Dissertations / Theses on the topic 'Molecular radiotherapy'

Many types of dosimeters are used in the clinic to measure radiation dose for therapy but none of them directly measures the biological effect of this dose. The overall purpose of this work was to develop a dosimeter that measures biological damage in the form of double-strand breaks to deoxyribonucleic acid. This dosimeter could provide a more biologically relevant measure of radiation damage than the currently utilized dosimeters. A pair of oligonucleotides was designed to fabricate this dosimeter. One is labeled with a 5’-end biotin and the other with a 5’-end 6 Fluorescein amidite (fluorescent dye excited at 495?nanometer, with a peak emission at 520 nanometer). These were designed to adhere to certain locations on the pRS316 vector and serve as the primers for polymerase chain reactions. The end product of this reaction is a 4 kilo-base pair double strands deoxyribonucleic acid fragment with biotin on one end and 6 Fluorescein amidite oligonucleotide on the other attached to streptavidin beads. The biotin end connects the double strands deoxyribonucleic acid to the streptavidin bead. These bead-connected double strands deoxyribonucleic acid were suspended in 50 microliter of phosphate-buffered saline and placed into a tube for irradiation. Following irradiation of the deoxyribonucleic acid dosimeter, we take advantage of the magnetic properties of the streptavidin bead by placing our sample microtube against a magnet. The magnetic field pulls the streptavidin beads against the side of the tube. If a double-strand-break has occurred for a double strands deoxyribonucleic acid, the fluorescein end of the double strands deoxyribonucleic acid becomes free and is no longer attached to the bead or held against the side of the microtube. The free fluorescein following a double-strand-break in double strands deoxyribonucleic acid is referred to here as supernatant. The supernatant is extracted and placed in another microtube, while the unbroken double strands deoxyribonucleic acid remain attached to the beads and stay in the microtube (Fig. 4). Those beads were re-suspended with 50 microliter of phosphate-buffered saline again (called beads), then we placed both supernatant and beads in a reader microplate and we read the fluorescence signal for both with a fluorescence reader (BioTek Synergy 2). These beads and supernatant fluorescence signals are denoted by B and S, respectively. The relative amount of supernatant fluorescence counts is proportional to the probability of a double-strand-break. The probability of double-strand-break was calculated with the following equation:

(S-BG)/(S+B-2BG) (1)

where S was the supernatant fluorescence intensity (related to the number of double strands deoxyribonucleic acid with double-strand breaks), B was the re-suspended beads fluorescence intensity (related to the number of double strands deoxyribonucleic acid without double-strand breaks), and BG was the phosphate-buffered saline fluorescence intensity (related to the background signal). There are two advantages that this type of dosimeter has over the gel separation technique. First, it is important to irradiate deoxyribonucleic acid in a solution that has similar osmolarity and ion concentrations to that in a human, such as phosphate-buffered saline. A gel dosimeter would require a transfer to gel to separate deoxyribonucleic acid, whereas our dosimeter can be separated in this solution. Currently, we use pipettes to manually perform this separation, but this step could be automated. Second, the magnetic deoxyribonucleic acid separation technique is much faster than that for gel electrophoresis. Calibration of radiotherapy equipment isn’t something that happens in national science laboratories, with only world-leading experts. This is something that happens locally at every cancer clinic, with physicists that do not have the luxury of focusing solely on this one measurement. For this reason, ease of use is critical for this type of technology. (Abstract shortened by ProQuest.)

Molecular radiotherapy (MRT) is a fast developing and promising treatment for metastasised neuroendocrine tumours. Efficacy of MRT is based on the capability to selectively "deliver" radiation to tumour cells, minimizing administered dose to normal tissues. Outcome of MRT depends on the individual patient characteristics. For that reason, personalized treatment planning is important to improve outcomes of therapy. Dosimetry plays a key role in this setting, as it is the main physical quantity related to radiation effects on cells. Dosimetry in MRT consists in a complex series of procedures ranging from imaging quantification to dose calculation. This doctoral thesis focused on several aspects concerning the clinical implementation of absorbed dose calculations in MRT. Accuracy of SPECT/CT quantification was assessed in order to determine the optimal reconstruction parameters. A model of PVE correction was developed in order to improve the activity quantification in small volume, such us lesions in clinical patterns. Advanced dosimetric methods were compared with the aim of defining the most accurate modality, applicable in clinical routine. Also, for the first time on a large number of clinical cases, the overall uncertainty of tumour dose calculation was assessed. As part of the MRTDosimetry project, protocols for calibration of SPECT/CT systems and implementation of dosimetry were drawn up in order to provide standard guidelines to the clinics offering MRT. To estimate the risk of experiencing radio-toxicity side effects and the chance of inducing damage on neoplastic cells is crucial for patient selection and treatment planning. In this thesis, the NTCP and TCP models were derived based on clinical data as help to clinicians to decide the pharmaceutical dosage in relation to the therapy control and the limitation of damage to healthy tissues. Moreover, a model for tumour response prediction based on Machine Learning analysis was developed.

Over half of the 120,000 patients diagnosed with a solid tumour in Australia annually require radiotherapy as part of their treatment. Despite significantly enhancing cancer survival, the damage done to healthy native tissues is inevitable and problematic. Radiotherapy soft tissue injury is progressive, may intensify years after treatment and is characterised by pain, contracture, tissue breakdown, recurrent infection and lymphoedema. Important implications of radiation injury for surgeons are that surgical procedures (whether for functional restoration or cancer recurrence) in irradiated tissues become technically difficult and more hazardous. Direct wound closure or local flaps are restricted by stiff, non-compliant tissue, and even if wound edges are opposable, they are frequently subject to poor wound healing or breakdown. Further, the ability of an irradiated wound bed to accept skin grafts is diminished, necessitating more complex reconstructive procedures such as free microvascular tissue transfer from distant sites, in which radiation injury is also a chief contributing factor to poor patient outcome. First, essential cell-specific functions were investigated to establish the effects of radiotherapy-injury on the ability of the cells in skin and subcutaneous tissues to survive or respond to subsequent injury or challenge. Next, molecular alterations resulting from irradiation were characterised at mRNA level using next generation sequencing and further investigated using pathway analysis. Finally, the reparative potential of the adipose-derived stem cells (ADSCs) to reduce radiotherapy injury was investigated in using a similar panel of cellular assays. The constituent proteins secreted by these cells that exerted a regenerative effect were then isolated for further analyses. The results detailed in this thesis demonstrate that each individual component of skin and subcutaneous tissue exhibits a unique response to radiotherapy injury - challenging the traditional dogma that large scale irreversible cell death is responsible for the manifestation of radiotherapy soft tissue injury. Notable findings include radiotherapy-induced hyper-migration of fibroblasts and pericytes, reduced apoptosis in endothelial and stem cell populations, global suppression of lymphatic endothelial cell (LEC) repair functions and significant alterations in the differentiation capacity of ADSCs. Next generation gene sequencing revealed key molecular alterations resulting from radiotherapy in a variety of cell types. Significant findings include dysregulation of extracellular matrix proteins and basement membrane collagens – changes likely to contribute to the hypermigratory, adhesive and highly contractile phenotype seen in irradiated fibroblasts. Up-regulation of intercellular adhesion molecule 1 (ICAM-1) in blood vessel endothelial cells found in acute radiotherapy injury was validated in irradiated human tissues and was demonstrated to remain persistently elevated months-to-years after completion of therapy. This finding suggested a key candidate for application to mitigate radiotherapy injury at both the micro and macrovascular level. In order to combat the impaired functions seen in LECs after irradiation, experiments were conducted using stimulation with known potent lymphangiogenic factors VEGF-C and VEGF-D. Irradiated LEC demonstrated an obliterated capacity for response to this stimulation, due to a unique profile of ablated VEGF receptor (VEGFR) -2 signaling and reduced VEGFR-3 activation. Concurrently, up-regulation of interleukin (IL) -8 and chemokine receptor CXCR7 in irradiated LEC was seen and validated in mouse and human tissues to remain upregulated in chronic radiotherapy injury. These two protein candidates, not typically associated with lymphangiogenic properties demonstrated selective lymphangiogenic effect in both normal and irradiated LECs. Together this novel set of data suggest that LECs attempt to regenerate after radiotherapy injury using parallel signaling axes to the traditional VEGF-C and VEGF-D signaling pathways, which are uniquely rendered impotent by radiotherapy injury. Overall, methods to salvage irradiated tissues to a point to which soft-tissue quality would permit simple wound closure or other tissue repair techniques is desperately needed by clinicians. Fat grafting has been reported as a promising avenue to achieve this when used in previously irradiated areas. It was incidentally noted that irradiated tissue overlying the fat graft became more compliant and less lymphoedematous. The diminished capacity of irradiated ADSC to migrate and differentiate to fat represented significant impairments in their regenerative function. Radiation not only impairs loco-regional ADSC function, but was also shown to block the recruitment and homing of functional ADSC from sites distant to the injury. This may be due to the mentioned presence of CXCR7 secreted by irradiated LEC. Therefore, to overcome injury and aid in regeneration of tissues the mechanical introduction of healthy ADSC, via fat grafting may be needed to override the failed ADSC recruitment mechanisms. In the fat grafting model using the introduction of the secretome of ADSCs, ADSC-conditioned media (ADSCCM) was able to reverse the effects of radiotherapy injury in both fibroblasts and LEC populations. The final section of this thesis investigated putative therapeutic mechanisms by which ADSCs reverse radiotherapy induced soft tissue injury. Examination of ADSCCM was performed using proteomics, exosome analysis and metabolomics approaches. Several key candidates were identified that may lead to promising therapeutic avenues by which radiotherapy injury can be mitigated. Understanding of the cellular and molecular mechanisms of radiotherapy induced soft tissue injury, methods by which ADSCCM mediates reversal of the resulting cell dysfunction will provide vital clues and putative therapeutic channels by which to reverse these pathological alterations, thereby reducing the devastating burden of chronic, debilitating side effects of radiotherapy such as fibrosis, lymphoedema and other related diseases, in cancer survivors.

La thérapie interne par radionucléides est encore aujourd’hui un domaine peu exploité parmi les différentes modalités de traitement contre le cancer. Son spectre d’applications est toutefois en pleine évolution grâce notamment à l'apparition de nouveaux radiopharmaceutiques émetteurs beta ou alpha (peptides, alpha-thérapie ²²³ Ra, alpha-immunothérapie ²²¹ As,...) (Ersahin 2011). Dans ce contexte, la grande hétérogénéité des doses délivrées et des effets observés, à la fois en terme de toxicité et de réponse, démontrent qu'une dosimétrie personnalisée est essentielle pour optimiser le traitement (Strigari 2011). En pratique clinique, la dosimétrie de la tumeur et des organes à risque (foie, rein, ...) repose sur l’image de la biodistribution et de la cinétique précise du radiopharmaceutique chez chaque patient. Ces images peuvent être réalisées avec un traceur pré-thérapeutique pour planifier le traitement ou après celui-ci, afin de corréler directement les effets observés aux doses délivrées de manière à optimiser le protocole (activité maximum à injecter, intervalle entre les injections). Les contraintes de détection imposées par les protocoles de traitement sont très différentes de celles associées à un examen diagnostique (Flux 2011, Konijnenberg 2011). Les gamma-caméras conventionnelles ne sont ainsi pas adaptées à la détection de fortes activités de rayonnements gamma d'énergies inférieures à 100 keV (²²³ Ra) ou supérieures à 300 keV (¹³¹I, ⁹⁰Y). D’autre part, les fortes activités des traceurs injectés exigent généralement que le patient reste isolé, ce qui le rend donc plus difficilement accessible par les techniques d’imagerie standard. Enfin, la disponibilité de ces systèmes est incompatible avec un échantillonnage temporel précis de la cinétique du traceur, qui joue un rôle très important dans la quantification des doses absorbées. L'objectif de ma thèse était de proposer de nouvelles approches instrumentales visant à renforcer le contrôle de la dose délivrée aux patients lors d'un traitement de radiothérapie moléculaire. Ceci est réalisé en réduisant les incertitudes associées à la quantification de l'activité (et donc au calcul de la dose absorbée) grâce à l'utilisation d'un système d'imagerie compact et hautement optimisé. Il consistait à mettre au point et à optimiser une gamma-caméra mobile miniaturisée à haute résolution spécialement conçue pour améliorer l'évaluation quantitative individuelle de la distribution hétérogène et de la bio-cinétique du radiotraceur avant et après administration du traitement. L'étude était axée sur le traitement des maladies bénignes et malignes de la thyroïde à l'aide de l'¹³¹ I. Le premier prototype de la caméra, avec un champ de vue de 5x5 cm² , consiste en un collimateur à trous parallèles à haute énergie, réalisé en impression 3D et optimisé par simulations Monte Carlo, couplé à un scintillateur inorganique continu, lu par une technologie récente basée sur des matrices de photomultiplicateurs au silicium (SiPM). Ses propriétés intrinsèques, en termes d'énergie et de réponse spatiale, ont été testées avec des sources ponctuelles de ⁵⁷ Co et ¹³³ Ba. Le premier prototype de la caméra a été calibré avec de l'¹³¹ I. La calibration du système conduit à une résolution spatiale globale de (3.14±0.03) mm et à une sensibilité moyenne de (1.23±0.01) cps/MBq, le deux à 5 cm de distance. Nous avons effectué les premières études précliniques avec l'utilisation de différents fantômes thyroïdiens imprimés en 3D, avec et sans nodules, remplis de ¹³¹ I. Des résultats très prometteurs ont été atteints (valeurs de RC proches de l’unité), qui mettent en évidence ses performances adaptées à une quantification précise dans un contexte clinique assez réaliste
Targeted radionuclide therapy is still a developing area among the different treatment modalities against cancer. However, its range of applications is rapidly expanding thanks to the emergence of new radiopharmaceuticals labeled with beta or alpha emitters (peptides, ²²³ Ra alpha-therapy, ²²¹ As alpha- immunotherapy, ...) (Ersahin 2011). In that context, the large heterogeneity of absorbed doses and the range of effects observed, both in terms of toxicity and response, demonstrate that individualized patient dosimetry is essential to optimize this therapy (Strigari 2011). In clinical practice, patient-specific dosimetry of tumors and organs-at-risk (liver, kidney, ...) is image-based and rely on the quantification of radio- pharmaceutical uptake as a function of time. These images can be obtained from either a pre-therapy tracer study or from a previous therapy procedure. The detection constraints imposed by the treatment protocols are very different from those associated with diagnostic imaging. (Flux 2011 Konijnenberg 2011). Thus, conventional gamma cameras are not suited for detecting high activity of gamma emitters with energy below 100 keV (²²³ Ra) or greater than 300 keV (¹³¹ I, ⁹⁰Y ). Moreover, high activities of the injected tracer typically require isolation of the patient, making the use of standard imaging devices difficult. Finally, the availability of these devices is incompatible with an accurate temporal sampling of the kinetics of the tracer, which is a key parameter for the quantification of the absorbed doses. The objective of my thesis was precisely to propose new instrumental and methodological approaches aiming to strengthen the control of the dose released to patients during molecular radiotherapy. This is achieved by reducing the uncertainties associated to activity quantification (and therefore to the absorbed dose calculation) through the use of a compact and highly optimized imaging system. Specifically, the work consisted in the development and optimization of a miniaturized, high-resolution mobile gamma camera specifically designed to improve the individual quantitative assessment of the heterogeneous distribution and biokinetics of the radiotracer before and after treatment administration. The study was focused on the treatment of benign and malign thyroid disease with ¹³¹ I. The first prototype of the mobile camera, with a field of view of 5x5 cm², consists of a high-energy parallel- hole collimator, optimized with Monte Carlo simulation and made with 3D printing, coupled to a 6 mm thick continuous CeBr3 scintillator readout by a recent and well-suited technology based on arrays of Silicon Pho- tomultiplier (SiPMs) detectors. Its intrinsic properties, in term of energy and spatial response, have been tested with collimated point source of ⁵⁷Co and ¹³³Ba. The first feasibility prototype has been then calibrated with a line and five cylindrical sources filled with ¹³¹ I. The system calibration leads to an overall spatial resolution of (3.14±0.03) mm at a distance of 5 cm and a sensitivity that decreases with distance and slightly changes with source size. An average sensitivity of (1.23±0.01) cps/MBq has been found at 5 cm. In order to test the quantification capability of the camera, the first preclinical planar studies involved the use of different 3D-printed thyroid phantoms filled with ¹³¹ I, with and without nodules. Although corresponding to a relatively ideal, but realistic, clinical situation (no superimposition of background activity), the optimized imaging features of the camera leads to very promising results, with activity recovery factors that deviate of around 2% from the unity

Object Rupture of intracranial arteriovenous malformations is a leading cause of stroke in children and young adults. Treatment options include surgery and highly focused radiation (stereotactic radiosurgery). For large and deep seated lesions, the risks of surgery may be prohibitively high, while radiosurgery has a disappointingly low efficacy and long latency. Radiosurgery carries the most promise for significant advances, however the process by which radiosurgery achieves obliteration is incompletely understood. Inflammation and thrombosis are likely to be important in the radiation response and may be amenable to pharmacological manipulation to improve radiosurgical efficacy. Materials and methods Immunohistochemistry and electron microscopy were used to study normal cerebral vessels, cavernous malformations and AVMs, some of which had previously been irradiated. An attempt was made to culture AVM endothelial cells to study the immediate response of AVM endothelium to radiosurgery. The effects of radiosurgery in a rat model of AVM were studied using immunohistochemistry and the results used to determine the choice of a pharmacological strategy to enhance the thrombotic effects of radiosurgery. Results Vascular malformations have a different endothelial inflammatory phenotype than normal cerebral vessels. Radiosurgery may cause long term changes in inflammatory molecule expression and leads to endothelial loss with exposure of pro-thrombotic molecules. Ultrastructural effects of irradiation include widespread cell loss, smooth muscle cell (SMC) proliferation and thrombosis. Endothelial culture from AVMs proved difficult due to SMC predominance in initial cultures. Radiosurgery upregulated several endothelial inflammatory molecules in the animal model and may induce pro-thrombotic cell membrane alterations. The administration of lipopolysaccharide and soluble tissue factor to rats following radiosurgery led to selective thrombosis of irradiated vessels. Conclusions Inflammation and thrombosis are important in the radiosurgical response of AVMs. Lumen obliteration appears to be mediated by proliferation of cells within the vessel wall and thrombosis. Upregulation of inflammatory molecules and perhaps disruption of the normal phospholipid asymmetry of the endothelial and SMC membranes are some of the earliest responses to radiosurgery. The alterations induced by radiation may be harnessed to selectively initiate thrombus formation. Stimulation of thrombosis may improve the efficacy of radiosurgery, increasing treatable lesion size and reducing latency.

Glioblastoma (GBM) is the most frequent and malignant primary brain tumor. The current standard of care for adult patient with diagnosed GBM is surgery followed by radiotherapy (RT) plus concomitant and adjuvant temozolomide (TMZ) chemotherapy. Despite intense patient management, conventional therapies are not able to achieve long-term remissions and eventually almost every tumor recurs. The impossibility of extensive tumor debulking, the marked heterogeneity of lesions, the poor drug delivery in the brain and the presence of cancer cells with stem features (Cancer Stem Cells, CSCs) within the bulk of the tumor contribute significantly to the lack of effective treatment options. We ought to develop an in-vitro model to investigate molecular mechanisms underlying GBM resistance based on two major cornerstones: (i) the key duality between Glioblastoma Initiating Cells (GICs) and the bulk of the tumor; and (ii) the intratumoral heterogeneity. Consequently, we conceived a paired model where both GICs and differentiated GBM cells depicting the bulk of the tumor were represented. Both culture models were derived from the same GBM post- surgical specimen, but were established and maintained in different culturing conditions. Moreover, we aimed to design a model that could preserve as much as possible the intratumoral heterogeneity of GBM, within the known limits of in-vitro cultures. Consequently, cultures obtained from GBM specimens were not sorted for expression of putative cancer stem cells markers. Six different GBM patients’ samples were processed and established in-vitro as both Differentiated Glial Cells (DGC) and GICs cultures. Established GICs cultures and corresponding tumor-of-origin were analysed according to the molecular subtypes defined on the basis of transcriptomic signature and both were classified as predominantly Mesenchymal. DGC and GICs deriving from the same patient and growing in cultures as monolayer and neurospheres respectively, were compared side-by-side and stem’s functional features and markers expression were investigated. Neurosphere cultures demonstrated to be enriched in GICs, whereas monolayer cultures were not, as indicated by their poor clonogenic capacity and absent CSCs markers expression. In addition, CSCs markers’ expression patterns highlighted the heterogeneous nature of GICs cultures. Consequently, we demonstated that the neurosphere culture method is a proper approach to isolate GICs within the GBM tumor mass, preserving GICs heterogenic nature. Radiosensitivity of four established culture pairs was investigated by means of clonogenic assay and all established unsorted GICs-enriched cultures ended up being more radioresistant than their differentiated counterparts. Importantly, radiation response of irradiated GICs, but not of DGC, correlates with patient’s outcome, thus supporting the GICs leading role in defining patient treatment response. In conclusion, we propose a quick and affordable method to faithfully determine cancer cells’ treatment response and potentially predict patient outcome based on empirical data. Following clinically relevant fractionated radiotherapy we detected, by means of transcriptomic analysis, marked activation of inflammatory-related pathways, ECM remodeling, cell migration and intercellular crosstalk in GICs. Strikingly, several genes pointed to epithelial/mesenchymal transition processes via IL6/JAK/STAT3 and TNF-α/NFkβ pathways. A small signature of radiation-induced Mes-associated genes was defined in GICs: ICAM1, COX2, CTGF, IL6, LIF and NNMT. In addition, the possible involvement of ITGA6 in GICs response to ionizing radiation was investigated. The knock-down of ITGA6 in GICs-enriched culture enhanced their radiosensitivity, potentially improving tumor radiocurability, and reported decreased capacity to retain stemness after radiotherapy.
El Glioblastoma (GBM) es el tumor cerebral primario maligno más frecuente en adultos. El tratamiento actual, consiste en cirugía seguida de radioterapia (RT) más quimioterapia, no evita las recidivas a largo plazo. Para investigar los mecanismos moleculares que subyacen a la resistencia de GBM a la RT, se ha desarrollado un modelo in-vitro basado en dos pilares fundamentales: (i) la dualidad entre las Glioblastoma Initiating Cells (GICs) y el resto de células neoplásicas (células diferenciadas, DGC); y (ii) la heterogeneidad intratumoral. Los cultivos de GICs y las muestras de tumor homólogas se clasificaron como de tipo mesenquimal. Se compararon los cultivos DGC y GICs por sus características funcionales y metabólicas, la expresión de marcadores de células madres tumorales y la respuesta a la RT. Los cultivos GICs demostraron estar enriquecidos en CSCs, y el patrón de expresión de marcadores de CSCs evidenció su heterogeneidad, a diferencia de lo observado en DGC. Además, todos los cultivos enriquecidos en GICs fueron, a largo plazo, más resistentes a la RT en comparación con sus homólogos diferenciados. Es importante destacar que la radioresistencia de las GICs, pero no de las DGC, se correlaciona con el pronóstico de los pacientes, apoyando así el papel de las GICs en la respuesta al tratamiento. En conclusión, se propone un método rápido y económico para determinar fielmente la respuesta al tratamiento con RT de las células tumorales y potencialmente predecir la evolución del paciente basado en datos empíricos. Para entender mejor el fenómeno de la resistencia a la RT de las GICs se realizó un análisis transcriptómico de DGC y GICs postirradiación. Exclusivamente en las GICs se detectó una activación significativa de las vías relacionadas con la inflamación, remodelación de la matriz extracelular, migración celular, interacción célula-célula y transición epitelio- mesénquima mediado por STAT3 y NF-κβ. Se identificó un grupo de genes asociados al perfil mesenquimal e inducidos por la radiación en GICs: ICAM1, COX2, CTGF, IL-6, LIF y NNMT. Finalmente, se investigó la posible implicación de ITGA6, previamente descrito como marcador de CSCs en GBM, en la respuesta de GICs a la RT. La inhibición de ITGA6 en los cultivos enriquecidos en GICs aumentó la sensibilidad a la RT, mejorando potencialmente la respuesta al tratamiento.

Breast cancer is a major cause of cancer-related death among women throughout the world. Treatment of breast cancer often fails due to the development of resistance to both chemo- and radiotherapy. The aim of this study was to analyze and compare the response to radiation of MCF-7 breast adenocarcinoma cells and MCF-7 cells that are resistant to doxorubicin (MCF-7/DOX). The results presented in this thesis show that drug-resistant MCF-7/DOX cells survive high doses of radiation exposure better than MCF-7 cells. Moreover, the chemo- and radioresistance of MCF-7/DOX cells share common molecular mechanisms and loss of sensitivity to radiation in chemo-resistant cells may be explained by alterations in their DNA methylation profile. The results of experiments presented in this thesis may, therefore, serve as a first step for future analysis of tumour resistance to radio- and chemotherapy and for the development of novel epigenetic strategies for reversal of breast cancer resistance to cytotoxic treatment regimens.
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Le potentiel des nanoparticules d'or (AuNPs) pour améliorer l’efficacité de la radiothérapie est démontré par de nombreuses études expérimentales in vivo et in vitro. Ces particules métalliques augmentent significativement l’effet de la radiosensibilisation. La réaction en jeu est la radiolyse de l’eau: une fois excitées par un rayon X, elles génèrent des espèces réactives oxygénées qui amplifient les dégâts d’ADN et mènent à une plus grande destruction des cellules cancéreuses. Cependant, pour une efficacité thérapeutique plus optimale, plusieurs propriétés des AuNPs doivent être prises en compte lors de la synthèse comme leur taille, leur forme et leur surface qui sont suspectibles d’influencer ses effets catalytiques dans l’environnement biologique (majoritairement de liquide d’eau). Ces aspects structuraux ne sont pas encore examinés dans l’état de l’art, ni expérimentalement ni théoriquement. Ce travail de thèse a pour but de rationaliser la stabilité de AuNPs dans un environnement chimique ou biologique avant l’irradiation par des outils de modélisation théorique. Dans un premier temps, nous nous sommes intéressés à la stabilité des AuNPs dans la gamme de 1- 3.4 nm. Nous étudions ensuite le comportement de ces nanoparticules dans un environnement biologique (hydratation) et chimique modèle (PEGylation), et la combinaison des deux environnements. Quand les nanoclusters de 0.9-1.8 nm sont en interaction avec une couche de molécules d’eau à saturation, nous avons montré qu’il y a une transformation de NPs métastables (dans le vide) telles que l'ino-décaèdre en NPs métastables plus favorables telles que l'icosaèdre. Alors qu’en présence d’une couche de ligands PEG, la liaison forte Au-S et les liaisons hydrogène entre les ligands entraînent une déformation significative de la morphologie de la nanoparticule, à savoir une stellation du décaèdre Au54. Par ailleurs, nous avons montré que les ligands PEG promeuvent le confinement de quelques molécules d’eau à proximité des AuNPs. Nos conclusions ouvrent des perspectives intéressantes pour la modélisation théorique de la radiolyse de l’eau. Parallèlement à ces études, nous nous sommes intéressés à la caractérisation de différents types de lésions d’ADN. Deux projets ont été menés: premièrement, nous démontrons l’interaction d’un peptide trilysine avec un oligonucléotide qui pourrait conduire à la formation de pontage d’ADN-polyamine. Ensuite, le deuxième projet porte sur la rationalisation de différents taux de réparation de dimères de cyclobutanemathide iochella pyrimidine en présence de l’enzyme de reconnaissance DDB2 à l’échelle atomique
The potential of gold nanoparticles (AuNPs) to improve the performance of radiotherapy is demonstrated by numerous in vivo and in vitro experimental studies. These metallic particles increase significantly the radiosensitization effect. Upon water radiolysis, AuNPs generate reactive oxygen species that amplify DNA damage and lead to a greater destruction of cancerous cells. Nevertheless, for a more optimal therapeutic efficacy, several properties of AuNPs must be taken into account during the synthesis, such as their size, shape and surface which can tune their catalytic effects in the biological environment (mainly liquid water). These structural aspects are not yet examined in the state-of-the-art, either experimentally or theoretically. This thesis aims to rationalize the stability of AuNPs in the presence of chemical or biological environment before irradiation by using theoretical approaches. Firstly, we have modeled the stability of AuNPs in the range 1- 3.4 nm. We have then studied the behavior of these nanoparticles in a biological (hydration) and chemical (PEGylation) model environment, and the combination of the two environments. When 0.9-1.8 nm nanoclusters interact with a complete shell of water molecules, we have shown that metastable ino-decahedrons (in vacuum) are transformed into more favourable metastable icosahedrons. While in the presence of monoshell of PEG ligands, the strong Au-S bond and the hydrogen bonds between the ligands induce a significant deformation on the nanoparticle morphology, i.e. stellation of the Au54 decahedron. In addition, we have shown that PEG ligands promote the confinement of a few water molecules in the vicinity of AuNPs. Our conclusions open interesting perspectives for the theoretical modeling of water radiolysis. In parallel with these studies, we have focused on the characterization of different types of DNA lesions. Two projects have been performed: firstly, we have studied the interaction of a trilysine peptide with an oligonucleotide which could lead to the formation of DNA-polyamine cross link. Then, the second project focuses on rationalizing different repair rates of cyclobutane pyrimidine dimers in the presence of the DDB2 recognition enzyme at the atomic scale

Prostate cancer is one of the most common types of cancer worldwide and claims hundreds of thousands of lives annually. Currently the most common treatment for prostate cancer is external beam radiotherapy, however, this treatment comes with serious side effects since it lacks selectivity for the cancer cells. Therefore, less harmful treatments are needed and sought for, such as targeted treatments that are intended to only affect cancer cells and thereby reduce the side effects. Targeted treatments require a target that differentiates the cancer cells from healthy cells. A promising target candidate that has gained attention in recent years is gastrin releasing peptide receptor (GRPR), a protein commonly overexpressed in prostate cancer cells. Furthermore, a targeting molecule intended to bind to the target is also required. For this purpose, the bombesin analogue RM26, a high affinity GRPR binder, shows promise. Previous studies have led to the development of RM26-conjugates for the purpose of targeted prostate cancer radiotherapy. In these conjugates RM26 has been linked to a DOTA-chelator for radiolabeling, and an albumin binding domain (ABD) to prolong the conjugate’s half-life in vivo by binding to human serum albumin (HSA). The idea is that the RM26-conjugate will bind to both HSA in the blood and to GRPR on the prostate cancer cells and eliminate the cancer cells with the radiation from the radionuclide attached to the DOTA-chelator. Although these earlier studied conjugates have been very promising some improvements of certain aspects need to be achieved, mainly to improve the biodistribution with retained GRPR binding affinity. Therefor the purpose of this project was to produce three new versions of previous RM26- conjugates and evaluate if they are suitable for further prostate cancer therapy studies. The three RM26-conjugates were developed with primarily recombinant expression in E. coli cells and solid phase peptide synthesis (SPPS). The characterization phase in this project was carried out with mainly five different methods: matrix-assisted laser desorption ionization time- of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization- mass spectrometry (ESI-MS), circular dichroism (CD), surface plasmon resonance (SPR) and flow cytometry. The results showed that all three new RM26-conjugates were possible to produce and yielded final products corresponding to the expected molecular weights. Furthermore, the results indicate that all three RM26-conjuagtes are stable and maintain their structural properties under in vivo- temperatures and that they have high binding affinity for HSA. Further studies need to be conducted before drawing any certain conclusions regarding GRPR binding affinity.
Prostatacancer är en av de mest vanligt förekommande cancertyperna världen över och skördar hundratusentals liv årligen. I nuläget är extern strålbehandling det vanligaste terapialternativet mot prostatacancer, men denna behandling kommer med allvarliga biverkningar på grund av att den saknar selektivitet för cancerceller. Därför finns ett stort behov av mindre skadliga behandlingsformer, såsom riktade behandlingar som endast är avsedda att påverka cancerceller och därigenom minska biverkningarna. Riktade behandlingar kräver ett mål som skiljer cancercellerna från friska celler. En lovande målkandidat som har uppmärksammats de senaste åren är gastrinfrisättande peptidreceptor (GRPR), ett protein som vanligtvis överuttrycks i prostatacancerceller. I tillägg så krävs också en målsökande molekyl avsedd att binda till målet. För detta ändamål visar bombesinanalogen RM26, en GRPR-bindare med hög affinitet, sig vara lovande. Tidigare studier har utvecklat RM26-konjugat för målinriktad strålbehandling av prostatacancer. Dessa konjugat består av en RM26-peptid bunden till en DOTA-kelator för radioinmärkning och en albuminbindande domän (ABD) för att förlänga konjugatens halveringstid in vivo genom att binda till humant serumalbumin (HSA). Syftet med RM26- konjugaten är att de ska binda till både HSA i blodet och GRPR på prostatacancercellerna, och därmed eliminera cancercellerna med strålning från den radioinmärkta DOTA-kelatorn. Även om de tidigare RM26-konjugaten har varit mycket lovande krävs det att vissa förbättringar av några aspekter uppnås, främst affiniteten för GRPR. Syftet med detta projekt var därför att producera tre nya versioner av tidigare RM26-konjugat och utvärdera ifall de uppvisar tillfredsställande egenskaper. De tre RM26-konjugaten utvecklades primärt rekombinant i E. coli-celler och fastfas- peptidsyntes (SPPS). Karaktäriseringsfasen i detta projekt genomfördes med huvudsakligen fem olika metoder: MALDI-TOF-MS, elektrosprejjonisering-masspektrometri (ESI-MS), cirkulär dikroism (CD), ytplasmonresonans (SPR) och flödescytometri. Resultaten visade att alla tre nya RM26-konjugat var möjliga att producera och gav slutprodukter motsvarande de förväntade molekylvikterna. Vidare indikerar resultaten att alla tre RM26-konjugat är stabila och bibehåller sina strukturella egenskaper under in vivo-temperaturer och att de har hög affinitet för HSA. Ytterligare studier bör utföras innan säkrare slutsatser kan dras angående GRPR-bindningsaffinitet.

L'objectif de ce manuscrit de thèse Thèse de doctorat par Validation des Acquis de l'Expérience (VAE) est de mettre en évidence les principales études de recherche menées à UCLA qui ont conduit à l'implémentation clinique des technique théranostique ciblant le PSMA aux États-Unis. Le manuscrit est divisé en 2 parties : l'imagerie et la thérapie.Dans la première partie sont fournis les essais pivots d'efficacité diagnostique utilisés pour l'autorisation par la FDA du 68Ga-PSMA-11 (Article #1: Performance diagnostique de la TEP-PSMA pour la localisation de la récidive du cancer de la prostate: un essai clinique d'imagerie prospective multicentrique de phase 3 (n=635), PMID 30920593; Article #2: Performance diagnostique de la TEP-PSMA pour la détection des métastases ganglionnaires pelviennes avant prostatectomie radicale et curage ganglionnaire pelvien: un essai clinique d'imagerie prospective multicentrique de phase 3 (n=764), PMID 34529005), des études comparatives comparant la TEP-PSMA aux techniques d'imagerie standard (Article #3: TEP/TDM a la 18F-Fluciclovine et au 68Ga-PSMA-11 chez les patients présentant une récidive biochimique après prostatectomie avec un taux de PSA ≤2,0 ng/ml: un essai d'imagerie comparative prospectif monocentrique (n=50), PMID 31375469; Article #4: Comparaison de la TEP/TDM-PSMA et de l'IRMmp avec référence par histopathologie dans la détection, la localisation intra-prostatique et la détermination de l'extension locale du cancer primitif de la prostate: résultats dans une cohorte monocentrique d'un essai prospectif (n=74), PMID 34649942) et une étude montrant un impact significatif sur la prise en charge (Article #5: Cartographie par TEP/TDM-PSMA de la récidive biochimique du cancer de la prostate après prostatectomie radicale chez 270 patients avec un taux de PSA<1,0 ng/ml : Impact sur la planification de la radiothérapie de rattrapage (n=270), PMID 29123013) qui a conduit à un essai randomisé visant à montrer une amélioration des résultats cliniques grâce à la TEP-PSMA (Article #6: Essai randomisé prospectif de phase 3 de radiothérapie de rattrapage du cancer de la prostate guidée par TEP-PSMA [PSMA-SRT] (n=193), PMID 30616601; Article #7: Mise à jour de l'essai PSMA-SRT NCT03582774: un Essai randomisé prospectif de phase 3 de radiothérapie de rattrapage du cancer de la prostate guidée par TEP-PSMA (n=193), PMID 33386288).Dans la deuxième partie sont présentés les résultats du premier essai de phase 2 de la thérapie au 177Lu-PSMA aux USA qui a précédé l'essai VISION (Article #8 : Essai prospectif de phase 2 de radiothérapie moléculaire au 177Lu-PSMA-617 pour cancer de la prostate métastatique résistant à la castration (RESIST-PC): Résultats d'efficacité de la cohorte UCLA (n=43), PMID 34016732; Article #9: Tolérance et sécurité de la radiothérapie moléculaire au 177Lu-PSMA-617: résultats de l'essai prospectif multicentrique de phase 2 RESIST-PC NCT03042312 (n=64), PMID 34272322), des études rétrospectives multicentriques visant à affiner les critères de sélection TEP-PSMA (Article #10: Suivi des patients négatifs au PSMA par critères TEP-PSMA de l'essai VISION traités par 177Lu-PSMA: une analyse rétrospective multicentrique (n=301), PMID 35273096; Article #11: Ratio tumeur/glande salivaire par TEP-PSMA pour prédire la réponse à la thérapie par 177Lu-PSMA: une étude rétrospective multicentrique internationale (n=237), PMID 36997329; Article #12: Nomogrammes pour prédire les résultats après traitement au 177Lu-PSMA chez les patients atteints d'un cancer de la prostate métastatique résistant à la castration: une étude rétrospective internationale multicentrique (n=270), PMID 34246328) et une revue narrative des mécanismes de résistance à la thérapie au 177Lu-PSMA (Article #13: Prédicteurs de réponse et utilisation dans le monde réel de la radiothérapie moléculaire ciblée du cancer de la prostate: PSMA et au-delà. PMID 35609224)
The aim of this manuscript for PhD by Accreditation of Prior Learning is to highlight the key research studies conducted at UCLA which lead to the clinical implementation of PSMA-theranostics in the US. The manuscript is divided in 2 parts: imaging and Therapy.In the first part are provided the pivotal trials of diagnostic efficacy used for the FDA approval of 68Ga-PSMA-11 (Article #1: Assessment of 68Ga-PSMA-11 PET Accuracy in Localizing Recurrent Prostate Cancer: A Prospective Multicenter Single-Arm Phase 3 Clinical Trial (n=635), WP Fendler at al. JAMA Oncol 2019 PMID 30920593; Article #2: Diagnostic Accuracy of 68Ga-PSMA-11 PET for Pelvic Nodal Metastasis Detection Prior to Radical Prostatectomy and Pelvic Lymph Node Dissection: A Multicenter Prospective Phase 3 Imaging Trial (n=764), TA Hope et al. JAMA Oncol 2021 PMID 34529005), head-to-head comparison trials comparing PSMA PET to standard of care imaging techniques (Article #3: 18F-Fluciclovine and 68Ga-PSMA-11 PET/CT in patients with biochemical recurrence after prostatectomy at PSA levels of ≤2.0ng/ml: a prospective single-center single-arm comparative imaging trial (n=50), J Calais et al. Lancet Oncol 2019 PMID 31375469; Article #4: Head-to-head comparison of 68Ga-PSMA-11 PET/CT and mpMRI with histopathology gold-standard in the detection, intra-prostatic localization and local extension of primary prostate cancer: results from a prospective single-center imaging trial (n=74), I Sonni et al. J Nucl Med 2022 PMID 34649942) and a study showing significant impact on management (Article #5: 68Ga-PSMA-11 PET/CT mapping of prostate cancer biochemical recurrence following radical prostatectomy in 270 patients with PSA<1.0ng/ml: Impact on Salvage Radiotherapy Planning (n=270), J Calais et al. J Nucl Med 2018 PMID 29123013) that lead to a randomized imaging trial powered for clinical outcome (Article #6: Randomized prospective phase 3 trial of 68Ga-PSMA-11 PET/CT molecular imaging for prostate cancer salvage radiotherapy planning [PSMA-SRT] (n=193), J Calais et al. BMC cancer 2019 PMID 30616601; Article #7: Update from PSMA-SRT Trial NCT03582774: A Randomized Phase 3 Imaging Trial of Prostate-specific Membrane Antigen Positron Emission Tomography for Salvage Radiation Therapy for Prostate Cancer Recurrence Powered for Clinical Outcome (n=193), J Calais et al. Eur Urol Focus PMID 33386288).In the second part are provided the results of the first phase 2 trial of 177Lu-PSMA therapy in the US that preceded the VISION trial (Article #8: Prospective phase 2 trial of PSMA-targeted molecular RadiothErapy with 177Lu-PSMA-617 for metastatic Castration-reSISTant Prostate Cancer (RESIST-PC): Efficacy results of the UCLA cohort (n=43), J Calais et al. J Nucl Med 2021 PMID 34016732; Article #9: Safety of PSMA-targeted molecular radioligand therapy with 177Lu-PSMA-617: results from the prospective multicenter phase 2 trial RESIST-PC NCT03042312 (n=64), J Calais et al. J Nucl Med 2021 PMID 34272322), multicenter retrospective studies aiming at refining the PSMA-PET selection criteria (Article #10: Outcome of patients with PSMA-PET/CT screen failure by VISION criteria and treated with 177Lu-PSMA therapy: a multicenter retrospective analysis (n=301), M Hotta et al. J Nucl Med 2022 PMID 35273096; Article #11: PSMA PET Tumor-to-Salivary Gland Ratio to Predict Response to 177Lu-PSMA Radioligand Therapy: An International Multicenter Retrospective Study (n=237), M Hotta et al. J Nucl Med 2023 PMID 36997329; Article #12: Nomograms to predict outcomes after 177Lu-PSMA therapy in men with metastatic castration-resistant prostate cancer: an international, multicenter, retrospective study (n=270), A Gafita et al. Lancet Oncol 2021 PMID 34246328) and a narrative review of the mechanisms of resistance to 177Lu-PSMA therapy (Article #13: Predictors and Real-World Use of Prostate-Specific Radioligand Therapy: PSMA and Beyond. A Gafita et al. Am Soc Clin Oncol Educ Book 2022 PMID 35609224)

The use of highly-energetic iOIl: beams for treating cancerous tumours is of increasing importance. In this study the damage caused to plasmid DNA by HO and H+ beams was investigated. Damage to dry DNA samples as a function of dose was assessed using gel electrophoresis. Results show enhanced levels of damage relative to that of x-rays and demonstrate an increase in damage with beam energy. Additionally, a considerable dependence on sample structure was shown. X-ray irradiation work has shown that doping samples with gold nanoparticles greatly enhances DNA damage and could enhance the effectiveness of cancer treatment. Hence in this study, damage to DNA samples containing gold nanoparticles was investigated, however, no enhancement was observed. TOF-MS was used to investigate fragment damage profiles at a molecular level. High-mass fragments corresponding to basal cleavage were not observed. Significant buffer residues in the dried samples were seen and identified. A model of DNA damage as a function of dose was constructed, providing quantitative conclusions about the effects of both gold nanoparticles and the different buffers used. Most of the energy deposited in the body by ionising radiation is absorbed by water molecules, leading to radical formation and, potentially, subsequent DNA damage. Current damage models use information on energy loss rates with penetration depth instead of information on radical production. In this work, fundamental fragmentation processes of water molecules irradiated with H+, HO and carbon ions and atoms were investigated. It was shown that it is imperative to consider the damage caused by fast neutrals in any dose profile modelling. Additionally, results for the ion production rate, to which free-radical production relates directly, show it is actually relatively uniform at a region where the ions come to rest and is not as sharply localised as suggested by the Bragg peak energy loss profiles.

The members of the epidermal growth factor (EGF) / ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this thesis work we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. The lack of EGFR-mediated growth-stimulatory activity prompted us to evaluate EGFt for targeted delivery of 111In, an Auger electron emitter, into EGFR-positive cancer cells. An 111In-DTPA-EGFt radioconjugate was developed and its properties were analyzed and compared to those of 111In-DTPA-hEGF. First we determined that 111In-DTPA-EGFt displays high specificity and affinity for EGFR. However, the cellular uptake of 111In-DTPA-EGFt resulted to be lower than that of 111In-DTPA-hEGF. Once internalized, 111In-DTPA-EGFt showed a high efficiency to accumulate into the cell nucleus, where the radioactivity emitted by 111In may damage the DNA. In accordance, 111In-DTPA-EGFt showed to be cytotoxic in vitro against breast cancer cells, although its cytotoxicity was lower compared to 111In-DTPA-hEGF. In vivo studies revealed a longer half-life in blood for 111In-DTPA-EGFt than for 111In-DTPA-hEGF and higher uptake in the kidney, with minor accumulation in other normal tissues. 111In-DTPA-EGFt accumulated in MDA-MB-468 tumors where, interestingly, 111In-DTPA-EGFt was detected in a great proportion in the cell nucleus. All the data obtained from this work indicate that EGFt may be a potential EGFR blocker for cancer therapy and also an attractive ligand for delivery of cytotoxic agents into the nucleus of EGFR-positive cancer cells.
Els receptors i lligands de la família del factor de creixement epidèrmic (EGF)/ErbB són dianes molt importants en el desenvolupament de teràpies contra el càncer. No obstant, l’eficàcia terapèutica dels fàrmacs dirigits a atacar aquesta via i que són utilitzats actualment en clínica és limitada. Per aquest motiu la recerca de noves molècules que inactivin els receptors d’aquesta família mitjançant noves estratègies és avui dia una de les vies més explorades. En aquesta tesi s’ha desenvolupat un pèptid idèntic al factor de creixement epidèrmic EGF que li manca la seva part C-terminal (EGFt) com a nou inhibidor de EGFR. El disseny d’aquest pèptid truncat s’ha basat en la superposició tridimensional de l’estructura de l’EGF i de l’inhibidor de la carboxipeptidasa de patata (PCI), un bloquejador de la via de l’EGFR descrit prèviament pel nostre grup. El pèptid ha estat produït en E.coli i s’ha aconseguit obtenir un alt rendiment de la proteïna i amb la seva conformació estructural correcta. Hem observat que l’EGFt in vitro té una capacitat molt menor per induir dímers del receptor i també la seva fosforilació si la comparem amb l’activitat que té l’hEGF natiu. Per altra banda, l’EGFt promou la internalització del receptor i la seva translocació al nucli cel·lular tal i com ho fa l’hEGF, tot i que no estimula el creixement cel·lular. A més, l’EGFt competeix amb els lligands natius de la família i inhibeix la proliferació cel·lular. La manca d’activitat estimuladora del creixement cel·lular d’aquest pèptid quan s’uneix a l’EGFR ens va portar a provar la utilització de l’EGFt com a vehicle de toxines dirigit a cèl·lules tumorals que sobreexpresessin EGFR. Concretament, es va produir un radioconjugat de EGFt amb l’isòtop radioactiu emissor d’electrons Auger Indi-111. Les propietats d’aquest radioconjugat es van analitzar i es van comparar amb el radioconjugat produït amb hEGF natiu. En primer lloc es va determinar que 111In-DTPA-EGFt té una alta especificitat i afinitat per EGFR. No obstant, la captació cel·lular de 111In-DTPA-EGFt va resultar ser menor que la de 111In-DTPA-hEGF. Un cop internalitzat, 111In-DTPA-EGFt va mostrar una alta eficiència per acumular-se en el nucli de la cèl·lula, on la radioactivitat emesa per 111In danya l'ADN. 111In-DTPA-EGFt va mostrar ser citotòxic in vitro contra cèl·lules de càncer de mama, encara que la seva citotoxicitat va ser menor en comparació amb 111In-DTPA-hEGF. Els estudis in vivo van revelar una vida mitjana més llarga en sang per 111In-DTPA-EGFt que per 111In-DTPA-hEGF, una major captació en el ronyó i una menor acumulació en altres teixits normals. 111In-DTPA-EGFt es va detectar en els tumors de cèl·lules MDA-MB-468 on el radiocompost es va acumular preferentment en el nucli de la cèl·lula. Les dades recollides en aquest treball indiquen que l’EGFt pot tenir un gran potencial com a bloquejador en teràpia pel càncer i a més pot ser un bon lligand per utilitzar com a vehicle d’agents citotòxics dirigits al nucli de cèl·lules tumorals positives en EGFR.

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Dissertação (Mestrado em Tecnologia Nuclear)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Mestrado em Biomedicina Molecular
Head and Neck Cancers (HNC) are a group of tumours located in the upper aero-digestive tract. Head and Neck Squamous Cell Carcinoma (HNSCC) represent about 90% of all HNC cases. It has been considered the sixth most malignant tumour worldwide and, despite clinical and technological advances, the five-year survival rate has not improved much in the last years. Nowadays, HNSCC is well established as a heterogeneous disease and that its development is due to accumulation of genetic events. Apart from the majority of the patients being diagnosed in an advanced stage, HNSCC is also a disease with poor therapeutic outcome. One of the therapeutic approaches is radiotherapy. However, this approach has different drawbacks like the radioresistance acquired by some tumour cells, leading to a worse prognosis. A major knowledge in radiation biology is imperative to improve this type of treatment and avoid late toxicities, maintaining patient quality of life in the subsequent years after treatment. Then, identification of genetic markers associated to radiotherapy response in patients and possible alterations in cells after radiotherapy are essential steps towards an improved diagnosis, higher survival rate and a better life quality. Not much is known about the radiation effects on cells, so, the principal aim of this study was to contribute to a more extensive knowledge about radiation treatment in HNSCC. For this, two commercial cell lines, HSC-3 and BICR-10, were used and characterized resorting to karyotyping, aCGH and MS-MLPA. These cell lines were submitted to different doses of irradiation and the resulting genetic and methylation alterations were evaluated. Our results showed a great difference in radiation response between the two cell lines, allowing the conclusion that HSC-3 was much more radiosensitive than BICR-10. Bearing this in mind, analysis of cell death, cell cycle and DNA damages was performed to try to elucidate the motifs behind this difference. The characterization of both cell lines allowed the confirmation that HSC-3 was derived from a metastatic tumour and the hypothesis that BICR-10 was derived from a dysplasia. Furthermore, this pilot study enabled the suggestion of some genetic and epigenetic alterations that cells suffer after radiation treatment. Additionally, it also allowed the association of some genetic characteristics that could be related to the differences in radiation response observable in this two cell lines. Taken together all of our results contribute to a better understanding of radiation effects on HNSCC allowing one further step towards the prediction of patients’ outcome, better choice of treatment approaches and ultimately a better quality of life.
Cancro da Cabeça e Pescoço refere-se a um grupo de tumores que aparecem no trato aerodigestivo superior, sendo que o carcinoma das células escamosas da cabeça e pescoço (CCECP) corresponde a mais de 90% de todos os casos de cancro nesta região. Foi considerado o sexto tumor mais maligno em todo o mundo e, apesar de todos os avanços tecnológicos e clínicos, a taxa de sobrevivência a cinco anos não melhorou significativamente nas últimas décadas. Atualmente sabe-se que o CCECP é uma doença bastante heterogénea que se desenvolve devido à acumulação de alterações genéticas e epigenéticas. Alguns dos grandes problemas associados a este tipo de cancro são o diagnóstico em fase tardia da doença e os poucos resultados terapêuticos. Uma das escolhas terapêuticas para o CCECP é a radioterapia, no entanto, esta tem diversos inconvenientes, como a radioresistência adquirida por algumas células tumorais, que se associam a piores prognósticos. Um aumento do conhecimento na área da biologia da radiação é necessário para melhorar esta opção terapêutica, evitando futuros efeitos tóxicos e fornecendo uma melhor qualidade de vida nos anos subsequentes ao tratamento. Desta forma, a identificação de marcadores moleculares associados quer a uma resposta à radioterapia, quer a possíveis alterações celulares após tratamento com radiação, é essencial para melhorar o diagnóstico, taxa de sobrevivência e qualidade de vida destes doentes. Adicionalmente, existe uma grande falha no conhecimento em relação aos efeitos da radiação nas células, como tal, o principal objetivo deste estudo foi o de contribuir para um conhecimento mais alargado do efeito da radiação em doentes com CCECP. Para isso foram utilizadas duas linhas comerciais celulares, HSC-3 (derivada de um tumor metastático da língua) e BICR-10 (derivada de um tumor da mucosa bucal), que foram caracterizadas com recurso a aCGH, MS-MLPA e citogenética convencional. Estas linhas foram submetidas a diferentes doses de radiação e as alterações genéticas e de metilação pós tratamento foram determinadas. Estes resultados demonstraram uma grande variação de resposta à radiação para estas duas linhas celulares, permitindo a conclusão que a linha HSC-3 é mais radiossensível que a linha BICR-10. Tendo isto em mente, procedeu-se a análise da morte celular, ciclo celular e danos no DNA de forma a tentar compreender esta diferença. A caracterização genética de ambas as linhas celulares permitiu corroborar que a linha HSC-3 era derivada de um tumor metastático e sugeriu que a linha celular BICR-10 estaria associada a um estado de displasia. Para além disto, foi possível analisar alterações genéticas e epigenéticas ocorridas após irradiação e associar determinados perfis genéticos a uma melhor ou pior resposta à radiação. Em suma, os nossos resultados contribuiram para um conhecimento mais aprofundado dos efeitos da radiação no CCECP possibilitando, no futuro, melhores opções de tratamento e uma melhor qualidade de vida para estes doentes.

La médecine nucléaire est une spécialité médicale qui utilise un radiopharmaceutique dont l'administration permet généralement de visualiser une fonction, en détectant les émissions gamma (γ) du radio-isotope vectorisé. Lorsque le but de cette pratique est la thérapie (radiothérapie moléculaire), on privilégie des isotopes qui émettent des radiation à courte portée (α, β ou électrons Auger). Les traitements utilisant 177Lu-DOTATATE ont obtenu leur autorisation de mise sur le marché (AMM) sur la base de l'administration de 7,4 GBq par cycle (activité fixe), sans tenir compte de la variabilité de fixation inter patient. Ceci entraîne une importante fluctuation de la dose absorbée délivrée aux organes à risque et aux cibles tumorales, et par conséquent, une grande difficulté à prédire les résultats du traitement. Des études récentes suggèrent que la planification basée sur une dosimétrie individuelle est une piste d'optimisation du traitement. L'objectif de ce travail est de participer au développement de la dosimétrie clinique en radiothérapie moléculaire, notamment par le développement d'un outil web dédié à la dosimétrie interne personnalisée de patients traités avec 177Lu et basé sur la méthode Monte-Carlo. Dans un premier temps, nous avons réalisé une étude sur la modélisation de systèmes SPECT avec le code Monte-Carlo GATE. L'optimisation des simulations a été réalisée par différentes méthodes pour réduire les temps de simulation. Ces techniques ont réduit le temps de simulation jusqu'à un facteur de 85. Certaines ont été utilisées dans la comparaison entre acquisitions tomographiques simulées et expérimentales. Cette comparaison a permis la modélisation du contexte expérimental utilisé dans la validation de l'outil web, Finalement, une page web a été conçue en utilisant le framework Django où une séquence de scripts en Python et Bash réalisent le calcul de la dose absorbée par simulation avec GATE. Les doses absorbée obtenues ont été comparées avec OLINDA (version 1 et 2). Nos résultats montrent des différences entre 0,3% et 6,1%, selon la version d'OLINDA
Nuclear medicine is a medical specialty that uses a radiopharmaceutical whose administration generally allows to visualize an organ function by detecting the gamma (γ) emissions of the targeted radioisotope. When the goal of this practice is molecular radiotherapy, isotopes emitting short-range radiation (α, β or electron Augers) are preferred. In general, treatments using 177Lu-DOTATATE still uses the historical practice of a fixed administration of 7.4 GBq per cycle, regardless the sex, age or inter-patient fixation variability. This causes a large fluctuation of the absorbed dose delivered to organs at risk and tumour targets, and therefore a great difficulty in predicting the treatment results. Recent studies suggest that treatment planning based on individual dosimetry is a way to optimize the treatment. The objective of this work is to contribute to the development of clinical dosimetry in molecular radiotherapy, in particular by developing a web tool based on the Monte Carlo method GATE dedicated to individualised internal dosimetry of patients treated with 177Lu. First of all, a study on the modelling of the SPECT systems by the Monte Carlo toolkit, GATE was realized, as well as the optimization of these simulations, where different methods were used in order to reduce simulation time. These techniques reduced simulation time by up to 85, and some of them were used in the comparison between simulated and experimental tomographic acquisitions. This comparison allowed the modelling of an experimental context which was used for the web tool validation. Finally, the web page was designed using the Django framework where a sequence of scripts in Python and Bash perform the calculation of the absorbed dose by GATE simulations. The absorbed doses obtained were compared with OLINDA versions 1 and 2, and the results show differences between 0.3% and 6.1%, depending on OLINDA's version

Ce travail de thèse nous a mené à apporter de nouveaux développements au code Monte-Carlo de simulation détaillée Geant4-DNA pour étudier les interactions des électrons de basse énergie dans l'eau liquide, principal constituant des organismes biologiques. La précision des résultats obtenus avec les codes Monte-Carlo repose sur le réalisme de leurs modèles physiques : les sections efficaces. CPA100 est un autre code Monte-Carlo de structure de trace. Il dispose de sections efficaces d'ionisation, d'excitation électronique et de diffusion élastique dont les méthodes de calculs sont indépendantes de celles utilisées pour les sections efficaces de Geant4-DNA (modèles physique " option 2 " et son amélioration " option 4 "). De plus, les sections efficaces de CPA100 sont en meilleur accord avec certaines données expérimentales. Nous avons implémenté les sections efficaces de CPA100 dans Geant4-DNA pour offrir aux utilisateurs l'opportunité d'utiliser des modèles physiques alternatifs désignés Geant4-DNA-CPA100. Ils sont disponibles en libre accès dans la plateforme Geant4 depuis juillet 2017. La vérification de l'implémentation correcte de ces modèles physiques dans Geant4-DNA a consisté à comparer la simulation de plusieurs grandeurs de base obtenues avec Geant4-DNA-CPA100 et CPA100 et des résultats très similaires ont été obtenus. Par exemple, un excellent accord entre les longueurs de trajectoire et les nombres d'interactions a été mis en évidence. Puis, nous avons évalué l'impact des sections efficaces en utilisant les modèles physiques originaux de Geant4-DNA (" option 2 " et " option 4 "), Geant4-DNA-CPA100 et le code PENELOPE, pour obtenir des grandeurs d'intérêt pour des calculs dosimétriques : les " dose-point kernels " (DPK, pour des électrons monoénergétiques) et les facteurs S (pour des électrons monoénergétiques et des émetteurs d'électrons Auger). Les calculs de DPK de Geant4-DNA avec les modèles physiques " option 2 " et " option 4 " sont similaires et une différence systématique a été mise en évidence avec Geant4-DNA-CPA100. Les DPK calculés par ce dernier ont montré un bon accord avec le code PENELOPE. Les facteurs S obtenus avec Geant4-DNA " option 2 " sont globalement proches de Geant4-DNA-CPA100. Enfin, nous avons cartographié les dépôts d'énergie dans un contexte de radioimmunothérapie. De telles simulations sont habituellement réalisées en considérant des tumeurs sphériques et des biodistributions uniformes d'anticorps monoclonaux. Nous avons extrait des données plus réalistes d'un modèle 3D innovant de lymphome folliculaire, incubé avec des anticorps. Les dépôts d'énergie ont été calculés pour différents émetteurs d'électrons Auger (111In et 125I) et de particules ß- (90Y, 131I et 177Lu). Ces calculs ont montré que les émetteurs de particules ß- délivrent plus d'énergie et irradient une plus grande fraction du volume que les émetteurs d'électrons Auger. L'émetteur de particule ß- le plus efficace dépend de la taille du modèle qui est utilisé
During this PhD thesis, new developments have been brought to Geant4-DNA step-by-step Monte Carlo code. They were used to study low-energy electron interactions in liquid water - the major component of living organisms. The accuracy of results obtained through Monte Carlo code is limited by the validity of their cross sections. CPA100 is another step-by-step Monte Carlo code. It is equipped with ionization, electronic excitation and elastic scattering cross sections. However, these cross sections are calculated according to methods independent of those used for Geant4-DNA cross section calculations, which consisted of two original physics models: "option 2" and its improvement, "option 4". Moreover, in some cases CPA100 cross sections are in better agreement with experimental data. Therefore, the first objective of this research was to implement CPA100 cross sections into Geant4-DNA in order to give users the choice of alternative physics models, known as Geant4-DNA-CPA100. They have been available to users since July 2017. The verification of the correct implementation of these physics models within Geant4-DNA involved a comparison of different basic quantities between Geant4-DNA-CPA100 and CPA100 and extremely similar results were obtained. For instance, a very good agreement was highlighted between the calculations of the track length and the number of interactions. Consequently, the impact of cross sections was assessed using the original Geant4-DNA physics models ("option 2" and "option 4"), the alternative Geant4-DNA-CPA100 physics models and PENELOPE code for calculations of useful quantities in nuclear medicine, such as dose-point kernels (DPKs for monoenergetic electrons) and S values (for monoenergetic electrons and Auger electron emitters). With regards to DPK calculations, Geant4-DNA with "option 2" and "option 4" physics models were in close agreement, showing a systematic difference with Geant4-DNA-CPA100, which in turn were close to those calculated with PENELOPE code. For S value calculations, however, Geant4-DNA results were in good agreement with Geant4-DNA-CPA100. Finally, in the context of radioimmunotherapy, energy depositions were mapped. Such simulations are usually performed assuming spherical tumor geometries and uniform monoclonal antibody distributions. Realistic data was extracted from an innovative 3D follicular lymphoma model incubated with antibodies. Energy depositions were calculated for Auger electron (111In and 125I) and ß- particle (90Y, 131I and 177Lu) emitters. It was demonstrated that ß- particle emitters delivered more energy and irradiated greater volume than Auger electron emitters. The most effective ß- particle emitter depends on the size of the model that is used

Malgré la découverte des antigènes tumoraux depuis quelques décennies dans les mélanomes humains, l’immunothérapie anticancéreuse reste encore relativement inefficace. En outre, les traitements anti-tumoraux classiques tels que la chimiothérapie et la radiothérapie ont longtemps été considérés comme des traitements affectant principalement les cellules tumorales en division. Actuellement, des études de plus en plus nombreuses suggèrent que le succès de la chimiothérapie dépendrait, en partie, du système immunitaire. En effet, d’une part, les agents chimio-thérapeutiques pourraient induire une mort immunogène des cellules tumorales et donc potentialiser les réponses immunitaires. D’autre part, certains rapports montrent que la présence préalable de cellules immunes dans l’environnement tumoral améliore le pronostic clinique observé après chimiothérapie notamment.

Au cours de ce travail, nous avons étudié l’effet du cyclophosphamide (CTX, un agent alkylant) sur le système immunitaire et la résistance anti-tumorale dans des souris porteuses du mastocytome P815. Nous avons remarqué qu’une seule injection de CTX dans des souris inoculées 10-20 jours plus tôt avec une dose létale de cellules tumorales induit la survie dans 100% des souris traitées. En outre, le rejet tumoral induit par le CTX est strictement dépendant des lymphocytes T CD4+ et CD8+, et permet une résistance tumorale à long terme spécifique du mastocytome P815. Le but de cette étude était d’appréhender les mécanismes cellulaires et moléculaires impliqués dans cette mémoire spécifique de la tumeur.

Nous avons premièrement montré que le CTX augmente les réponses de type Th1 et Th17 dans des souris immunisées. L’activation de ces réponses requiert l’IL-12p40 et corrèle avec une augmentation du nombre de cellules CD11b+/F4/80+/Ly6C+, suggérant que ces DCs inflammatoires présumées pourraient être une source potentielle d’IL-12 et/ou d’IL-23. Des résultats similaires ont été observés dans des souris porteuses de la tumeur P815 et traitées au CTX. Nous avons également caractérisé les cellules T anti-tumorales effectrices qui infiltrent la tumeur et en particulier, nous avons étudié le rôle des cellules T auxiliaires CD4+ dans la migration des lymphocytes T CD8+ spécifiques de la tumeur. Nous avons observé que la déplétion des cellules T CD4+ semble induire un blocage des lymphocytes T CD8+ dans les ganglions drainant la tumeur qui ne migrent alors plus vers le foyer tumoral. Nous avons donc évalué le rôle de chimiokines/récepteurs aux chimiokines qui pourraient être impliqués dans ce processus tels que les couples CXCR3/CXCL9-10-11.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Within the field of radiotherapy treatments of tumour diseases, the hunt for more accurate and effective treatment methods is a continuous process. For some years ion-beam based radiotherapy, especially the proton-beam based applications, has increased in popularity and availability. The main reason behind this is the fact that ion-beam based applications make it possible to modulate the dose after the planning target volume (PTV) defined by the radiation oncologist. This means that it becomes possible to spare tissue in another way, which might result in more effective treatments, especially in the vicinity of radio sensitive organs. Ion-beam based treatments are however more sensitive to uncertainties in PTV position and beam range as ion-beams have a fixed range depending on target media and initial energy, as opposed to the conventional x-ray beams that do not really have a defined range. Instead their intensity decreases exponentially at a rate dependent of the initial energy and target media. Therefore density heterogeneities result in uncertainties in the planned treatments. As the plans normally are created using a CT-images, for which metallic implants can yield increased heterogeneities both from the implants themselves and so called metal artifacts (distortions in the images caused by different processes as the X-rays used in image acquisition goes through metals). Metallic implants affects the accuracy of a treatment, and therefore also the related risks, so it is important to have an idea of the magnitude of the impact. Therefore the aim of this study is to estimate the impact on a proton-beam based treatment plan for six cranial implants. These were one Ti-mesh implant, one temporal plate implant, one burr-hole cover implant and three craniofix implants of different sizes, which all are commonly seen at the Skandion clinic. Also the ability of the treatment planning system (TPS), used at the clinic, to simulate the effects on the plans caused by the implants is to be studied. From this result it should be estimated if the margins and practices in place at the clinic, for when it is required to aim the beam through the implant, are sufficient or if they should be changed. This study consisted of one test on the range shift effects and one test on the lateral dose distribution changes, with one preparational test in the form of a calibration of Gafchromic EBT3 films. The range shift test was performed on three of the implants, excluding the three craniofix implants using a water phantom and a treatment plan created to represent a standard treatment in the cranial area. The lateral dose distribution change test was performed as a solid phantom study using radiochromic film, for two treatment plans (one where the PTV was located \SI{2}{\centi\metre} below surface, for all implants, and one where it was located at the surface, only for the Ti-mesh and the temporal plate). The results of both tests were compared to simulations performed in the Eclipse treatment planing system (TPS) available at Skandion. The result of the range shift test showed a maximum range shift of \SI{-1.03 +- 0.01}{\milli\metre}, for the burr-hole cover implant, and as the related Eclipse simulations showed a maximal shift of \SI{-0.17 +- 0.01}{\milli\metre} there was a clear problem with the simulation. However, this might not be because of the TPS but due to errors in the CT-image reconstruction, such as, for example, geometrical errors in the representation of the implants. As the margin applied for a similar situation at the Skandion clinic (in order to correct for several uncertainty factors) is \SI{4.2}{\milli\metre} there might be a need to increase this margin depending on the situation. For the lateral distribution effects no definite results were found as the change varied in magnitude, even if it tended to manifest as a decreasing dose for the first plan and a increasing dose for the second. It was therefore concluded that further studies are needed before anything clear can be said.

Le cancer de la vessie est un problème majeur de santé publique. Il est le quatrième cancer le plus fréquent chez l’homme en termes d’incidence. 25% des cancers diagnostiqués sont des tumeurs envahissant le muscle (TVIM) présentant un mauvais pronostic. La cystectomie est le traitement standard de référence pour les TVIM, même si elle présente des inconvénients importants. La radiothérapie, associée à une chimiothérapie et à une résection transurétrale de la tumeur, émerge comme traitement conservateur alternatif. La chimiothérapie n’épargne pas les tissus sains et présentent de nombreux effets secondaires indésirables. Il est donc d’importance de découvrir de nouvelles stratégies de radiosensibilisation pour les tumeurs de la vessie. TYRO3 est un récepteur à activité tyrosine kinase de la famille TAM (qui comprend TYRO3, AXL et MERTK). TYRO3 est surexprimé dans de nombreux types de cancers et favorise la prolifération, la survie et la résistance des cellules tumorales à la chimiothérapie. De plus, la surexpression de TYRO3 a été associée à une diminution de la survie globale des patients. Cependant, le rôle de TYRO3 dans le cancer de la vessie n'a pas encore été étudié. Dans cette thèse, je me suis intéressée : (1) Au rôle de TYRO3 dans le cancer de la vessie; (2) A l'effet radiosensibilisant de la perte d’expression ou de l’inhibition de TYRO3 dans les cellules cancéreuses de la vessie; (3) A l'effet de l'inhibition ou de la perte d’expression de TYRO3 sur l’urothélium humain sain.Nous avons démontré que TYRO3 est surexprimé dans 50% des TVIM. De plus, nous avons mis en évidence que les cellules tumorales de vessie surexprimant TYRO3 développaient une dépendance à ce récepteur pour leur survie et leur croissance. Les résultats des données transcriptomiques suggèrent que la perte d’expression de TYRO3 induit des modifications importantes dans le contrôle du cycle cellulaire et de l’apoptose, ce qui laisse supposer que TYRO3 pourrait augmenter la sensibilité de ces tumeurs à la radiothérapie. La perte d’expression ou l’inhibition de TYRO3 dans les cellules dérivées de tumeur de vessie induit une radiosensibilisation significative des cellules traitées. A l’inverse, la surexpression de TYRO3 par transfection plasmidique sur des cellules exprimant peu TYRO3 induit une radiorésistance. En association avec le rayonnement, la perte d’expression de TYRO3 conduit à un arrêt du cycle cellulaire et à une persistance à 24h des foyers de réparation (détectés par immunofluorescence). Enfin, les travaux sur les cellules dérivées de l’urothélium sain ont montré que la perte d’expression de TYRO3 n'affectait pas leur viabilité suggérant que le ciblage de TYRO3 pourrait améliorer l'efficacité de la radiothérapie tout en épargnant les tissus normaux environnants
Bladder cancer (BCa) is a major global health problem. It is the fourth most common cancer in men in industrialized countries. 25% of all diagnosed BCa are Muscle-invasive bladder cancers (MIBC) which have poor prognosis. Cystectomy is the standard treatment for MIBC, but for patients with comorbidities it presents significant drawbacks including increased risk of infection and impacted quality of life. Radiotherapy coupled with chemotherapy and tumor transurethral resection has emerged as a promising bladder sparing. Chemotherapy does not spare normal tissue and results in side effects. Therefore, it is of great interest to discover novel radiosensitisation strategies for bladder tumors.TYRO3 is a receptor tyrosine kinase of the TAM family (comprising TYRO3, AXL and MERTK) and is known to regulate diverse biological. TYRO3 is overexpressed in many types of cancer and promotes tumor cell proliferation, survival and resistance to chemotherapy. In addition, higher levels of TYRO3 expression have been associated with decreased overall survival in patients of diverse cancers. However, the role of TYRO3 in BCa has so far not been studied. In this thesis, I investigated:(1)The role of TYRO3 in BCa; (2) The radiosensitising effect of TYRO3 downregulation and inhibition in BCa cells; (3) The effect of TYRO3 downregulation and inhibition on normal human urothelial tissue.We first demonstrated that TYRO3 is overexpressed in 50% of MIBCs. TYRO3 overexpression conferred a TYRO3-dependance to bladder tumor cells for cell growth and viability. Transcriptomic analysis of TYRO3-downregulated cells suggested that TYRO3 signaling controlled cell cycle and protected from apoptosis, which indicated a potential to improve radiation response. TYRO3 downregulation lead to a significantly increased radiosensitivity of BCa cells and conversely, TYRO3-overexpression induced radioresistance. In combination with radiotherapy, TYRO3 dowregulation lead to a cell cycle arrest and a long term persistence of Ionizing Radiation-Induced Foci (IRIF). Finally, I demonstrated that TYRO3 downregulation and inhibition did not impact viability of normal human bladder cells suggesting that inhibiting TYRO3 could improve radiotherapy efficiency while sparing normal surrounding tissues

Dissertação para obtenção do Grau de Doutor em Engenharia Física
The role of ionising radiation as a source of damage to living tissues and cells has been recognized as a key issue regarding cellular DNA integrity and, ultimately, mutagenesis. The lethal effect of radiation, despite being most of the time undesired, can sometimes be useful, as is the case of radiation therapy. However, still the major concern in medicine is that only the cancerous cell material should be destroyed, keeping as much as possible healthy tissue unaffected. One way to control this damage seems to be the application of radiosensitizers that are incorporated into cancer cells. The cancer tissue doped with these radiation sensitizing molecules may be destroyed preferentially under radiation exposure, in very well defined places and even with radiation doses which may be low enough to prevent healthy cell material to be affected in the surrounding medium. This leads to nanodosimetry and so the sorts of interactions have now to be described at the molecular level. Upon irradiation, the most abundant secondary species produced along the radiation track are low energy electrons and so the study of electron induced damage to biological relevant molecules seems indubitably relevant. The research described in this thesis covers for the first time the study of electron transfer on two halouracils (5-chlorouracil and 5-fluorouracil) and isolated DNA/RNA basis (thymine and uracil)by atom-molecule collisions. In order to investigate such molecules, a crossed beam experiment, comprising a neutral potassium beam and a biolomecular effusive beam, was improved and a time-offlight mass spectrometer implemented allowing for the detection of negative ion formation following electron transfer processes in atom-molecule collisions. In these experiments the anionic fragmentation patterns and formation yields were obtained. These results are shown to be significantly different from the dissociative electron attachment (free electrons) results, unveiling that the damaging potential of secondary electrons to biomolecules can be somewhat underestimated. In addition, the halouracils sensibility to electron induced damage appears to be enhanced with respect to thymine and uracil, which may be extremely relevant as it reinforces their effectiveness as radiosensitizer molecules.
Portuguese Foundation for Science and Technology - SFRH/BD/32271/2006

Telomerase is expressed in the majority (>85%) of tumours but not in differentiated normal tissue. This enzyme catalyses the elongation of telomeres – a process critical for continued cell proliferation. Telomerase is a potential novel target for molecularly-targeted radiotherapy (mRT), due to its nuclear localization and expression profile. The radiolabelling of telomerase inhibitors may accelerate and enhance the cytotoxicity of such molecules, as a result of irradiation of the DNA. An oligonucleotide targeting telomerase RNA (hTR), shown to inhibit enzyme activity in vitro, was selected for study. Complementary and non-targeting control oligonucleotides were conjugated to a metal chelator (DTPA) to allow radiolabelling with indium-111. The radioiodination of MST-312, BIBR-1532 and flavonoid-derived small molecule inhibitors of telomerase was also pursued. The inhibitory activity of the candidate molecules was analysed using the telomeric repeat amplification protocol (TRAP). The internalization of inhibitors was assessed by gamma-counting following cell lysis. The clonogenic assay was employed to measure the effect of modified inhibitors on cell survival. Small molecule telomerase inhibitors were modified for labelling with iodine-123, which led to a modest decrease in inhibitory potency, compared to the parent molecules. Radiolabelled small molecules exhibited poor stability and internalization into cancer cells, so were unsuitable for mRT. Modified oligonucleotides potently inhibited telomerase activity, whereas a non-targeting oligonucleotide exhibited no inhibitory activity. Indium-111 radiolabelled oligonucleotides decreased the clonogenic survival of telomerase-positive breast cancer cells but not telomerase-negative cells, in a sequence-specific manner. Accordingly, complementary radiolabelled oligonucleotides were found to induce the DNA damage marker γH2AX. Oligonucleotides localized to nuclear Cajal bodies, the sites of telomerase assembly, in a proportion of cancer cells. Telomerase inhibitors of different classes were radiolabelled with Auger electron-emitting radionuclides, and delivered to cells. Radiolabelled oligonucleotides targeting telomerase significantly reduced the clonogenicity of cancer cells in vitro. This study represents a novel approach for the mRT of telomerase-positive cancers.

Bone marrow dosimetry is a topic of high interest in molecular radiotherapy. Predicting the level of hematological toxicity is one of the most important goals of nuclear medicine radiation dosimetry. To achieve this, it is necessary to quantify the absorbed dose to the active bone marrow, thus aiming at administering the most efficient therapy with a minimum level of adverse effects in the patient. The anatomical complexity of trabecular bone and bone marrow leads to the need of applying non-nuclear medicine imaging methods for determining the spatial distribution of soft tissue, adipose tissue, and bone in spongiosa. Therefore, the two objectives of this dissertation are: i) to apply magnetic resonance imaging (MRI) for quantification of the fat volume fraction, and ii) to validate a method based on dual-energy quantitative computed tomography (DEQCT) for quantification of the trabecular bone volume fraction. In a first step, an MRI sequence (two-point Dixon) for fat-water separation was validated in a 3 Tesla system by quantifying the fat volume fraction in a phantom and the lumbar vertebrae of volunteers and comparing with magnetic resonance spectroscopy (MRS). After successful validation, the fat volume fraction was retrospectively measured in the five lumbar vertebrae of 44 patient images acquired in the clinical routine. The two-point Dixon showed a good quantification of the fat volume fraction in the phantom experiment (-9.8% maximum relative error with respect to the nominal values). In the volunteers, a non-significant difference between MRI and MRS was found for the quantification of the fat volume fraction in volumes-of-interest with similar dimensions and position in both quantification methodologies (MRI and MRS). In the study with patient data, the marrow conversion (red → yellow marrow) was found to be age-dependent, and slower in males (0.3% per year) than in females (0.5% per year). Also, considerable variability of the fat volume fraction in patients of similar ages and the same gender was observed. These results enable the use of two-point Dixon MRI in the quantification of the fat volume fraction in the bone marrow. Additionally, the constant marrow conversion during adulthood suggests that a patient-specific approach should replace the assumption of a constant cellularity volume fraction of 0.7 (reference man) (1,2) as proposed by the International Commission on Radiological Protection (ICRP). In a second step, a quantification method based on DEQCT was validated in two CT systems: i) a clinical CT integrated into a SPECT/CT and ii) a dual-source computed tomography (DSCT) system. The method was applied in two phantoms: the first was used to validate the DEQCT method by the quantification of the hydroxyapatite volume fraction in three vials of 50 ml each and three different hydroxyapatite concentrations (100 mg/cm3, 200 mg/cm3, 300 mg/cm3). The second phantom was the European spine phantom (ESP), an anthropomorphic spine phantom. It was used to quantify the bone mineral content (BMC) on the whole vertebra and the hydroxyapatite volume fraction (VFHA) in the spongiosa region of each vertebra of the phantom. Lastly, the BMC of lumbar vertebrae 1 (LV1) and 2 (LV2) was measured in a patient using DEQCT and dual-energy X-ray absorptiometry (DEXA). Furthermore, the hydroxyapatite volume fraction (VFHA) and the bone volume fraction (VFB) was calculated for both the whole vertebrae and the spongiosa region of LV1 and LV2. The measured and nominal hydroxyapatite volume fraction in the vial phantom showed a good correlation (maximum relative error: 14.2%). The quantification of the BMC on the whole vertebra and the VFHA on the spongiosa region showed larger relative errors than in the validation phantom. The quantification of BMC on LV1 and LV2 showed relative errors between DEXA and DSCT equal to 7.6% (LV1) and -8.4% (LV2). Also, the values of the VFHA (mineral bone) were smaller than the VFB. This result is consistent with the bone composition (mineral bone plus organic material). The DEQCT method enables the quantification of hydroxyapatite (mineral bone) and bone (mineral bone plus organic material) in a clinical setting. However, the method showed an overestimation of the quantified mineral bone volume fraction. This overestimation might be related to the lack of detailed information on the CT X-ray spectra and detector sensitivity. Also, the DEQCT method showed a dependency on the CT reconstruction kernel and the chemical description of the materials to be quantified. Based on the results of this work, the feasibility for quantifying the fat volume fraction and the bone volume fraction in the spongiosa in a clinical setting has been demonstrated/proven. Furthermore, the differences in fat volume fraction in females and males, as well as the variability of the fat volume fraction in subjects of similar ages, questions the approximation of the cellularity volume fraction by only a single ICRP reference value in bone marrow dosimetry for molecular radiotherapy. Lastly, this study presents the first approach for non-invasive quantification of the bone volume fraction (mineral bone plus organic material) for improved bone marrow dosimetry
Die Knochenmarkdosimetrie ist von großem Interesse für die Radionuklidtherapie. Die Vorhersage des Grades der hämatologischen Toxizität ist eines der wichtigsten Ziele der nuklearmedizinischen Dosimetrie. Um dieses Ziel zu erreichen, ist es erforderlich, die Energiedosis des aktiven Knochenmarks zu quantifizieren, um dem Patienten so eine möglichst effiziente Therapie mit einem minimalen Maß an unerwünschten Nebenwirkungen verabreichen zu können. Die anatomische Komplexität von Knochentrabekel und Knochenmark macht es erforderlich, nicht-nuklearmedizinische bildgebende Verfahren anzuwenden, um die räumliche Verteilung von Weichgewebe, Fettgewebe und Knochen in der Spongiosa zu bestimmen. Daher sind die zwei Ziele dieser Dissertation: i) die Anwendung der Magnetresonanztomographie (MRT) zur Quantifizierung des Fettvolumenanteils und ii) die Validierung einer auf der quantitativen Dual-Energy Computertomographie (engl. Dual-energy quantitative computed tomography, DEQCT) basierenden Methode zur Quantifizierung des Knochentrabekelvolumenanteils. In einem ersten Schritt wurde eine Zweipunkt-Dixon-Sequenz der MRT zur Fett-Wasser-Trennung in einem 3 Tesla-System validiert, indem der Fettvolumenanteil in einem Phantom und in den Lendenwirbeln von Probanden quantifiziert und mit mittels der Magnetresonanzspektroskopie (MRS) ermittelten Werten verglichen wurde. Nach erfolgreicher Validierung wurde der Fettvolumenanteil retrospektiv an den fünf Lendenwirbeln von 44 in der im klinischen Routine aufgenommenen Patientendatensätzen gemessen. Die Zweipunkt-Dixon-Methode zeigte eine gute Quantifizierung des Fettvolumenanteils im Phantomexperiment (-9,8% maximaler relativer Fehler in Bezug auf die Nennwerte). Bei den Probanden wurde ein nicht signifikanter Unterschied zwischen MRT und MRS für die Quantifizierung des Fettvolumenanteils in einem Zielvolumen mit ähnlichen Dimensionen und ähnlicher Orientierung festgestellt. In der Patientenstudie wurde festgestellt, dass die Umwandlung des Knochenmarks (rotes Knochenmark → gelbes Knochenmark) altersabhängig und bei Männern (0,3% pro Jahr) langsamer als bei Frauen (0,5% pro Jahr) voranschreitet. Es wurde allerdings auch eine beträchtliche Variabilität des Fettvolumenanteils bei Patienten ähnlichen Alters und gleichen Geschlechts beobachtet. Diese Ergebnisse ermöglichen die Verwendung der Zweipunkt-Dixon-MRT zur Quantifizierung des Fettvolumenanteils im Knochenmark. Darüber hinaus legt die konstante Umwandlung des Knochenmarks im Erwachsenenalter nahe, dass der von der Internationalen Strahlenschutzkommission (engl. International Commission on Radiological Protection, ICRP) vorgeschlagene konstante Zellvolumenanteil von 0,7 (Referenzwert für einen männlichen Erwachsenen) (1,2) durch einen patientenspezifischen Ansatz ersetzt werden sollte. In einem zweiten Schritt wurde eine auf DEQCT basierende Quantifizierungsmethode in zwei CT-Systemen validiert: i) ein in ein SPECT/CT integriertes klinisches CT und ii) ein Dual-Source-Computertomographie-System (DSCT). Die Methode wurde an zwei Phantomen erprobt: Das erste diente zur Validierung der DEQCT-Methode, wobei der Hydroxylapatit-Volumenanteil in drei 50-Milliter-Phiolen mit drei verschiedenen Hydroxylapatit-Konzentrationen (100 mg/cm3, 200 mg/cm3, 300 mg/cm3) quantifiziert wurde. Das zweite Phantom war das European Spine Phantom (ESP), ein anthropomorphes Wirbelsäulenphantom. Es wurde verwendet, um den Knochenmineralgehalt (engl. Bone Mineral Content, BMC) des gesamten Wirbels und den Hydroxylapatit-Volumenanteil (VFHA) in der Spongiosa-Region jedes Phantomwirbels zu quantifizieren. Schließlich wurde der BMC der Lendenwirbel 1 (LV1) und 2 (LV2) bei einem Patienten unter Verwendung von DEQCT und Dual-Röntgen-Absorptiometrie (engl. dual-energy X-ray absorptiometry, DEXA) gemessen. Darüber hinaus wurden der Hydroxylapatit-Volumenanteil (VFHA) und der Knochenvolumenanteil (VFB) sowohl für die gesamten Wirbel als auch für die Spongiosa-Region von LV1 und LV2 berechnet. Der gemessene und der nominelle Hydroxylapatit-Volumenanteil in den Phiolen zeigten eine gute Korrelation (maximaler relativer Fehler: 14,2%). Die Quantifizierung des BMC im gesamten Wirbel und des VFHA in der Spongiosa-Region zeigten größere relative Fehler als im Validierungsphantom: Die BMC-Quantifizierung für LV1 und LV2 ergaben relative Fehler zwischen DEXA und DSCT in Höhe von 7,6% (LV1) und -8,4% (LV2). Auch die Werte des VFHA (mineralischer Knochen) waren kleiner als die des VFB. Dieses Ergebnis steht im Einklang mit der Knochenzusammensetzung (Knochenmineral plus organisches Material). Die DEQCT-Methode ermöglicht die Quantifizierung von Hydroxylapatit (mineralischer Knochen) und Knochen (mineralischer Knochen plus organisches Material) in einem klinischen Umfeld. Die Methode zeigte jedoch eine Überschätzung des quantifizierten mineralischen Knochenvolumenanteils. Diese Überschätzung könnte mit dem Mangel an detaillierten Informationen über die CT-Röntgenspektren und die Detektorempfindlichkeit zusammenhängen. Auch die DEQCT-Methode zeigte eine Abhängigkeit vom verwendeten CT-Rekonstruktionsalgorithmus und der chemischen Beschreibung der zu quantifizierenden Materialien. Die Ergebnisse dieser Dissertation zeigen die Machbarkeit einer Quantifizierung des Fettvolumenteils und des Knochenvolumenteils in der Spongiosa in einem klinischen Kontext. Darüber hinaus geben die Unterschiede im Fettvolumenanteil von Frauen und Männern sowie die Variabilität des Fettvolumenanteils bei Individuen ähnlichen Alters Grund zur kritischen Auseinandersetzung mit der Näherung des Zellvolumenanteils durch nur einen einzelnen ICRP-Referenzwert in der Knochenmarkdosimetrie bei Radionuklidtherapien. Zusätzlich wird in dieser Arbeit der erste Ansatz für eine nicht-invasive Quantifizierung des Volumenanteils des Knochens (Knochenmineral plus organisches Material) für eine verbesserte Dosimetrie des Knochenmarks vorgestellt

A new concept quickly gaining ground in the field of cancer research is that the inflammatory process plays a key role in cancer development and metastasis; however, the molecular mechanisms of such an involvement in cancer progression remain largely unspecified. YKL-40, also known as human cartilage glycoprotein 39, is a secreted heparin-binding protein with ties to both cancers and inflammatory disease. In these diseases, YKL-40 has been suggested to play a role in regulating tissue and extracellular matrix remodeling. It has been found that in certain cancers, including breast, colorectal and brain, that high YKL-40 serum levels correlate with poor outcome, and consequently it may serve as a biomarker. Our recent study has shown that tumor-derived YKL-40 acts as an angiogenic factor due to its ability to up-regulate vessel formation and metastasis during tumor development. However, blockade of the function of YKL-40, which implicates therapeutic value, has not been explored yet. The goal of this project was to better understand the importance of tumor-derived YKL-40 in angiogenesis through both functional and structural studies. By establishing a monoclonal YKL-40 antibody for blocking YKL-40, the function of tumor-derived YKL-40 in inducing endothelial cell angiogenesis and tumor cell survival was uncovered, confirming YKL-40's importance in tumor signaling as well as offering evidence in the benefit of its neutralization. Additionally, a postulated heparin-binding domain on YKL-40 was mutated in hopes of revealing the relevance of this binding ability on YKL-40's function and whether this could serve as a target in inhibiting YKL-40 signaling.

Salivary glands (SGs) are important exocrine glands of the craniofacial region, whose main role is to produce and secrete saliva, a seromucous solution necessary for a diverse spectrum of critical functions, such as the preliminary digestion and swallowing of solid food, the articulation of speech, the maintenance of dental enamel and the prevention of oral infections. The production and secretion of saliva is orchestrated by a large and diverse collection of epithelial cell populations. Although many of the cell types that form the SG epithelium can be recognized morphologically and investigated using histological assays, it is currently impossible to achieve their differential purification from primary tissues as live cells, due to the lack of surface markers known to be either selectively or preferentially expressed by various cell subsets. This critical gap in knowledge limits our capacity to conduct functional studies in many areas of SG biology, including studies aimed at elucidating the developmental relationships that link different cell types (e.g. testing whether selected cell types can act as progenitors for the generation of others), studies elucidating the roles played by different cell types during regeneration of the SG epithelium following injury (e.g. radiotherapy), and studies investigating the biology of SG malignancies characterized by a heterogeneous cell composition, such as Adenoid Cystic Carcinomas (ACCs). In this work, we aimed to advance our understanding of the cell composition of the salivary gland epithelium and to identify surface markers that enable the differential purification of its various cell types by fluorescence-activated cell sorting (FACS), in order to facilitate functional investigations of their individual capacity to act as stem/progenitor cells in prospective assays. In the first portion of our studies, we leveraged single-cell RNA sequencing (scRNA-seq) to dissect the transcriptional identities of various epithelial cell populations found in normal murine SGs, and discovered surface markers that allowed us to purify eight distinct cell types by FACS. We then used bulk RNA sequencing to generate high-resolution transcriptomic profiles of seven of these populations, and annotated their identity (e.g. acinar, ductal, basal, myoepithelial) in terms of anatomical location and differential expression of lineage-specific biomarkers. Furthermore, using a three-dimensional (3D) in vitro organoid tissue culture assay, we tested each of the newly identified SG populations for stem/progenitor properties, and demonstrated that organoid forming capacity is primarily restricted to only one of them, characterized by a basal phenotype, and able to function as a bipotent progenitor in vitro. Finally, we used FACS to examine the effects of radiotherapy on the cell composition of the mouse SG epithelium, and demonstrated that, of the eight newly identified populations, at least four display preferential sensitivity to radiation injury. In the second portion of our studies, we tested whether the surface markers that we identified as differentially expressed between different subtypes of SG epithelial cells could also be leveraged to achieve the purification of the two subsets of malignant cells known to co-exist in Adenoid Cystic Carcinoma (ACC), one of the most common and lethal forms of human SG malignancy. A defining feature of ACC is the presence of two distinct cell populations, resembling myoepithelial and ductal cell types found in the normal salivary gland epithelium. However, little is known about the developmental relationship linking these two cell populations, their individual capacity to sustain the growth of malignant tissues upon xeno-transplantation, as well as their distinct behavior in terms of responses to therapeutic manipulations. By utilizing cell surface markers identified as differentially expressed in the mouse SG epithelium, we developed a sorting strategy that enabled us to isolate the two major subtypes of malignant cells found in ACCs. By conducting prospective xeno-transplantation experiments in immunodeficient mice, we demonstrated that, contrary to common belief, myoepithelial-like cells are highly tumorigenic (i.e. do not represent an indolent component of the tumor) and can act as progenitors of ductal-like cells. Furthermore, by investigating differences in the transcriptional profiles of myoepithelial-like and ductal-like cells, we discovered that the two cell types differ in the expression of multiple components of the biochemical pathways that control retinoic acid (RA) signaling. We find that RA direct and inverse agonism have opposing effects on cell composition through distinct molecular mechanisms, whereby direct agonism facilitates differentiation of myoepithelial-like to ductal-like cells, and inverse agonism induces selective cell death of ductal-like cells. Finally, we demonstrate that inhibition of RA signaling with inverse agonists is able to profoundly impair in vivo growth of human ACCs implanted in immunodeficient mice. Overall, the findings reported in this study advance our understanding of the cellular composition of both normal and malignant SG epithelia, establish novel and robust analytical assays for the purification of multiple subtypes of SG epithelial cells, and reveal novel strategies for the therapeutic manipulation of differentiation programs in human ACCs.

Despite advances in technology and understanding of biological systems in the past two decades, modern drug discovery is still a lengthy, expensive, difficult and inefficient process with low rate of new therapeutic discovery. The search for new effective drugs remains a somewhat empirical process. There is compelling need for a more fundamental, mechanistic understanding of human cancers and anticancer drugs to design more appropriate drugs. Radiotherapy is still the major therapy of cancer. It uses high-energy ionizing radiation such as x-rays and charged particle beams to destroy cancer cells. DNA is well known to be the principal biological target of radiotherapy, but the molecular mechanism of ionizing radiation induced DNA damage was elusive. The conventional thought of the ???OH radical as the major origin for ionizing radiation induced DNA damage is questionable. Although various strategies and types of compounds have been designed and developed as potential radiosensitizers to enhance the radiosensitizing efficiency of radiotherapy, none of them have been approved for clinical use. The general outcomes of clinical trials have been disappointing. This thesis presents an innovative molecular-mechanism-based drug discovery project to develop novel drugs for effective radiotherapy of cancer through the emerging femtomedicine approach. Its ultimate goal is to develop more effective radiosensitizers, based on our unique molecular understandings of ionizing radiation induced DNA damage and halopyrimidines as a family of potential radiosensitizers. Direct, real-time observation of molecular reactions is of significant importance in diverse fields from chemistry and biology, environmental sciences to medicine. Femtosecond time-resolved laser spectroscopy (fs-TRLS) is a very powerful, direct technique for real-time observation of molecular reactions. Its key strength lies in short duration laser flashes of a time scale at which reactions actually happen - femtoseconds (fs) (1fs = 10???15 second). Since the late 1980s, its application to study chemical and biological systems led to the births of new subfields of science, called femtochemistry and femtobiology. Recently, femtomedicine has been proposed as a new transdisciplinary frontier to integrate ultrafast laser techniques with biomedical methods for advances in fundamental understandings and treatments of major human diseases. This the remarkable opportunity afforded through real-time observation of biochemical reactions at the molecular level. Femtomedicine holds the promise of advances in the radiotherapy of cancer. Several important findings were made in this thesis. First, our results of careful and high-quality fs-TRLS measurements have resolved the long existing controversies about the physical nature and lifetimes of a novel ultrashort-lived electron species (epre???) generated in radiolysis of water. These results have not only resolved the large discrepancies existing in the literature but provided new insights into electron hydration dynamics in bulk water. Such information is important for quantitative understanding and modeling of the role of non-equilibrium epre??? in electron-driven reactions in diverse environmental and biological systems, from radiation chemistry and radiation biology to atmospheric ozone depletion. Second, our fs-TRLS results have unraveled how epre??? plays a crucial role in ionizing radiation induced DNA damage. We found that among DNA bases, only T and especially G are vulnerable to a dissociative electron transfer (DET) reaction with epre??? leading to bond breaks, while the electron can be stably trapped at C and especially A to form stable anions. The results not only challenge the conventional notion that damage to the genome by ionizing radiation is mainly induced by the oxidizing ???OH radical, but provide a deeper fundamental understanding of the molecular mechanism of the DNA damage caused by a reductive agent (epre???). Our findings have led to a new molecular mechanism of reductive DNA damage. Third, halopyrimidines, especially BrdU and IdU, have passed Phase I to II clinical trials as potential hypoxic radiosensitizers, but the outcome of Phase III clinical trials was disappointing. Our results of fs-TRLS studies have provided a new molecular mechanism of action of halopyrimidines (XdUs, X=F, Cl, Br and I) in liquid water under ionizing radiation. We found that it is the ultrashort-lived epre???, rather than the long-lived ehyd???, that is responsible for DET reactions of XdUs. This reaction leads to the formation of the reactive dU??? radical, which then causes DNA strand breaks and cancer cell death. Our results have challenged a long accepted mechanism that long-lived ehyd??? would be responsible for the radical formation from halogenated molecules. Furthermore, we found that the DET reaction efficacy leading to the formation of the reactive dU??? radical is in the order of FdU << CldU < BrdU < IdU. Thus, only BrdU and IdU could be explored as potential radiosensitizers, in agreement with the results of bioactivity tests and clinical trials. Fourth, our fs-TRLS studies have provided a molecular mechanism for the DNA sequence selectivity of BrdU and IdU in radiosensitization. We found the DET reactions of BrdU/ IdU with dAMP*??? and dGMP*??? formed by attachment of epre??? generated by radiolysis of water in aqueous BrdU-dAMP/dGMP and IdU-dAMP/dGMP complexes under ionizing radiation. This new mechanistic insight into the interaction of BrdU and IdU with DNA provides clues to improve the halogen familty as potential radiosensitizers and to develop more effective radiosensitizers for clinical applications. Fifth, based on our molecular mechanistic understandings of DNA damage induced by ionizing radiation and halopyrimidines as potential radiosensitizers, we develop more effective new radisensitizing drug candidates through the femtomedicine approach. We have performed a fs-TRLS study of the DET reaction of a candidate compound (RS-1) with epre???, and found that the DET reaction of epre??? with RS-1 is much stronger than that of IdU (and certainly BrdU and CldU). Moreover, we have tested the radiosensitizing effect of RS-1 against human cervical cancer (HeLa) cells exposed to various doses of x-ray irradiation through DNA damage measurements by gel electrophoresis and cell viability/death assays by MTT. Our results have confirmed that RS-1 can largely enhance the radiosensitivity of treated human cervical cancer (HeLa) cells to x-ray (ionizing) radiation. It is clearly demonstrated that RS-1 has a much better radiosensitizing effect than IdU. Although these are just preliminary results, our results have shown promise of developing more effective radiosensitizers. In summary, our studies have demonstrated the potential of femtomedicine as an exciting new frontier to bring breakthroughs in understanding fundamental biological processes and to provide an efficient and economical strategy for development of new anticancer drugs.

Replacement of thymidine in DNA by halopyrimidines, such as bromodeoxyuridine (BrdU) and iododeoxyuridine (IdU), has long been known to enhance DNA damage and cell death induced by ionizing/UV radiation, but the mechanism of action of halopyrimidines at the molecular level is poorly understood. We have applied advanced time-resolved femtosecond laser spectroscopy to this molecular system of biological, chemical and medical significance. We obtained the first real-time observations of the transition states of the ultrafast electron transfer (UET) reactions of halopyrimidines with the ultrashort-lived precursor to the hydrated electron, which is a general product in ionizing/UV radiation. Our results provide a mechanistic understanding of these photo-/radiosensitizing drugs at the molecular level. We found that the UET reaction of BrdU is completed within 0.2 picosecond (ps) after the electronic exciataion, leading to the formation of the transition state BrdU* with a lifetime of ~1.5 ps that then dissociates into Br and a high reactive radical dU•. We have also demonstrated that the reaction efficiency for the formation of the reactive radical dU• to cause DNA damage and cell death is in the order of IdU>>BrdU>CldU>>FdU. This is due to the availability of two precursor states of ~0.2 ps and ~ 0.54 ps lifetimes for dissociative electron attachment (DEA) to IdU, of one precursor state of ~0.2 ps lifetime for DEAs to BrdU and CldU, and no precursors for DEA to FdU. This explains why BrdU and IdU were found to be effective radio-/photosensitizers and indicates that IdU should be explored as the most effective radiosensitizer among halopyrimidines. Moreover, as a by-product of this project, these halopyrimidines have been employed as quantum-state-specific molecular probes to resolve a long-standing controversy about the nature and lifetimes of prehydrated electrons. These findings also have a broader significance as they indicated that nonequilibrium precursor electrons may play an important role in electron-initiated reactions in many biological, chemical and environmental systems. We have also demonstrated UET reactions of nucleotides with the precursor to the hydrated electrons. Our results indicate that among DNA bases, adenine is the most efficient electron trapper and an effective electron transfer promoter, while guanine is the most effective in dissociative electron attachment. These results not only primarily explain the sequence selectivity of duplex DNA containing BrdU/IdU, but imply that the DEA of guanine is an important mechanism for radiation-induced DNA damage in ionizing radiation and radiotherapy of cancer.

La sénescence cellulaire est un mécanisme naturel de suppression tumorale défini par un arrêt stable de la prolifération. Bien que presque toujours mutées dans au moins une des voies déclenchant la sénescence (ex : p53/p21 ou p16/Rb), les cellules cancéreuses conservent souvent la capacité d’y entrer en réponse au traitement. Cette sénescence induite par la thérapie (SIT) peut être ciblée pharmacologiquement pour en renforcer les effets positifs. Une approche émergente consiste à combiner un traitement anticancéreux induisant la sénescence à un sénolytique, agent éliminant spécifiquement les cellules sénescentes. Dans le contexte du cancer de la prostate (CP), différents types de SIT ont déjà été observées dans de multiples modèles exposés à différents traitements. Cependant, le manque de données comparant ces phénotypes souligne le besoin d’analyses plus systématiques. De plus, la sensibilité aux sénolytiques des cellules du CP en état de SIT n’a pas encore été évaluée dans ces contextes. Dans cette étude, nous avons évalué les destins cellulaires des lignées du CP après exposition à trois traitements pertinents au niveau clinique : l’irradiation et l’olaparib, deux inducteurs de dommages à l’ADN et l’enzalutamide, un anti-androgène. Dépendamment de la lignée, les traitements par irradiation et olaparib ont mené à une réponse dirigée principalement vers la sénescence ou vers une réponse mixte de mort cellulaire, de catastrophe mitotique et de sénescence. Dans tous les cas, ceux-ci ont déclenché un phénotype sénescent classique et convertible en mort cellulaire par des sénolytiques inhibiteurs des antiapoptotiques de la famille Bcl-2. D’autre part, le traitement à l’enzalutamide a déclenché un phénotype semblable à la sénescence se distinguant par sa réversibilité, son absence de dommages à l’ADN et son insensibilité à ces mêmes sénolytiques. Globalement, nos résultats soulignent l’importance du contexte thérapeutique dans l’élaboration des stratégies de manipulation de la SIT du CP. Ils constituent également une justification robuste à l’étude préclinique des traitements combinant la radiothérapie ou l’olaparib à des inhibiteurs des antiapoptotiques de la famille Bcl-2 dans le contexte du CP.
Cellular senescence is a natural tumor suppression mechanism defined by a stable proliferation arrest. Although almost always mutated in at least one of the senescence pathways genes (e.g., p53/p21 or p16/Rb), cancer cells often retain the ability to become senescent in response to treatment. This therapy-induced senescence (TIS) can be pharmacologically targeted to enhance its positive effects. An emerging approach is to combine senescence-inducing cancer treatment with senolytics, compounds that specifically eliminate senescent cells. In the context of prostate cancer (PCa), different types of TIS have already been observed in multiple models exposed to different treatments. However, the lack of data comparing these phenotypes highlights the need for more systematic analyses. In addition, the senolytic sensitivity of TIS PCa cells has not yet been evaluated in these settings. In this study, we evaluated the cell fates of PCa cell lines after exposure to three clinically relevant treatments: irradiation and olaparib, two DNA damage inducers, and enzalutamide, an anti-androgen. Depending on the cell line, irradiation and olaparib treatments led to a response mainly directed towards senescence or toward a mixed response of cell death, mitotic catastrophe and senescence. In all cases, these treatments triggered a classic senescent phenotype that was convertible to cell death by senolytic inhibitors of the Bcl-2 family antiapoptotics. On the other hand, treatment with enzalutamide triggered a senescence-like phenotype, distinguishable by its reversibility, absence of DNA damage and insensitivity to these same senolytics. Overall, our results underscore the importance of the therapeutic context in the development of PCa-TIS manipulation strategies. They also provide a robust rationale for the preclinical study of treatments that combine radiotherapy or olaparib with Bcl-2 family antiapoptotic inhibitors in the PCa context.

Tese de Doutoramento do Programa de Doutoramento em Ciências da Saúde, ramo de Tecnologias da Saúde, apresentada à Faculdade de Medicina da Universidade de Coimbra.
A radioterapia é uma das modalidades de tratamento mais comummente utilizada para o tratamento do cancro, tanto de tumores sólidos como de tumores com origem hematopoiética. O cancro do pulmão é um tumor sólido, com uma das taxas de incidência e de mortalidade mais elevadas em todo mundo. O linfoma difuso de grandes células B é um tumor hematopoiético e o tipo mais comum de linfoma- não Hodgkin. Este trabalho teve como um dos objetivos determinar e caraterizar os efeitos intracelulares da radioterapia em linhas celulares de cancro do pulmão de pequenas células (H69) e de não pequenas células (A549 e H1299) e de linfoma difuso de grandes células B (Farage). Para este propósito, foram determinados os efeitos da radiação ionizante na viabilidade, na proliferação, na sobrevivência e nos mecanismos de morte celular. Realizaram-se os ensaios do azul tripano, do Alamar Blue®, clonogénico, estudos de citometria de fluxo, através da dupla marcação com anexina V e com iodeto de propídeo, da marcação das proteínas BAX e BCL-2, das alterações no potencial de membrana mitocondrial e estudos de morfologia celular com a coloração May-Grünwald Giemsa. Para além disto, foram também avaliados, por citometria de fluxo, os efeitos da radiação ionizante no stresse oxidativo, no que respeita à produção intracelular de peróxidos, de radical superóxido e da defesa anti-oxidante glutationa reduzida. Os danos no DNA foram determinados pelo ensaio cometa, e a expressão da proteína P53 total e fosforilada foi avaliada por western blot. Paralelamente constituiu outro objetivo deste trabalho a avaliação dos efeitos da radioterapia no sistema imunitário de doentes com cancro do pulmão e com linfoma difuso de grandes células B. Para a concretização deste objetivo foram incluídos neste estudo 8 doentes de cancro do pulmão e 9 doentes de linfoma difuso de grandes células B. Após a colheita de sangue periférico foram determinadas as contagens leucocitárias pela realização de um leucograma, as contagens linfocitárias e das células T reguladoras com recurso a Lymphogram® e imunofenotipagem, respetivamente. Foram ainda avaliadas trinta e quatro citocinas e quimiocinas, relevantes na resposta do sistema imunitário, através do kit comercial ProcartaPlexTM Immunoassay Magnetic Beads. A radiação ionizante induziu diminuição da proliferação, da viabilidade e da sobrevivência celular em todas as linhas celulares. No entanto, a sobrevivência ajustou-se a modelos de agressão celular distintos. Enquanto as linhas celulares H1299 e Farage apresentaram uma resposta correspondente que se ajustou ao modelo linear quadrático, as linhas celulares H69 e A549 apresentaram uma resposta correspondente ao modelo de um só alvo um só toque, portanto, ao modelo linear. Após exposição à radiação ionizante, o tipo de morte celular preferencial foi dependente da dose e, presumivelmente, do perfil de expressão de P53. Assim a expressão P53Wild, das células Farage e A549, foi associada a morte celular por apoptose, enquanto a ausência de expressão, verificada nas células H1299, e a produção de uma proteína mutada, descrita nas H69, foi associada a morte celular por necrose, particularmente com as doses mais elevadas. Além disso, nas linhas celulares Farage e A549 os níveis de expressão da proteína P53 com a irradiação tanto no que respeita à proteína total como à forma fosforilada aumentam. De forma complementar verificou-se que a despolarização de membrana mitocondrial e o aumento da razão BAX/BCL-2 indicam que a morte celular por apoptose, típica das células P53Wild, parece ser ativada pela via intrínseca. Relativamente ao ciclo celular verificou-se bloqueio nas fases G0/G1 e na fase S nas linhas celulares Farage e A549 enquanto as linhas celulares H1299 e H69 progrediram nas referidas fases mas ficaram comprometidas mais tardiamente na fase G2/M. Estes dados reforçam a importância do perfil molecular na resposta à radiação ionizante. Finalmente, o stresse oxidativo e os danos no DNA destacaram-se como processos de grande importância associados aos efeitos da radiação ionizante. Paralelamente, o estudo de patologia humana realizado no âmbito desta Tese revelou que a resposta do sistema imunitário ao tratamento por radioterapia foi dependente do tipo de cancro em estudo. A avaliação do leucograma dos doentes com cancro do pulmão evidenciou alterações nos leucócitos, linfócitos e monócitos a meio do tratamento com radioterapia. No entanto, não se observaram diferenças significativas no leucograma dos doentes com linfoma difuso de grandes células B. Do mesmo modo, nos doentes com cancro do pulmão observaram-se alterações no linfograma no que respeita às células B, às células Natural Killer e às células Natural Killer citotóxicas. Por outro lado, nos doentes com linfoma difuso de grandes células B verificaram-se alterações a nível das células T reguladoras induzidas. Relativamente às citocinas e às quimiocinas associadas ao perfil Th1 destacou- se o aumento da concentração de interferão γ nos doentes com linfoma difuso de grandes células B. Os doentes com cancro do pulmão apresentaram um perfil Th1 mais acentuado do que os doentes com linfoma difuso de grandes células B, o que se concluiu pelos níveis superiores de IL-2, de INF-γ e de IL-1β. Paralelamente, o perfil Th2 caracterizou-se pela maior concentração de IL-5, no sangue periférico dos doentes com linfoma difuso de grandes células B. Finalmente, destacou-se o aumento da concentração de IL-27 e de IL-7 nos doentes com cancro do pulmão. Com este trabalho foi possível concluir que a resposta à radioterapia é dependente das caraterísticas celulares e moleculares das células tumorais em estudo. O melhor conhecimento e compreensão das caraterísticas moleculares do tumor e dos mecanismos de resposta ao tratamento constituem uma mais-valia na decisão terapêutica e na avaliação do prognóstico. Para além disto, o microambiente tumoral, bem como a atuação do sistema imunitário sobre células tumorais no contexto da radioterapia, podem condicionar a resposta à terapêutica e a sobrevivência a longo termo.
Radiotherapy is one of the most common modalities used for treating both solid tumors and hematopoietic origintumors. Lung cancer is a solid tumor, with on of the higher incidence and mortality rates worldwide. Diffuse large B-cell lymphoma is a hematopoietic tumor being the most common type of lymphoma non-Hodgkin. One of the aims of this work was to determine and characterize the intracellular effects of radiotherapy in cell lines of small cell lung cancer (H69), non-small cell lung cancer (A549 and H1299) and diffuse large B cell lymphoma (Farage). For this purpose, we determined the effects of ionizing radiation on viability, proliferation, survival and cell death mechanisms. For this, trypan blue assay, Alamar Blue® assay, clonogenic studies, flow cytometry studies, using the double staining with Annexin V and propidium iodide, labelling BAX and BCL-2 proteins, alteration of mitochondrial membrane potential, and cell morphology studies, with May Grünwald-Giemsa staining, were performed. Furthermore, we also evaluated by flow cytometry, the effects of ionizing radiation in the oxidative stress regarding production of intracellular peroxides, superoxide radicals and of the anti-oxidant defense glutathione. The DNA damage was determined by the comet assay, and the expression of phosphorylated and total P53 protein was assessed by Western blot. Alongside, another aim of this study was to evaluate the effects of radiation on the immune system of patients with lung cancer and with diffuse large B cell lymphoma. To achieve this goal eight lung cancer patients and nine diffuse large B cell lymphoma patients were included in this study. After collecting peripheral blood, leukocyte counts were determined as well as the lymphocyte counts and the regulatory T cells using the Lymphogram® and immunophenotyping, respectively. We also evaluated thirty four cytokines and chemokines relevant to the response of the immune system through the commercial kit ProcartaPlex™ Immunoassay Magnetic Beads. Ionizing radiation induced a decrease in proliferation, viability and cell survival in all cell lines. However, survival was adjusted to different cellular injury models. While H1299 and Farage cell lines had a response that correspondes to the linear quadratic model, H69 and A549 cell lines showed a response set to the model of one touch one target, íe the linear model. After exposure to ionizing radiation, the preferred type of cell death observed was dose dependent and, presumably, P53 profile expression dependent. Thus, expression of P53Wild in Farage and A549 cells was associated with cell death by apoptosis while no P53 expression observed in H1299 cells, and production of a mutated protein, described for H69, was associated with cell death by necrosis, particularly at higher doses. In addition, the A549 and Farage cell lines the level of P53 protein and of phospohorilated P53 protein augmented. Complementarily, the depolarization of the mitochondrial membrane potential and the increase in BAX/BCL-2 ratio indicates that cell death by apoptosis, typical of P53Wild cells, seem to be activated by the intrinsic pathway. For the cell cycle a blockage in G0/G1 and S phase was observed in A549 and in Farage cell lines while H69 and H1299 cell lines progressed in these phases but were compromised later in G2/M phase. These findings underscore the importance of the molecular profile in response to ionizing radiation. Finally, the oxidative stress and DNA damage stood out as very important processes in the effects of ionizing radiation. Parallelly, the study of human pathology performed within this Thesis revealed that the immune system response to radiotherapy was dependent on cancer type. The assessment of leucocyte cout of lung cancer patients showed changes in leukocytes, lymphocytes and monocytes during treatment with radiation. However, no significant differences in leucocytes were found in diffuse large B cell lymphoma patients. Similarly, in lung cancer patients changes were observed in limphogram with respect to B cells, Natural Killer cells and cytotoxic Natural Killer cells. Furthermore in patients with diffuse large B cell lymphoma there were changes in the induced regulatory T cells. Regarding cytokines and chemokines associated with Th1 profile, we observed an increase in interferon-γ concentration in diffuse large B cell lymphoma patients. Lung cancer patients showed a stronger Th1 profile than patients with diffuse large B cell lymphoma, which is confirmed by the higher levels of IL-2, INF-γ and IL-1β. In parallel, the Th2 profile was characterized by the highest concentration of IL-5 in the peripheral blood of diffuse large B cell lymphoma. Finally, there was an increase in IL-27 concentration and in IL-7 in lung cancer patients. We can concluded that the response to radiation is dependent on the cellular and molecular characteristics of the tumor cells under study. Better knowledge and understanding of the molecular characteristics of the tumor and mechanisms of response to treatment is an asset concerning the therapeutic decision and prognosis evaluation. In addition, tumor microenvironment, as well as the activity of the immune system on tumor cells in the context of radiotherapy, may contribute to better therapeutic outcome and survival in the long term.

Le cancer du col utérin (CCU) est dans plus de 99% des cas provoqué par une infection avec le virus du papillome humain (VPH), dont le potentiel oncogénique réside dans l'expression des proto-oncogènes viraux E6/E7. Le potentiel carcinogénique de ces protéines virales réside essentiellement dans leurs actions sur les produits des gènes suppresseurs de tumeur p53 et RB. Les produits de ces gènes, p53 et Rb, font parti des voies de signalisation de réponse aux dommages de l'ADN cellulaire (RDA) et leur perte entraine une perte de fonctionnalité qui mène à une instabilité génomique. À long terme et en présence de d'autres facteurs ceux-ci mèneront au développement d'un cancer. Les protéines E6 et E7 sont constitutivement exprimées dans les cellules du CCU ainsi que dans les cellules de tout autre cancer induit par le VPH et seulement dans ces dernières. La prise en charge des cas avancés de ces cancers se fait principalement par radiothérapie et chimiothérapie concomitante. La chimio-radiothérapie utilisée en traitement est efficace mais résulte en un taux élevé de morbidité et un nombre important de patientes récidiveront. Nous proposons que l'exploitation de l'expression spécifique d’E6 et d’E7 dans les cellules du CCU permette d’envisager une stratégie de létalité synthétique afin d'amplifier l'effet létal de l'irradiation sur les cellules CCU. Ceci permettrait potentiellement d'augmenter l'efficacité du traitement et de diminuer les récidives, ainsi que la morbidité liée au traitement. En s'appuyant sur cette hypothèse, notre objectif est d’identifier des composés dont l'action seule ou couplée à l'irradiation provoquerait préférentiellement la mort des cellules exprimant les protéines E6 et E7 du VPH. Les cellules testées comprennent des cellules isogéniques humaines issues de kératinocytes normaux que nous avons modifiées séquentiellement pour obtenir les modifications associées aux cellules CCU (hTERT, E6 et E7), ainsi que les lignées de cellules de CCU HeLa et CaSki .Nous avons procédé à la mise au point et à la validation du protocole de criblage et des méthodes d’évaluation de la sensibilisation, qui se définit comme une perte de viabilité, un arrêt ou ralentissement de la croissance, par détection d’ATP ainsi que par coloration d’ADN génomique au DRAQ5. Suite à un criblage ciblé impliquant des inhibiteurs connus de la voie de réparation des dommages à l’ADN, nous avons identifié l’inhibiteur de mdm2, Nutlin-3, comme étant un composé sensibilisant et radio-sensibilisant préférentiellement les cellules exprimant E6 et E7 du VPH. La Nutlin-3 a été testée sur des cellules HEKn-hTERT-E6-E7, des cellules CaSki et HeLa. L’effet de sensibilisation et de radio-sensibilisation a été confirmé dans ces trois lignées. Tel que suggéré par son action sur mdmd2, la Nutlin-3 permet la stabilisation de p53 dans les cellules HEKn-hTERT-E6-E7 et CaSki et sa réactivation dans les lignées cellulaires HeLa et CaSki. Malgré cette stabilisation de p53, de façon surprenante, l’effet de la Nutlin-3 sur la sensibilisation et la radio-sensibilisation des cellules HeLa et CaSki semble indépendant de p53, tel qu’observé en utilisant des cellules HeLa-GSE et CaSki-GSE dont le p53 est déficient. In vivo la Nutlin-3a montre dans un essai préliminaire l’inhibition de la croissance tumorale des xénogreffes HeLa chez des souris RAG2γc. Ce résultat reste à confirmer avec un essai impliquant un nombre d’échantillons plus grand. À plus long terme, nous comptons étudier l’implication de mdm2 dans l’effet de sensibilisant de la Nutlin-3 dans les cellules CCUs, ainsi que les autres cibles pouvant être impliquées dans la création de cet effet sensibilisant observé.
More than 99% of uterine cervical cancer (UCC) are caused by human papillomavirus (HPV) infections. The oncogenic potential of this virus lies in the expression of the proto-oncogenes E6/E7. These viral proteins are considered carcinogenic because of their effects on tumor suppressor proteins p53 and Rb. E6 and E7 promote p53 and Rb inactivation resulting in a loss of function in the DNA damage response pathways (DDR), genomic instability, and cancer development. The E6 and E7 proteins are expressed constitutively and specifically in cervical cancer cells and in the cells of other HPV-induced cancers. The treatment of advanced UCC is based on simultaneous radiotherapy and chemotherapy. Although these strategies are somewhat efficacious, there are still significant co-morbidities and cancer relapses. We hypothesized that the specific expression of E6 and E7 in cervical cancer cells can be exploited in a synthetic lethality strategy to amplify the lethal effect of irradiation. Thus, the efficacy of treatment could be increased, while reducing the cancer recurrence and treatment-related morbidities. Our objective is to identify chemical compounds that if used alone or coupled with irradiation, would preferentially induce the death of cells expressing proteins E6 and E7 of HPV. We used a cellular model of human keratinocytes that were modified to obtain the genetic signature associated with cervical cancer cells (the expression of E6 and E7). We then proceeded to the optimization and validation of the methods used to evaluate the sensitization of the tested cells. To measure sensitization, we evaluated the quantity of cellular ATP by ATPlite assay and the cellular DNA content with the DNA stain DRAQ5. After establishing a screening protocol, we proceeded to a low-density screening to identify a compound that can sensitize or radio-sensitize cervical cancer cells expressing the HPV proteins E6 and E7. We identified Nutlin-3 a mdm2, inhibitor, as a radio-sensitizing component for cells expressing E6 and E7 of HPV. Nutlin-3 was tested and sensitization confirmed in HEKn-hTERT-E6-E7 and in the cervical cancer cell lines HeLa and CaSki. We confirmed by Western-Blot the stabilisation of p53 in HEKn-hTERT-E6-E7 and CaSki cells treated with Nutlin-3. Moreover, Nutlin-3 promotes p53 reactivation in the HeLa and CaSki cell lines. Surprisingly, the effect of Nutlin-3 on the sensitization and radio-sensitization of the HeLa and CaSki cell lines appears to be p53-independent. This is based on the observations made using p53-deficient HeLa-GSE and CaSki-GSE cell lines, which were also sensitized by Nutlin-3. In addition, preliminary experiments showed that Nutlin-3a inhibits in vivo tumor growth, as seen using xenografts of HeLa in mice with a RAG2γc genetic background. This remains to be confirmed using an extended cohort of mice. In the future, it will be important to examine the implication of mdm2 in the sensitization effect of Nutlin-3 in cervical cancer cells and to find others possible targets that may play a role in the sensitization effect of Nutlin-3 observed in cervical cancer cells.

For any drug candidate to be approved by the U.S. Food and Drug Administration, it must meet strict standards for safety and efficacy. While the field of nuclear medicine is over 100 years old, traditional methods such as external beams or systematic administration have rarely met these standards or have limited application. Ligand-targeted therapy and diagnostics, or “theranostics,” has emerged in the past several decades as an exciting field that offers new possibilities to design drugs that are both safe and effective. When applied to nuclear medicine, the field of ligand-targeted radioactive theranostics is younger still, with many critical lessons being discovered and applied currently. This dissertation outlines the necessary principles of radioactive theranostic drug design, then demonstrates the application of several more recent techniques to improve both the efficacy and safety of radioactive theranostics targeting two high priority oncological targets: fibroblast activation protein alpha and folate receptor.