Chalcone JAI‐51 improves efficacy of synchrotron microbeam radiation therapy of brain tumors

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 (<2c) and a proportional increase of endoreplicative cells (>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.     

Radiobiological features of the anti‐cancer strategies involving synchrotron X‐rays

Synchrotrons are opening new paths in innovative anti‐cancer radiotherapy strategies. Indeed, the fluence of X‐rays induced by synchrotrons is so high (10⁶ times higher than standard medical irradiators) that it enables the production of X‐ray beams tunable in energy (monochromatic beams) and in size (micrometric beams). Monochromatic synchrotron X‐ray beams theoretically permit photoactivate high‐Z elements to be introduced in or close to tumours in order to increase the yield of damage by enhanced energy photoabsorption. This is notably the case of attempts with iodinated contrast agents used in tumour imaging (the computed tomography therapy approach) and with platinated agents used in chemotherapy (the PAT‐Plat approach). Micrometric synchrotron X‐ray beams theoretically permit very high radiation doses to accumulate in tumours by using arrays of parallel microplanar beams that spare the surrounding tissues (the microbeam radiation therapy approach). These anti‐cancer applications of synchrotron radiation have been developed at the European Synchrotron Radiation Facility to be applied to glioma, one of the tumour tissues most refractory to standard treatments. In the present paper the molecular and cellular mechanisms involved in these three approaches are reviewed, in the context of recent advances in radiobiology. Furthermore, by considering the unavoidable biases, an attempt to propose a comparison of the different results obtained in preclinical trials dealing with rats bearing tumours is given.     

Fluorescence imaging of reactive oxygen species by confocal laser scanning microscopy for track analysis of synchrotron X‐ray photoelectric nanoradiator dose: X‐ray pump–optical probe

Bursts of emissions of low‐energy electrons, including interatomic Coulomb decay electrons and Auger electrons (0–1000 eV), as well as X‐ray fluorescence produced by irradiation of large‐Z element nanoparticles by either X‐ray photons or high‐energy ion beams, is referred to as the nanoradiator effect. In therapeutic applications, this effect can damage pathological tissues that selectively take up the nanoparticles. Herein, a new nanoradiator dosimetry method is presented that uses probes for reactive oxygen species (ROS) incorporated into three‐dimensional gels, on which macrophages containing iron oxide nanoparticles (IONs) are attached. This method, together with site‐specific irradiation of the intracellular nanoparticles from a microbeam of polychromatic synchrotron X‐rays (5–14 keV), measures the range and distribution of OH radicals produced by X‐ray emission or superoxide anions () produced by low‐energy electrons. The measurements are based on confocal laser scanning of the fluorescence of the hydroxyl radical probe 2‐[6‐(4′‐amino)phenoxy‐3H‐xanthen‐3‐on‐9‐yl] benzoic acid (APF) or the superoxide probe hydroethidine‐dihydroethidium (DHE) that was oxidized by each ROS, enabling tracking of the radiation dose emitted by the nanoradiator. In the range 70 µm below the irradiated cell, radicals derived mostly from either incident X‐ray or X‐ray fluorescence of ION nanoradiators are distributed along the line of depth direction in ROS gel. In contrast, derived from secondary electron or low‐energy electron emission by ION nanoradiators are scattered over the ROS gel. ROS fluorescence due to the ION nanoradiators was observed continuously to a depth of 1.5 mm for both oxidized APF and oxidized DHE with relatively large intensity compared with the fluorescence caused by the ROS produced solely by incident primary X‐rays, which was limited to a depth of 600 µm, suggesting dose enhancement as well as more penetration by nanoradiators. In conclusion, the combined use of a synchrotron X‐ray microbeam‐irradiated three‐dimensional ROS gel and confocal laser scanning fluorescence microscopy provides a simple dosimetry method for track analysis of X‐ray photoelectric nanoradiator radiation, suggesting extensive cellular damage with dose‐enhancement beyond a single cell containing IONs.     

Preclinical radiotherapy at the Australian Synchrotron's Imaging and Medical Beamline: instrumentation,dosimetry and a small‐animal feasibility study

Therapeutic applications of synchrotron X‐rays such as microbeam (MRT) and minibeam (MBRT) radiation therapy promise significant advantages over conventional clinical techniques for some diseases if successfully transferred to clinical practice. Preclinical studies show clear evidence that a number of normal tissues in animal models display a tolerance to much higher doses from MRT compared with conventional radiotherapy. However, a wide spread in the parameters studied makes it difficult to make any conclusions about the associated tumour control or normal tissue complication probabilities. To facilitate more systematic and reproducible preclinical synchrotron radiotherapy studies, a dedicated preclinical station including small‐animal irradiation stage was designed and installed at the Imaging and Medical Beamline (IMBL) at the Australian Synchrotron. The stage was characterized in terms of the accuracy and reliability of the vertical scanning speed, as this is the key variable in dose delivery. The measured speed was found to be within 1% of the nominal speed for the range of speeds measured by an interferometer. Furthermore, dose measurements confirm the expected relationship between speed and dose and show that the measured dose is independent of the scan direction. Important dosimetric parameters such as peak dose, valley dose, the collimator output factor and peak‐to‐valley dose ratio are presented for 5 mm × 5 mm, 10 mm × 10 mm and 20 mm × 20 mm field sizes. Finally, a feasibility study on three glioma‐bearing rats was performed. MRT and MBRT doses were prescribed to achieve an average dose of 65 Gy in the target, and magnetic resonance imaging follow‐up was performed at various time points after irradiation to follow the tumour volume. Although it is impossible to draw conclusions on the different treatments with such a small number of animals, the feasibility of end‐to‐end preclinical synchrotron radiotherapy studies using the IMBL preclinical stage is demonstrated.     

Adaption of a diffractometer for time‐resolved X‐ray resonant magnetic scattering

A new set‐up is presented to measure element‐selective magnetization dynamics using the ALICE chamber [Grabis et al. (2003), Rev. Sci. Instrum. 74 , 4048–4051] at the BESSY II synchrotron at the Helmholtz‐Zentrum Berlin. A magnetic‐field pulse serves as excitation, and the magnetization precession is probed by element‐selective X‐ray resonant magnetic scattering. With the use of single‐bunch‐generated X‐rays a temporal resolution well below 100 ps is reached. The ALICE diffractometer environment enables investigations of thin films, described here, multilayers and laterally structured samples in reflection or diffuse scattering geometry. The combination of the time‐resolved set‐up with a cryostat in the ALICE chamber will allow temperature‐dependent studies of precessional magnetization dynamics and of damping constants to be conducted over a large temperature range and for a large variety of systems in reflection geometry.     

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, 1 × 10⁵ 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.     

Specific molecular and cellular events induced by irradiated X‐ray photoactivatable drugs raise the problem of co‐toxicities: particular consequences for anti‐cancer synchrotron therapy

Synchrotrons are capable of producing intense low‐energy X‐rays that enable the photoactivation of high‐Z elements. Photoactivation therapy (PAT) consists of loading tumors with photoactivatable drugs and thereafter irradiating them at an energy, generally close to the K‐edge of the element, that enhances the photoelectric effect. To date, three major photoactivatable elements are used in PAT: platinum (cisplatin and carboplatin), iodine (iodinated contrast agents and iododeoxyuridine) and gadolinium (motexafin gadolinium). However, the molecular and cellular events specific to PAT and the radiobiological properties of these photoactivatable drugs are still misknown. Here, it is examined how standard and synchrotron X‐rays combined with photoactivatable drugs impact on the cellular response of human endothelial cells. These findings suggest that the radiolysis products of the photoactivatable drugs may participate in the synergetic effects of PAT by increasing the severity of radiation‐induced DNA double‐strand breaks. Interestingly, subpopulation of highly damaged cells was found to be a cellular pattern specific to PAT. The data show that the efficiency of emerging anti‐cancer modalities involving synchrotron photoactivation strongly depends on the choice of photoactivatable drugs, and important series of experiments are required to secure their clinical transfer before applying to humans.     

A scanning Kelvin probe for synchrotron investigations: the in situ detection of radiation‐induced potential changes

A wide range of high‐performance X‐ray surface/interface characterization techniques are implemented nowadays at every synchrotron radiation source. However, these techniques are not always `non‐destructive' because possible beam‐induced electronic or structural changes may occur during X‐ray irradiation. As these changes may be at least partially reversible, an in situ technique is required for assessing their extent. Here the integration of a scanning Kelvin probe (SKP) set‐up with a synchrotron hard X‐ray interface scattering instrument for the in situ detection of work function variations resulting from X‐ray irradiation is reported. First results, obtained on bare sapphire and sapphire covered by a room‐temperature ionic liquid, are presented. In both cases a potential change was detected, which decayed and vanished after switching off the X‐ray beam. This demonstrates the usefulness of a SKP for in situ monitoring of surface/interface potentials during X‐ray materials characterization experiments.     

Morphological study of early‐stage lung cancer using synchrotron radiation

In the present study the feasibility of applying synchrotron radiation to the morphological study of early‐stage lung cancer has been investigated. Lewis lung cancer was implanted and grown in a nude mouse for different periods, and imaged using phase‐contrast synchrotron X‐rays. Morphological differences were clearly shown between the normal lung and cancerous tissues at this early stage. Irregular and tortuous angiogenesis were found in the periphery region of the developing lung cancer. Results from this study indicate that synchrotron X‐rays can be used for imaging cancer development and progression with minimal invasion.     

Characterization of Micro‐ and Nanoscale LuPO4:Pr3+,Nd3+ with Strong UV‐C Emission to Reduce X‐Ray Doses in Radiation Therapy

UV‐C emitting nanoscale scintillators can be used to sensitize cancer cells selectively against X‐rays during radiation therapy, due to the lethal DNA lesions caused by UV‐C photons. Unfortunately, nanoscale particles (NPs) show decreased UV‐C emission intensity. In this paper, the influence of different Nd³⁺ concentrations on the UV‐C emission of micro‐ and nanoscale LuPO₄:Pr³⁺ is investigated upon X‐ray irradiation and vacuum UV excitation (160 nm). Co‐doped LuPO₄ results in increased UV‐C emission independent of excitation source due to energy transfer from Nd³⁺ to Pr³⁺. The highest UV‐C emission intensity is observed for LuPO₄:Pr³⁺,Nd³⁺(1%,2.5%) upon X‐ray irradiation. Finally, LuPO₄ NPs co‐doped with different dopant concentrations are synthesized, and the biological efficacy of the combined approach (X‐rays and UV‐C) is assessed using the colony formation assay. Cell culture experiments confirm increased cell death compared to X‐rays alone due to the formation of UV‐specific DNA damages, supporting the feasibility of this approach.     

Densification of sol–gel silica thin films induced by hard X‐rays generated by synchrotron radiation

In this article the effects induced by exposure of sol–gel thin films to hard X‐rays have been studied. Thin films of silica and hybrid organic–inorganic silica have been prepared via dip‐coating and the materials were exposed immediately after preparation to an intense source of light of several keV generated by a synchrotron source. The samples were exposed to increasing doses and the effects of the radiation have been evaluated by Fourier transform infrared spectroscopy, spectroscopic ellipsometry and atomic force microscopy. The X‐ray beam induces a significant densification on the silica films without producing any degradation such as cracks, flaws or delamination at the interface. The densification is accompanied by a decrease in thickness and an increase in refractive index both in the pure silica and in the hybrid films. The effect on the hybrid material is to induce densification through reaction of silanol groups but also removal of the organic groups, which are covalently bonded to silicon via Si—C bonds. At the highest exposure dose the removal of the organic groups is complete and the film becomes pure silica. Hard X‐rays can be used as an efficient and direct writing tool to pattern coating layers of different types of compositions.     

Pump−probe experiments at the TEMPO beamline using the low‐α operation mode of Synchrotron SOLEIL

The SOLEIL synchrotron radiation source is regularly operated in special filling modes dedicated to pump–probe experiments. Among others, the low‐α mode operation is characterized by shorter pulse duration and represents the natural bridge between 50 ps synchrotron pulses and femtosecond experiments. Here, the capabilities in low‐α mode of the experimental set‐ups developed at the TEMPO beamline to perform pump–probe experiments with soft X‐rays based on photoelectron or photon detection are presented. A 282 kHz repetition‐rate femtosecond laser is synchronized with the synchrotron radiation time structure to induce fast electronic and/or magnetic excitations. Detection is performed using a two‐dimensional space resolution plus time resolution detector based on microchannel plates equipped with a delay line. Results of time‐resolved photoelectron spectroscopy, circular dichroism and magnetic scattering experiments are reported, and their respective advantages and limitations in the framework of high‐time‐resolution pump–probe experiments compared and discussed.     

Spatial and temporal distribution of γH2AX fluorescence in human cell cultures following synchrotron‐generated X‐ray microbeams: lack of correlation between persistent γH2AX foci and apoptosis

Formation of γH2AX foci (a marker of DNA double‐strand breaks), rates of foci clearance and apoptosis were investigated in cultured normal human fibroblasts and p53 wild‐type malignant glioma cells after exposure to high‐dose synchrotron‐generated microbeams. Doses up to 283 Gy were delivered using beam geometries that included a microbeam array (50 µm wide, 400 µm spacing), single microbeams (60–570 µm wide) and a broad beam (32 mm wide). The two cell types exhibited similar trends with respect to the initial formation and time‐dependent clearance of γH2AX foci after irradiation. High levels of γH2AX foci persisted as late as 72 h post‐irradiation in the majority of cells within cultures of both cell types. Levels of persistent foci after irradiation via the 570 µm microbeam or broad beam were higher when compared with those observed after exposure to the 60 µm microbeam or microbeam array. Despite persistence of γH2AX foci, these irradiation conditions triggered apoptosis in only a small proportion (<5%) of cells within cultures of both cell types. These results contribute to the understanding of the fundamental biological consequences of high‐dose microbeam irradiations, and implicate the importance of non‐apoptotic responses such as p53‐mediated growth arrest (premature senescence).     

Hydrothermal formation of tobermorite studied by in situ X‐ray diffraction under autoclave condition

Hydrothermal formation of tobermorite from a pre‐cured cake has been investigated by transmission X‐ray diffraction (XRD) using high‐energy X‐rays from a synchrotron radiation source in combination with a newly designed autoclave cell. The autoclave cell has a large and thin beryllium window for wide‐angle X‐ray diffraction; nevertheless, it withstands a steam pressure of more than 1.2 MPa, which enables in situ XRD measurements in a temperature range of 373 to 463 K under a saturated steam pressure. Formation and/or decomposition of several components has been successfully observed during 7.5 h of reaction time. From the intensity changes of the intermediate materials, namely non‐crystalline C–S–H and hydroxylellestadite, two pathways for tobermorite formation have been confirmed. Thus, the newly developed autoclave cell can be used for the analyses of reaction mechanisms under specific atmospheres and temperatures.     

Comparison of in vitro breast cancer visibility in analyser‐based computed tomography with histopathology,mammography, computed tomography and magnetic resonance imaging

High‐resolution analyser‐based X‐ray imaging computed tomography (HR ABI‐CT) findings on in vitro human breast cancer are compared with histopathology, mammography, computed tomography (CT) and magnetic resonance imaging. The HR ABI‐CT images provided significantly better low‐contrast visibility compared with the standard radiological images. Fine cancer structures indistinguishable and superimposed in mammograms were seen, and could be matched with the histopathological results. The mean glandular dose was less than 1 mGy in mammography and 12–13 mGy in CT and ABI‐CT. The excellent visibility of in vitro breast cancer suggests that HR ABI‐CT may have a valuable role in the future as an adjunct or even alternative to current breast diagnostics, when radiation dose is further decreased, and compact synchrotron radiation sources become available.     

Direct nano‐scale patterning of Ag films using hard X‐ray induced oxidation

The morphological change of silver nano‐particles (AgNPs) exposed to an intense synchrotron X‐ray beam was investigated for the purpose of direct nano‐scale patterning of metal thin films. AgNPs irradiated by hard X‐rays in oxygen ambient were oxidized and migrated out of the illuminated region. The observed X‐ray induced oxidation was utilized to fabricate nano‐scale metal line patterns using sectioned WSi₂/Si multilayers as masks. Lines with a width as small as 21 nm were successfully fabricated on Ag films on silicon nitride. Au/Ag nano‐lines were also fabricated using the proposed method.     

Defect engineering by synchrotron radiation X‐rays in CeO2 nanocrystals

This work reports an unconventional defect engineering approach using synchrotron‐radiation‐based X‐rays on ceria nanocrystal catalysts of particle sizes 4.4–10.6 nm. The generation of a large number of oxygen‐vacancy defects (OVDs), and therefore an effective reduction of cations, has been found in CeO₂ catalytic materials bombarded by high‐intensity synchrotron X‐ray beams of beam size 1.5 mm × 0.5 mm, photon energies of 5.5–7.8 keV and photon fluxes up to 1.53 × 10¹² photons s^?1. The experimentally observed cation reduction was theoretically explained by a first‐principles formation‐energy calculation for oxygen vacancy defects. The results clearly indicate that OVD formation is mainly a result of X‐ray‐excited core holes that give rise to valence holes through electron down conversion in the material. Thermal annealing and subvalent Y‐doping were also employed to modulate the efficiency of oxygen escape, providing extra control on the X‐ray‐induced OVD generating process. Both the core‐hole‐dominated bond breaking and oxygen escape mechanisms play pivotal roles for efficient OVD formation. This X‐ray irradiation approach, as an alternative defect engineering method, can be applied to a wide variety of nanostructured materials for physical‐property modification.     

Grazing‐incidence small‐angle X‐ray scattering from Ge nanodots self‐organized on Si(001) examined with soft X‐rays

Grazing‐incidence small‐angle X‐ray scattering (GISAXS) measurements with soft X‐rays have been applied to Ge nanodots capped with a Si layer. Spatially anisotropic distribution of nanodots resulted in strongly asymmetric GISAXS patterns in the q_y direction in the soft X‐ray region, which have not been observed with conventional hard X‐rays. However, such apparent differences were explained by performing a GISAXS intensity calculation on the Ewald sphere, i.e. taking the curvature of Ewald sphere into account.     

L‐series X‐ray emission spectra of 3d‐metal compounds with various excitation conditions

L‐series emissions of manganese, iron, and zinc oxides were studied using electron beam excitation and highly brilliant synchrotron radiation excitation. We showed that manganese and iron oxides show different Lβ/Lα intensity ratio because of their oxidation states and excitation electron voltages. On the other hand, we could not get any L‐series emissions from those bulk samples when excited by normal incident high‐energy monochromatic X‐rays, while samples of thin films and samples excited by grazing incident monochromatic X‐rays showed clear emissions. It is suggested that the difference of Lβ/Lα intensity ratio due to the oxidized states mainly concerns with the Coster–Kronig transition ratio of the samples, while self‐absorption effects should also deeply contribute the ratio, considering the experimental results. Copyright © 2016 John Wiley & Sons, Ltd.     

SOLEIL shining on the solution‐state structure of biomacromolecules by synchrotron X‐ray footprinting at the Metrology beamline

Synchrotron X‐ray footprinting complements the techniques commonly used to define the structure of molecules such as crystallography, small‐angle X‐ray scattering and nuclear magnetic resonance. It is remarkably useful in probing the structure and interactions of proteins with lipids, nucleic acids or with other proteins in solution, often better reflecting the in vivo state dynamics. To date, most X‐ray footprinting studies have been carried out at the National Synchrotron Light Source, USA, and at the European Synchrotron Radiation Facility in Grenoble, France. This work presents X‐ray footprinting of biomolecules performed for the first time at the X‐ray Metrology beamline at the SOLEIL synchrotron radiation source. The installation at this beamline of a stopped‐flow apparatus for sample delivery, an irradiation capillary and an automatic sample collector enabled the X‐ray footprinting study of the structure of the soluble protein factor H (FH) from the human complement system as well as of the lipid‐associated hydrophobic protein S3 oleosin from plant seed. Mass spectrometry analysis showed that the structural integrity of both proteins was not affected by the short exposition to the oxygen radicals produced during the irradiation. Irradiated molecules were subsequently analysed using high‐resolution mass spectrometry to identify and locate oxidized amino acids. Moreover, the analyses of FH in its free state and in complex with complement C3b protein have allowed us to create a map of reactive solvent‐exposed residues on the surface of FH and to observe the changes in oxidation of FH residues upon C3b binding. Studies of the solvent accessibility of the S3 oleosin show that X‐ray footprinting offers also a unique approach to studying the structure of proteins embedded within membranes or lipid bodies. All the biomolecular applications reported herein demonstrate that the Metrology beamline at SOLEIL can be successfully used for synchrotron X‐ray footprinting of biomolecules.