Dosimetric and biologic intercomparison between electron and proton FLASH beams.

Background and purpose: The FLASH effect has been validated in different preclinical experiments with electrons (eFLASH) and protons (pFLASH) operating at a mean dose rate above 40 Gy/s. However, no systematic intercomparison of the FLASH effect produced by e vs. pFLASH has yet been performed and constitutes the aim of the present study. Materials and methods: The electron eRT6/Oriatron/CHUV/5.5 MeV and proton Gantry1/PSI/170 MeV were used to deliver conventional (0.1 Gy/s eCONV and pCONV) and FLASH (≥100 Gy/s eFLASH and pFLASH) irradiation. Protons were delivered in transmission. Dosimetric and biologic intercomparisons were performed with previously validated models. Results: Doses measured at Gantry1 were in agreement (± 2.5%) with reference dosimeters calibrated at CHUV/IRA. The neurocognitive capacity of e and pFLASH irradiated mice was indistinguishable from the control while both e and pCONV irradiated cohorts showed cognitive decrements. Complete tumor response was obtained with the two beams and was similar between e and pFLASH vs. e and pCONV. Tumor rejection was similar indicating that T-cell memory response is beam-type and dose-rate independent. Conclusion: Despite major differences in the temporal microstructure, this study shows that dosimetric standards can be established. The sparing of brain function and tumor control produced by the two beams were similar, suggesting that the most important physical parameter driving the FLASH effect is the overall time of exposure which should be in the range of hundreds of milliseconds for WBI in mice. In addition, we observed that immunological memory response is similar between electron and proton beams and is independent off the dose rate.

Novel unconventional radiotherapy techniques: Current status and future perspectives - Report from the 2nd international radiation oncology online seminar.

•Improvement of therapeutic ratio by novel unconventional radiotherapy approaches.•Immunomodulation using high-dose spatially fractionated radiotherapy.•Boosting radiation anti-tumor effects by adding an immune-mediated cell killing.

Distinct nitrogen isotopic compositions of healthy and cancerous tissue in mice brain and head&neck micro-biopsies.

BACKGROUND: Cancerous cells can recycle metabolic ammonium for their growth. As this ammonium has a low nitrogen isotope ratio (15N/14N), its recycling may cause cancer tissue to have lower 15N/14N than surrounding healthy tissue. We investigated whether, within a given tissue type in individual mice, tumoral and healthy tissues could be distinguished based on their 15N/14N. METHODS: Micro-biopsies of murine tumors and adjacent tissues were analyzed for 15N/14N using novel high-sensitivity methods. Isotopic analysis was pursued in Nude and C57BL/6 mice models with mature orthotopic brain and head&neck tumors generated by implantation of H454 and MEERL95 murine cells, respectively. RESULTS: In the 7 mice analyzed, the brain tumors had distinctly lower 15N/14N than healthy neural tissue. In the 5 mice with head&neck tumors, the difference was smaller and more variable. This was at least partly due to infiltration of healthy head&neck tissue by tumor cells. However, it may also indicate that the 15N/14N difference between tumoral and healthy tissue depends on the nitrogen metabolism of the healthy organ in question. CONCLUSIONS: The findings, coupled with the high sensitivity of the 15N/14N measurement method used here, suggest a new approach for micro-biopsy-based diagnosis of malignancy as well as an avenue for investigation of cancer metabolism.

Isodose 20 Gy found as a threshold dose for radiation recall dermatitis.

Radiation recall is a rare phenomenon that can be observed in the field of radiotherapy, months or years after irradiation when a patient is exposed to certain pharmaceutical agents. In this report, we relate a case of radiation recall dermatitis induced after the application of a topical natural cream, 2 years after the initial radiotherapy treatment. Skin reactions were severe and limited to the irradiated volume, whereas a large part of the skin where the cream was applied outside the radiation field was strictly normal. More precisely, the radiation recall dermatitis matched with the isodose 20 Gy, whereas no recall reaction was observed in the lower dose areas (5, 10 or 15 Gy) despite these areas were also largely exposed to the cream. In conclusion, this is the first report that could provide a threshold dose for the occurrence of a radiation recall dermatitis, which was not observed below 20 Gy, in the context of this topical reagent.

Biological Benefits of Ultra-high Dose Rate FLASH Radiotherapy: Sleeping Beauty Awoken.

FLASH radiotherapy (FLASH-RT) is a technology that could modify the way radiotherapy is delivered in the future. This technique involves the ultra-fast delivery of radiotherapy at dose rates several orders of magnitude higher than those currently used in routine clinical practice. This very short time of exposure leads to the striking observation of relative protection of normal tissues that are exposed to FLASH-RT as compared with conventional dose rate radiotherapy. Here we summarise the current knowledge about the FLASH effect and provide a synthesis of the observations that have been reported on various experimental animal models (mice, zebrafish, pig, cats), various organs (lung, gut, brain, skin) and by various groups across 40 years of research. We also propose possible mechanisms for the FLASH effect, as well as possible paths for clinical application.

[Ultrahigh dose-rate, "flash" irradiation minimizes the side-effects of radiotherapy]. / Radiothérapie « flash ¼ à très haut débit de dose : un moyen d'augmenter l'indice thérapeutique par minimisation des dommages aux tissus sains ?

PURPOSE: Pencil beam scanning and filter free techniques may involve dose-rates considerably higher than those used in conventional external-beam radiotherapy. Our purpose was to investigate normal tissue and tumour responses in vivo to short pulses of radiation. MATERIAL AND METHODS: C57BL/6J mice were exposed to bilateral thorax irradiation using pulsed (at least 40 Gy/s, flash) or conventional dose-rate irradiation (0.03 Gy/s or less) in single dose. Immunohistochemical and histological methods were used to compare early radio-induced apoptosis and the development of lung fibrosis in the two situations. The response of two human (HBCx-12A, HEp-2) tumour xenografts in nude mice and one syngeneic, orthotopic lung carcinoma in C57BL/6J mice (TC-1 Luc+), was monitored in both radiation modes. RESULTS: A 17 Gy conventional irradiation induced pulmonary fibrosis and activation of the TGF-beta cascade in 100% of the animals 24-36 weeks post-treatment, as expected, whereas no animal developed complications below 23 Gy flash irradiation, and a 30 Gy flash irradiation was required to induce the same extent of fibrosis as 17 Gy conventional irradiation. Cutaneous lesions were also reduced in severity. Flash irradiation protected vascular and bronchial smooth muscle cells as well as epithelial cells of bronchi against acute apoptosis as shown by analysis of caspase-3 activation and TUNEL staining. In contrast, the antitumour effectiveness of flash irradiation was maintained and not different from that of conventional irradiation. CONCLUSION: Flash irradiation shifted by a large factor the threshold dose required to initiate lung fibrosis without loss of the antitumour efficiency, suggesting that the method might be used to advantage to minimize the complications of radiotherapy.

Normal tissues toxicities triggered by combined anti-angiogenic and radiation therapies: hurdles might be ahead.

BACKGROUND: Combined-modality therapy is a promising approach to improve the therapeutic index of radiotherapy. However, these improvements could come at the cost of increased toxicities. Clinical trials evaluating anti-tumour efficacy of bevacizumab combined with radiotherapy have encountered unexpected side effects. This study is the first systematic evaluation of normal tissue toxicity triggered by anti-angiogenic agents combined with radiation therapy in mice. METHODS: Effect of a mouse anti-VEGF antibody was monitored on acute toxicity studying radiation-induced intestinal ulceration (12 Gy TBI); on subacute toxicity using a model of oral mucositis (16.5 Gy); on late radiation injuries by monitoring lung fibrosis (bleomycin and 19 Gy). RESULTS: Combination of irradiation with anti-VEGF antibody enhanced intestinal damages with severe epithelial ulcerations, had no adverse impact on oral mucositis and dramatically worsened the fibrotic picture induced by bleomycin and irradiation to the lung. INTERPRETATION: These reports bring to light the important questions about safety and underscore the need for appropriate preclinical modelling of the impact on normal tissues of novel drug-radiation regimens. Our findings also highlight the complexity of anti-VEGF action, which could in defined conditions exert tissue-specific protection. The findings indicate that the combination of targeted drugs with radiotherapy should be approached with caution.

[Cancer stem cells: a new target for lung cancer treatment]. / Cellules souches cancéreuses : nouvelle cible thérapeutique dans le traitement des cancers bronchopulmonaires.

Lung cancer remains the leading cause of cancer death. Understanding lung tumours physiopathology should provide opportunity to prevent tumour development or/and improve their therapeutic management. Cancer stem cell theory refers to a subpopulation of cancer cells also named tumour initiating cells that can drive cancer development. Cells presenting these characteristics have been identified and isolated from lung cancer. Exploring cell markers and signalling pathways specific to lung cancer stem cells may lead to progress in therapy and improve the prognosis of patients with lung cancer. Continuous efforts in developing in vitro and in vivo models may yield reliable tools to better understand cancer stem cell abilities and to test new therapeutic targets. Even if some data are in favour of a higher chemo and radioresistance of cancer stem cells this issue remains disputed. Preclinical data on putative cancer stem cell targets are emerging by now. These preliminary studies are critical for the next generation of lung cancer therapies.

[Pharmacological modulation of late radio-induced side effects]. / Modulation pharmacologique des effets tardifs de l'irradiation.

After normal tissue exposure to radiation therapy, late side effects can occur and may reduce patients' quality of life due to their progressive nature. Late toxicities occurrence is the main limiting factor of radiotherapy. Various biological disorders related to irradiation are involved in the development of late toxicities including fibrosis. The present review will focus on the recent physiopathological and molecular mechanisms described to be involved in the development of late radio-induced toxicities, that provide therapeutic perspective for pharmacomodulation.

[Reirradiation of normal tissues: preclinical radiobiological data]. / Réirradiation des tissus sains: données radiobiologiques précliniques.

Reirradiation represent an unfrequent particular clinical situation. The risk/benefit ratio assessment must be taken into account, considering both clinical and dosimetric aspects. There is a relatively limited amount of preclinical data available to date and clinicians should cautiously perform reirradiations in selected indications. This review summarizes the experimental data available on reirradiation of normal tissues, the consequences on early and late toxicities as well as the intrinsic limitations of these models.

Fc-receptor-mediated intracellular delivery of Cu/Zn-superoxide dismutase (SOD1) protects against redox-induced apoptosis through a nitric oxide dependent mechanism.

BACKGROUND: Using specific antibodies against bovine Cu/Zn-superoxide dismutase (EC 1.15.1.1, SOD1) we demonstrated that anti-SOD antibodies (IgG1) are able to promote the intracellular translocation of the antioxidant enzyme. The transduction signalling mediated by IgG1 immune complexes are known to promote a concomitant production of superoxide and nitric oxide leading to the production of peroxynitrites and cell death by apoptosis. The Fc-mediated intracellular delivery of SOD1 thus limited the endogenous production of superoxide. It was thus of interest to confirm that in the absence of superoxide anion, the production of nitric oxide protected cells against apoptosis. Study in greater detail clearly stated that under superoxide anion-free conditions, nitric oxide promoted the cell antioxidant armature and thus protected cells against redox-induced apoptosis. MATERIALS AND METHODS: The murine macrophage cell-lines J774 A1 were preactivated or not with interferon-gamma and were then stimulated by IgG1 immune complexes (IC), free SOD1 or SOD1 IC and superoxide anion, nitric oxide, peroxynitrite, and tumor necrosis factor-alpha (TNF-alpha) production was evaluated. The redox consequences of these activation processes were also evaluated on mitochondrial respiration and apoptosis as well as on the controlled expression of the cellular antioxidant armature. RESULTS: We demonstrated that SOD1 IC induced a Fcgamma receptor (FcgammaR)-dependent intracellular delivery of the antioxidant enzyme in IFN-gamma activated murine macrophages (the J774 AI cell line). The concomitant stimulation of the FcyR and the translocation of the SOD1 in the cytoplasm of IFN-gamma-activated macrophages not only reduced the production of superoxide anion but also induced the expression of the inducible form of nitric oxide synthase (iNOS) and the related NO production. This inducing effect in the absence of superoxide anion production reduced mitochondrial damages and cell death by apoptosis and promoted the intracellular antioxidant armature. CONCLUSIONS: To define the pharmacologic mechanism of action of bovine SOD1, we attempted to identify the second messengers that are induced by SOD1 IC. In this work, we propose that Fc-mediated intracellular delivery of the SOD1 that reduced the production of superoxide anion and of peroxynitrite, promoted a NO-induced protective effect in inducing the antioxidant armature of the cells. Taken together, these data suggested that specific immune responses against antigenic SOD1 could promote the pharmacological properties of the antioxidant enzyme likely via a NO-dependent mechanism.

Striking regression of subcutaneous fibrosis induced by high doses of gamma rays using a combination of pentoxifylline and alpha-tocopherol: an experimental study.

PURPOSE: To establish a successful treatment of subcutaneous fibrosis developing after high doses of gamma rays, suitable for use in clinical practice. METHODS AND MATERIALS: We used an animal model of acute localized gamma irradiation simulating accidental overexposure in humans. Three groups of 5 Large White pigs were irradiated using a collimated 192Ir source to deliver a single dose of 160 Gy onto the skin surface (100%) of the outer side of the thigh. A well-defined block of necrosis developed within a few weeks which had healed after 26 weeks to leave a block of subcutaneous fibrosis involving skin and skeletal muscle. One experimental group of 5 pigs was dosed orally for 26 weeks starting 26 weeks after irradiation with 1600 mg/120 kg body weight of pentoxifylline (PTX) included in the reconstituted food during its fabrication, and another group of 5 was dosed orally for the same period with a daily dose of 1600 mg/120 kg body weight of PTX combined with 2000 IU/120 kg body weight of alpha-tocopherol. Five irradiated control pigs were given normal food only. Animals were assessed for changes in the density of the palpated fibrotic block and in the dimensions of the projected cutaneous surface. Depth of scar tissue was determined by ultrasound. Physical and sonographic findings were confirmed by autopsy 26 weeks after treatment started. The density, length, width, and depth of the block of fibrotic scar tissue, and the areas and volume of its projected cutaneous surface, were compared before treatment, 6 and 13 weeks thereafter, and at 26 weeks. RESULTS: The experimental animals exhibited no change in behavior and no abnormal clinical or anatomic signs. No modifications were observed in the block of fibrotic scar tissue of pigs dosed with PTX alone. However, significant softening and shrinking of this block were noted in the pigs dosed with PTX + alpha-tocopherol 13 weeks after treatment started and at autopsy, when mean regression was approximately 30% for length, approximately 50% for width and depth, and approximately 70% for area and volume. Histologic examination showed completely normal muscle and subcutaneous tissue surrounding the residual scar tissue. The 50% decrease in the linear dimensions of the scar tissue, were comparable to the results obtained in our previous clinical studies, and were highly significant compared to the clinical and autopsy results for the controls. Histologic examination of the residual scar tissue revealed tissue which was more homogenous and less cellular and inflammatory than in control and PTX-dosed pigs. The tissular and cellular immunolocalization of tumor necrosis factor alpha (TNFalpha) was similar in the residual fibrotic tissues of all three groups of pigs, whereas the immunostaining of transforming growth factor beta-1(TGFbeta-1) diminished much more in the residual fibrotic scar tissue of the PTX + alpha-tocopherol-dosed pigs than in the two other groups. CONCLUSIONS: The present results showed a striking regression of the subcutaneous fibrotic scar tissue that develops as a consequence of high doses of gamma rays.

Coactivation of AP-1 activity and TGF-beta1 gene expression in the stress response of normal skin cells to ionizing radiation.

Activation of the AP-1 transcription factor and TGF-beta1 growth factor by ionizing radiation was studied both in vivo in pig skin, and in vitro in human fibroblasts and keratinocytes. Three and 6 h after irradiation, the Fos and Jun proteins and their binding activity to an AP-1 consensus sequence were strongly induced by high doses of gamma-rays. c-Fos, c-Jun and JunB proteins were found to be present in gel-shift complexes by probing with specific antibodies. Both keratinocytes and fibroblasts exhibited heightened AP-1 activity following irradiation. As we previously found that TGF-beta1 is involved in the development of skin lesions induced by radiation, TGF-beta1 gene expression was also examined. Two and 6 h after irradiation, the levels of TGF-beta1 transcripts were increased in skin. By immunostaining, TGF-beta1 protein levels were found to be increased in fibroblasts, keratinocytes and endothelial cells. As the TGF-beta1 promoter contains AP-1 binding sites, the relation between AP-1 activity and TGF-beta1 induction was addressed. The -365 TGF-beta1 promoter fragment, which contains a high affinity AP-1 site, exhibited increased binding to Jun and Fos proteins following irradiation. These results suggest that stress-inducible TGF-beta1 expression is mediated by the activation of AP-1 transcription factor.