Good Timing Matters: The Spatially Fractionated High Dose Rate Boost Should Come First.

Monoplanar microbeam irradiation (MBI) and pencilbeam irradiation (PBI) are two new concepts of high dose rate radiotherapy, combined with spatial dose fractionation at the micrometre range. In a small animal model, we have explored the concept of integrating MBI or PBI as a simultaneously integrated boost (SIB), either at the beginning or at the end of a conventional, low-dose rate schedule of 5x4 Gy broad beam (BB) whole brain radiotherapy (WBRT). MBI was administered as array of 50 µm wide, quasi-parallel microbeams. For PBI, the target was covered with an array of 50 µm × 50 µm pencilbeams. In both techniques, the centre-to-centre distance was 400 µm. To assure that the entire brain received a dose of at least 4 Gy in all irradiated animals, the peak doses were calculated based on the daily BB fraction to approximate the valley dose. The results of our study have shown that the sequence of the BB irradiation fractions and the microbeam SIB is important to limit the risk of acute adverse effects, including epileptic seizures and death. The microbeam SIB should be integrated early rather than late in the irradiation schedule

Microbeam Irradiation as a Simultaneously Integrated Boost in a Conventional Whole-Brain Radiotherapy Protocol.

Microbeam radiotherapy (MRT), an experimental high-dose rate concept with spatial fractionation at the micrometre range, has shown a high therapeutic potential as well as good preservation of normal tissue function in pre-clinical studies. We investigated the suitability of MRT as a simultaneously integrated boost (SIB) in conventional whole-brain irradiation (WBRT). A 174 Gy MRT SIB was administered with an array of quasi-parallel, 50 µm wide microbeams spaced at a centre-to-centre distance of 400 µm either on the first or last day of a 5 × 4 Gy radiotherapy schedule in healthy adult C57 BL/6J mice and in F98 glioma cell cultures. The animals were observed for signs of intracranial pressure and focal neurologic signs. Colony counts were conducted in F98 glioma cell cultures. No signs of acute adverse effects were observed in any of the irradiated animals within 3 days after the last irradiation fraction. The tumoricidal effect on F98 cell in vitro was higher when the MRT boost was delivered on the first day of the irradiation course, as opposed to the last day. Therefore, the MRT SIB should be integrated into a clinical radiotherapy schedule as early as possible

TRANSMISSION OF SIGNALS FROM RATS RECEIVING HIGH DOSES OF MICROBEAM RADIATION TO CAGE MATES: AN INTER-MAMMAL BYSTANDER EFFECT

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

SYRA3 COST Action – Microbeam radiation therapy: Roots and prospects

AbstractMicrobeam radiation therapy (MRT) is an irradiation modality for therapeutic purposes which uses arrays of collimated quasi parallel microbeams, each up to 100 μm wide, to deliver high radiation doses. Several studies have reported the extraordinary tolerance of normal tissues to MRT irradiation; conversely, MRT has been shown to be highly efficient on tumor growth control. The original and most widely developed application of MRT, yet in the preclinical phase, consists in using spatially fractionated X-ray beams issued from a synchrotron radiation source in the treatment of brain tumors. More recently, MRT has been tested in successful pioneering assays to reduce or interrupt seizures in preclinical models of epilepsy. The MRT concept has also been extended to proton therapy. The development of MRT towards its clinical implementation is presently driven by an EU-supported consortium of laboratories from 16 countries within the COST Action TD1205 (SYRA3). The results of the first SYRA3 workshop on “Radiation Therapy with Synchrotron Radiation: Achievements and Challenges” held in Krakow (Poland) during March 25–26 2014 are summarized in this issue with an overview presented in this paper. The papers reflect the multidisciplinary international activities of SYRA3. The topics covered in this focus issue include medical physics aspects, pre-clinical studies, clinical applications, and an industrial perspective; finally an outlook towards future prospects of compact sources and proton microbeams

3D Imaging of Striatal Transplants in a Small Animal Model of Huntington’s Disease

High-resolution imaging in small animal models of neurologic disease is a technical challenge. In a pilot project, we have explored a non-destructive synchrotron imaging technique for the 3D visualization of intracerebral tissue transplants in a well-established small animal model of Huntington’s disease. Four adult female Sprague Dawley rats each received injections of 0.12 M quinolinic acid (QA) into two target positions in the left striatum, thus creating unilateral left-sided striatal lesions similar to those frequently seen in patients suffering from Huntington’s disease. One week after lesioning, the animals received transplants prepared from whole ganglionic eminences (wGEs) obtained from 13- to 14-day-old rat embryos. Of the four lesioned animals, three received transplants of GNP-loaded cells and one animal received a transplant of naïve cells, serving as control. Post-mortem synchrotron-based microCT was used to obtain images of the neurotransplants. The images obtained of GNP-loaded tissue transplants at the synchrotron corresponded in size and shape to the histological images of transplants developed from naïve cells. Thus, we conclude that non-destructive synchrotron imaging techniques such as phase-contrast imaging are suitable to obtain high-resolution images of GNP-loaded tissue transplants

γ-H2AX as a Marker for Dose Deposition in the Brain of Wistar Rats after Synchrotron Microbeam Radiation

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 γ-H2AX antibody as a marker for dose deposition in the brain of rats after synchrotron microbeam radiation therapy (MRT).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 γ-H2AX primary antibody.While the presence of the C6 glioma does not seem to modulate the formation of γ-H2AX in normal tissue, the irradiation dose and the recovery versus time are the most important factors affecting the development of γ-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 γ-H2AX biomarker does not only represent DNA damage produced by radiation, but also damage caused by bystander effects.In conclusion, we suggest that the γ-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

Investigation of Abscopal and Bystander Effects in Immunocompromised Mice After Exposure to Pencilbeam and Microbeam Synchrotron Radiation.

Out-of-field effects are of considerable interest in radiotherapy. The mechanisms are poorly understood but are thought to involve signaling processes, which induce responses in non-targeted cells and tissues. The immune response is thought to play a role. The goal of this research was to study the induction of abscopal effects in the bladders of NU-Foxn1 mice after irradiating their brains using Pencil Beam (PB) or microbeam (MRT) irradiation at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Athymic nude mice injected with F98 glioma cells into their right cerebral hemisphere 7 d earlier were treated with either MRT or PB. After recovery times of 2, 12, and 48 h, the urinary bladders were extracted and cultured as tissue explants for 24 h. The growth medium containing the potential signaling factors was harvested, filtered, and transferred to HaCaT reporter cells to assess their clonogenic survival and calcium signaling potential. The results show that in the tumor-free mice, both treatment modalities produce strong bystander/abscopal signals using the clonogenic reporter assay; however, the calcium data do not support a calcium channel mediated mechanism. The presence of a tumor reduces or reverses the effect. PB produced significantly stronger effects in the bladders of tumor-bearing animals. The authors conclude that immunocompromised mice produce signals, which can alter the response of unirradiated reporter cells; however, a novel mechanism appears to be involved

Pencilbeam irradiation technique for whole brain radiotherapy: technical and biological challenges in a small animal model.

We have conducted the first in-vivo experiments in pencilbeam irradiation, a new synchrotron radiation technique based on the principle of microbeam irradiation, a concept of spatially fractionated high-dose irradiation. In an animal model of adult C57 BL/6J mice we have determined technical and physiological limitations with the present technical setup of the technique. Fifty-eight animals were distributed in eleven experimental groups, ten groups receiving whole brain radiotherapy with arrays of 50 µm wide beams. We have tested peak doses ranging between 172 Gy and 2,298 Gy at 3 mm depth. Animals in five groups received whole brain radiotherapy with a center-to-center (ctc) distance of 200 µm and a peak-to-valley ratio (PVDR) of ∼ 100, in the other five groups the ctc was 400 µm (PVDR ∼ 400). Motor and memory abilities were assessed during a six months observation period following irradiation. The lower dose limit, determined by the technical equipment, was at 172 Gy. The LD50 was about 1,164 Gy for a ctc of 200 µm and higher than 2,298 Gy for a ctc of 400 µm. Age-dependent loss in motor and memory performance was seen in all groups. Better overall performance (close to that of healthy controls) was seen in the groups irradiated with a ctc of 400 µm

Single-cell resolution in high-resolution synchrotron X-ray CT imaging with gold nanoparticles

Gold nanoparticles are excellent intracellular markers in X-ray imaging. Having shown previously the suitability of gold nanoparticles to detect small groups of cells with the synchrotron-based computed tomography (CT) technique both ex vivo and in vivo, it is now demonstrated that even single-cell resolution can be obtained in the brain at least ex vivo. Working in a small animal model of malignant brain tumour, the image quality obtained with different imaging modalities was compared. To generate the brain tumour, 1x10^5 C6 glioma cells were loaded with gold nanoparticles and implanted in the right cerebral hemisphere of an adult rat. Raw data were acquired with absorption X-ray CT followed by a local tomography technique based on synchrotron X-ray absorption yielding single-cell resolution. The reconstructed synchrotron X-ray images were compared with images obtained by small animal magnetic resonance imaging. The presence of gold nanoparticles in the tumour tissue was verified in histological sections

Use of synchrotron medical microbeam irradiation to investigate radiation-induced bystander and abscopal effects in vivo

AbstractThe 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