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Purpose In the last three decades, Synchrotron Microbeam Radiation Therapy (S-MRT) has been shown to achieve both good tumour control and normal tissue sparing in a range of pre-clinical animal models. However, the use of S-MRT for the treatment of lung tumours has not yet been investigated. This study is the first to evaluate the therapeutic efficacy of S-MRT for the treatment of lung carcinoma, using a new syngeneic and orthotopic mouse model. Methods and materials Lewis Lung carcinoma-bearing mice were irradiated with two cross-fired arrays of S-MRT or Synchrotron Broad-Beam (S-BB) radiotherapy. S-MRT consisted of 17 microbeams with a width of 50 µm and centre-to-centre spacing of 400 µm. Each microbeam delivered a peak entrance dose of 400 Gy while S-BB delivered a homogeneous entrance dose of 5.16 Gy (corresponding to the S-MRT valley dose). Results Both treatments prolonged the survival of mice relative to the untreated controls (CTR). However, mice in the S-MRT group developed severe pulmonary oedema around the irradiated carcinomas and did not have improved survival relative to the S-BB group. Subsequent post-mortem examination of tumour size revealed that the mice in the S-MRT group had notably smaller tumour volume compared to the S-BB group, despite the presence of oedema. Mice that were sham-implanted did not display any decline in health following S-MRT, experiencing only mild and transient oedema between 4 days and 3 months post-irradiation which disappeared after 4 months. Finally, a parallel study investigating the lungs of healthy mice showed the complete absence of radiation-induced pulmonary fibrosis 6 months after S-MRT. Conclusions S-MRT is a promising tool for the treatment of lung carcinoma, reducing tumour size compared to mice treated with S-BB and sparing healthy lungs from pulmonary fibrosis. Future experiments should focus on optimising S-MRT parameters to minimise pulmonary oedema and maximise the therapeutic ratio.

Conventional radiotherapy is a widely used non-invasive form of treatment for many types of cancer. However,due to a low threshold in the lung for radiation-induced normal tissue damage, it is of less utility in treating lung cancer. For this reason, surgery is the preferred treatment for lung cancer, which has the detriment of being highly invasive. Non-conventional ultra-high dose rate (FLASH) radiotherapy is currently of great interest in the radiotherapy community due to demonstrations of reduced normal tissue toxicity in lung and other anatomy. This study investigates the effects of FLASH microbeam radiotherapy, which in addition to ultra-high dose rate incorporates a spatial segmentation of the radiation field, on the normal lung tissue of rats. With a focus on fibrotic damage, this work demonstrates that FLASH microbeam radiotherapy provides an order of magnitude increase in normal tissue radio-resistance compared to FLASH radiotherapy. This result suggests FLASH microbeam radiotherapy holds promise for much improved non-invasive control of lung cancer.

Purpose Synchrotron microbeam radiation therapy (MRT) is a method that spatially distributes the x-ray beam into several microbeams of very high dose (peak dose), regularly separated by low-dose intervals (valley dose). MRT selectively spares normal tissues, relative to conventional (uniform broad beam [BB]) radiation therapy. Methods and Materials To evaluate the effect of MRT on radioresistant melanoma, B16-F10 murine melanomas were implanted into mice ears. Tumors were either treated with MRT (407.6 Gy peak; 6.2 Gy valley dose) or uniform BB irradiation (6.2 Gy). Results MRT induced significantly longer tumor regrowth delay than did BB irradiation. A significant 24% reduction in blood vessel perfusion was observed 5 days after MRT, and the cell proliferation index was significantly lower in melanomas treated by MRT compared with BB. MRT provoked a greater induction of senescence in melanoma cells. Bio-Plex analyses revealed enhanced concentration of monocyte-attracting chemokines in the MRT group: MCP-1 at D5, MIP-1α, MIP-1β, IL12p40, and RANTES at D9. This was associated with leukocytic infiltration at D9 after MRT, attributed mainly to CD8 T cells, natural killer cells, and macrophages. Conclusions In light of its potential to disrupt blood vessels that promote infiltration of the tumor by immune cells and its induction of senescence, MRT could be a new therapeutic approach for radioresistant melanoma.

Purpose This study provides the first experimental application of multiscale three-dimensional (3D) X-ray Phase Contrast Imaging Computed Tomography (XPCI-CT) virtual histology for the inspection and quantitative assessment of the late stage effects of radio-induced lesions on lungs in a small animal model. Methods and Materials Healthy male Fischer rats were irradiated with X-ray standard broad beams and Microbeam Radiation Therapy (MRT), a high dose rate (14 kGy/s), FLASH spatially-fractionated X-ray therapy to avoid the beamlets smearing due to cardiosynchronous movements of the organs during the irradiation. After organ dissection, ex-vivo XPCI-CT was applied to all the samples and the results were quantitatively analysed and correlated to histologic data. Results XPCI-CT enables the 3D visualization of lung tissues with unprecedented contrast and sensitivity allowing alveoli, vessels and bronchi hierarchical visualization. XPCI-CT discriminates in 3D radio-induced lesions such as fibrotic scars, Ca/Fe deposits and, in addition, allows a full-organ accurate quantification of the fibrotic tissue within the irradiated organs. The radiation-induced fibrotic tissue content is less than 10% of the analyzed volume for all the MRT treated organs while it reaches the 34% in the case of irradiations with 50 Gy using a broad beam. Conclusions XPCI-CT is an effective imaging technique able to provide detailed 3D information for the assessment of lung pathology and treatment efficacy in a small animal model.

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Background: Microbeam Radiation Therapy (MRT) is an innovative approach in radiation oncology where a collimator subdivides the homogeneous radiation field into an array of co-planar, high-dose beams which are tens of micrometres wide and separated by a few hundred micrometres. Objective: This scoping review was conducted to map the available evidence and provide a comprehensive overview of the similarities, differences, and outcomes of all experiments that have employed animal models in MRT. Methods: We considered articles that employed animal models for the purpose of studying the effects of MRT. We searched in seven databases for published and unpublished literature. Two independent reviewers screened citations for inclusion. Data extraction was done by three reviewers. Results: After screening 5688 citations and 159 full-text papers, 95 articles were included, of which 72 were experimental articles. Here we present the animal models and pre-clinical radiation parameters employed in the existing MRT literature according to their use in cancer treatment, non-neoplastic diseases, or normal tissue studies. Conclusions: The study of MRT is concentrated in brain-related diseases performed mostly in rat models. An appropriate comparison between MRT and conventional radiotherapy (instead of synchrotron broad beam) is needed. Recommendations are provided for future studies involving MRT.

Background: Synchrotron Microbeam Radiotherapy (MRT) significantly improves local tumour control with minimal normal tissue toxicity. MRT delivers orthovoltage X-rays at an ultra-high &ldquo, FLASH&rdquo, dose rate in spatially fractionated beams, typically only few tens of micrometres wide. One of the biggest challenges in translating MRT to the clinic is its use of high peak doses, of around 300&ndash, 600 Gy, which can currently only be delivered by synchrotron facilities. Therefore, in an effort to improve the translation of MRT to the clinic, this work studied whether the temporal fractionation of traditional MRT into several sessions with lower, more clinically feasible, peak doses could still maintain local tumour control. Methods: Two groups of twelve C57Bl/6J female mice harbouring B16-F10 melanomas in their ears were treated with microbeams of 50 &micro, m in width spaced by 200 &micro, m from their centres. The treatment modality was either (i) a single MRT session of 401.23 Gy peak dose (7.40 Gy valley dose, i.e., dose between beams), or (ii) three MRT sessions of 133.41 Gy peak dose (2.46 Gy valley dose) delivered over 3 days in different anatomical planes, which intersected at 45 degrees. The mean dose rate was 12,750 Gy/s, with exposure times between 34.2 and 11.4 ms, respectively. Results: Temporally fractionated MRT ablated 50% of B16-F10 mouse melanomas, preventing organ metastases and local tumour recurrence for 18 months. In the rest of the animals, the median survival increased by 2.5-fold in comparison to the single MRT session and by 4.1-fold with respect to untreated mice. Conclusions: Temporally fractionating MRT with lower peak doses not only maintained tumour control, but also increased the efficacy of this technique. These results demonstrate that the solution to making MRT more clinically feasible is to irradiate with several fractions of intersecting arrays with lower peak doses. This provides alternatives to synchrotron sources where future microbeam radiotherapy could be delivered with less intense radiation sources.

The functional roles of the Caudate nucleus (Cd) are well known. Selective Cd lesions can be found in neurological disorders. However, little is known about the dynamics of the behavioral changes during progressive Cd ablation. Current stereotactic radiosurgery technologies allow the progressive ablation of a brain region with limited adverse effects in surrounding normal tissues. This could be of high interest for the study of the modified behavioral functions in relation with the degree of impairment of the brain structures. Using hypofractionated stereotactic radiotherapy combined with synchrotron microbeam radiation, we investigated, during one year after irradiation, the effects of unilateral radio-ablation of the right Cd on the behavior of Yucatan minipigs. The right Cd was irradiated to a minimal dose of 35.5 Gy delivered in three fractions. MRI-based morphological brain integrity and behavioral functions, i.e. locomotion, motivation/hedonism were assessed. We detected a progressive radio-necrosis leading to a quasi-total ablation one year after irradiation, with an additional alteration of surrounding areas. Transitory changes in the motivation/hedonism were firstly detected, then on locomotion, suggesting the influence of different compensatory mechanisms depending on the functions related to Cd and possibly some surrounding areas. We concluded that early behavioral changes related to eating functions are relevant markers for the early detection of ongoing lesions occurring in Cd-related neurological disorders.

International audience; Purpose: To analyze the effects of micro-beam irradiation (MBI) on the normal tissues of the mouse ear.Methods and Materials: Normal mouse ears are a unique model, which in addition to skin contain striated muscles, cartilage, blood and lymphatic vessels, and few hair follicles. This renders the mouse ear an excellent model for complex tissue studies. The ears of C57BL6 mice were exposed to MBI (50-mu m-wide micro-beams, spaced 200 mu m between centers) with peak entrance doses of 200, 400, or 800 Gy (at ultra-high dose rates). Tissue samples were examined histopathologically, with conventional light and electron microscopy, at 2, 7, 15, 30, and 240 days after irradiation (dpi). Sham-irradiated animals acted as controls.Results: Only an entrance dose of 800 Gy caused a significant increase in the thickness of both epidermal and dermal ear compartments seen from 15 to 30 dpi; the number of sebaceous glands was significantly reduced by 30 dpi. The numbers of apoptotic bodies and infiltrating leukocytes peaked between 15 and 30 dpi. Lymphatic vessels were prominently enlarged at 15 up to 240 dpi. Sarcomere lesions in striated muscle were observed after all doses, starting from 2 dpi; scar tissue within individual beam paths remained visible up to 240 dpi. Cartilage and blood vessel changes remained histologically inconspicuous.Conclusions: Normal tissues such as skin, cartilage, and blood and lymphatic vessels are highly tolerant to MBI after entrance doses up to 400 Gy. The striated muscles appeared to be the most sensitive to MBI. Those findings should be taken into consideration in future micro-beam radiation therapy treatment schedules. (C) 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

International audience; Microbeam radiation therapy (MRT) is a new form of preclinical radiotherapy using quasi-parallel arrays of synchrotron X-ray microbeams. While the deposition of several hundred Grays in the microbeam paths, the normal brain tissues presents a high tolerance which is accompanied by the permanence of apparently normal vessels. Conversely, the efficiency of MRT on tumor growth control is thought to be related to a preferential damaging of tumor blood vessels.The high resistance of the healthy vascular network was demonstrated in different animal models by in vivo biphoton microscopy, magnetic resonance imaging, and histological studies. While a transient increase in permeability was shown, the structure of the vessels remained intact. The use of a chick chorioallantoic membrane at different stages of development showed that the damages induced by microbeams depend on vessel maturation. In vivo and ultrastructural observations showed negligible effects of microbeams on the mature vasculature at late stages of development; nevertheless a complete destruction of the immature capillary plexus was found in the microbeam paths. The use of MRT in rodent models revealed a preferential effect on tumor vessels. Although no major modification was observed in the vasculature of normal brain tissue, tumors showed a denudation of capillaries accompanied by transient increased permeability followed by reduced tumor perfusion and finally, a decrease in number of tumor vessels. Thus, MRT is a very promising treatment strategy with pronounced tumor control effects most likely based on the anti-vascular effects of MRT.

Microbeam radiation therapy (MRT), a novel form of spatially fractionated radiotherapy (RT), uses arrays of synchrotron-generated X-ray microbeams (MB). MRT has been identified as a promising treatment concept that might be applied to patients with malignant central nervous system (CNS) tumours for whom, at the current stage of development, no satisfactory therapy is available yet. Preclinical experimental studies have shown that the CNS of healthy rodents and piglets can tolerate much higher radiation doses delivered by spatially separated MBs than those delivered by a single, uninterrupted, macroscopically wide beam. High-dose, high-precision radiotherapies such as MRT with reduced probabilities of normal tissue complications offer prospects of improved therapeutic ratios, as extensively demonstrated by results of experiments published by many international groups in the last two decades. The significance of developing MRT as a new RT approach cannot be understated. Up to 50% of cancer patients receive conventional RT, and any new treatment that provides better tumour control whilst preserving healthy tissue is likely to significantly improve patient outcomes.

Microbeam irradiation is spatially fractionated radiation on a micrometer scale. Microbeam irradiation with therapeutic intent has become known as microbeam radiation therapy (MRT). The basic concept of MRT was developed in the 1980s, but it has not yet been tested in any human clinical trial, even though there is now a large number of animal studies demonstrating its marked therapeutic potential with an exceptional normal tissue sparing effect. Furthermore, MRT is conceptually similar to macroscopic grid based radiation therapy which has been used in clinical practice for decades. In this review, the potential clinical applications of MRT are analysed for both malignant and non-malignant diseases.

Synchrotron microbeam radiation therapy (MRT) relies on the spatial fractionation of a synchrotron beam into parallel micron-wide beams allowing deposition of hectogray doses. MRT controls the intracranial tumor growth in rodent models while sparing normal brain tissues. Our aim was to identify the early biological processes underlying the differential effect of MRT on tumor and normal brain tissues. The expression of 28,000 transcripts was tested by microarray 6 hr after unidirectional MRT (400 Gy, 50 µm-wide microbeams, 200 µm spacing). The specific response of tumor tissues to MRT consisted in the significant transcriptomic modulation of 431 probesets (316 genes). Among them, 30 were not detected in normal brain tissues, neither before nor after MRT. Areg, Trib3 and Nppb were down-regulated, whereas all others were up-regulated. Twenty-two had similar expression profiles during the 2 weeks observed after MRT, including Ccnb1, Cdc20, Pttg1 and Plk1 related to the mitotic role of the Polo-like kinase (Plk) pathway. The up-regulation of Areg expression may indicate the emergence of survival processes in tumor cells triggered by the irradiation; while the modulation of the "mitotic role of Plk1" pathway, which relates to cytokinetic features of the tumor observed histologically after MRT, may partially explain the control of tumor growth by MRT. The identification of these tumor-specific responses permit to consider new strategies that might potentiate the antitumoral effect of MRT.

International audience; Synchrotron-generated X-ray (SRX) microbeams deposit high radiation doses to submillimetric targets whilst minimizing irradiation of neighboring healthy tissue. We developed a new radiosurgical method which demonstrably transects cortical brain tissue without affecting adjacent regions. We made such image-guided SRX microtransections in the left somatosensory cortex in a rat model of generalized epilepsy using high radiation doses (820 Gy) in thin (200 μm) parallel slices of tissue. This procedure, targeting the brain volume from which seizures arose, altered the abnormal neuronal activities for at least 9 weeks, as evidenced by a decrease of seizure power and coherence between tissue slices in comparison to the contralateral cortex. The brain tissue located between transections stayed histologically normal, while the irradiated micro-slices remained devoid of myelin and neurons two months after irradiation. This pre-clinical proof of concept highlights the translational potential of non-invasive SRX transections for treating epilepsies that are not eligible for resective surgery. Surgical resections for intractable epilepsies have reached impressive success rates in recent years 1,2. However, the management of seizures arising from eloquent cortical regions remains challenging due to high rates (up to 50%) of post-operative neurological deficits 3–6. Non-resective procedures such as vagus nerve stimulation 7 , scheduled deep brain stimulation 8 or closed-loop neurostimulation 9 , did not achieve seizure freedom in most cases. Multiple Subpial Transection (MST), developed by Morrell et al. 10 , is currently the most effective surgical technique applicable to eloquent cortex. Transections made with a special surgical knife at intervals of 5 mm interrupt horizontal intracortical fibers, while preserving vertical fibers. The procedure is based upon experimental evidence indicating that epileptogenic discharges require substantial side-to-side or horizontal interaction of cortical neurons 11 , whereas major functional properties of cortical tissue rely greatly on vertical fiber connections of the columnar units 12. MST reduces synchronized discharges from the epileptic focus and limits their spread without impairing the function of the transected cortex 13. However, MST is an invasive procedure that only reaches moderate rates of seizure freedom because of the limited access to deep cortical tissue/sulci 14,15 and the risk of post-operative deficits, such as brain edema, infections, and bleeding 16. Radiosurgery is an attractive alternative to surgical MST but radio-transections are currently unachievable with conventional radiosurgical devices and/or mega-voltage machines since their lateral dose profile does not provide the required sharp dose demarcations between irradiated and non-irradiated tissue slices 17,18. Further, the transection procedure requires focused delivery of hectogray doses 19 , currently only achievable at 3 rd generation synchrotron facilities. The assessment of the efficacy of synchrotron radiation for seizure management in rodents has been studied during the last few years using different methods and irradiation configurations 20–23. The purpose of the present study was to perform a novel method of non-invasive cortical transections in epileptic rats using synchrotron x-rays (SRX) and to quantify the induced changes of normal and pathological electrical activities. To achieve

Our goal was the visualizing the vascular damage and acute inflammatory response to micro- and minibeam irradiation in vivo. Microbeam (MRT) and minibeam radiation therapies (MBRT) are tumor treatment approaches of potential clinical relevance, both consisting of parallel X-ray beams and allowing the delivery of thousands of Grays within tumors. We compared the effects of microbeams (25–100 μm wide) and minibeams (200–800 μm wide) on vasculature, inflammation and surrounding tissue changes during zebrafish caudal fin regeneration in vivo. Microbeam irradiation triggered an acute inflammatory response restricted to the regenerating tissue. Six hours post irradiation (6 hpi), it was infiltrated by neutrophils and fli1a+ thrombocytes adhered to the cell wall locally in the beam path. The mature tissue was not affected by microbeam irradiation. In contrast, minibeam irradiation efficiently damaged the immature tissue at 6 hpi and damaged both the mature and immature tissue at 48 hpi. We demonstrate that vascular damage, inflammatory processes and cellular toxicity depend on the beam width and the stage of tissue maturation. Minibeam irradiation did not differentiate between mature and immature tissue. In contrast, all irradiation-induced effects of the microbeams were restricted to the rapidly growing immature tissue, indicating that microbeam irradiation could be a promising tumor treatment tool.

International audience; Rationale: An alternative approach for the improvement of radiotherapy consists in increasing differentially the radiation dose between tumors and healthy tissues using nanoparticles (NPs) that have been beforehand internalized into the tumor. These high-Z NPs can be photo-activated by monochromatic synchrotron X-rays, leading to a local dose enhancement delivered to the neighboring tumor cells[1]. This enhancement is due to secondary and Auger electrons expelled from the NPs by the radiations. In order to carry the NPs into the tumor center, macrophages are currently under study for their phagocytosis and diapedesis abilities[2] (cf. Figure adapted from [3] and [4]). In this study we characterized J774A.1 macrophages’ internalization kinetics and subcellular distribution of iron NPs and compared them to the internalization abilities of the F98 glioblastoma cell line.Materials and Methods: Three aspects of internalization were examined: first, the location of internalized NPs in J774A.1 macrophages and F98 glioblastoma cells following a 24h incubation with iron NPs (0.3 mg/mL in the cell culture medium) was determined by optical microscopy after cellslicing. Subsequently, the iron intake after a 24h incubation with NPs (0.3 mg/mL and 0.06 mg/mL in the cell culture medium) was characterized for the two types of cells using ICP-MS. Finally, the internalization dynamics were studied by live phase-contrast microscopy imagining for 11 hours and by absorbance measurements for 24 hours using a plate reader.Results: F98 tumor cells and J774A.1 macrophages are both able to endocytose NPs: we measured ~61±10 pg of internalized iron per macrophage compared with ~33±5 pg per F98 cell (initial iron concentration: 0.3 mg/mL in culture medium). F98 internalizing NPs for 10 hours showed stress signs during the first minutes after the NPs injection, but behaved like F98 control cells during the rest of the experiment. Finally, we determined that the internalization kinetics for J774A.1 had a typical saturation time of one hour.Conclusion: Macrophages seem to be promising vectors for NPs, being able to endocytose and retain in their cytoplasm larger quantities of NPs than tumor cells. Our following studies will attempt to shed light on their other potential abilities as “Trojan Horses”.

Grid (or sieve) therapy ("Gitter-" oder "Siebtherapie"), spatially fractionated kilo- and megavolt X-ray therapy, was invented in 1909 by Alban Kohler, a radiologist in Wiesbaden, Germany. He tested it on several patients before 1913 using approximately 60-70kV Hittorf-Crookes tubes. Kohler pushed the X-ray tube's lead-shielded housing against a stiff grid of 1 mm-square iron wires woven 3.0-3.5mm on center, taped tightly to the skin over a thin chamois. Numerous islets unshielded by iron in the pressure-blanched skin were irradiated with up to about 6 erythema doses (ED). The skin was then thoroughly cleansed, disinfected, and bandaged; delayed punctate necrosis healed in several weeks. Although grid therapy was disparaged or ignored until the 1930s, it has been used successfully since then to shrink bulky malignancies. Also, advanced cancers in rats and mice have been mitigated or ablated using Kohler's concept since the early 1990s by unidirectional or stereotactic exposure to an array of nearly parallel microplanar (25-75μm-wide) beams of very intense, moderately hard (median energy approximately 100 keV) synchrotron-generated X rays spaced 0.1-0.4mm on center. Such beams maintain sharp edges at high doses well beneath the skin yet confer little toxicity. They could palliate some otherwise intractable malignancies, perhaps in young children too, with tolerable sequelae. There are plans for such studies in larger animals.

To evaluate microbeam radiation therapy (MRT), for brain tumor treatment, the bystander effect in nonirradiated companion animals was investigated. Adult rats were irradiated with 35 or 350 Gy at the European Synchrotron Research Facility using homogenous irradiation (HR) or MRT to the right brain hemisphere. The irradiated rats were housed with nonirradiated rats. After 48 hours, all rats were euthanized and the frontal lobe proteome was analyzed using 2-dimensional electrophoresis and mass spectrometry. Proteome changes were determined by analysis of variance ( P < .05). Homogenous irradiation increased serum albumin, heat shock protein 71 (HSP-71), triosephosphate isomerase (TPI), fructose bisphosphate aldolase (FBA), and prohibitin and decreased dihydrolipoyl dehydrogenase (DLD) and pyruvate kinase. Microbeam radiation therapy increased HSP-71, FBA, and prohibitin, and decreased aconitase, dihydropyrimidinase, TPI, tubulin DLD, and pyruvate kinase. Cage mates with HR irradiated rats showed increased HSP-71 and FBA and decreased pyruvate kinase, DLD, and aconitase. Cage mates with MRT irradiated rats showed increased HSP-71, prohibitin, and FBA and decreased aconitase and DLD. Homogenous irradiation proteome changes indicated tumorigenesis, while MRT proteome changes indicated an oxidative stress response. The bystander effect of proteome changes appeared antitumorigenic and inducing radioresistance. This investigation also supports the need for research into prohibitin interaction with HSP-70/71 chaperones and cancer therapy.

International audience; The technical feasibility of temporal and spatial fractionations of the radiation dose has been evaluated using synchrotron microbeam radiation therapy for brain tumors in rats. A significant increase in lifespan (216%, p < 0.0001) resulted when three fractions of microbeam irradiation were applied to the tumor through three different ports, orthogonal to each other, at 24 h intervals. However, there were no long-term survivors, and immunohistological studies revealed that 9 L tumors were not entirely ablated.

Microbeam radiation therapy (MRT), a form of experimental radiosurgery of tumours using multiple parallel, planar, micrometres-wide, synchrotron-generated X-ray beams ('microbeams'), can safely deliver radiation doses to contiguous normal animal tissues that are much higher than the maximum doses tolerated by the same normal tissues of animals or patients from any standard millimetres-wide radiosurgical beam. An array of parallel microbeams, even in doses that cause little damage to radiosensitive developing tissues, for example, the chick chorioallantoic membrane, can inhibit growth or ablate some transplanted malignant tumours in rodents. The cerebella of 100 normal 20 to 38g suckling Sprague-Dawley rat pups and of 13 normal 5 to 12kg weanling Yorkshire piglets were irradiated with an array of parallel, synchrotron-wiggler-generated X-ray microbeams in doses overlapping the MRT-relevant range (about 50-600Gy) using the ID17 wiggler beamline tangential to the 6GeV electron synchrotron ring at the European Synchrotron Radiation Facility in Grenoble, France. Subsequent favourable development of most animals over at least 1 year suggests that MRT might be used to treat children's brain tumours with less risk to the development of the central nervous system than is presently the case when using wider beams.

International audience; Objective Synchrotron radiation has shown high therapeutic potential in small animal models of malignant brain tumours. However, more studies are needed to understand the radiobiological effects caused by the delivery of high doses of spatially fractionated x-rays in tissue. The purpose of this study was to explore the use of the gamma-H2AX antibody as a marker for dose deposition in the brain of rats after synchrotron microbeam radiation therapy (MRT). Methods Normal and tumour-bearing Wistar rats were exposed to 35, 70 or 350 Gy of MRT to their right cerebral hemisphere. The brains were extracted either at 4 or 8 hours after irradiation and immediately placed in formalin. Sections of paraffin-embedded tissue were incubated with anti gamma-H2AX primary antibody. Results While the presence of the C6 glioma does not seem to modulate the formation of gamma-H2AX in normal tissue, the irradiation dose and the recovery versus time are the most important factors affecting the development of gamma-H2AX foci. Our results also suggest that doses of 350 Gy can trigger the release of bystander signals that significantly amplify the DNA damage caused by radiation and that the gamma-H2AX biomarker does not only represent DNA damage produced by radiation, but also damage caused by bystander effects. Conclusion In conclusion, we suggest that the gamma-H2AX foci should be used as biomarker for targeted and non-targeted DNA damage after synchrotron radiation rather than a tool to measure the actual physical doses

The question of whether bystander and abscopal effects are the same is unclear. Our experimental system enables us to address this question by allowing irradiated organisms to partner with unexposed individuals. Organs from both animals and appropriate sham and scatter dose controls are tested for expression of several endpoints such as calcium flux, role of 5HT, reporter assay cell death and proteomic profile. The results show that membrane related functions of calcium and 5HT are critical for true bystander effect expression. Our original inter-animal experiments used fish species whole body irradiated with low doses of X-rays, which prevented us from addressing the abscopal effect question. Data which are much more relevant in radiotherapy are now available for rats which received high dose local irradiation to the implanted right brain glioma. The data were generated using quasi-parallel microbeams at the biomedical beamline at the European Synchrotron Radiation Facility in Grenoble France. This means we can directly compare abscopal and “true” bystander effects in a rodent tumour model. Analysis of right brain hemisphere, left brain and urinary bladder in the directly irradiated animals and their unirradiated partners strongly suggests that bystander effects (in partner animals) are not the same as abscopal effects (in the irradiated animal). Furthermore, the presence of a tumour in the right brain alters the magnitude of both abscopal and bystander effects in the tissues from the directly irradiated animal and in the unirradiated partners which did not contain tumours, meaning the type of signal was different.

International audience; Epilepsy is one of the most important neurological diseases. It concerns about 1% of the population worldwide. Despite the discovery of new molecules, one third of epileptic patients are resistant to anti-epileptic drugs and among them only a few can benefit from resective surgery. In this context, radiotherapy is an interesting alternative to the other treatments and several clinical devices exist (e.g., Gamma Knife®). The European Synchrotron Radiation Facility offers the possibility to develop new methods of radiosurgery and to study their antiepileptic effects. Here, we discuss several studies that we performed recently to test and try to understand the antiepileptic effects of X-ray synchrotron microbeams in different animal models of epilepsy. We showed a decrease of seizures after Interlaced Microbeam Radiotherapy (IntMRT) of the somatosensory cortex, known as the seizure generator, in a genetic model of absence epilepsy. These antiepileptic effects were stable over 4 months and with low tissular and functional side-effects. The irradiated pyramidal neurons still displayed their physiological activity but did not synchronize anymore. We also obtained a lasting suppression of seizures after IntMRT of the dorsal hippocampus in a mouse model of mesiotemporal lobe epilepsy. However, an important variability of antiepileptic efficiency was observed probably due to the small size of the targeted structure. Despite these encouraging proofs-of-concepts, there is now a need to adapt IntMRT to other models of epilepsy in rodents which are close to refractory forms of epilepsy in human patients and to implement this approach to non-human primates, before moving to clinical trials.

International audience; Among rodent models for brain tumors, the 9L gliosarcoma is one of the most widely used. Our 9L-European Synchrotron Radiation Facility (ESRF) model was developed from cells acquired at the Brookhaven National Laboratory (NY, USA) in 1997 and implanted in the right caudate nucleus of syngeneic Fisher rats. It has been largely used by the user community of the ESRF during the last decade, for imaging, radiotherapy, and chemotherapy, including innovative treatments based on particular irradiation techniques and/or use of new drugs. This work presents a detailed study of its characteristics, assessed by magnetic resonance imaging (MRI), histology, immunohistochemistry, and cytogenetic analysis. The data used for this work were from rats sampled in six experiments carried out over a 3-year period in our lab (total number of rats = 142). The 9L-ESRF tumors were induced by a stereotactic inoculation of 10(4) 9L cells in the right caudate nucleus of the brain. The assessment of vascular parameters was performed by MRI (blood volume fraction and vascular size index) and by immunostaining of vessels (rat endothelial cell antigen-1 and type IV collagen). Immunohistochemistry and regular histology were used to describe features such as tumor cell infiltration, necrosis area, nuclear pleomorphism, cellularity, mitotic characteristics, leukocytic infiltration, proliferation, and inflammation. Moreover, for each of the six experiments, the survival of the animals was assessed and related to the tumor growth observed by MRI or histology. Additionally, the cytogenetic status of the 9L cells used at ESRF lab was investigated by comparative genomics hybridization analysis. Finally, the response of the 9L-ESRF tumor to radiotherapy was estimated by plotting the survival curves after irradiation. The median survival time of 9L-ESRF tumor-bearing rats was highly reproducible (19-20 days). The 9L-ESRF tumors presented a quasi-exponential growth, were highly vascularized with a high cellular density and a high proliferative index, accompanied by signs of inflammatory responses. We also report an infiltrative pattern which is poorly observed on conventional 9 L tumor. The 9L-ESRF cells presented some cytogenetic specificities such as altered regions including CDK4, CDKN2A, CDKN2B, and MDM2 genes. Finally, the lifespan of 9L-ESRF tumor-bearing rats was enhanced up to 28, 35, and 45 days for single doses of 10, 20, and 2 × 20 Gy, respectively. First, this report describes an animal model that is used worldwide. Second, we describe few features typical of our model if compared to other 9L models worldwide. Altogether, the 9L-ESRF tumor model presents characteristics close to the human high-grade gliomas such as high proliferative capability, high vascularization and a high infiltrative pattern. Its response to radiotherapy demonstrates its potential as a tool for innovative radiotherapy protocols.

International audience; Inter-animal signaling from irradiated to non-irradiated organisms has been demonstrated for whole body irradiated mice and also for fish. The aim of the current study was to look at radiotherapy style limited exposure to part of the body using doses relevant in preclinical therapy. High dose homogenous field irradiation and the use of irradiation in the microbeam radiation therapy mode at the European Synchrotron Radiation Facility (ESRF) at Grenoble was tested by giving high doses to the right brain hemisphere of the rat. The right and left cerebral hemispheres and the urinary bladder were later removed to determine whether abscopal effects could be produced in the animals and also whether effects occurred in cage mates housed with them. The results show strong bystander signal production in the contra-lateral brain hemisphere and weaker effects in the distant bladder of the irradiated rats. Signal strength was similar or greater in each tissue in the cage mates housed for 48hrs with the irradiated rats. Our results support the hypothesis that proximity to an irradiated animal induces signalling changes in an unirradiated partner. If similar signaling occurs between humans, the results could have implications for caregivers and hospital staff treating radiotherapy patients

The distribution of the sst2A receptor was investigated, using immunohistochemistry, with the specific antipeptide antibody R2-88, in a total of 120 tumors of the nervous and the neuroendocrine systems, including small-cell lung carcinomas, medulloblastomas, neuroblastomas, pheochromocytomas, and paragangliomas. The great majority of the tumor samples, frozen or formalin-fixed, showed a positive immunohistochemical staining with R2-88, and an excellent correlation with receptor autoradiography using 125I[Tyr3]-octreotide. Whereas small-cell lung carcinomas and medulloblastomas had a predominantly plasma membrane staining, pheochromocytomas and neuroblastomas had variable ratios of cell surface and intracellular staining. Strikingly, a preferentially cytoplasmic staining was seen in tumors with a high level of somatostatin gene expression, whereas a more plasma membranous staining was seen in tumors lacking somatostatin messenger RNA. Specificity of both the plasma membrane and the cytoplasmic staining pattern was confirmed in immunoblots, which showed the immunoreactive receptor migrating as a characteristic 70-kDa broad band. In both immunohistochemical and immunoblotting experiments, staining was abolished by antibody blockade with 100 nM antigen peptide. These results describe, for the first time, the localization of the sst2A receptor protein in human small-cell lung carcinomas, medulloblastomas, neuroblastomas, and paragangliomas. Moreover, it is the first report investigating possible causes for distinct subcellular localizations of sst2A in human tissues. We show that the subcellular distribution of the receptor may be dependent on the surrounding somatostatin concentration, consistent with both the known effect of somatostatin to cause sst2A receptor internalization and an autocrine regulation of tumors by the peptide they produce. Moreover, our demonstration that the sst2A receptor can be identified in this group of tumors using simple immunohistochemical methods in formalin-fixed, paraffin-embedded material opens numerous diagnostic, therapeutic, and prognostic opportunities.

Infection with Helicobacter pylori (Hp) induces the formation of lymphoid tissue in the stomach and the occasional development of primary gastric B-cell lymphomas. We have studied the expression of 2 chemokines that attract B lymphocytes, BCA-1 and SLC, in gastric tissue samples obtained from patients with chronic gastritis induced by Hp infection or nonsteroidal anti-inflammatory drugs, as well as from patients with Hp-associated low-grade and high-grade gastric lymphomas. High-level expression of BCA-1 and its receptor, CXCR5, was observed in all mucosal lymphoid aggregates and in the mantle zone of all secondary lymphoid follicles in Hp-induced gastric mucosa-associated lymphoid tissue (MALT). Follicular dendritic cells and B lymphocytes are possible sources of BCA-1, which is not expressed by T lymphocytes, macrophages, or CD1a(+) dendritic cells. Strong expression of BCA-1 and CXCR5 was also detected in the transformed B cells of gastric MALT lymphomas. By contrast, SLC was confined almost exclusively to endothelial cells in and outside the lymphoid tissue. Only scant, occasional SLC expression was observed in the marginal zone of MALT follicles. Our findings indicate that BCA-1, which functions as a homing chemokine in normal lymphoid tissue, is induced in chronic Hp gastritis and is involved in the formation of lymphoid follicles and gastric lymphomas of the MALT type.

Receptors for regulatory peptides, such as somatostatin or vasoactive intestinal peptide (VIP), expressed at high density by neoplastic cells, can be instrumental for tumor diagnosis and therapy. Little is known about the expression of neurotensin receptors in human tumors. In the present study, 464 human neoplasms of various types were investigated for their neurotensin receptor content by in vitro receptor autoradiography on tissue sections using 125I-[Tyr3]-neurotensin as radioligand. Neurotensin receptors were identified and localized in tumor cells of 11/17 Ewing's sarcomas, 21/40 meningiomas, 10/23 astrocytomas, 5/13 medulloblastomas, 7/24 medullary thyroid cancers and 2/8 small cell lung cancers. They were rarely found in non-small cell lung cancers and breast carcinomas; they were absent in prostate, ovarian, renal cell and hepatocellular carcinomas, neuroendocrine gut tumors, pituitary adenomas, schwannomas, neuroblastomas and lymphomas. When present, the receptors bound with nanomolar affinity neurotensin and acetyl-neurotensin-(8-13), with lower affinity neuromedin N, diethylenetriamine penta-acetic acidneurotensin-(8-13) and SR 48692, but not neurotensin-(1-11). They were all of the NT1 type, without high affinity for levocabastine. Further, in 2 receptor-positive Ewing's sarcomas, neurotensin mRNA was detected by in situ hybridization techniques. Since neurotensin is known to stimulate cell proliferation, the presence of neurotensin receptors in human neoplasia may be of biological relevance, possibly as an integrative part of an autocrine feedback mechanism of tumor growth stimulation.

Thymomas are usually found in the anterior mediastinum, the normal location of the thymus. Involvement of the pericardium by thymic tumors is seen in invasive or metastasized thymoma. Very rarely, thymomas arise primarily in the pericardium. These tumors are believed to derive from thymic tissue which was misplaced in the pericardium during embryologic development. In contrast to patients with orthotopic thymoma who commonly suffer from paraneoplastic diseases, especially myasthenia gravis, patients with intrapericardial thymoma manifestations mainly have symptoms of congestive heart failure which are caused by local complications of tumor growth. In this study, we present two cases of thymoma involving the pericardium. Both tumors were polygonal-oval cell thymomas. In one of the cases diagnosis of an entirely intrapericardial thymoma was established by autopsy. In the other case, explorative thoracotomy revealed massive pericardial and pleural tumor manifestations. The latter tumor showed a peculiar histological pattern with multiple glomeruloid bodies, a finding reported only once for thymomas.

Receptors for cholecystokinin (CCK), gastrin, neurotensin, somatostatin and vasoactive intestinal peptide (VIP) are over-expressed in several human tumors, where they have diagnostic and therapeutic implications. Since reports on the expression of these peptide receptors in primary gastric and colonic adenocarcinomas are either non-existent or conflicting, a detailed evaluation with particular emphasis on the tissue localization was undertaken. CCK-A, CCK-B, neurotensin, somatostatin and VIP receptors were localized by in vitro receptor autoradiography with iodinated radioligands on histological sections of surgical samples of 27 gastric and 25 colonic adenocarcinomas. CCK-A, CCK-B and neurotensin-1 receptors were found in a minority of both tumor types. Somatostatin receptors were found in 18/27 gastric and 2/25 colonic cancers. VIP receptors were found in 14/26 gastric and 23/25 colonic cancers; subtype characterization suggests VIP1 receptors. In addition, resected tumor samples contained non-malignant tissues (mucosa, smooth muscle, nerves or vessels) with high amounts of the various peptide receptors. Therefore, regulatory peptide receptors are expressed differentially in gastric and colonic cancers but also very frequently in "contaminating" non-malignant tissues. Since results using morphological techniques are superior to those using homogenates, we recommend that localization of these receptors to the tissues should always be attempted, to minimize receptor over-estimation in tumors and to prevent spurious results.

Regulatory peptides, such as vasoactive intestinal peptide (VIP), somatostatin (SS), or substance P (SP), are considered to play a role in immune regulation. To localize the targets of these peptides in the human immune system, their receptors have been evaluated with in vitro receptor autoradiography in lymph nodes, tonsils, appendix, Peyer's patches, spleen, and thymus. The three peptide receptors were detected in all lymphoid tissues tested, but, unexpectedly, usually in distinct compartments. In lymph nodes, palatine tonsils, vermiform appendix, and Peyer's patches, VIP receptors were found in the CD3 positive zone around lymphoid follicles; SS receptors in the germinal centers of secondary follicles; and SP receptors mainly in interfollicular blood vessels. In the spleen, VIP receptors were detected in periarterial lymphatic sheaths, SS receptors in the red pulp, and SP receptors in the central arteries. In the thymus, VIP receptors were present in cortex and medulla, SS receptors in the medulla, and SP receptors in blood vessels. For comparison, cholecystokinin (CCK)-A and -B receptors were not demonstrated in any of these tissues. These results suggest a strong compartmentalization of the three peptide receptors in human lymphoid tissues and represent the molecular basis for the understanding of a very complex and interactive mode of action of these peptides.

International audience; PURPOSE: Synchrotron microbeam radiation therapy (MRT) is an innovative irradiation modality based on spatial fractionation of a high-dose X-ray beam into lattices of microbeams. The increase in lifespan of brain tumor-bearing rats is associated with vascular damage but the physiological consequences of MRT on blood vessels have not been described. In this manuscript, we evaluate the oxygenation changes induced by MRT in an intracerebral 9L gliosarcoma model. METHODS: Tissue responses to MRT (two orthogonal arrays (2×400Gy)) were studied using magnetic resonance-based measurements of local blood oxygen saturation (MR_SO2) and quantitative immunohistology of RECA-1, Type-IV collagen and GLUT-1, marker of hypoxia. RESULTS: In tumors, MR_SO2 decreased by a factor of 2 in tumor between day 8 and day 45 after MRT. This correlated with tumor vascular remodeling, i.e. decrease in vessel density, increases in half-vessel distances (×5) and GLUT-1 immunoreactivity. Conversely, MRT did not change normal brain MR_SO2, although vessel inter-distances increased slightly. CONCLUSION: We provide new evidence for the differential effect of MRT on tumor vasculature, an effect that leads to tumor hypoxia. As hypothesized formerly, the vasculature of the normal brain exposed to MRT remains sufficiently perfused to prevent any hypoxia.

International audience; Radiotherapy has shown some efficacy for epilepsies but the insufficient confinement of the radiation dose to the pathological target reduces its indications. Synchrotron-generated X-rays overcome this limitation and allow the delivery of focalized radiation doses to discrete brain volumes via interlaced arrays of microbeams (IntMRT). Here, we used IntMRT to target brain structures involved in seizure generation in a rat model of absence epilepsy (GAERS). We addressed the issue of whether and how synchrotron radiotherapeutic treatment suppresses epileptic activities in neuronal networks. IntMRT was used to target the somatosensory cortex (S1Cx), a region involved in seizure generation in the GAERS. The antiepileptic mechanisms were investigated by recording multisite local-field potentials and the intracellular activity of irradiated S1Cx pyramidal neurons in vivo. MRI and histopathological images displayed precise and sharp dose deposition and revealed no impairment of surrounding tissues. Local-field potentials from behaving animals demonstrated a quasi-total abolition of epileptiform activities within the target. The irradiated S1Cx was unable to initiate seizures, whereas neighboring non-irradiated cortical and thalamic regions could still produce pathological oscillations. In vivo intracellular recordings showed that irradiated pyramidal neurons were strongly hyperpolarized and displayed a decreased excitability and a reduction of spontaneous synaptic activities. These functional alterations explain the suppression of large-scale synchronization within irradiated cortical networks. Our work provides the first post-irradiation electrophysiological recordings of individual neurons. Altogether, our data are a critical step towards understanding how X-ray radiation impacts neuronal physiology and epileptogenic processes.

The present study examined whether growth characteristics of diffusely growing non-Hodgkin's lymphomas (NHL) may differ as a function of stage. Among 105 NHL of various types and sub-types (REAL [Revised European-American Lymphoma] classification), localized (Ann Arbor pathologic stages I + II) lymphomas exhibited clearly higher indices for mitotic activity, apoptosis and cell turnover, as well as a significantly lower percentage of cells containing immunohistochemically detectable bcl.2 protein, than disseminated (stages III + IV) NHL A similar pattern emerged when high-grade (Kiel classification) lymphomas only were evaluated. Low-grade NHL showed analogous, but less marked, stage-dependent characteristics, with the exception of median percentages of bcl-2 + cells, which remained comparable in all stages. Our findings are consistent with the notion that dissemination of diffusely growing NHL is usually associated with reduced cell turnover and, in high-grade lymphomas, with the generation of longer-lived cells.

Besides a prominent mononuclear cell infiltration of the islets of Langerhans, nonobese diabetic (NOD) mice also show massive cellular infiltrates of the submandibular and lacrimal glands concomitant with histological signs of tissue damage. To obtain insights into the mechanisms operative during the initiation and progression of tissue damage, we followed by in situ hybridization the appearance of cells containing mRNA of the gene encoding the proinflammatory cytokine TNF-alpha in the cellular infiltrates. Cells expressing TNF-alpha are mainly located in infiltrates, are absent in nonaffected glands, and are preferentially found among CD4 T cells. Secretion of TNF-alpha by gland-infiltrating cells was confirmed by an ELISPOT procedure. Direct evidence for an instrumental role of TNF-alpha in initiation and progression of submandibular and lacrimal gland infiltration is provided by the observed significant reduction in the extent of infiltration in nonobese diabetic mice transgenic for a soluble TNF receptor p55 fused to the Fc part of human IgG3. This protection from infiltration is paralleled by decreased expression of the adhesion molecules ICAM-1 and VCAM-1 in submandibular and lacrimal glands. These data suggest a central role of TNF-alpha in the initiation and progression of autoimmune tissue destruction of salivary glands and indicate beneficial effects of soluble TNF receptors in the treatment of organ-specific autoimmune diseases.

BACKGROUND & AIMS: Neuropeptide receptors, expressed at high density by neoplastic cells, can be instrumental for diagnosis and therapy. However, little is known about neuropeptides and neuropeptide receptors in the tumor bed. The aim of this study was to study the expression of two neuropeptide receptors in peritumoral blood vessels. METHODS: Somatostatin and substance P receptors were measured using in vitro receptor autoradiography in well-defined submucosal and subserosal vessels located in the immediate surroundings of human colorectal carcinomas or in normal colon taken at increasing distances from the tumor. RESULTS: Both receptors were 3-5-fold overexpressed in the host veins within a 2-cm-wide area surrounding human primary colorectal carcinomas compared with veins located at 5-10-cm distance in normal gut tissue. The expression of peritumoral vascular somatostatin receptor and substance P receptor appears independent of receptor expression in the tumor, of tumor stage, and of the grade of local inflammation. CONCLUSIONS: These biochemical alterations of veins in the immediate vicinity of tumors represent a novel tumor-related site of expression for the two receptors and may point to a new biological principle of host-tumor interaction. Thus, vascular regulatory mechanisms in the tumor bed may be important for tumor development and metastasis and may be used as a target for tumor therapy. (Gastroenterology 1996 Jun;110(6):1719-26)

International audience; Microbeam radiation therapy (MRT), a preclinical form of radiosurgery, uses spatially fractionated micrometre-wide synchrotron-generated X-ray beams. As MRT alone is predominantly palliative for animal tumors, the effects of the combination of MRT and a newly synthesized chemotherapeutic agent JAI-51 on 9L gliosarcomas have been evaluated. Fourteen days (D14) after implantation (D0), intracerebral 9LGS-bearing rats received either MRT, JAI-51 or both treatments. JAI-51, alone or immediately after MRT, was administered three times per week. Animals were kept up to ∼20 weeks after irradiation or sacrificed at D16 or D28 after treatment for cell cycle analysis. MRT plus JAI-51 increased significantly the lifespan compared with MRT alone (p = 0.0367). JAI-51 treatment alone had no effect on rat survival. MRT alone or associated with JAI-51 induced a cell cycle blockade in G2/M (p < 0.01) while the combined treatment also reduced the proportion of G0/G1 cells. At D28 after irradiation, MRT and MRT/JAI-51 had a smaller cell blockade effect in the G2/M phase owing to a significant increase in tumor cell death rate (8c). The combination of MRT and JAI-51 increases the survival of 9LGS-bearing rats by inducing endoreduplication of DNA and tumor cell death; further, it slowed the onset of tumor growth resumption two weeks after treatment.

Grid (or sieve) therapy ("Gitter-" oder "Siebtherapie"), spatially fractionated kilo- and megavolt X-ray therapy, was invented in 1909 by Alban Köhler, a radiologist in Wiesbaden, Germany. He tested it on several patients before 1913 using approximately 60-70kV Hittorf-Crookes tubes. Köhler pushed the X-ray tube's lead-shielded housing against a stiff grid of 1 mm-square iron wires woven 3.0-3.5mm on center, taped tightly to the skin over a thin chamois. Numerous islets unshielded by iron in the pressure-blanched skin were irradiated with up to about 6 erythema doses (ED). The skin was then thoroughly cleansed, disinfected, and bandaged; delayed punctate necrosis healed in several weeks. Although grid therapy was disparaged or ignored until the 1930s, it has been used successfully since then to shrink bulky malignancies. Also, advanced cancers in rats and mice have been mitigated or ablated using Köhler's concept since the early 1990s by unidirectional or stereotactic exposure to an array of nearly parallel microplanar (25-75μm-wide) beams of very intense, moderately hard (median energy approximately 100 keV) synchrotron-generated X rays spaced 0.1-0.4mm on center. Such beams maintain sharp edges at high doses well beneath the skin yet confer little toxicity. They could palliate some otherwise intractable malignancies, perhaps in young children too, with tolerable sequelae. There are plans for such studies in larger animals.

International audience; PURPOSE: The purpose is to evaluate effects of a new radiotherapy protocol, microbeam radiation therapy, on the artery wall. In previous studies on animal models, it was shown that capillaries recover well from hectogray doses of X-rays delivered in arrays of narrow (< or = 50 microm) beams with a minimum spacing of 200 microm. Here, short- and long-term effects of comparable microplanar beam configurations on the saphenous artery of the mouse hind leg were analyzed in situ by use of nonlinear optics and compared with histopathologic findings. METHODS AND MATERIALS: The left hind leg of normal mice including the saphenous artery was irradiated by an array of 26 microbeams of synchrotron X-rays (50 microm wide, spaced 400 microm on center) with peak entrance doses of 312 Gy and 2,000 Gy. RESULTS: The artery remained patent, but narrow arterial smooth muscle cell layer segments that were in the microplanar beam paths became atrophic and fibrotic in a dose-dependent pattern. The wide tunica media segments between those paths hypertrophied, as observed in situ by two-photon microscopy and histopathologically. CONCLUSIONS: Clinical risks of long-delayed disruption or occlusion of nontargeted arteries from microbeam radiation therapy will prove less than corresponding risks from broad-beam radiosurgery, especially if peak doses are kept below 3 hectograys.

Purpose The aim of this study is to explore the effects of Microbeam Radiation (MR) on vascular biology using the chick chorio-allantoic membrane (CAM). Methods CAMs were irradiated with multiple planar synchrotron X-ray beams at doses between 100 and 300 Gy and were evaluated morphologically and in vivo at days 8 and 12. Results In vivo monitoring and morphological investigations of day 8 CAM immature vasculature 6 hr after MR revealed a near total destruction of the capillary plexus within the beams’ width. Surprisingly, arteries and veins were not affected. Conversely, at day 12, only well defined lesions in the microvasculature (mature ones, covered by pericytes) were observed. After 300 Gy MR, TEM revealed enlargement of the interendothelial cell junctions which could explain the oedema, the microvessels along the beam path showed signs of disruption and apoptosis. The remaining vasculature recovered rapidly and CAM regained its normal thickness. Between 1 hr and 6 hr no additional morphological changes were observed. Conclusions The effects of MR are most likely mediated by capillary damage. The vascular toxicity of MR depends on the stage of capillary maturation: the immature vessels are much more vulnerable than the mature ones. The physiological effects of MR appear within a short time, the most important structural alterations being present between 15 and 60 minutes after irradiation.

International audience; PURPOSE: Synchrotron microbeam radiation therapy (MRT) relies on spatial fractionation of the incident photon beam into parallel micron-wide beams. Our aim was to analyze the effects of MRT on normal brain and 9L gliosarcoma tissues, particularly on blood vessels. METHODS AND MATERIALS: Responses to MRT (two arrays, one lateral, one anteroposterior (2 × 400 Gy), intersecting orthogonally in the tumor region) were studied during 6 weeks using MRI, immunohistochemistry, and vascular endothelial growth factor Western blot. RESULTS: MRT increased the median survival time of irradiated rats (×3.25), significantly increased blood vessel permeability, and inhibited tumor growth; a cytotoxic effect on 9L cells was detected 5 days after irradiation. Significant decreases in tumoral blood volume fraction and vessel diameter were measured from 8 days after irradiation, due to loss of endothelial cells in tumors as detected by immunochemistry. Edema was observed in the normal brain exposed to both crossfired arrays about 6 weeks after irradiation. This edema was associated with changes in blood vessel morphology and an overexpression of vascular endothelial growth factor. Conversely, vascular parameters and vessel morphology in brain regions exposed to one of the two arrays were not damaged, and there was no loss of vascular endothelia. CONCLUSIONS: We show for the first time that preferential damage of MRT to tumor vessels versus preservation of radioresistant normal brain vessels contributes to the efficient palliation of 9L gliosarcomas in rats. Molecular pathways of repair mechanisms in normal and tumoral vascular networks after MRT may be essential for the improvement of such differential effects on the vasculature.

In the thymus, based on the reactivity of their T-cell receptor with self-MHC and antigenic peptides, developing immature T-cells undergo positive and negative selection. Cells recognising self-peptides and MHC with high affinity are considered autoreactive, and thus potentially harmful, and are eliminated by induction of apoptotic cell death. Thymic negative selection is, however, only incomplete and autoreactive T-cells escape into the periphery. It is not the presence of autoreactive mature T- and B-lymphocytes as the underlying cause of tissue destruction and development of autoimmune diseases, but their uncontrolled and excessive clonal expansion upon activation by self-antigen. Thus, potent regulatory mechanisms must keep these autoreactive cells under control to avoid their inappropriate activation. Recent evidence indicates that death receptors of the tumour necrosis factor receptor family play a central role in mediating antigen receptor-induced suicide of autoreactive T-lymphocytes. Defects in these apoptosis-inducing regulatory mechanisms may result in the development of autoimmune diseases. Therefore, enhancing the cell's own suicide program, offers a most attractive therapeutic target for the treatment of autoimmune diseases.

Background & Aims: The localization and functional role of cholecystokinin (CCK) receptor proteins in normal and diseased human pancreas, particularly in ductal pancreatic carcinomas, remain unclear. Methods: Tissue samples of normal human pancreas, chronic pancreatitis, and ductal pancreatic carcinomas were investigated under carefully controlled conditions for expression of CCK1 and CCK2 receptor messenger RNA (mRNA) and proteins using in situ hybridization and in vitro CCK receptor autoradiography by means of subtype-selective analogues. Synaptophysin immunohistochemistry was used concomitantly for optimal identification of islets, nerves, and tumor areas with neuroendocrine features. Results: CCK2 receptor mRNA and proteins were found abundantly in human pancreatic islets in normal pancreas and chronic pancreatitis. CCK1 receptor proteins were found occasionally in small-sized pancreatic nerves, whereas acini expressed a low density of CCK2 receptors in a few cases of chronic pancreatitis. Ductal pancreatic carcinomas rarely expressed CCK receptors; a few receptor-positive tumors, often characterized by neuroendocrine differentiation, expressed the CCK2 receptor at the mRNA or protein level. However, the main source of CCK receptors in the pancreatic tumor samples consisted of CCK2-expressing islets and/or CCK1-expressing nerves rather than neoplastic tissue. Conclusions: These data indicate that the presence of CCK receptors in human ductal pancreatic tumor samples is mainly due to CCK2 expression in residual pancreatic islets and CCK1 in pancreatic nerves. Pancreatic acini and ductal pancreatic tumor cells very rarely express CCK2 receptors. These observations suggest that CCK analogues may not be of clinical use to target most of these cancers.

Preclinical experiments are carried out with approximately 20-30 microm wide, approximately 10 mm high parallel microbeams of hard, broad-"white"-spectrum x rays (approximately 50-600 keV) to investigate microbeam radiation therapy (MRT) of brain tumors in infants for whom other kinds of radiotherapy are inadequate and/or unsafe. Novel physical microdosimetry (implemented with MOSFET chips in the "edge-on" mode) and Monte Carlo computer-simulated dosimetry are described here for selected points in the peak and valley regions of a microbeam-irradiated tissue-equivalent phantom. Such microbeam irradiation causes minimal damage to normal tissues, possible because of rapid repair of their microscopic lesions. Radiation damage from an array of parallel microbeams tends to correlate with the range of peak-valley dose ratios (PVDR). This paper summarizes comparisons of our dosimetric MOSFET measurements with Monte Carlo calculations. Peak doses at depths22 mm are 18% less than Monte Carlo values, whereas those depths22 mm and valley doses at all depths investigated (2 mm-62 mm) are within 2-13% of the Monte Carlo values. These results lend credence to the use of MOSFET detector systems in edge-on mode for microplanar irradiation dosimetry.

Among patients with differentiated thyroid carcinoma (diffTCa), the rare hyperfunctioning or toxic TCa (ToxTCa) was diagnosed when scintigraphic hot thyroid areas were attributable only to diffTCa (diameter1 cm by pathological examination) and/or total thyroidectomy failed to induce hypothyroidism. Of 924 cases of all TCa (papillary diffTCa 47.3%, follicular diffTCa 44.2%, others 8.5%), 19 had ToxTCa (2.1%, 15 of 19 follicular, 4 of 19 papillary, P = 0.008). These received a more intensive radioiodine therapy (average cumulated (131)I activities 21.8 vs. 15.2 GBq, P0.01). Five-year survival rates for ToxTCa (n = 19, 56%) and diffTCa (n = 545, 94.5%) differed [hazard ratio 4.8, 95% confidence interval (CI) 2.8-8.1, P = 0.001]. However, the differences were attenuated by matching ToxTCa and diffTCa (n = 57, 5-yr survival rate 74%) for age, sex, and histopathologic type (hazard ratio 2.1, 95% CI 1.13-3.9, P = 0.02). Correcting statistically for M(1) against M(0) stage distribution resulted in a further reduction of the hazard ratio (hazard ratio 1.8, 95% CI 0.93-3.48, P = 0.08). An M(1) stage is an important prognostic factor in ToxTCa patients. Thus, ToxTCa, treated with higher activities of (131)I, has a survival prognosis close to that of matched diffTCa cases, both groups consisting mainly (79%) of follicular subtypes.

International audience; Since approximately two thirds of epileptic patients are non-eligible for surgery, local axonal fiber transections might be of particular interest for them. Micrometer to millimeter wide synchrotron-generated X-ray beamlets produced by spatial fractionation of the main beam could generate such fiber disruptions non-invasively. The aim of this work was to optimize irradiation parameters for the induction of fiber transections in the rat brain white matter by exposure to such beamlets. For this purpose, we irradiated cortex and external capsule of normal rats in the antero-posterior direction with a 4 mm×4 mm array of 25 to 1000 µm wide beamlets and entrance doses of 150 Gy to 500 Gy. Axonal fiber responses were assessed with diffusion tensor imaging and fiber tractography; myelin fibers were examined histopathologically. Our study suggests that high radiation doses (500 Gy) are required to interrupt axons and myelin sheaths. However, a radiation dose of 500 Gy delivered by wide minibeams (1000 µm) induced macroscopic brain damage, depicted by a massive loss of matter in fiber tractography maps. With the same radiation dose, the damage induced by thinner microbeams (50 to 100 µm) was limited to their paths. No macroscopic necrosis was observed in the irradiated target while overt transections of myelin were detected histopathologically. Diffusivity values were found to be significantly reduced. A radiation dose ≤ 500 Gy associated with a beamlet size of < 50 µm did not cause visible transections, neither on diffusion maps nor on sections stained for myelin. We conclude that a peak dose of 500 Gy combined with a microbeam width of 100 µm optimally induced axonal transections in the white matter of the brain.

Lymph nodes with Hodgkin disease (HD) harbor few neoplastic cells in a marked leukocytic infiltrate. Since chemokines are likely to be involved in the recruitment of these leukocytes, the expression of potentially relevant chemokines and chemokine receptors were studied in lymph nodes from 24 patients with HD and in 5 control lymph nodes. The expression of regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta was analyzed by in situ hybridization and that of CCR3 and CCR5 by immunohistochemistry and flow cytometry. It was found that, overall, the expression of all 4 chemokines was markedly enhanced, but the cellular source was different. RANTES was expressed almost exclusively by T cells whereas the expression of MCP-1, MIP-1alpha, and MIP-1beta was confined largely to macrophages. In control lymph nodes, chemokine expression was low, with the exception of MIP-1alpha in macrophages. CCR3 and CCR5 were highly expressed in T cells of HD involved but not of control lymph nodes. CCR3 was equally distributed in CD4+ and CD8+ cells, but CCR5 was associated largely with CD4+ cells. In HD lymph nodes, CCR3 and CCR5 were also expressed in B cells, which normally do not express these receptors. All these chemokines and receptors studied, by contrast, were absent in the neoplastic cells. It was concluded that chemokines are involved in the formation of the HD nonneoplastic leukocytic infiltrate. Expression of CCR3 and CCR5 appears to be characteristic of HD, but the roles of these receptors' up-regulation for the disease process remain unclear.