Multiparametric radiobiological assays show that variation of X-ray energy strongly impacts relative biological effectiveness: comparison between 220 kV and 4 MV

International audienceBased on classic clonogenic assay, it is accepted by the scientific community that, whatever the energy, the relative biological effectiveness of X-rays is equal to 1. However, although X-ray beams are widely used in diagnosis, interventional medicine and radiotherapy, comparisons of their energies are scarce. We therefore assessed in vitro the effects of low- and high-energy X-rays using Human umbilical vein endothelial cells (HUVECs) by performing clonogenic assay, measuring viability/mortality, counting γ-H2AX foci, studying cell proliferation and cellular senescence by flow cytometry and by performing gene analysis on custom arrays. Taken together, excepted for γ-H2AX foci counts, these experiments systematically show more adverse effects of high energy X-rays, while the relative biological effectiveness of photons is around 1, whatever the quality of the X-ray beam. These results strongly suggest that multiparametric analysis should be considered in support of clonogenic assay

The TGF-β/Smad Repressor TG-Interacting Factor 1 (TGIF1) Plays a Role in Radiation-Induced Intestinal Injury Independently of a Smad Signaling Pathway

Despite advances in radiation delivery protocols, exposure of normal tissues during the course of radiation therapy remains a limiting factor of cancer treatment. If the canonical TGF-β/Smad pathway has been extensively studied and implicated in the development of radiation damage in various organs, the precise modalities of its activation following radiation exposure remain elusive. In the present study, we hypothesized that TGF-β1 signaling and target genes expression may depend on radiation-induced modifications in Smad transcriptional co-repressors/inhibitors expressions (TGIF1, SnoN, Ski and Smad7). In endothelial cells (HUVECs) and in a model of experimental radiation enteropathy in mice, radiation exposure increases expression of TGF-β/Smad pathway and of its target gene PAI-1, together with the overexpression of Smad co-repressor TGIF1. In mice, TGIF1 deficiency is not associated with changes in the expression of radiation-induced TGF-β pathway-related transcripts following localized small intestinal irradiation. In HUVECs, TGIF1 overexpression or silencing has no influence either on the radiation-induced Smad activation or the Smad3-dependent PAI-1 overexpression. However, TGIF1 genetic deficiency sensitizes mice to radiation-induced intestinal damage after total body or localized small intestinal radiation exposure, demonstrating that TGIF1 plays a role in radiation-induced intestinal injury. In conclusion, the TGF-β/Smad co-repressor TGIF1 plays a role in radiation-induced normal tissue damage by a Smad-independent mechanism

Time-course analysis of mouse serum proteome changes following exposure of the skin to ionizing radiation.

Radiation-induced lesion outcomes of normal tissues are difficult to predict. In particular, radiotherapy or local exposure to a radioactive source by accident can trigger strong injury to the skin. The finding of biomarkers is of fundamental relevance for the prediction of lesion apparition and its evolution, and for the settlement of therapeutic strategies. In order to study radiation-induced cutaneous lesions, we developed a mouse model in which the dorsal skin was selectively exposed to ionizing radiation (IR). 2-D difference gel electrophoresis (2-D DIGE) coupled with MS was used to investigate proteins altered in expression and/or PTM in serum. Proteome changes were monitored from 1 day to 1 month postirradiation, at a dose of 40 Gy, in this specific model developing reproducible clinical symptoms ranging from erythema to skin ulceration with wound healing. About 60 proteins (including some isoforms and likely post-translational variants), representing 20 different proteins, that exhibited significant and reproducible kinetic expression changes, were identified using MS and database searches. Several proteins, down- or up-regulated from day one, could prove to be good candidates to prognosticate the evolution of a skin lesion such as necrosis. In addition, we observed shifts in pI of several spot trains, revealing potential PTM changes, which could also serve as indicators of irradiation or as predictors of lesion severity

Preclinical model of stereotactic ablative lung irradiation using arc delivery in the mouse:effect of beam size changes and dose effect at constant collimation

International audiencePurpose: Stereotactic body radiation therapy is a therapeutic option offered to high surgical risk cancer patients with lung cancer. Focal lung irradiation in mice is a new preclinical model to help understand the development of lung damage in this context. Here we developed a mouse model of lung stereotactic therapy using arc delivery and monitored the development of lung damage while varying beam size and dose delivered.Methods and Materials: C57BL/6JRj mice were exposed to 90 Gy focal irradiation on the left lung, using 1 mm diameter, 3 x 3 mm2, 7 x 7 mm2 or 10 x 10 mm2 beam collimation for beam size effect, and using 3 x 3 mm2 beam collimation delivering 20 to 120 Gy for dose effect. Long-term lung damage was monitored with micro-CT imaging together with anatomopathological and gene expression measurements in the injured patch and the ipsilateral and contralateral lungs. Results: Both 1 mm diameter and 3 x 3 mm2 beam collimation allow long-term studies, but only 3 mm beam collimation generates lung fibrosis when delivering 90 Gy. Dose-effect studies with constant 3 mm beam collimation revealed a dose of 60 Gy as the minimum to obtain lung fibrosis 6 months post-exposure. Lung fibrosis development was associated with club cell depletion and increased type II pneumocyte numbers. Lung injury developed with ipsilateral and contralateral consequences such as parenchymal thickening and gene expression modifications. Conclusions: Arc therapy allows long-term studies and dose escalation without lethality. In our dose-delivering conditions, dose-effect studies revealed that 3 x 3 mm2 beam collimation to a minimum single dose of 60 Gy enables preclinical models for the assessment of lung injury within a 6-month period. This model of lung tissue fibrosis in a time length compatible with mouse life span may offer good prospects for future mechanistic studies

Preclinical model of stereotactic ablative lung irradiation using arc delivery in the mouse:fractionation effect

International audiencePurpose: pre-clinical modelling of radiation-induced lung damage is being challenged by emerging radiation therapies, such as stereotactic body radiation therapy. The aim of the present study was to implement previously published data on focal lung irradiation in mice using different fractionation schedules, with the objective of developing lung fibrosis in a time lapse that is compatible with the lifespan of a rodent.Methods and materials: the left lungs of C57BL/6JRj mice were exposed to ionizing radiation using arc therapy and 3 x 3 mm beam collimation. Three-fraction schedules delivered in 1 week were used with 20, 28, 40 and 50 Gy doses per fraction. Lung tissue opacification, global histological damage and the numbers of type II pneumocytes and club cells were assessed 6 months post-exposure, together with the gene expression of several lung cells and inflammation markers.Results: administration of 3 x 40 or 3 x 50 Gy generated focal lung fibrosis after 6 months, with tissue opacification visible by cone beam computed tomography, tissue scarring and consolidation, decreased club cell numbers and a reactive increase in the number of type II pneumocytes.Conclusions: a fractionation schedule using an arc-therapy-delivered 3 fraction/1 week regimen with 3 x 3 mm beam collimation was complex but feasible, with satisfying image-guided animal repositioning. Such a schedule generates focal lung fibrosis within 6 months when using 40 or 50 Gy per fraction. A comparison with previously published laboratory data suggests that, in this focal lung irradiation configuration, administrating a Biological Effective Dose ≥ 1000 Gy should be recommended to obtain lung fibrosis within 6 months

Detecting time periods of differential gene expression using Gaussian processes: An application to endothelial cells exposed to radiotherapy dose fraction

International audienceMotivation: Identifying the set of genes differentially expressed along time is an important task in two-sample time course experiments. Furthermore, estimating at which time periods the differential expression is present can provide additional insight into temporal gene functions. The current differential detection methods are designed to detect difference along observation time intervals or on single measurement points, warranting dense measurements along time to characterize the full temporal differential expression patterns. Results: We propose a novel Bayesian likelihood ratio test to estimate the differential expression time periods. Applying the ratio test to systems of genes provides the temporal response timings and durations of gene expression to a biological condition. We introduce a novel non-stationary Gaussian process as the underlying expression model, with major improvements on model fitness on perturbation and stress experiments. The method is robust to uneven or sparse measurements along time. We assess the performance of the method on realistically simulated dataset and compare against state-of-the-art methods. We additionally apply the method to the analysis of primary human endothelial cells under an ionizing radiation stress to study the transcriptional perturbations over 283 measured genes in an attempt to better understand the role of endothelium in both normal and cancer tissues during radiotherapy. As a result, using the cascade of differential expression periods, domain literature and gene enrichment analysis, we gain insights into the dynamic response of endothelial cells to irradiation

HIF-1α Deletion in the Endothelium, but Not in the Epithelium, Protects From Radiation-Induced Enteritis.

Radiation therapy in the pelvic area is associated with side effects that impact the quality of life of cancer survivors. Interestingly, the gastrointestinal tract is able to adapt to significant changes in oxygen availability, suggesting that mechanisms related to hypoxia sensing help preserve tissue integrity in this organ. However, hypoxia-inducible factor (HIF)-dependent responses to radiation-induced gut toxicity are unknown. Radiation-induced intestinal toxicity is a complex process involving multiple cellular compartments. Here, we investigated whether epithelial or endothelial tissue-specific HIF-1α deletion could affect acute intestinal response to radiation.Using constitutive and inducible epithelial or endothelial tissue-specific HIF-1α deletion, we evaluated the consequences of epithelial or endothelial HIF-1α deletion on radiation-induced enteritis after localized irradiation. Survival, radiation-induced tissue injury, molecular inflammatory profile, tissue hypoxia, and vascular injury were monitored.Surprisingly, epithelium-specific HIF-1α deletion does not alter radiation-induced intestinal injury. However, irradiated VECad-Cre+/-HIF-1αFL/FL mice present with lower radiation-induced damage, showed a preserved vasculature, reduced hypoxia, and reduced proinflammatory response compared with irradiated HIF-1αFL/FL mice.We demonstrate in vivo that HIF-1α impacts radiation-induced enteritis and that this role differs according to the targeted cell type. Our work provides a new role for HIF-1α and endothelium-dependent mechanisms driving inflammatory processes in gut mucosae. Results presented show that effects on normal tissues have to be taken into account in approaches aiming to modulate hypoxia or hypoxia-related molecular mechanisms

Variation of 4 MV X-ray dose rate in fractionated irradiation strongly impacts biological endothelial cell response in vitro

International audienceComparisons of X-ray beam dose rates are scarce although these beams are widely used in medical diagnosis or radiotherapy. We have recently demonstrated in vitro and in vivo, that for a single dose of irradiation, the relative biological effectiveness (RBE) deviates from 1 when changing the dose rate of high energy X-ray beams. To further investigate the impact of the dose rate on RBE, in this study we performed in vitro fractionated irradiations by using the same two dose rates (0.63 and 2.5 Gy.min-1) of high-energy X-rays (both at 4 MV) on normal endothelial cells (HUVECs). We studied the viability/mortality, measured cellular senescence by flow cytometry and performed gene analysis on custom arrays. Taken together, these experiments show that the RBE of photons deviates from 1 when varying the dose rate of high-energy X-rays in fractionated irradiations. These results strengthen the interest of multiparametric analysis approaches in providing an accurate evaluation of the outcomes of irradiated cells in support of clonogenic assays, especially when such assays are not feasible

HIF-1α Deletion in the Endothelium, but Not in the Epithelium, Protects From Radiation-Induced Enteritis

International audienceBackground & Aims: Radiation therapy in the pelvic area is associated with side effects that impact the quality of life of cancer survivors. Interestingly, the gastrointestinal tract is able to adapt to significant changes in oxygen availability, suggesting that mechanisms related to hypoxia sensing help preserve tissue integrity in this organ. However, hypoxia-inducible factor (HIF)-dependent responses to radiation-induced gut toxicity are unknown. Radiation-induced intestinal toxicity is a complex process involving multiple cellular compartments. Here, we investigated whether epithelial or endothelial tissue-specific HIF-1α deletion could affect acute intestinal response to radiation.Methods: Using constitutive and inducible epithelial or endothelial tissue-specific HIF-1α deletion, we evaluated the consequences of epithelial or endothelial HIF-1α deletion on radiation-induced enteritis after localized irradiation. Survival, radiation-induced tissue injury, molecular inflammatory profile, tissue hypoxia, and vascular injury were monitored.Results: Surprisingly, epithelium-specific HIF-1α deletion does not alter radiation-induced intestinal injury. However, irradiated VECad-Cre+/-HIF-1αFL/FL mice present with lower radiation-induced damage, showed a preserved vasculature, reduced hypoxia, and reduced proinflammatory response compared with irradiated HIF-1αFL/FL mice.Conclusions: We demonstrate in vivo that HIF-1α impacts radiation-induced enteritis and that this role differs according to the targeted cell type. Our work provides a new role for HIF-1α and endothelium-dependent mechanisms driving inflammatory processes in gut mucosae. Results presented show that effects on normal tissues have to be taken into account in approaches aiming to modulate hypoxia or hypoxia-related molecular mechanisms