Cellular Composition and Contribution of Tertiary Lymphoid Structures to Tumor Immune Infiltration and Modulation by Radiation Therapy

Immune-based anti-cancer strategies combined with radiation therapy (RT) are actively being investigated but many questions remain, such as the ideal treatment scheme and whether a potent immune response can be generated both locally and systemically. In this context, tumor-associated tertiary lymphoid structures (TLS) have become a subject of research. While TLS are present in several types of cancer with strong similarities, they are especially relevant in medullary breast carcinoma (MBC). This suggests that MBC patients are ideally suited for investigating this question and may benefit from adapted therapeutic options. As RT is a corner-stone of MBC treatment, investigating interactions between RT and TLS composition is also clinically relevant. We thus first characterized the lymphoid structures associated with MBC in a patient case report and demonstrated that they closely resemble the TLS observed in a genetical mouse model. In this model, we quantitatively and qualitatively investigated the cellular composition of the tumor-associated TLS. Finally, we investigated TLS regulation after hypo-fractionated RT and showed that RT induced their acute and transient depletion, followed by a restoration phase. This study is the first work to bring a comprehensive and timely characterization of tumor-associated TLS in basal conditions and after RT. It highlights cellular targets (i.e., Tregs) that could be selectively modulated in subsequent studies to optimize anti-tumor immune response. The study of TLS modulation is worth further investigation in the context of RT and personalized medicine

Professional development through mentoring: Final evaluation of the pilot mentoring programme of the European society of radiotherapy and oncology

: The European SocieTy for Radiotherapy and Oncology (ESTRO) organized a one-year pilot mentoring programme. At evaluation after one year, both mentors and mentees scored the programme with a median score of 9 on a scale of 10. All of the mentors indicated that they wanted to participate again as mentors

Normal Brain, Neural Stem Cells and Brain Tumors response to FLASH radiotherapy.

De nos jours, plus de 50% des patients porteurs de tumeur bénéficient d’un traitement de radiothérapie. Malgré de récentes avancées technologiques augmentant de la précision des traitements, la radiothérapie encéphalique induit toujours des effets secondaires invalidants et irréversibles. Ce constat justifie le développement de nouvelles techniques de radiothérapie. Des études précliniques réalisées sur l’irradiation FLASH ont montré la possibilité de maintenir un effet anti-tumoral tout en réduisant drastiquement les effets secondaires sur le tissu sain. Cet effet a été appelé « l’effet FLASH ». Cette technologie consistant à délivrer des doses à des débits supérieurs à 40 Gy/s a généré un intérêt important pour l’augmentation de l’index thérapeutique de la radiothérapie.Ce travail de thèse vise à étudier l’effet anti-tumoral de l’irradiation FLASH sur des modèles précliniques de glioblastome, tout en évaluant ses effets sur le tissu cérébral sain. Des modèles murins de glioblastome sous-cutané, orthotopique et transgénique ont été développés et irradiés grâce à un prototype d’accélérateur linéaire d’électrons délivrant une irradiation FLASH ou conventionnelle. De plus, des modèles murins d’irradiation encéphalique ont été mis au point afin d’investiguer les effets cellulaires et les altérations fonctionnelles induites par l’irradiation FLASH. La division cellulaire et la structure neuronale dans l’hippocampe ont été évaluées, ainsi que des aspects plus physiopathologiques comme la neuroinflammation ou l’astrogliose. Un panel de tests cognitifs a également été utilisé afin d’étudier les altérations cognitives induites par l’irradiation encéphalique. Enfin, les évènements physico-chimiques engendrés par l’irradiation FLASH et plus particulièrement le rôle de la consommation de dioxygène lors de l’irradiation, ont été analysés afin d’élucider les mécanismes qui supportent l’effet FLASH.Dans tous les modèles étudiés, l’irradiation FLASH a présenté un effet anti-tumoral au minimum similaire à celui de l’irradiation conventionnelle. Les modèles d’irradiation encéphalique ont montré une innocuité de l’irradiation FLASH sur le tissu cérébral sain, avec une absence de déficits cognitifs pour des débits de dose supérieurs à 100 Gy/s, couplée à une absence d’altération de la division cellulaire et de la structure neuronale dans l’hippocampe, une absence de neuroinflammation et d’astrogliose. De plus, des résultats similaires ont été observés avec l’utilisation de rayons X délivrés à ultra-haut débit par un rayonnement synchrotron. Sur le plan mécanistique, la réversion des effets protecteurs de l’irradiation FLASH par l’induction d’une hyperoxie, l’absence d’effet de l’anoxie sur l’effet anti-tumoral et la production de moins de radicaux libres souligne le rôle primaire du dioxygène dans l’effet FLASH.L’ensemble de ces résultats illustre la possibilité d’augmenter l’index thérapeutique de la radiothérapie en utilisant l’irradiation FLASH. En effet, cette nouvelle technologie permet de préserver le tissu sain contre les toxicités radio-induites lorsque l’irradiation est délivrée à des débits supérieurs à 100 Gy/s, tout en gardant un effet anti-tumoral équivalent à l’irradiation conventionnelle. D’après ces résultats précliniques et un transfert clinique dans un futur proche, l’irradiation FLASH pourrait devenir une technique de choix dans le traitement des tumeurs par radiothérapie.Nowadays, more than 50% of cancer patients can benefit from a radiation-therapy treatment. Despite important technological advance and dose delivery precision, encephalic radiation-therapy still induces large and irreversible side effects in pediatric and adult cancer patients, justifying the urge to develop new radiation-therapy techniques. Preclinical studies on FLASH irradiation (FLASH-RT) showed a possibility to efficiently treat the tumors, without inducing drastic side-effects on the normal tissue, by increasing the dose-rate over 40 Gy/s. This so called “FLASH effect” set off an important interest in this new irradiation technology to increase the therapeutic ratio of radiation-therapy.This PhD work aimed at investigating the antitumor effect of FLASH-RT on brain tumor models along with the assessment of the ultra-high dose-rate irradiation effects on the normal brain tissue. In this context, subcutaneous, orthotopic and transgenic glioblastoma murine models were used to investigate the curative effect of FLASH irradiation delivered with an experimental LINAC available at the CHUV, and able to deliver both conventional and FLASH irradiation. Moreover, murine models of whole brain irradiation were developed to investigate the radiation-induced cellular and functional alterations at early and late time-points post-FLASH-RT. These models were used to decipher the cellular effectors involved in the brain’s radiation response including hippocampal cell-division and neuronal responses but also more physio pathological aspects as radiation-induced reactive astrogliosis and neuroinflammation. A panel of well-defined cognitive tests was also developed to investigate the radiation-induced cognitive alterations. Eventually, the physio-chemical primary events induced by FLASH-RT, and particularly the role of dioxygen consumption, were investigated to decipher the mechanisms that underlie the FLASH effect.In all investigated tumor models, FLASH-RT displayed an efficient antitumor effect at least similar to the conventional irradiation. The whole brain irradiation models showed an innocuousness of FLASH-RT on the normal brain tissue, with an absence of cognitive deficit several months after irradiation at dose-rates above 100 Gy/s, coupled with a preservation of hippocampal cell division and neuronal structure. This protection was also observed at the physio pathological level with an absence of astrogliosis and neuroinflammation. Moreover, these results were reproduced with ultra-high dose-rate X-Rays delivered with a synchrotron light source. On the mechanistic side, the reversion of the protective effects of FLASH-RT by hyperoxia, and the absence of effect of anoxia on the antitumor effect, along with a decreased ROS production underlies the primary role of dioxygen consumption during ultra-high dose-rate irradiation.Altogether, these unique results depict the possibility to increase the therapeutic index of radiation-therapy by the use of FLASH-RT. Indeed, this new irradiation technology preserves the normal brain tissue from radiation-induced toxicities by increasing the dose-rate over 100 Gy/s, while keeping an antitumor effect equivalent to the conventional dose-rate irradiation. According to these preclinical results and an upcoming clinical translation, FLASH-RT might become a major contributor to the cancer treatment by radiation therapy

Ultra-high dose rate electron beams and the FLASH effect: From preclinical evidence to a new radiotherapy paradigm.

In their seminal paper from 2014, Fauvadon et al. coined the term FLASH irradiation to describe ultra-high-dose rate irradiation with dose rates greater than 40 Gy/s, which results in delivery times of fractions of a second. The experiments presented in that paper were performed with a high-dose-per-pulse 4.5 MeV electron beam, and the results served as the basis for the modern-day field of FLASH radiation therapy (RT). In this article, we review the studies that have been published after those early experiments, demonstrating the robust effects of FLASH RT on normal tissue sparing in preclinical models. We also outline the various irradiation parameters that have been used. Although the robustness of the biological response has been established, the mechanisms behind the FLASH effect are currently under investigation in a number of laboratories. However, differences in the magnitude of the FLASH effect between experiments in different labs have been reported. Reasons for these differences even within the same animal model are currently unknown, but likely has to do with the marked differences in irradiation parameter settings used. Here, we show that these parameters are often not reported, which complicates large multistudy comparisons. For this reason, we propose a new standard for beam parameter reporting and discuss a systematic path to the clinical translation of FLASH RT

Infrared microspectroscopy to elucidate the underlying biomolecular mechanisms of FLASH radiotherapy

Altres ajuts: acords transformatius de la UABFLASH-radiotherapy (FLASH-RT) is an emerging modality that uses ultra-high dose rates of radiation to enable curative doses to the tumor while preserving normal tissue. The biological studies showed the potential of FLASH-RT to revolutionize radiotherapy cancer treatments. However, the complex biological basis of FLASH-RT is not fully known yet. Aim: Within this context, our aim is to get deeper insights into the biomolecular mechanisms underlying FLASH-RT through Fourier Transform Infrared Microspectroscopy (FTIRM). C57Bl/6J female mice were whole brain irradiated at 10 Gy with the eRT6-Oriatron system. 10 Gy FLASH-RT was delivered in 1 pulse of 1.8μs and conventional irradiations at 0.1 Gy/s. Brains were sampled and prepared for analysis 24 h post-RT. FTIRM was performed at the MIRAS beamline of ALBA Synchrotron. Infrared raster scanning maps of the whole mice brain sections were collected for each sample condition. Hyperspectral imaging and Principal Component Analysis (PCA) were performed in several regions of the brain. PCA results evidenced a clear separation between conventional and FLASH irradiations in the 1800-950 cm region, with a significant overlap between FLASH and Control groups. An analysis of the loading plots revealed that most of the variance accounting for the separation between groups was associated to modifications in the protein backbone (Amide I). This protein degradation and/or conformational rearrangement was concomitant with nucleic acid fragmentation/condensation. Cluster separation between FLASH and conventional groups was also present in the 3000-2800 cm region, being correlated with changes in the methylene and methyl group concentrations and in the lipid chain length. Specific vibrational features were detected as a function of the brain region. This work provided new insights into the biomolecular effects involved in FLASH-RT through FTIRM. Our results showed that beyond nucleic acid investigations, one should take into account other dose-rate responsive molecules such as proteins, as they might be key to understand FLASH effect