Ces dernières années, le paradigme de la radiobiologie selon lequel les effets biologiques des rayonnements ionisants ne concernent strictement que les dommages à l'ADN et les conséquences liées à leur non réparation ou à leur réparation défectueuse, a été remis en question. Ainsi, plusieurs études suggèrent que des mécanismes «non centrés » sur l'ADN ont une importance significative dans les réponses radio-induites. Ces effets doivent donc être identifiés et caractérisés afin d’évaluer leurs contributions respectives dans des phénomènes tels que la radiorésistance, les risques associés au développement de cancers radio-induits, les conséquences des expositions aux faibles doses. Pour ce faire, il est nécessaire : (i) d'analyser la contribution de ces différentes voies de signalisation et réparation induites en fonction de la dose et de la zone d’irradiation; (ii) d’’étudier les réponses radio-induites suite à l’irradiation exclusive de compartiments subcellulaires spécifiques (exclure les dommages spécifiques à l'ADN nucléaire); (iii) d’améliorer la connaissance des mécanismes moléculaires impliqués dans les phénomènes de radiosensibilité/radiorésistance dans la perspective d’optimiser les protocoles de radiothérapie et d’évaluer in vitro de nouvelles thérapies associant par exemple les effets des rayonnements ionisants et de nanoparticules d’oxydes métalliques. Les microfaisceaux de particules chargées offrent des caractéristiques uniques pour répondre à ces questions en permettant (i) des irradiations sélectives et en dose contrôlée de populations cellulaires et donc l’étude in vitro des effets « ciblés » et « non ciblés » à l'échelle cellulaire et subcellulaire, (ii) de caractériser l’homéostasie de cultures cellulaires en réponses à des expositions aux rayonnements ionisants et/ou aux nanoparticules d’oxydes métalliques (micro-analyse chimique multi-élémentaire). Ainsi, au cours de ma thèse, j'ai validé et exploité des méthodes d’évaluation qualitatives et quantitatives (i) in cellulo et en temps réel de la réponse radio-induite de compartiments biologiques spécifiques (ADN, mitochondrie, …) ; (ii) in vitro de la radiosensibilité de lignées sarcomateuses issues de patients; et (iii) in vitro des effets induits par des expositions à des nanoparticules d'oxydes métalliques afin d’évaluer leur potentiel thérapeutique et anti-cancéreux. / Few years ago, the paradigm of radiation biology was that the biological effects of ionizing radiations occurred only if cell nuclei were hit, and that cell death/dysfunction was strictly due to unrepaired/misrepaired DNA. Now, next this “DNA-centric” view several results have shown the importance of “non-DNA centered” effects. Both non-targeted effects and DNA-targeted effects induced by ionizing radiations need to be clarified for the evaluation of the associated radiation resistance phenomena and cancer risks. A complete overview on radiation induced effects requires the study of several points: (i) analyzing the contribution of different signaling and repair pathways activated in response to radiation-induced injuries; (ii) elucidating non-targeted effects to explain cellular mechanisms induced in cellular compartments different from DNA; and (iii) improving the knowledge of sensitivity/resistance molecular mechanisms to adapt, improve and optimize the radiation treatment protocols combining ionizing radiations and nanoparticles. Charged particle microbeams provide unique features to answer these challenge questions by (i) studying in vitro both targeted and non-targeted radiation responses at the cellular scale, (ii) performing dose-controlled irradiations on a cellular populations and (iii) quantifying the chemical element distribution in single cells after exposure to ionizing radiations or nanoparticles. By using this tool, I had the opportunity to (i) use an original micro-irradiation setup based on charged particles microbeam (AIFIRA) with which the delivered particles are controlled in time, amount and space to validate in vitro methodological approaches for assessing the radiation sensitivity of different biological compartments (DNA and cytoplasm); (ii) assess the radiation sensitivity of a collection of cancerous cell lines derived from patients in the context of radiation therapy; (iii) study metal oxide nanoparticles effects in cells in order to understand the potential of nanoparticles in emerging cancer therapeutic approaches.

Radiotherapy remains one of the most important treatment modalities for cancer therapy. Malignant tumors show an extended spectrum of intrinsic radiation sensitivity even among tumors of the same entity or with similar histological features. Predicting intrinsic radiation sensitivity might improve treatment outcome and allow individualized treatment. Hence, an assay that provides a predictive information of the tumor’s intrinsic radiation sensitivity is of great need. Histone H2AX, a histone variant of histone H2A family, is rapidly phosphorylated upon DNA double strand break (DSB) induction resulting in gamma H2AX (γH2AX). Gamma H2AX accumulates at DNA DSB sites and subsequently recruits DNA damage repair factors. Formation of γH2AX is visualized by an immunohistology-based approach and detected as foci under an epifluorescent microscope. Gamma H2AX foci represent DNA DSBs, while residual γH2AX foci (foci detected 24 h post irradiation) are considered as unrepaired damages. In previous studies, the γH2AX foci assay showed a high potential as a predictive method for radiosensitivity. This thesis aims to further translate and optimize the ex vivo γH2AX foci assay for a clinical applicability. In this study, all experiments were performed using human head and neck squamous cell carcinoma (hHNSCC) xenograft models. For ex vivo investigations, tumors on the hind legs of nude mice were excised and cut into multiple pieces, or fine-needle biopsies of the tumors were taken. Tumor biopsies were reoxygenated in culture medium for 10 h or 24 h followed by radiation exposure of 0 8 Gy. Tumor biopsies were fixed and embedded in paraffin 24 h post irradiation. For the γH2AX foci assay under in vivo conditions, tumors-bearing mice were irradiated with single doses of 0 8 Gy. Tumors were excised, fixed, and paraffin embedded 24 h post irradiation. Manual quantification of γH2AX foci was performed exclusively in perfused areas, which were identified by pimonidazole (hypoxic marker) and BrdU (proliferation marker) staining. Foci number was corrected, normalized, and statistically analyzed by a linear mixed effects model (LMEM), linear regression model or analysis of covariance. To investigate tumor heterogeneity in the ex vivo γH2AX foci assay, γH2AX foci were enumerated in four equally treated tumor specimens per group i.e. unirradiated and ex vivo irradiated with 4 Gy. Strong intratumoral heterogeneity in γH2AX foci was determined with a minor intertumoral heterogeneity. No significant effect of reoxygenation between 10 h or 24 h was observed, enhancing clinical practicability of the assay. The effect of experimental settings was studied by analyzing data from this study (ex vivo) and from comparable published data (in vivo) with LMEM. Radiation induced nuclear area alteration was detected in some of the evaluated tumor models in under both experimental conditions. A greater intra and intertumoral heterogeneity were observed in the ex vivo set up compared to the in vivo set up. Radiation response determined by the γH2AX foci assay in ex vivo irradiated biopsies and in the corresponding in vivo irradiated tumors was evaluated. Between in vivo and ex vivo, four out of five tumor models showed comparable slopes of dose response curves (SDRC) of normalized and corrected γH2AX foci. SDRC of normalized γH2AX foci was able to classify tumors according to their intrinsic radiation sensitivity (TCD50). In conclusion, the ex vivo γH2AX foci assay holds a promising potential for predicting radiation sensitivity in solid tumors. The comparable radiation response assessed by γH2AX foci of in vivo irradiated tumors and the matching ex vivo irradiated tumor biopsies supports clinical applicability of the assay. Using SDRC of γH2AX foci as a predictor of radiosensitivity, radioresistant and radiosensitive tumors could be classified. The significant intratumoral heterogeneity in the ex vivo γH2AX foci assay suggests a limited representativeness of a single biopsy for radiosensitivity prediction. Additionally, the change of tumor microenvironment modulated cellular adaptation and DNA damage repair capability. The outcomes suggested that a sufficient number of cells, regions of interest, and biopsies are required to obtain a solid prediction.:Contents List of Abbreviations List of Figures List of Tables 1. Introduction 1.1 Effect of ionizing radiation on cellular level 1.1.1 Radiation induces cell death 1.1.2 Cell-cycle arrest mediated by radiation 1.2 DNA damage repair 1.2.1 Non homologous end joining (NHEJ) 1.2.2 Homologous recombination (HR) 1.2.3 Base damage repair and single strand break repair 1.2.4 Role of γH2AX in DNA damage repair 1.3 Prediction of tumor radioresponsiveness 1.3.1 Prediction of tumor radiation sensitivity by γH2AX 2 Tumor heterogeneity determined with a γH2AX foci assay: A study in human head and neck squamous cell carcinoma (hHNSCC) 2.1 Summary of the publication 3 Heterogeneity of γH2AX foci increases in ex vivo biopsies relative to in vivo tumors. 3.1 Summary of the publication 4 Comparable radiation response of ex vivo and in vivo irradiated tumor samples determined by residual γH2AX foci 4.1 Summary of the manuscript 5 Discussion 5.1 Tumor heterogeneity in γH2AX foci assay 5.2 Alteration of nuclear area post irradiation 5.3 Clinical relevance of the γH2AX foci assay 5.4 Technical challenges and limitations of the assay 5.5 Conclusion and Outlook 6 Abstract 7 Zusammenfassung 8 Bibliography Acknowledgement Appendices Part A: Materials A.1 Tumor lines A.2 Chemicals and Materials A.3 Devices and Software Part B: Supplementary materials B.1 Supplementary materials of publication I B.2 Supplementary materials of publication II B.3 Supplementary materials of manuscript

The nucleotide excision repair (NER) pathway is essential for repair of UV-induced bulky DNA lesions. NER is divided into two subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). UVC radiation has been shown to result in the formation of bulky DNA lesions, which are removed by NER. Previous published reports have shown a role for the p53 tumour suppressor protein in GGR and TCR, but the involvement of p53 in TCR has been controversial. In addition, it has also been suggested that hypoxia affects NER and expression of p53. In the present work, the role of p53, hypoxia and HIF-lα in NER was investigated. It was determined that p53 overexpression in primary human fibroblasts resulted in up-regulation of both the GGR and TCR subpathways of a UV -damaged reporter gene. Pre-treatment of cells with low UVC-fluence and p53 overexpression also induced an upregulation of GGR and TCR. These results are consistent with a p53-dependent upregulation of TCR and GGR of the UVC-damaged reporter gene, as well with a UV-inducible TCR and GGR that is dependent on p53 expression prior to UV treatment. Hypoxia coupled to low pH induced a transient up-regulation of p53 expression and NER in human primary normal fibroblasts and a concomitant decrease in UVC sensitivity. In contrast, in tumour cells hypoxia coupled to low pH resulted in a delayed, but not absent up-regulation of NER, which was p53-independent and did not result in a decrease in UVC sensitivity. We report here that it is the early transient p53-dependent up-regulation induced by hypoxia coupled to acidosis in human primary normal fibroblasts that may play a significant role in cellular UVC sensitivity. These data suggest a different cellular NER response to hypoxia compared to hypoxia coupled to low pH. The NER response to hypoxia and hypoxia coupled with acidosis was also different in primary cells when compared to tumour-derived cells. It was demonstrated that expression of dominant-negative HIF-lα in rat prostate tumour cells results in a reduction in host cell reactivation (HCR) of a UV-damaged reporter gene when compared to that in wild-type HIF-lα cells under normoxic conditions suggesting that basal HIF-lα expression may play an important role in NER. In addition we showed that hypoxia induced an up-regulation of NER in human primary normal fibroblasts that was delayed, but not absent in TCR-deficient CSB cells, suggesting a role for hypoxia in up-regulation of the GGR pathway of NER of a UVdamaged reporter gene. In contrast, HIF-lα-overexpression under conditions of hypoxia resulted in a down-regulation of NER in normal fibroblasts, which was delayed, but not absent in CSB fibroblasts. These results suggest that HIF-1α and CSB are involved in a hypoxia-induced NER response. This work provides further evidence that both GGR and TCR are p53-dependent. In addition, this study provides evidence that hypoxia and hypoxia coupled to acidosis can up-regulate NER in both primary and tumour cells, and that HIF-lα and the CSB protein play an important role in a hypoxia-induced NER response. / Thesis / Doctor of Philosophy (PhD)