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International audience; Synchrotron-generated microplanar beams (microbeams) provide the most stereo-selective irradiation modality known today. This novel irradiation modality has been shown to control seizures originating from eloquent cortex causing no neurological deficit in experimental animals. To test the hypothesis that application of microbeams in the hippocampus, the most common source of refractory seizures, is safe and does not induce severe side effects, we used microbeams to induce transections to the hippocampus of healthy rats. An array of parallel microbeams carrying an incident dose of 600 Gy was delivered to the rat hippocampus. Immunohistochemistry of phosphorylated gamma-H2AX showed cell death along the microbeam irradiation paths in rats 48 hours after irradiation. No evident behavioral or neurological deficits were observed during the 3-month period of observation. MR imaging showed no signs of radio-induced edema or radionecrosis 3 months after irradiation. Histological analysis showed a very well preserved hippocampal cytoarchitecture and confirmed the presence of clear-cut microscopic transections across the hippocampus. These data support the use of synchrotron-generated microbeams as a novel tool to slice the hippocampus of living rats in a minimally invasive way, providing (i) a novel experimental model to study hippocampal function and (ii) a new treatment tool for patients affected by refractory epilepsy induced by mesial temporal sclerosis.

Purpose: Microbeam radiation therapy is an innovative treatment approach in radiation therapy that uses arrays of a few tens of micrometer wide and a few hundreds of micrometer spaced planar x-ray beams as treatment fields. In preclinical studies these fields efficiently eradicated tumors while normal tissue could effectively be spared. However, development and clinical application of microbeam radiation therapy is impeded by a lack of suitable small scale sources. Until now, only large synchrotrons provide appropriate beam properties for the production of microbeams. Methods: In this work, a conventional x-ray tube with a small focal spot and a specially designed collimator are used to produce microbeams for preclinical research. The applicability of the developed source is demonstrated in a pilot in vitro experiment. The properties of the produced radiation field are characterized by radiochromic film dosimetry. Results: 50 mu m wide and 400 mu m spaced microbeams were produced in a 20x20 mm(2) sized microbeam field. The peak to valley dose ratio ranged from 15.5 to 30, which is comparable to values obtained at synchrotrons. A dose rate of up to 300 mGy/s was achieved in the microbeam peaks. Analysis of DNA double strand repair and cell cycle distribution after in vitro exposures of pancreatic cancer cells (Panc1) at the x-ray tube and the European Synchrotron leads to similar results. In particular, a reduced G2 cell cycle arrest is observed in cells in the microbeam peak region. Conclusions: At its current stage, the source is restricted to in vitro applications. However, moderate modifications of the setup may soon allow in vivo research in mice and rats. (C) 2016 Author(s).

Introduction: X-ray microbeam radiation therapy (MRT) as a novel tumor treatment strategy deposits high doses in spatially fractionated X-ray beamlets promising reduced normal tissue toxicity, compared to conventional irradiation, and a better tumor control. Radiobiological studies of MRT with kilovoltage X-rays are mainly performed at synchrotron radiation facilities with high costs and space requirements. The Munich Compact Light Source (MuCLS) is a laboratory-sized and cost-effective source based on inverse Compton scattering of infrared laser photons [1]. Currently the most widely accepted method for assessment of treatment efficiencies is tumor growth delay with subcutaneous tumors in the hind leg of small animals. However, a new model is required for MRT with kilovoltage X-ray beams which only allow for short penetration depths. Therefore, we successfully developed a setup for a growth delay study in a tumor-bearing mouse ear model for investigation of MRT at the MuCLS. In addition, we successfully established a protocol to isolate tumor cells from irradiated tumors for evaluation of radiobiological effect on cellular level. Materials & Methods: The dose rate of the MuCLS was improved with the installation of a polycapillary collimation optic. A W-Air collimator was inserted to get a collimated X-ray beam with 50 μm wide microbeams and a center-to-center distance of 350 μm. We implemented the mouse ear tumor model with a human head and neck cancer cell line FaDu [2] suspended in extracellular matrix and subcutaneously injected into the right ear of NMRI (nu/nu) mice. After reaching a size of 2x2 mm2 tumors were irradiated using doses of either 3 and 5 Gy with 25 keV X-rays at the MuCLS. Tumor growth delay was determined with a caliper over a follow-up period of 30 days and compared between MRT, homogeneous and control mice. Animals were sacrificed when tumors reached the 15-fold initial volume. A single tumor cell suspension was prepared from excised tumors for in-vitro studies. The analysis of radiosensitivity by colony formation assay and stable chromosomal aberrations by two-color fluorescence in-situ hybridization is still on-going. Results: We successfully installed a setup at the MuCLS which allows irradiation of tumors in small animals and implemented a xenograft tumor model in mouse ears. Homogeneously irradiated tumors showed a growth delay at 5 Gy compared to control mice. There was no tumor growth delay after MRT and homogeneous irradiation at 3 Gy. Tumor cells from irradiated tumors were successfully isolated and cultured. Preliminary data shows an increased radiosensitivity of tumor cells originating from homogeneously and MRT-irradiated tumors compared to control tumor cells. Conclusion: This innovative approach allowed the irradiation of tumors in a mouse ear model at a novel laser-based X-ray source, the MuCLS. Homogeneous irradiation at MuCLS induced a tumor growth delay at 5 Gy. In addition, we successfully validated a protocol for tumor cell isolation for investigations of radiation-induced effects. Supported by the DFG Cluster of Excellence: Munich-Centre for Advanced Photonics. References: [1] Eggl et al., J. Synchrotron Rad. (2016) 23: 1137 [2] Beyreuther et al., PLos One (2017)

Microbeam Radiation Therapy is an innovative pre-clinical strategy which uses arrays of parallel, tens of micrometres wide kilo-voltage photon beams to treat tumours. These x-ray beams are typically generated on a synchrotron source. It was shown that these beam geometries allow exceptional normal tissue sparing from radiation damage while still being effective in tumour ablation. A final biological explanation for this enhanced therapeutic ratio has still not been found, some experimental data support an important role of the vasculature. In this work, the effect of microbeams on a normal microvascular network of the cerebral cortex was assessed in computer simulations and compared to the effect of homogeneous, seamless exposures at equal energy absorption. The anatomy of a cerebral microvascular network and the inflicted radiation damage were simulated to closely mimic experimental data using a novel probabilistic model of radiation damage to blood vessels. It was found that the spatial dose fractionation by microbeam arrays significantly decreased the vascular damage. The higher the peak-to-valley dose ratio, the more pronounced the sparing effect. Simulations of the radiation damage as a function of morphological parameters of the vascular network demonstrated that the distribution of blood vessel radii is a key parameter determining both the overall radiation damage of the vasculature and the dose-dependent differential effect of microbeam irradiation.

Introduction: The tumor growth delay assay is a well-accepted technique in experimental animal tumor models for the measurement of the response to treatments. Tumor growth delay assays were mostly performed with subcutaneous xenograft tumors in the hind leg of small animals. However, some radiation qualities with low energy and/or very small irradiation fields cannot use this method. This study was performed to test a new irradiation setup at the Small Animal Radiation Research Platform (SARRP, Xtrahl Ltd.) which can be especially used to irradiate very small tumors with low energy X-rays. Methods: This study was performed with a human head and neck cancer cell line (FaDu). 100 000 FaDu cells were suspended in Matrigel® and subcutaneously injected at the right ear of immunocompromised NMRI nu/nu mice. Tumors with a size of 2x2 mm2 were irradiated with 3 Gy and 6 Gy operating the SARRP at 70 kVp X-rays. Tumor growth was determined over a follow-up of 20 days with a caliper. The tumor growth delay was compared between homogeneously and non-irradiated mice. 20 days after irradiation tumor cells were transferred in cell culture. Results: In this pilot study using 70 kVp X-rays, six tumor-bearing mice were irradiated with either 3 or 6 Gy. Three tumor-bearing mice served as a control. The tumor volume doubling time of unirradiated tumors was 2.75 ± 0.4 days. Out of three, one mouse showed an obvious tumor growth delay at 3 Gy. However, all tumors irradiated with 6 Gy were controlled. The tumor cells which were transferred into cell culture medium showed normal growth characteristics. Conclusion and Outlook: We successfully implemented a xenograft tumor system in mouse ears and irradiations of 2x2 mm2 tumors at the SARRP. The mouse ear tumor model allows an accurate and simple method to determine the tumor volume. In future, this tumor-bearing mouse ear model will enable irradiations which are limited due to small irradiation fields and/or low X-ray energies. Moreover, it is possible to isolate tumor cells out of the mouse ear for future in-vitro analysis. This new method could be used at the first brilliant and compact synchrotron X-ray source (Munich Compact Light Source) where the dose can be deposited by spatially fractionated X-ray beamlets like microbeam radiation therapy (MRT). Acknowledgements: This work was supported by the DFG-Cluster of Excellence ‘‘Munich-Centre for Advanced Photonics’’.

The first microbeam irradiation study at the biomedical beamline SYRMEP of the Elettra-Sincrotrone Trieste, the technical setup of the experiment and the results of the associated cell culture study in murine melanoma cells are reported., It has been shown previously both in vitro and in vivo that microbeam irradiation (MBI) can control malignant tumour cells more effectively than the clinically established concepts of broad beam irradiation. With the aim to extend the international capacity for microbeam research, the first MBI experiment at the biomedical beamline SYRMEP of the Italian synchrotron facility ELETTRA has been conducted. Using a multislit collimator produced by the company TECOMET, arrays of quasi-parallel microbeams were successfully generated with a beam width of 50 µm and a centre-to-centre distance of 400 µm. Murine melanoma cell cultures were irradiated with a target dose of approximately 65 Gy at a mean photon energy of ∼30 keV with a dose rate of 70 Gy s−1 and a peak-to-valley dose of ∼123. This work demonstrated a melanoma cell reduction of approximately 80% after MBI. It is suggested that, while a high energy is essential to achieve high dose rates in order to deposit high treatment doses in a short time in a deep-seated target, for in vitro studies and for the treatment of superficial tumours a spectrum in the lower energy range might be equally suitable or even advantageous.