouvoir déposer de très hautes doses d'irradiation (plusieurs 100aines de Gy) au niveau de la masse tumorale. En effet, le fractionnement spatial des rayons X en microfaisceaux parallèles de quelques dizaines de micromètres s'est montré efficace dans le traitement des tumeurs cérébrales du rongeur tout en préservant le tissu cérébral péritumoral. Pour autant, son mode d'action sur le plan biologique n'est qu'en partie connu. Si l'effet différentiel de cette irradiation sur les vaisseaux sains et tumoraux a pu être démontré ces dernières années, il ne peut expliquer à lui seul l'efficacité de la MRT. Dans ce travail, nous avons établi une description de la réponse transcriptomique précoce des tissus sains et tumoraux (gliosarcome 9L) à la MRT et les fonctions biologiques et voies de signalisation associées. Ces résultats constituent une base de données interrogeable à partir d'hypothèses précises. Cette base a ainsi permis d'identifier des transcrits impliqués dans la réponse de la tumeur à la MRT et dont l'inhibition n'interfèrerait pas avec la réparation des tissus sains : nous avons proposé 3 cibles potentielles qui permettraient d'augmenter l'index thérapeutique de la MRT. (i) L'inhibition radio-induite d'un groupe de 13 gènes (Plk1, Cdc20, Ccnb1, Pttg1, Bub1, Dlgap5, Cenpf, Kif20a, Traf4af1, Depdc1b, Mxd3, Cenpe et Cenpf), participerait au contrôle tumoral précoce après MRT par la perturbation de la division cellulaire et pourrait être amplifié pour prolonger l'inhibition de la croissance tumorale. (ii) La mise à profit de l'activation du promoteur de Clecsf6 au sein des tumeurs irradiées permettrait la surexpression locale, via les monocytes modifiés et infiltrés, de protéine d'intérêt thérapeutique. (iii) Areg (codant pour l'Amphiréguline) est surexprimé au sein du tissu tumoral après MRT et son implication connue dans la chimio/radiorésistance nous conduit à considérer que son inhibition pourrait être une stratégie de renforcement des effets de la MRT. Par ailleurs, nous avons montré que la MRT engendrait de meilleurs résultats sur le contrôle tumoral et la survie animale qu'une irradiation synchrotron en champ plein (avec une dose équivalente à la vallée MRT). Cependant, aucune différence transcriptomique ne pouvant soutenir cet effet n'a pu être mis en évidence. / Synchrotron Microbeam Radiation Therapy (MRT) is a novel form of radiosurgery of brain tumors which allows high dose deposition (few hundreds of Gy) in pathologic tissues. The spatial fractionation of the incident beam into arrays of near-parallel microbeams has shown efficiency on brain tumors implanted in rodents while sparing normal tissues. The preferential effects observed on tumor vessels could not entirely explain the efficiency of MRT and other biological mechanisms might be involved in tumor control. In this work, we described the early whole transcriptomic responses of normal and tumoral (9L gliosarcoma) tissues to MRT and the associated biofunctions and pathways. This provides a questionable data base which can be used by the whole MRT community. This base allows to identify transcripts involved in tumor response to MRT and which inhibition would have no consequence in healthy tissue repair. We identified 3 relevant targets which might increase the therapeutic index of MRT. (i) The radio-induced inhibition of a cluster of 13 genes (Plk1, Cdc20, Ccnb1, Pttg1, Bub1, Dlgap5, Cenpf, Kif20a, Traf4af1, Depdc1b, Mxd3, Cenpe and Cenpf) may be involved in tumor control after MRT through the deregulation of cell division and could be amplified to continue the tumor growth inhibition. (ii) We might benefit from the activation of the Clecsf6 promoter in irradiated tumors by delivering, via modified and injected monocytes, some therapeutic proteins. (iii) Finally, Areg (encoding for Amphiregulin) is overexpressed in tumors after MRT and its involvement described in chimio/radioresistance enable to consider that its inhibition might help in tumor control after irradiation. We also showed that MRT induces a greater tumor control and survival rates compared with similar broad beam irradiations but no differences in transcriptomic responses have been highlighted.

La Thérapie par MicroFaisceaux (MRT) et la Thérapie Stéréotaxique par Rayonnement Synchrotron (SSRT) sont des techniques innovantes de radiothérapie expérimentale développées actuellement à l'ESRF. L'utilisation de modèles tumoraux différents pour chaque technique limite leur comparaison. <br />En MRT, sur rats porteurs de tumeur 9L, la médiane de survie des rats contrôle est doublée (de 20 jours à 40 jours) lors d'irradiation avec un espacement de 200 µm entre les microfaisceaux voire triplée (67 jours) à 100 µm d'espacement (mais provoquant alors d'importantes lésions du tissu sain). L'influence importante du collimateur multifentes, a également été démontrée. La combinaison de diverses drogues avec la technique de MRT a été testée. Des résultats prometteurs (médiane de survie de 40 jours et 30% de survivants à long terme) sont obtenus en injectant du gadolinium en intracérébral avant une irradiation MRT en faisceaux croisés à 460 Gy. De plus, l'irradiation MRT de tumeurs à stade plus précoce permet de quadrupler la médiane de survie (79 jours) et d'obtenir 30% de survivants à long terme. La mise en place d'un ciblage de la tumeur par imagerie avant l'irradiation et l'utilisation d'un collimateur adapté permettront d'améliorer encore ces résultats. Les différences entre les deux modèles tumoraux utilisés en MRT (modèle 9L) et en SSRT (modèle F98) étant importantes des expériences comparatives MRT/SSRT ont été réalisées sur ces deux modèles. Les résultats obtenus montrent une efficacité proche des 2 techniques sur le modèle F98 et une meilleure efficacité de la MRT sur le modèle 9L. Ces résultats pourront permettre d'orienter le type tumoral adapté à chaque technique.

Microbeam radiation therapy (MRT) is an experimental form of radiation treatment which causes less damage to normal tissue in comparison with customary broad-beam radiation treatment. In this method the synchrotron generated X-ray beam is passed through a multislit collimator and applied to the tumor in the form of an array of planar microbeams. MRT dosimetry is an extremely challenging task and no current detector can provide the required wide dynamic rang and high spatial resolution. In this thesis, fluorophosphate (FP) and fluoroaluminate (FA) glass plates doped with trivalent samarium (Sm3+) are characterized towards developing a potential X-ray detector suitable for MRT dosimetry. The detection is based on the difference in the photoluminescence signatures of Sm3+ ions and Sm2+ ions; the latter are formed under X-ray irradiation. This valency conversion is accompanied by the formation of defects including hole centers (HCs) and electron centers (ECs) in the glass structure which absorb light in the UV and visible regions (induced absorbance). Both FP and FA glasses show promising dynamic range for MRT and may be used as a linear sensor up to ~150 Gy and as a nonlinear sensor up to ∼2400 Gy, where saturation is reached. X-ray induced defects saturate at the same dose. The optimum doping concentration is in the 0.001˗ 0.2 at.% range. Doping with higher concentrations will decrease the conversion efficiency. The glass plates also show a very promising spatial resolution (as high as a few microns) for recording the dose profile of microbeams which is readout using a confocal fluorescence microscopy technique. These plates are restorable as well and the response is reproducible. The effects of previous X-ray exposure including samarium valency conversion as well as induced absorbance may be erased by annealing at temperatures exceeding the glass transition temperature Tg while annealing at TA < Tg enhances the response. This enhancement is explained by a thermally stimulated relaxation of host glass ionic matrix surrounding X-ray induced Sm2+ ions. Optical erasure is another practical means to erase the recorded data. Nearly complete Sm2+ to Sm3+ reconversion (erasure) is achieved by intense optical illumination at 405 nm. While, existing X-ray induced bands would be only partially erased. Electron spin resonance (ESR) and optical absorbance spectroscopy are used to investigate the nature of X-ray induced defects and their correlation with Sm valency conversion. A model based on competition between defect center formation and the Sm3+ ⇆ Sm2+ conversion successfully explains the different processes occurring in the glass matrix under X-ray irradiation.

Two innovative radiotherapy (RT) approaches are under development at the ID17 Biomedical Beamline of the European Synchrotron Radiation Facility (ESRF): microbeam radiation therapy (MRT) and minibeam radiation therapy (MBRT). The two main distinct characteristics with respect to conventional RT are the use of submillimetric field sizes and spatial fractionation of the dose. This PhD work deals with different features related to small-field dosimetry involved in these techniques. Monte Carlo (MC) calculations and several experimental methods are used with this aim in mind. The core of this PhD Thesis consisted of the development and benchmarking of an MC-based computation engine for a treatment planning system devoted to MRT within the framework of the preparation of forthcoming MRT clinical trials. Additional achievements were the definition of safe MRT irradiation protocols, the assessment of scatter factors in MRT, the further improvement of the MRT therapeutic index by injecting a contrast agent into the tumour and the definition of a dosimetry protocol for preclinical trials in MBRT.

<p>Microbeam radiation therapy (MRT) is an experimental radiotherapy concept that has been primarily developed for the treatment of malignant brain tumours. MRT uses high flux synchrotron x-rays delivered as an array of parallel microbeams in high doses of irradiation in fractions of seconds. The aims of this study were to 1) investigate the induction of bystander effects after normal and tumour-bearing rat brains were exposed to MRT and homogenous radiation; 2) validate a brain bystander proteome by detecting protein expression throughout immunohistochemistry: and 3) to investigate whether communication of bystander signals can be produced between animals.</p> <p>Healthy and tumour-bearing Wistar rats were exposed to 17.5, 35, 70 or 350 Gy of MRT or homogenous field of synchrotron radiation to the right brain hemisphere. To study the communication of bystander effects between animals, irradiated rats shared the same cage with non-irradiated rats over a period of 48 hours. After euthanasia of the animals, brains and bladders were dissected, and samples for immunohistochemistry and bystander clonogenic assays were set up.</p> <p>Clonogenic survival of the reporter HPVG cells showed that bystander effects occurred in both the non-irradiated hemisphere and bladder of normal and tumour-bearing rats, while the irradiated hemisphere showed the direct effects of radiation. Moreover, communication of bystander signals was confirmed in the non-irradiated rats.</p> <p>In conclusion, the results suggest that the MRT and homogenous radiation of unilateral normal and tumour-bearing rat brains produce bystander signals that affect the whole organism and that those signals also can be transmitted to non-irradiated animals.</p> / Master of Science (MSc)