Search

Background and PurposeCardiac arrhythmia is a recognised potential complication of thoracic radiotherapy, but the responsible cardiac substructures for arrhythmogenesis have not been identified. Arrhythmogenic tissue is commonly located in the pulmonary veins (PVs) of cardiology patients with arrhythmia, however these structures are not currently considered organs-at-risk during radiotherapy planning. A standardised approach to their delineation was developed and evaluated.Materials and MethodsThe gross and radiological anatomy relevant to atrial fibrillation was derived from cardiology and radiology literature by a multidisciplinary team. A region of interest and contouring instructions for radiotherapy computed tomography scans were iteratively developed and subsequently evaluated. Radiation oncologists (n = 5) and radiation technologists (n = 2) contoured the PVs on the four-dimensional planning datasets of five patients with locally advanced lung cancer treated with 1.8–2.75 Gy fractions. Contours were compared to reference contours agreed by the researchers using geometric and dosimetric parameters.ResultsThe mean dose to the PVs was 35% prescription dose. Geometric and dosimetric similarity of the observer contours with reference contours was fair, with an overall mean Dice of 0.80 ± 0.02. The right superior PV (mean DSC 0.83 ± 0.02) had better overlap than the left (mean DSC 0.80 ± 0.03), but the inferior PVs were equivalent (mean DSC of 0.78). The mean difference in mean dose was 0.79 Gy ± 0.71 (1.46% ± 1.25).ConclusionA PV atlas with multidisciplinary approval led to reproducible delineation for radiotherapy planning, supporting the utility of the atlas in future clinical radiotherapy cardiotoxicity research encompassing arrhythmia endpoints.

BACKGROUND AND PURPOSE: The effects of radiation on the heart are dependent on dose, fractionation, overall treatment time, and pre-existing cardiovascular pathology. Murine models have played a central role in improving our understanding of the radiation response of the heart yet a wide range of exposure parameters have been used. We evaluated the study design of published murine cardiac irradiation experiments to assess gaps in the literature and to suggest guidance for the harmonisation of future study reporting.METHODS AND MATERIALS: A systematic review of mouse/rat studies published 1981-2021 that examined the effect of radiation on the heart was performed. The protocol was published on PROSPERO (CRD42021238921) and the findings were reported in accordance with the PRISMA guidance. Risk of bias was assessed using the SYRCLE checklist.RESULTS: 159 relevant full-text original articles were reviewed. The heart only was the target volume in 67% of the studies and simulation details were unavailable for 44% studies. Dosimetry methods were reported in 31% studies. The pulmonary effects of whole and partial heart irradiation were reported in 13% studies. Seventy-eight unique dose-fractionation schedules were evaluated. Large heterogeneity was observed in the endpoints measured, and the reporting standards were highly variable.CONCLUSIONS: Current murine models of radiation cardiotoxicity cover a wide range of irradiation configurations and latency periods. There is a lack of evidence describing clinically relevant dose-fractionations, circulating biomarkers and radioprotectants. Recommendations for the consistent reporting of methods and results of in vivo cardiac irradiation studies are made to increase their suitability for informing the design of clinical studies.

Radiomics image analysis has the potential to uncover disease characteristics for the development of predictive signatures and personalised radiotherapy treatment. Inter-observer and inter-software delineation variabilities are known to have downstream effects on radiomics features, reducing the reliability of the analysis. The purpose of this study was to investigate the impact of these variabilities on radiomics outputs from preclinical cone-beam computed tomography (CBCT) scans. Inter-observer variabilities were assessed using manual and semi-automated contours of mouse lungs (n = 16). Inter-software variabilities were determined between two tools (3D Slicer and ITK-SNAP). The contours were compared using Dice similarity coefficient (DSC) scores and the 95th percentile of the Hausdorff distance (HD95p) metrics. The good reliability of the radiomics outputs was defined using intraclass correlation coefficients (ICC) and their 95% confidence intervals. The median DSC scores were high (0.82–0.94), and the HD95p metrics were within the submillimetre range for all comparisons. the shape and NGTDM features were impacted the most. Manual contours had the most reliable features (73%), followed by semi-automated (66%) and inter-software (51%) variabilities. From a total of 842 features, 314 robust features overlapped across all contouring methodologies. In addition, our results have a 70% overlap with features identified from clinical inter-observer studies

Figure legends for Supplementary Figures

Caspase-8 dependent apoptosis induced by IR

IR-induced changes in cell surface expression of death receptors

Response of H460 and A549 cells to IR

Combination of SAHA with IR in H460 and A549 cells using different treatment schedules

Supplementary Methods and Table 1. Supplementary Table 1: siRNA target genes

Ataxia telangiectasia mutated (ATM) is an important signaling molecule in the DNA damage response (DDR). ATM loss of function can produce a synthetic lethal phenotype in combination with tumor-associated mutations in FA/BRCA pathway components. In this study, we took an siRNA screening strategy to identify other tumor suppressors that, when inhibited, similarly sensitized cells to ATM inhibition. In this manner, we determined that PTEN and ATM were synthetically lethal when jointly inhibited. PTEN-deficient cells exhibited elevated levels of reactive oxygen species, increased endogenous DNA damage, and constitutive ATM activation. ATM inhibition caused catastrophic DNA damage, mitotic cell cycle arrest, and apoptosis specifically in PTEN-deficient cells in comparison with wild-type cells. Antioxidants abrogated the increase in DNA damage and ATM activation in PTEN-deficient cells, suggesting a requirement for oxidative DNA damage in the mechanism of cell death. Lastly, the ATM inhibitor KU-60019 was specifically toxic to PTEN mutant cancer cells in tumor xenografts and reversible by reintroduction of wild-type PTEN. Together, our results offer a mechanistic rationale for clinical evaluation of ATM inhibitors in PTEN-deficient tumors. Cancer Res; 75(11); 2159–65. ©2015 AACR.

Supplementary Figures 1-4. Figure 1: ATM is a potential drug target for PTEN-deficient cells Figure 2: The synthetic lethality with PTEN loss and ATM inhibition is independent of AKT function Figure 3: The synthetic lethality with PTEN loss and ATM inhibition is independent of RAD51 function Supplementary Figure 4: In vivo efficacy of ATM inhibition with PTEN loss

This Roadmap paper covers the field of precision preclinical x-ray radiation studies in animal models. It is mostly focused on models for cancer and normal tissue response to radiation, but also discusses other disease models. The recent technological evolution in imaging, irradiation, dosimetry and monitoring that have empowered these kinds of studies is discussed, and many developments in the near future are outlined. Finally, clinical translation and reverse translation are discussed.

Lung cancer's radiomic phenotype may potentially inform clinical decision-making with respect to radical radiotherapy. At present there are no validated biomarkers available for the individualisation of radical radiotherapy in lung cancer and the mortality rate of this disease remains the highest of all other solid tumours. MEDLINE was searched using the terms 'radiomics' and 'lung cancer' according to the Preferred Reporting Items for Systematic Reviews and Met-Analyses (PRISMA) guidance. Radiomics studies were defined as those manuscripts describing the extraction and analysis of at least 10 quantifiable imaging features. Only those studies assessing disease control, survival or toxicity outcomes for patients with lung cancer following radical radiotherapy ± chemotherapy were included. Study titles and abstracts were reviewed by two independent reviewers. The Radiomics Quality Score was applied to the full text of included papers. Of 244 returned results, 44 studies met the eligibility criteria for inclusion. End points frequently reported were local (17%), regional (17%) and distant control (31%), overall survival (79%) and pulmonary toxicity (4%). Imaging features strongly associated with clinical outcomes include texture features belonging to the subclasses Gray level run length matrix, Gray level co-occurrence matrix and kurtosis. The median cohort size for model development was 100 (15-645); in the 11 studies with external validation in a separate independent population, the median cohort size was 84 (21-295). The median number of imaging features extracted was 184 (10-6538). The median Radiomics Quality Score was 11% (0-47). Patient-reported outcomes were not incorporated within any studies identified. No studies externally validated a radiomics signature in a registered prospective study. Imaging-derived indices attained through radiomic analyses could equip thoracic oncologists with biomarkers for treatment response, patterns of failure, normal tissue toxicity and survival in lung cancer. Based on routine scans, their non-invasive nature and cost-effectiveness are major advantages over conventional pathological assessment. Improved tools are required for the appraisal of radiomics studies, as significant barriers to clinical implementation remain, such as standardisation of input scan data, quality of reporting and external validation of signatures in randomised, interventional clinical trials.

Purpose: Radiation cardiotoxicity (RC) is a clinically significant adverse effect of treatment for patients with thoracic malignancies. Clinical studies in lung cancer have indicated that heart substructures are not uniformly radiosensitive, and that dose to the heart base drives RC. In this study, we aimed to characterize late changes in gene expression using spatial transcriptomics in a mouse model of base regional radiosensitivity. Methods and Materials: An aged female C57BL/6 mouse was irradiated with 16 Gy delivered to the cranial third of the heart using a 6 × 9 mm parallel opposed beam geometry on a small animal radiation research platform, and a second mouse was sham-irradiated. After echocardiography, whole hearts were collected at 30 weeks for spatial transcriptomic analysis to map gene expression changes occurring in different regions of the partially irradiated heart. Cardiac regions were manually annotated on the capture slides and the gene expression profiles compared across different regions. Results: Ejection fraction was reduced at 30 weeks after a 16 Gy irradiation to the heart base, compared with the sham-irradiated controls. There were markedly more significant gene expression changes within the irradiated regions compared with nonirradiated regions. Variation was observed in the transcriptomic effects of radiation on different cardiac base structures (eg, between the right atrium [n = 86 dysregulated genes], left atrium [n = 96 dysregulated genes], and the vasculature [n = 129 dysregulated genes]). Disrupted biological processes spanned extracellular matrix as well as circulatory, neuronal, and contractility activities. Conclusions: This is the first study to report spatially resolved gene expression changes in irradiated tissues. Examination of the regional radiation response in the heart can help to further our understanding of the cardiac base's radiosensitivity and support the development of actionable targets for pharmacologic intervention and biologically relevant dose constraints.

Microbeam radiotherapy (MRT) is the delivery of spatially fractionated beams that have the potential to offer significant improvements in the therapeutic ratio due to the delivery of micron-sized high dose and dose rate beams. They build on longstanding clinical experience of GRID radiotherapy and more recently lattice-based approaches. Here we briefly overview the preclinical evidence for MRT efficacy and highlight the challenges for bringing this to clinical utility. The biological mechanisms underpinning MRT efficacy are still unclear, but involve vascular, bystander, stem cell and potentially immune responses. There is probably significant overlap in the mechanisms underpinning MRT responses and FLASH radiotherapy that needs to be further defined. (c) 2021 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

Purpose The recent implementation of MR-Linacs has highlighted theranostic opportunities of contrast agents in both imaging and radiotherapy. There is a lack of data exploring the potential of superparamagnetic iron oxide nanoparticles (SPIONs) as radiosensitisers. Through preclinical 225 kVp exposures, this study aimed to characterise the uptake and radiobiological effects of SPIONs in tumour cell models in vitro and to provide proof-of-principle application in a xenograft tumour model. Methods SPIONs were also characterised to determine their hydrodynamic radius using dynamic light scattering and uptake was measured using ICP-MS in 6 cancer cell lines; H460, MiaPaCa2, DU145, MCF7, U87 and HEPG2. The impact of SPIONs on radiobiological response was determined by measuring DNA damage using 53BP1 immunofluorescence and cell survival. Sensitisation Enhancement Ratios (SERs) were compared with the predicted Dose Enhancement Ratios (DEFs) based on physical absorption estimations. In vivo efficacy was demonstrated using a subcutaneous H460 xenograft tumour model in SCID mice by following intra-tumoural injection of SPIONs. Results The hydrodynamic radius was found to be between 110 and 130 nm, with evidence of being monodisperse in nature. SPIONs significantly increased DNA damage in all cell lines with the exception of U87 cells at a dose of 1 Gy, 1 h post-irradiation. Levels of DNA damage correlated with the cell survival, in which all cell lines except U87 cells showed an increased sensitivity (P P Conclusions SPIONs showed radiosensitising effects in 5 out of 6 cancer cell lines. No correlation was found between the cell-specific uptake of SPIONs into the cells and DNA damage levels. The in vivo study found a significant decrease in the tumour growth rate.

Background: Preclinical models of radiotherapy (RT) response are vital for the continued success and evolution of RT in the treatment of cancer. The irradiation of tissues in mouse models necessitates high levels of precision and accuracy to recapitulate clinical exposures and limit adverse effects on animal welfare. This requirement has been met by technological advances in preclinical RT platforms established over the past decade. Small animal RT systems use onboard computed tomography (CT) imaging to delineate target volumes and have significantly refined radiobiology experiments with major 3Rs impacts. However, the CT imaging is limited by the differential attenuation of tissues resulting in poor contrast in soft tissues. Clinically, radio-opaque fiducial markers (FMs) are used to establish anatomical reference points during treatment planning to ensure accuracy beam targeting, this approach is yet to translate back preclinical models. Methods: We report on the use of a novel liquid FM BioXmark® developed by Nanovi A/S (Kongens Lyngby, Denmark) that can be used to improve the visualisation of soft tissue targets during beam targeting and minimise dose to surrounding organs at risk. We present descriptive protocols and methods for the use of BioXmark® in experimental male and female C57BL/6J mouse models. Results: These guidelines outline the optimum needle size for uptake (18-gauge) and injection (25- or 26-gauge) of BioXmark® for use in mouse models along with recommended injection volumes (10-20 µl) for visualisation on preclinical cone beam CT (CBCT) scans. Injection techniques include subcutaneous, intraperitoneal, intra-tumoral and prostate injections. Conclusions: The use of BioXmark® can help to standardise targeting methods, improve alignment in preclinical image-guided RT and significantly improve the welfare of experimental animals with the reduction of normal tissue exposure to RT.

Objective. The delivery of intensity-modulated radiation fields has improved the conformity of dose to tumour targets during radiotherapy (RT). Previously, it has been shown that intercellular communication between cells positioned in- and outside of the radiation field impacts cellular radiosensitivity under hypoxic and normoxic conditions. However, the mechanism of intercellular communication in hypoxia remains to be fully understood. In this study, the cell-killing effects of intercellular communication in hypoxia were modelled in an effort to better understand the underlying mechanisms of response. Approach. By irradiating a 50% area of the culture dish (half-field exposure), experimental dose-response curves for cell survival and residual DNA double-strand breaks (DSBs) were generated in prostate (DU145) and non-small cell lung cancer (H1299) cells. The oxygen enhancement ratio (OER) was determined from early DSB yields (corresponding to relative direct damage) and used to model the in- and out-of-field radiosensitivity. Main results. The developed integrated microdosimetric-kinetic (IMK) model successfully predicted the experimental dose responses for survival and lethal lesions, and provides a mechanistic interpretation that the probability of hits for releasing cell-killing signals is dependent on oxygen. This experimental and modelling study also suggests that residual DSBs correspond to logarithmic survival fraction (meaning lethal lesions) for in- and out-of-field cells. Our data suggest that the OER value determined using uniform-field exposure can be applied to predict the in- and out-of-field radiosensitivity of cells following exposure to intensity modulated beams. Significance. The developed IMK model facilitates a more precise understanding of intercellular signalling following exposure to intensity-modulated radiation fields.

The National Cancer Institute's Radiation Research Program, in collaboration with the Radiosurgery Society, hosted a workshop called Understanding High-Dose, Ultra-High Dose Rate and Spatially Fractionated Radiotherapy on August 20 and 21, 2018 to bring together experts in experimental and clinical experience in these and related fields. Critically, the overall aims were to understand the biological underpinning of these emerging techniques and the technical/physical parameters that must be further defined to drive clinical practice through innovative biologically based clinical trials.

Radiation exposures at ultra-high dose rates (UHDR) at several orders of magnitude greater than in current clinical radiotherapy have been shown to manifest differential radiobiological responses compared to conventional dose rates (CONV). This has led to studies investigating the application of UHDR for therapeutic advantage (FLASH-RT) which have gained significant interest since the initial discovery in 2014 that demonstrated reduced lung toxicity with equivalent levels of tumour control compared with conventional dose-rate radiotherapy. Many subsequent studies have demonstrated the potential protective role of FLASH-RT in normal tissues, yet the underlying molecular and cellular mechanisms of the FLASH effect remain to be fully elucidated. Here, we summarise the current evidence of the FLASH effect and review FLASH-RT studies performed in preclinical models of normal tissue response. To critically examine the underlying biological mechanisms of responses to UHDR radiation exposures, we evaluate in vitro studies performed with normal and tumour cells. Differential responses to UHDR vs CONV irradiation recurrently involve reduced inflammatory processes and differential expression of pro- and anti-inflammatory genes. In addition, frequently reduced levels of DNA damage or misrepair products are seen after UHDR irradiation. So far, it is not clear what signal elicits these differential responses, but there are indications for involvement of reactive species. Different susceptibility to FLASH effects observed between normal and tumour cells may result from altered metabolic and detoxification pathways and/or repair pathways used by tumour cells. We summarize the current theories that may explain the FLASH effect and highlight important research questions which are key to a better mechanistic understanding and, thus, the future implementation of FLASH-RT in the clinic. This article is protected by copyright. All rights reserved.

Mouse models are essential tools in cancer research that have been used to understand the genetic basis of tumorigenesis, cancer progression and to test the efficacies of anticancer treatments including radiotherapy. They have played a critical role in our understanding of radiotherapy response in tumours and normal tissues and continue to evolve to better recapitulate the underlying biology of humans. In addition, recent developments in small animal irradiators have significantly improved in vivo irradiation techniques, allowing previously unimaginable experimental approaches to be explored in the laboratory. The combination of contemporary mouse models with small animal irradiators represents a major step forward for the radiobiology field in being able to much more accurately replicate clinical exposure scenarios. As radiobiology studies become ever more sophisticated in reflecting developments in the clinic, it is increasingly important to understand the basis and potential limitations of extrapolating data from mice to humans. This review provides an overview of mouse models and small animal radiotherapy platforms currently being used as advanced radiobiological research tools towards improving the translational power of preclinical studies.

Radiation-induced bystander effects (RIBE) may have potential implications for radiotherapy, yet the radiobiological impact and underlying mechanisms in hypoxic tumor cells remain to be determined. Using two human tumor cell lines, hepatoma HepG2 cells and glioblastoma T98G cells, the present study found that under both normoxic and hypoxic conditions, increased micronucleus formation and decreased cell survival were observed in non-irradiated bystander cells which had been co-cultured with X-irradiated cells or treated with conditioned-medium harvested from X-irradiated cells. Although the radiosensitivity of hypoxic tumor cells was lower than that of aerobic cells, the yield of micronucleus induced in bystander cells under hypoxia was similar to that measured under normoxia indicating that RIBE is a more significant factor in overall radiation damage of hypoxic cells. When hypoxic cells were treated with dimethyl sulfoxide (DMSO), a scavenger of reactive oxygen species (ROS), or aminoguanidine (AG), an inhibitor of nitric oxide synthase (NOS), before and during irradiation, the bystander response was partly diminished. Furthermore, when only hypoxic bystander cells were pretreated with siRNA hypoxia-inducible factor-1α (HIF-1α), RIBE were decreased slightly but if irradiated cells were treated with siRNA HIF-1α, hypoxic RIBE decreased significantly. In addition, the expression of HIF-1α could be increased in association with other downstream effector molecules such as glucose transporter 1 (GLUT-1), vascular endothelial growth factor (VEGF), and carbonic anhydrase (CA9) in irradiated hypoxic cells. However, the expression of HIF-1α expression in bystander cells was decreased by a conditioned medium from isogenic irradiated cells. The current results showed that under hypoxic conditions, irradiated HepG2 and T98G cells showed reduced radiosensitivity by increasing the expression of HIF-1α and induced a syngeneic bystander effect by decreasing the expression of HIF-1α and regulating its downstream target genes in both the irradiated or bystander cells.

Prostate cancer (PCa) is the most common non-cutaneous cancer in men and a notable cause of cancer mortality when it metastasises. The unfolded protein response (UPR) can be cytoprotective but when acutely activated can lead to cell death. In this study, we sought to enhance the acute activation of the UPR using radiation and ONC201, an UPR activator. Treating PCa cells with ONC201 quickly increased the expression of all the key regulators of the UPR and reduced the oxidative phosphorylation, with cell death occurring 72 h later. We exploited this time lag to sensitize prostate cancer cells to radiation through short-term treatment with ONC201. To understand how priming occurred, we performed RNA-Seq analysis and found that ONC201 suppressed the expression of cell cycle and DNA repair factors. In conclusion, we have shown that ONC201 can prime enhanced radiation response.

Radical radiotherapy, often in combination with hormone ablation, is a safe and effective treatment option for localised or locally-advanced prostate cancer. However, up to 30% of patients with locally advanced PCa will go on to develop biochemical failure, within 5 years, following initial radiotherapy. Improving radiotherapy response is clinically important since patients exhibiting biochemical failure develop castrate-resistant metastatic disease for which there is no curative therapy and median survival is 8&ndash, 18 months. The aim of this research was to determine if loss of PTEN (highly prevalent in advanced prostate cancer) is a novel therapeutic target in the treatment of advanced prostate cancer. Previous work has demonstrated PTEN-deficient cells are sensitised to inhibitors of ATM, a key regulator in the response to DSBs. Here, we have shown the role of PTEN in cellular response to IR was both complex and context-dependent. Secondly, we have confirmed ATM inhibition in PTEN-depleted cell models, enhances ionising radiation-induced cell killing with minimal toxicity to normal prostate RWPE-1 cells. Furthermore, combined treatment significantly inhibited PTEN-deficient tumour growth compared to PTEN-expressing counterparts, with minimal toxicity observed. We have further shown PTEN loss is accompanied by increased endogenous levels of ROS and DNA damage. Taken together, these findings provide pre-clinical data for future clinical evaluation of ATM inhibitors as a neoadjuvant/adjuvant in combination with radiation therapy in prostate cancer patients harbouring PTEN mutations.

International audience; This roadmap outlines the potential roles of metallic nanoparticles (MNPs) in the field of radiation therapy. MNPs made up of a wide range of materials (from Titanium, Z = 22, to Bismuth, Z = 83) and a similarly wide spectrum of potential clinical applications, including diagnostic, therapeutic (radiation dose enhancers, hyperthermia inducers, drug delivery vehicles, vaccine adjuvants, photosensitizers, enhancers of immunotherapy) and theranostic (combining both diagnostic and therapeutic), are being fabricated and evaluated. This roadmap covers contributions from experts in these topics summarizing their view of the current status and challenges, as well as expected advancements in technology to address these challenges.

The effects of various forms of ionising radiation are known to be mediated by interactions with cellular and molecular targets in irradiated and in some cases non-targeted tissue volumes. Despite major advances in advanced conformal delivery techniques, the probability of normal tissue complication (NTCP) remains the major dose-limiting factor in escalating total dose delivered during treatment. Potential strategies that have shown promise as novel delivery methods in achieving effective tumour control whilst sparing organs at risk involve the modulation of critical dose delivery parameters. This has led to the development of techniques using high dose spatial fractionation (GRID) and ultra-high dose rate (FLASH) which have translated to the clinic. The current review discusses the historical development and biological basis of GRID, microbeam and FLASH radiotherapy as advanced delivery modalities that have major potential for widespread implementation in the clinic in future years.

Spatially fractionated radiation therapy (SFRT) has been based on the delivery of a single high-dose fraction to a large treatment area that has been divided into several smaller fields, reducing the overall toxicity and adverse effects. Complementary microbeam studies have also shown an effective tissue-sparing effect (TSE) in various tissue types and species after spatially fractionated irradiation at the microscale level; however, the underlying biological mechanism remains elusive. In the current study, using the combination of an ex vivo mouse spermatogenesis model and high-precision X-ray microbeams, we revealed the significant TSE for maintaining spermatogenesis after spatially fractionated microbeam irradiation. We used the following ratios of the irradiated to nonirradiated areas: 50:50, 150:50 and 350:50 μm-slit, where approximately 50, 75 and 87.5% of the sample was irradiated (using center-To-center distances of 100, 200 and 400 μm, respectively). We found that the 50 and 75% micro-slit irradiated testicular tissues showed an almost unadulterated TSE for spermatogenesis, whereas the 87.5% micro-slit irradiated tissues showed an incomplete TSE. This suggests that the TSE efficiency for spermatogenesis is dependent on the size of the nonirradiated spermatogonial stem cell pool in the irradiated testicular tissues. In addition, there would be a spatiotemporal limitation of stem cell migration/competition, resulting in the insufficient TSE for 87.5% micro-slit irradiated tissues. These stem cell characteristics are essential for the accurate prediction of tissue-level responses during or after SFRT, indicating the clinical potential for achieving better outcomes while preventing adverse effects.

Functional impairment of the tumour suppressor PTEN is common in primary prostate cancer and has been linked to relapse post-radiotherapy (post-RT). Pre-clinical modelling supports elevated CXC chemokine signalling as a critical mediator of PTEN-depleted disease progression and therapeutic resistance. We assessed the correlation of PTEN deficiency with CXC chemokine signalling and its association with clinical outcomes. Gene expression analysis characterized a PTENLOW/CXCR1HIGH/CXCR2HIGH cluster of tumours that associates with earlier time to biochemical recurrence [hazard ratio (HR) 5.87 and 2.65, respectively] and development of systemic metastasis (HR 3.51). In vitro, CXCL signalling was further amplified following exposure of PTEN-deficient prostate cancer cell lines to ionizing radiation (IR). Inhibition of CXCR1/2 signalling in PTEN-depleted cell-based models increased IR sensitivity. In vivo, administration of a CXCR1/2-targeted pepducin (x1/2pal-i3), or CXCR2-specific antagonist (AZD5069), in combination with IR to PTEN-deficient xenografts attenuated tumour growth and progression compared to control or IR alone. Post-mortem analysis confirmed that x1/2pal-i3 administration attenuated IR-induced CXCL signalling and anti-apoptotic protein expression. Interventions targeting CXC chemokine signalling may provide an effective strategy to combine with RT in locally advanced prostate cancer patients with known presence of PTEN-deficient foci.

Background and purpose: Radiation-induced cardiac toxicity (RICT) remains one of the most critical dose limiting constraints in radiotherapy. Recent studies have shown higher doses to the base of the heart are associated with worse overall survival in lung cancer patients receiving radiotherapy. This work aimed to investigate the impact of sub-volume heart irradiation in a mouse model using small animal image-guided radiotherapy. Materials and methods: C57BL/6 mice were irradiated with a single fraction of 16 Gy to the base, middle or apex of the heart using a small animal radiotherapy research platform. Cone beam CT and echocardiography were performed at baseline and at 10 week intervals until 50 weeks post-treatment. Structural and functional parameters were correlated with mean heart dose (MHD) and volume of heart receiving 5 Gy (V5). Results: All irradiated mice showed a time dependent increase in left ventricle wall thickness in diastole of ~0.2 mm detected at 10 weeks post-treatment, with the most significant and persistent changes occurring in the heart base-irradiated animals. Similarly, statistically different functional effects (p < 0.01) were observed in base-irradiated animals which showed the most significant decreases compared to controls. The observed functional changes did not correlate with MHD and V5 (R 2 < 0.1), indicating that whole heart dosimetry parameters do not predict physiological changes resulting from cardiac sub-volume irradiation. Conclusions: This is the first report demonstrating the structural and functional consequences of sub-volume targeting in the mouse heart and reverse translates clinical observations indicating the heart base as a critical radiosensitive region.

BioXmark&reg, (Nanovi A/S, Denmark) is a novel fiducial marker based on a liquid, iodine-based and non-metallic formulation. BioXmark&reg, has been clinically validated and reverse translated to preclinical models to improve cone-beam CT (CBCT) target delineation in small animal image-guided radiotherapy (SAIGRT). However, in phantom image analysis and in vivo evaluation of radiobiological response after the injection of BioXmark&reg, are yet to be reported. In phantom measurements were performed to compare CBCT imaging artefacts with solid fiducials and determine optimum imaging parameters for BioXmark&reg, In vivo stability of BioXmark&reg, was assessed over a 5-month period, and the impact of BioXmark&reg, on in vivo tumour response from single-fraction and fractionated X-ray exposures was investigated in a subcutaneous syngeneic tumour model. BioXmark&reg, was stable, well tolerated and detectable on CBCT at volumes &le, 10 &micro, L. Our data showed imaging artefacts reduced by up to 84% and 89% compared to polymer and gold fiducial markers, respectively. BioXmark&reg, was shown to have no significant impact on tumour growth in control animals, but changes were observed in irradiated animals injected with BioXmark&reg, due to alterations in dose calculations induced by the sharp contrast enhancement. BioXmark&reg, is superior to solid fiducials with reduced imaging artefacts on CBCT. With minimal impact on the tumour growth delay, BioXmark&reg, can be implemented in SAIGRT to improve target delineation and reduce set-up errors.

Functional impairment of the tumour-suppressorPTENis common in primary-prostate cancer and has been linked to relapse post-radiotherapy (RT). Pre-clinical modelling supports elevated CXC-chemokine signaling as a critical mediator ofPTEN-depleted disease progression and therapeutic resistance. We assessed the correlation ofPTEN-deficiency with CXC-chemokine signaling and its association with clinical outcomes. Gene expression analysis characterized aPTENLOW/CXCR1HIGH/CXCR2HIGHcluster of tumors that associates with earlier time-to-biochemical recurrence (HR 5.87 and HR 2.65 respectively) and development of systemic metastasis (HR 3.51).In vitro, CXCL-signaling was further amplified following exposure ofPTEN-deficient prostate cancer cell lines to ionizing radiation (IR). Inhibition of CXCR1/2-signaling inPTEN-depleted cell-based models increased IR-sensitivity.In vivo, administration of a CXCR1/2-targeted pepducin (x1/2pal-i3), or CXCR2-specific antagonist (AZD5069), in combination with IR toPTEN-deficient xenografts attenuated tumor growth and progression compared to control or IR alone. Post-mortem analysis confirmed that x1/2pal-i3 administration attenuated IR-induced CXCL-signaling and anti-apoptotic protein expression. Interventions targeting CXC-chemokine signaling may provide an effective strategy to combine with radiotherapy, in both locally-advanced and oligometastatic-prostate cancers, with known presence ofPTEN-deficient foci.

The formation of sperm by the testes through the process of spermatogenesis is highly radiosensitive and can be affected by environmental, occupational and therapeutic radiation exposures. In this study, we applied an ex vivo mouse testis organ culture as an experimental model of spermatogenesis to investigate the radiobiological effects and to demonstrate its feasibility as a tool to determine response to complex, modulated radiation fields. This model uses Acr-GFP transgenic mice, which express the marker green fluorescent proteins specific for meiosis to allow observation of functional changes in real-time that can be used to analyze radiation-induced changes in the process of spermatogenesis. Our results showed that the model can accurately reproduce radiation-induced male germ cell toxicity, such as temporary infertility and permanent sterility. Furthermore, using a monochromatic X-ray microbeam, we applied this model to investigate the effects of heterogeneous radiation fields on testis tissue ex vivo. Our model represents a unique application in the field, which offers significant potential for gaining further mechanistic insight into radiation effects on the process of spermatogenesis.

Background and purpose: To evaluate the impact of ATR inhibition using AZD6738 in combination with radiotherapy on the response of non-small cell lung cancer (NSCLC) tumour models and a murine model of radiation induced fibrosis. Materials and methods: AZD6738 was evaluated as a monotherapy and in combination with radiation in vitro and in vivo using A549 and H460 NSCLC models. Radiation induced pulmonary fibrosis was evaluated by cone beam computed tomography (CBCT) and histological staining. Results: AZD6738 specifically inhibits ATR kinase and enhanced radiobiological response in NSCLC models but not in human bronchial epithelial cells (HBECs) in vitro. Significant tumour growth delay was observed in cell line derived xenografts (CDXs) of H460 cells (p < 0.05) which were less significant in A549 cells. Combination of AZD6738 with radiotherapy showed no significant change in lung tissue density by CBCT (p > 0.5) and histological scoring of radiation induced fibrosis (p > 0.5). Conclusion: Inhibition of ATR with AZD6738 in combination with radiotherapy increases tumour growth delay without observable augmentation of late radiation induced toxicity further underpinning translation towards clinical evaluation in NSCLC.

Purpose Recent demonstrations of normal tissue sparing by high-dose, high-dose-rate FLASH radiation therapy have driven considerable interest in its application to improve clinical outcomes. However, significant uncertainty remains about the underlying mechanisms of FLASH sparing and how deliveries can be optimized to maximize benefit from this effect. Rapid oxygen depletion has been suggested as a potential mechanism by which these effects occur, but this has yet to be quantitatively tested against experimental data. Methods and Materials Models of oxygen kinetics during irradiation were used to develop a time-dependent model of the oxygen enhancement ratio in mammalian cells that incorporates oxygen depletion. The characteristics of this model were then explored in terms of the dose and dose-rate dependence of the oxygen enhancement ratio. This model was also fit to experimental data from both in vitro and in vivo data sets. Results In cases of FLASH radiation therapy, this model suggests that oxygen levels can be depleted by amounts that are sufficient to affect radiosensitivity only in conditions of intermediate oxygen tension, with no effect seen at high or very low initial oxygen levels. The model also effectively reproduced the dose, dose rate, and oxygen tension dependence of responses to FLASH radiation therapy in a range of systems, with model parameters compatible with published data. Conclusions Oxygen depletion provides a credible quantitative model to understand the biological effects of FLASH radiation therapy and is compatible with a range of experimental observations of FLASH sparing. These results highlight the need for more detailed quantification of oxygen depletion under high-dose-rate radiation exposures in relevant systems and the importance of oxygen tension in target tissues for FLASH sparing to be observed.

Microbeam radiotherapy (MRT) is based on a spatial fractionation of synchrotron X-ray microbeams at the microscale level. Although the tissue-sparing effect (TSE) in response to non-uniform radiation fields was recognized more than one century ago, the TSE of MRT in the testes and its clinical importance for preventing male fertility remain to be determined. In this study, using the combination of MRT techniques and a unique ex vivo testes organ culture, we show, for the first time, the MRT-mediated TSE for the preservation of spermatogenesis. Furthermore, our high-precision microbeam analysis revealed that the survival and potential migration steps of the non-irradiated germ stem cells in the irradiated testes tissue would be needed for the effective TSE for spermatogenesis. Our findings indicated the distribution of dose irradiated in the testes at the microscale level is of clinical importance for delivering high doses of radiation to the tumor, while still preserving male fertility.

Prostate cancer (PCa) is the most common non-cutaneous cancer in men and a notable cause of cancer mortality when it metastasises. Localised disease is mostly treated with surgery or radiotherapy. As PCa develops and treatment resistance emerges, the unfolded protein response (UPR) arises as an important adaptive biology co-amplifying with key cancer drivers [1]. The UPR can be cytoprotective but when acutely activated can lead to cell death. In this study we sought to enhance the acute activation of the UPR using radiation and ONC201, previously reported to be an UPR activator [2]. We found that treating PCa cells with ONC201 quickly increases the expression of components in all arms of the UPR – ATF4, ATF6 and IRE1-XBP1 – culminating in the subsequent cell death. During this time window between UPR activation and cell death we tested the priming effect of short-term administration of ONC201 on radiation responses. Pre-treatment with ONC201 for 24 hours prior to irradiation led to enhanced cytotoxicity compared to radiation alone assessed by cell viability and clonogenic assays. With priming, RNA-Seq analysis showed a sustained suppression of transcripts encoding cell cycle regulators as well as components of the DNA damage response pathways. Phenotypically this was reflected in enhanced cell cycle arrest and induction of necrosis and apoptosis. Furthermore, we demonstrated that short-term administration of inhibitors of cell cycle regulators (Dinaciclib and BI2536), could replicate this priming effect. Thus, we propose future studies to assess the impact of the short-term administration of drugs targeting the UPR and cell cycle regulation to enhance radiotherapy response.

Precision medicine is a rapidly developing area that aims to deliver targeted therapies based on individual patient characteristics. However, current radiation treatment is not yet personalized; consequently, there is a critical need for specific patient characteristics of both tumor and normal tissues to be fully incorporated into dose prescription. Furthermore, current risk assessment following environmental, occupational, or accidental exposures to radiation is based on population effects, and does not account for individual diversity underpinning radiosensitivity. The lack of personalized approaches in both radiotherapy and radiation risk assessment resulted in the current situation where a population-based model, effective dose, is being used. In this review article, to stimulate scientific discussion for precision medicine in both radiotherapy and radiation risk assessment, we propose a novel radiological concept and metric - the personalized dose and the personalized risk index - that incorporate individual physiological, lifestyle-related and genomic variations and radiosensitivity, outlining the potential clinical application for precision medicine. We also review on recent progress in both genomics and biobanking research, which is promising for providing novel insights into individual radiosensitivity, and for creating a novel conceptual framework of precision radiotherapy and radiation risk assessment.

Translational research aims to provide direct support for advancing novel treatment approaches in oncology towards improving patient outcomes. Preclinical studies have a central role in this process and the ability to accurately model biological and physical aspects of the clinical scenario in radiation oncology is critical to translational success. The use of small animal irradiators with disease relevant mouse models and advanced in vivo imaging approaches offers unique possibilities to interrogate the radiotherapy response of tumors and normal tissues with high potential to translate to improvements in clinical outcomes. The present review highlights the current technology and applications of small animal irradiators, and explores how these can be combined with molecular and functional imaging in advanced preclinical radiotherapy research.

Despite a major paradigm shift in radiotherapy planning and delivery over the past three decades with continuing refinements, radiation induced lung damage (RILD) remains a major dose limiting toxicity in patients receiving thoracic irradiations. Our current understanding of the biological processes involved in RILD which includes DNA damage, inflammation, senescence and fibrosis, is based on clinical observations and experimental studies in mouse models using conventional radiation exposures. Whilst these studies have provided vital information on the pulmonary radiation response, the current implementation of small animal irradiators is enabling refinements in the precision and accuracy of dose delivery to mice which can be applied to studies of RILD. This review presents the current landscape of preclinical studies in RILD using small animal irradiators and highlights the challenges and opportunities for the further development of this emerging technology in the study of normal tissue damage in the lung.