Synchrotron X-Ray Radiation-Induced Bystander Effect: An Impact of the Scattered Radiation, Distance From the Irradiated Site and p53 Cell Status.

Synchrotron radiation, especially microbeam radiotherapy (MRT), has a great potential to improve cancer radiotherapy, but non-targeted effects of synchrotron radiation have not yet been sufficiently explored. We have previously demonstrated that scattered synchrotron radiation induces measurable γ-H2AX foci, a biomarker of DNA double-strand breaks, at biologically relevant distances from the irradiated field that could contribute to the apparent accumulation of bystander DNA damage detected in cells and tissues outside of the irradiated area. Here, we quantified an impact of scattered radiation to DNA damage response in "naïve" cells sharing the medium with the cells that were exposed to synchrotron radiation. To understand the effect of genetic alterations in naïve cells, we utilised p53-null and p53-wild-type human colon cancer cells HCT116. The cells were grown in two-well chamber slides, with only one of nine zones (of equal area) of one well irradiated with broad beam or MRT. γ-H2AX foci per cell values induced by scattered radiation in selected zones of the unirradiated well were compared to the commensurate values from selected zones in the irradiated well, with matching distances from the irradiated zone. Scattered radiation highly impacted the DNA damage response in both wells and a pronounced distance-independent bystander DNA damage was generated by broad-beam irradiations, while MRT-generated bystander response was negligible. For p53-null cells, a trend for a reduced response to scattered irradiation was observed, but not to bystander signalling. These results will be taken into account for the assessment of genotoxic effects in surrounding non-targeted tissues in preclinical experiments designed to optimise conditions for clinical MRT and for cancer treatment in patients.

Synchrotron radiation external beam rotational radiotherapy of breast cancer: proof of principle.

The principle of rotational summation of the absorbed dose for breast cancer treatment with orthovoltage X-ray beams was proposed by J. Boone in 2012. Here, use of X-ray synchrotron radiation for image guided external beam rotational radiotherapy treatment of breast cancer is proposed. Tumor irradiation occurs with the patient in the prone position hosted on a rotating bed, with her breast hanging from a hole in the bed, which rotates around a vertical axis passing through the tumor site. Horizontal collimation of the X-ray beam provides for whole breast or partial breast irradiation, while vertical translation of the bed and successive rotations allow for irradiation of the full tumor volume, with dose rates which permit also hypofractionated treatments. In this work, which follows a previous preliminary report, results are shown of a full series of measurements on polyethylene and acrylic cylindrical phantoms carried out at the Australian Synchrotron, confirmed by Geant4 Monte Carlo simulations, intended to demonstrate the proof of principle of the technique. Dose measurements were carried out with calibrated ion chambers, radiochromic films and thermoluminescence dosimeters. The photon energy investigated was 60 keV. Image guidance may occur with the transmitted beam for contrast-enhanced breast computed tomography. For a horizontal beam collimation of 1.5 cm and rotation around the central axis of a 14 cm-diameter polyethylene phantom, a periphery-to-center dose ratio of 14% was measured. The simulations showed that under the same conditions the dose ratio decreases with increasing photon energy down to 10% at 175 keV. These values are comparable with those achievable with conventional megavoltage radiotherapy of breast cancer with a medical linear accelerator. Dose painting was demonstrated with two off-center `cancer foci' with 1.3 Gy and 0.6 Gy target doses. The use of a radiosensitizing agent for dose enhancement is foreseen.

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.

Assessment and Implications of Scattered Microbeam and Broadbeam Synchrotron Radiation for Bystander Effect Studies.

Synchrotron radiation is an excellent tool for investigating bystander effects in cell and animal models because of the well-defined and controllable configuration of the beam. Although synchrotron radiation has many advantages for such studies compared to conventional radiation, the contribution of dose exposure from scattered radiation nevertheless remains a source of concern. Therefore, the influence of scattered radiation on the detection of bystander effects induced by synchrotron radiation in biological in vitro models was evaluated. Radiochromic XRQA2 film-based dosimetry was employed to measure the absorbed dose of scattered radiation in cultured cells at various distances from a field exposed to microbeam radiotherapy and broadbeam X-ray radiation. The level of scattered radiation was dependent on the distance, dose in the target zone and beam mode. The number of γ-H2AX foci in cells positioned at the same target distances was measured and used as a biodosimeter to evaluate the absorbed dose. A correlation of absorbed dose values measured by the physical and biological methods was identified. The γ-H2AX assay successfully quantitated the scattered radiation in the range starting from 10 mGy and its contribution to the observed radiation-induced bystander effect.

Revisiting Bragg's X-ray microscope: scatter based optical transient grating detection of pulsed ionising radiation.

Transient optical gratings for detecting ultrafast signals are routine for temporally resolved photochemical investigations. Many processes can contribute to the formation of such gratings; we indicate use of optically scattering centres that can be formed with highly variable latencies in different materials and devices using ionising radiation. Coherent light scattered by these centres can form the short-wavelength-to-optical-wavelength, incoherent-to-coherent basis of a Bragg X-ray microscope, with inherent scope for optical phasing. Depending on the dynamics of the medium chosen, the way is open to both ultrafast pulsed and integrating measurements. For experiments employing brief pulses, we discuss high-dynamic-range short-wavelength diffraction measurements with real-time optical reconstructions. Applications to optical real-time X-ray phase-retrieval are considered.

Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause serious infection in those with deficient or impaired phagocytes. We have developed the optically transparent and genetically tractable zebrafish embryo as a model for systemic P. aeruginosa infection. Despite lacking adaptive immunity at this developmental stage, zebrafish embryos were highly resistant to P. aeruginosa infection, but as in humans, phagocyte depletion dramatically increased their susceptibility. The virulence of an attenuated P. aeruginosa strain lacking a functional Type III secretion system was restored upon phagocyte depletion, suggesting that this system influences virulence through its effects on phagocytes. Intravital imaging revealed bacterial interactions with multiple blood cell types. Neutrophils and macrophages rapidly phagocytosed and killed P. aeruginosa, suggesting that both cell types play a role in protection against infection. Intravascular aggregation of erythrocytes and other blood cells with resultant circulatory blockage was observed immediately upon infection, which may be relevant to the pathogenesis of thrombotic complications of human P. aeruginosa infections. The real-time visualization capabilities and genetic tractability of the zebrafish infection model should enable elucidation of molecular and cellular details of P. aeruginosa pathogenesis in conditions associated with neutropenia or impaired phagocyte function.

Zebrafish granulocyte colony-stimulating factor receptor signaling promotes myelopoiesis and myeloid cell migration.

Granulocyte colony-stimulating factor receptor (GCSFR) signaling participates in the production of neutrophilic granulocytes during normal hematopoietic development, with a particularly important role during emergency hematopoiesis. This study describes the characterization of the zebrafish gcsf and gcsfr genes, which showed broad conservation and similar regulation to their mammalian counterparts. Morpholino-mediated knockdown of gcsfr and overexpression of gcsf revealed the presence of an anterior population of myeloid cells during primitive hematopoiesis that was dependent on GCSF/GCSFR for development and migration. This contrasted with a posterior domain that was largely independent of this pathway. Definitive myelopoiesis was also partially dependent on a functional GCSF/GCSFR pathway. Injection of bacterial lipopolysaccharide elicited significant induction of gcsf expression and emergency production of myeloid cells, which was abrogated by gcsfr knockdown. Collectively, these data demonstrate GCSF/GCSFR to be a conserved signaling system for facilitating the production of multiple myeloid cell lineages in both homeostatic and emergency conditions, as well as for early myeloid cell migration, establishing a useful experimental platform for further dissection of this pathway.

Intestinal differentiation in zebrafish requires Cdx1b, a functional equivalent of mammalian Cdx2.

BACKGROUND & AIMS: The ParaHox transcription factor Cdx2 is an essential determinant of intestinal phenotype in mammals throughout development, influencing gut function, homeostasis, and epithelial barrier integrity. Cdx2 expression demarcates the zones of intestinal stem cell proliferation in the adult gut, with deregulated expression implicated in intestinal metaplasia and cancer. However, in vivo analysis of these prospective roles has been limited because inactivation of Cdx2 in mice leads to preimplantation embryonic lethality. We used the zebrafish, a valuable model for studying gut development, to generate a system to further understanding of the role of Cdx2 in normal intestinal function and in disease states. METHODS: We isolated and characterized the zebrafish cdx1b ortholog and analyzed its function by antisense morpholino gene knockdown. RESULTS: We showed that zebrafish Cdx1b replaces the role of Cdx2 in gut development. Evolutionary studies have indicated that the zebrafish cdx2 loci were lost following the genome-wide duplication event that occurred in teleosts. Zebrafish Cdx1b is expressed exclusively in the developing intestine during late embryogenesis and regulates intestinal cell proliferation and terminal differentiation. CONCLUSIONS: This work established an in vivo system to explore further the activity of Cdx2 in the gut and its impact on processes such as inflammation and cancer.