Les cellules tumorales circulantes (CTC) représentent une source de matériel tumoral accessible de manière non invasive, susceptible de fournir des informations cliniques et fondamentales. Ces cellules issues de tumeurs primitives ou métastatiques représentent une population hétérogène d’éléments très rares circulant dans le sang. La personnalisation des traitements en oncologie repose sur la caractérisation moléculaire de biopsies tumorales mais celles-ci peuvent être difficiles à réaliser ou peu informatives. De ce fait, la caractérisation moléculaire et fonctionnelle des CTC présente un double intérêt, clinique pour identifier des biomarqueurs de sensibilité à des traitements, et fondamental pour étudier les mécanismes qui sous-tendent leur potentiel à initier des tumeurs.Les objectifs de ma thèse ont été d’une part de caractériser par séquençage de l’exome (WES) les CTC à l’échelle de cellule unique de patients atteints de cancers de la prostate (PCa) métastatiques et d’autre part d’établir puis caractériser des modèles de xénogreffes dérivés de CTC (CDX) chez des patients atteints de cancers bronchiques non à petites cellules (CBNPC) ou de PCa.Pour répondre au premier objectif, nous avons développé une méthode expérimentale globale incluant trois approches technologiques permettant d’enrichir et d’isoler des CTC individuelles de différents phénotypes (épithélial, épithélio-mésenchymateux et mésenchymateux), d’amplifier la totalité du génome (WGA) et de le séquencer. Le WES a été réalisé pour 34 échantillons de CTC sélectionnés sur des critères de qualité du WGA, ainsi que pour les biopsies de métastases correspondantes chez sept patients. Deux patients présentant une hétérogénéité phénotypique de leurs CTC, ont été analysés en profondeur. Nous avons mis en évidence des mutations partagées entre les CTC et les biopsies tumorales correspondantes ainsi que des mutations uniquement retrouvées dans les CTC. Ces mutations spécifiques aux CTC sont présentes dans tous les phénotypes et affectent particulièrement les gènes impliqués dans le remodelage du cytosquelette, la réparation de l’ADN ou l’invasion. L’existence de mutations communes entre les CTC de différents phénotypes suggère une relation phylogénique entre ces cellules mais une évolution divergente pendant le processus métastatique. Ce travail est soumis pour publication.Dans la seconde partie de ma thèse, nous avons implantés les CTC de 67 patients atteints de CBNPC et 24 patients atteints de PCa chez des souris immunodéprimées. Nous avons établis quatre CDX de CBNPC et un CDX de PCa. La caractérisation de ces modèles, des biopsies tumorales, des CTC collectées au moment de la xénogreffe, des CDX et des lignées cellulaires établies à partir du CDX, ont révélé que les CTC, le CDX et les lignées cellulaires « miment » le phénotype et le profil mutationnel des biopsies tumorales. La caractérisation plus approfondie de l’une des lignées cellulaires montre la présence d’un stress réplicatif et d’une instabilité génomique élevée. Ce résultat nous oriente sur l’hypothèse d’un rôle éventuel de l’instabilité génomique dans la tumorigénicité des CTC.Dans ce travail, nous avons montré que le profil mutationnel des CTC présente de fortes similitudes avec les biopsies tumorales des patients dans les patients atteints de PCa étudiés. De plus, nous avons observé l’existence de mutations spécifiques aux CTC, non détectées dans les biopsies tumorales. Également, nous montrons que des CTC issues de CBNPC et de PCa sont tumorigéniques in vivo et qu’elles reflètent le profil mutationnel des biopsies tumorales des patients. Ces modèles constituent des outils originaux et intéressants pour identifier de nouvelles cibles thérapeutiques et stratégies anti-cancéreuses, et comprendre les mécanismes qui supportent le potentiel des CTC à initier des tumeurs. / Circulating tumor cells (CTCs) represents an non invasive source of tumor material which may provide clinical and basic information. These cells derived from primary or metastatic tumors represents an heterogeneous population of very rare events which circulates in the blood. Oncology personnalized medicine is based on biopsies molecular characterization but these are sometimes which difficult to realize and poorly informative. Thereby molecular and functional characterization of CTCs presents a double interest, clinical to identify treatments biomarkers sensitivity and basic to study mechanisms underlying their tumor inititiating cell (TIC) potential. The two goals of my thesis were on the one hand to characterize by whole-exome sequencing (WES) at the single level the CTCs from patients with metastatic prostate cancers (mPCa) and on the other hand to establish and characterize CTC-derived xenografts (CDX) from patients with non-small-cell lung cancer (NSCLC) or mPCa. For the first goal we developped a global workflow which include three technological approaches to enrich and isolate individual CTCs from different phenotype (epithelial, epithelial and mesenchymal, mesenchymal), to perform whole genome amplification (WGA) and to sequence them. WES was performed on 34 CTC samples selected according to WGA quality and on corresponding metastasis biopsies from seven patients. Two patients with phenotypic heterogeneity of CTCs were deeply analyzed. We highlighted shared mutations between CTCs and matched biopsies as well as mutations only detected in CTCs. These private CTC mutations are detected in all phenotype and particularly affect genes invlved in cytoskeleton remodeling, DNA repair or invasion. The existence of common mutations between CTCs from various phenotype suggests a phylogenic link between these cells but a divergent evolution during metastatic process. This work is submitted for publication. For the second goal, we implanted CTCs from 67 NSCLC patients and 28 mPCa patients in immunocompromised mice. We established four NSCLC CDX and one mPCa CDX. The characterization of tumor biopsies, CTCs collected at the time of xenograft, CDX and CDX-derived cell lines revealed that CTCs, CDX and cell lines miror the phenotype and mutational landscape of tumor biopsies. The more deeply characterization of one cell line show the presence of a high replicative stress and genomic instability. This result directs us to the hypothesis of a possible role of the genomic instability in CTC tumorigenicity.We demonstrated in this work that CTCs mutational landscape harbors high similairities with patients tumor biopsies in mPCa. Furthermore we observed CTC private mutations not detected in tumor biopsies. Also we showed that some CTCs from NSCLC and mPCa are tumorigenic in vivo and that these CTCs mirror mutational profile of patients tumor biopsies. These models are original and interesting tools to identify new therapeutic targets and anti-tumoral strategies and understand mechanisms underlying the TIC potential of CTCs.

Tumor microenvironmental conditions play a vital role in promoting metastasis and tumor recurrence. Due to inefficient vasculature, cancer cells experience hypoxia, glucose deprivation and low pH even during the early stages of tumor growth. Tumor cells are proposed to adapt to these microenvironmental conditions by acquiring increased migratory and invasion potential and tumor initiating ability. Our research addresses the effect of these biochemical factors of the tumor microenvironment (TME) on motility, epithelial to mesenchymal transition (EMT) and stemness of non-small cell lung cancer (NSCLC). NCI-H292 and NCI-H1650 NSCLC cell lines were used to measure the effect of the above mentioned TME conditions. Apart from acidic pH, low glucose and hypoxia, the effect of high glucose conditions was also measured on H292 and H1650 cell lines. Acidic pH, high and low glucose conditions were observed to have no effect on the motility, EMT and stemness of H1650 cell line. Hence, use of this cell line was discontinued and no further treatment conditions were tested on this cell line. In H292 cell line, acidic pH, low glucose and tumor like conditions combined together (acidic pH + low glucose + hypoxia) [AP+LG+HYP] significantly decreased motility whereas hypoxia significantly increased the motility of H292 cells. High glucose did not affect the motility of H292 cells. Although N-cadherin, a mesenchymal marker, expression was significantly upregulated by acidic pH, high and low glucose conditions, no direct correlation was observed between N-cadherin expression and motility. E-cadherin expression was not affected by acidic pH, high and low glucose conditions. An increase in N-cadherin expression and no change in E-cadherin expression under these conditions might be an indication of partial EMT. Hypoxia and AP+LG+HYP did not alter the expression of E-cadherin and N-cadherin. Although expression of vimentin, another mesenchymal marker, and Sox2, a cancer stem cell marker (CSC), was observed at the mRNA level, no expression of vimentin and Sox2 proteins was observed in H292 cells under any of these treatment conditions. The expression of OCT4, another CSC marker, was also not observed at the protein level in H292 cells. HIF-1α expression was observed in H292 cells under normoxic conditions and was unaffected by hypoxia and AP+LG+HYP. Therefore our research indicates that the effect of these TME conditions might be different on different cancer cell lines or cancer types. Not all cancers may depend on EMT for metastasis. An increase in metastasis under hypoxia may be independent of HIF-1α.

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Deregulation of gene expression caused by genetic or epigenetic changes plays an important role in pathogenesis of cancer. The thesis is a commented collection of ten publications dealing with the molecular biology of tumours. The author has significantly contributed to all of them. All the articles contained in the thesis are linked to the topic of assessment of molecules involved in gene expression regulation (microRNAs) or DNA alterations that affect gene expression (promoter methylation, presence of a fusion gene). MicroRNAs are short single-stranded RNA molecules involved in posttranscriptional regulation of gene expression by triggering mRNA degradation or inhibiting translation. It is a basic mechanism with an impact on all cellular processes including the pathogenesis of various diseases. MicroRNAs can either act as oncogenes by decreasing the expression of tumour-suppressor genes or as tumour-suppressor genes by decreasing the expression of oncogenes. However, the network of microRNA - RNA interactions is much more complex. Our published results that are part of this thesis are focused on colorectal carcinoma (CRC), prostate cancer, head and neck squamous cell carcinoma (HNSCC), gastric cancer and non-small cell lung cancer (NSCLC). In patients with CRC, we demonstrated the prognostic...

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L’avènement des inhibiteurs du point de contrôle immunitaire (ICIs) ciblant l’axe PD-1/PD-L1 a révolutionné le traitement des patients avec un carcinome pulmonaire non à petites cellules (CPNPC). Ce mémoire consolide les conclusions de trois études distinctes visant à analyser et à améliorer l'efficacité des ICIs en monothérapie chez le CPNPC. La première section explore les bénéfices associés au durvalumab suivant une chimioradiothérapie chez les patients présentant un CPNPC localement avancé, confirmant le bénéfice de survie associé aux ICI et élargissant les perspectives émises depuis l'étude PACIFIC, y compris au niveau de la valeur prédictive du PD-L1. Dans l’optique de caractériser de nouveaux biomarqueurs d’efficacité, la deuxième section souligne le rôle crucial du microbiome intestinal dans la modulation de la réponse aux ICIs, spécifiquement au niveau de la dysbiose intestinale liée aux antibiotiques. La méta-analyse proposée confirme l’impact délétère des antibiotiques sur la survie des patients traités avec un ICI, tout particulièrement lorsque l’antibiothérapie précède l’inhibition du PD-1/PD-L1. Dans le domaine des stratégies immunomodulatrices émergentes, la troisième section explore l’impact de la cryoablation chez les patients présentant un CPNPC avec PD-L1≥50% et traités avec le pembrolizumab. Sans relever un signal d’efficacité supérieure, cette étude de phase I/II confirme la faisabilité et l’innocuité de la cryoablation, une technique permettant la libération d’antigènes tumoraux en circulation sans dénaturation thermique. Ultimement, ce mémoire propose un survol des bénéfices de survie, biomarqueurs prédictifs et stratégies immunomodulatrices liés à l’utilisation des ICIs chez les patients avec un CPNPC avec l’espoir d’optimiser les paradigmes thérapeutiques existants. / The advent of immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis has revolutionized the therapeutic landscape for patients diagnosed with non-small cell lung cancer (NSCLC). This thesis consolidates the findings of three distinct studies aiming to analyze and enhance the efficacy of ICI monotherapy in NSCLC. The first section delves into the real-world use of durvalumab following chemoradiotherapy in stage III NSCLC, confirming the survival benefit associated with ICI administration in this context and broadening the insights derived from the PACIFIC study, particularly regarding the predictive value of PD-L1. With the aim of characterizing new biomarkers of efficacy, the second section sheds light on the crucial role of the gut microbiome in modulating responses to ICIs, particularly intestinal dysbiosis related to antibiotics. The meta-analysis confirms the detrimental impact of antibiotics on the overall survival of patients with advanced cancer treated with ICI monotherapy, especially when antibiotic therapy precedes PD-1/PD-L1 inhibition. In the realm of emerging immunomodulatory strategies, the third section explores the impact of cryoablation in patients with NSCLC and PD-L1≥50% treated with pembrolizumab. Although the procedure did not translate into a signal of superior efficacy, the proposed phase I/II study confirms the feasibility and safety of cryoablation, a technique allowing the release of circulating tumor antigens without heat-related denaturation. Ultimately, this thesis presents a contemporary overview of survival benefits, predictive biomarkers, and immunomodulatory strategies associated with the use of ICIs in monotherapy in patients with NSCLC, with the hope of optimizing existing therapeutic paradigms.

Background Primary radiochemotherapy with photons is the standard treatment for locally advanced-stage non-small cell lung cancer (NSCLC) patients. Acute radiation-induced side effects such as oesophagitis and radiation pneumonitis limit patients’ quality of life, and the latter can be potentially life-threatening. Due to its distinct physical characteristics, proton therapy enables better sparing of normal tissues, which is supposed to translate into a reduction of radiation-induced side effects. Methods/design This is a single-centre, prospective, randomised controlled, phase II clinical trial to compare photon to proton radiotherapy up to 66 Gy (RBE) with concomitant standard chemotherapy in patients with locally advanced-stage NSCLC. Patients will be allocated in a 1:1 ratio to photon or proton therapy, and treatment will be delivered slightly accelerated with six fractions of 2 Gy (RBE) per week. Discussion The overall aim of the study is to show a decrease of early and intermediate radiation-induced toxicity using proton therapy. For the primary endpoint of the study we postulate a decrease of radiation-induced side effects (oesophagitis and pneumonitis grade II or higher) from 39 to 12%. Secondary endpoints are locoregional and distant failure, overall survival and late side effects. Trial registration Registered at ClinicalTrials.gov with Identifier NCT02731001 on 1 April 2016.

Les immunothérapies de type immune checkpoint blockade (ICB) ont révolutionné les approches thérapeutiques en oncologie médicale et ont largement contribué au fait que l’immunothérapie est maintenant considérée comme le quatrième pilier des traitements anticancer, aux côtés d’approches traditionnelles telles que la chirurgie, la radiothérapie et la chimiothérapie. Malgré les résultats encourageants des études cliniques évaluant ce type d’immunothérapie, la majorité des patients décèderont des suites de leur maladie. Conséquemment, le domaine de recherche visant à comprendre les mécanismes de résistance aux immunothérapies est en expansion constante. Plusieurs stratégies visant à améliorer les issues cliniques ont été proposées, parmi lesquelles figurent: 1) la recherche de nouvelles cibles thérapeutiques dans le microenvironnement immun tumoral et; 2) les études de combinaisons thérapeutiques où une immunothérapie est jumelée à d’autres types de modalités thérapeutiques potentiellement synergiques. Chacune des études présentées dans cette thèse de recherche s’apparente à l’une ou l’autre de ces stratégies. Dans le cadre de notre première étude, nous démontrons que la protéine WISP1 représente une cible prometteuse à l’intérieur du microenvironnement de plusieurs types de tumeurs solides étant donné son association avec différentes variables pronostiques et proinflammatoires, ainsi qu’avec un programme épigénétique complexe, la transition épithélialemésenchymateuse (Epithelial-Mesenchymal Transition; EMT). De plus, nous démontrons que les niveaux d’expression de WISP1 sont significativement plus élevés au sein des tumeurs démontrant une résistance primaire aux immunothérapies de type ICB, particulièrement lorsque qu’une signature reliée à l’EMT peut être retrouvée de façon concomitante. Pour notre deuxième étude, nous avons utilisé des modèles murins in vivo de cancer pulmonaire non à petites cellules KRAS-mutés afin de tester différentes combinaisons thérapeutiques jumelant une thérapie dite ciblée (i.e., un inhibiteur de MEK) a différentes immunothérapies de type ICB. Nos résultats démontrent que l’ajout d’une immunothérapie anti-CTLA-4 à l’inhibiteur de MEK AZD6244 (selumetinib) et une immunothérapie anti-PD-L1 augmente significativement la survie, et que ces bénéfices sont associés à une diminution de marqueurs reliés à l’EMT. Il existe donc un lien commun entre ces deux études qui repose sur l’importance de l’EMT comme facteur favorisant la résistance thérapeutique aux immunothérapies. De plus, nous démontrons pour la première fois que les bénéfices associés à la triple combinaison thérapeutique susmentionnée peuvent être corrélés à une diminution d’expression de marqueurs liés à l’EMT. Par conséquent, nos résultats sont discutés en tant que base potentielle pour de futures études visant à réduire la résistance thérapeutique reliée à l’EMT. Nous discutons également de la valeur translationnelle de nos résultats à travers le développement d’une étude clinique. / Immune checkpoint blockade (ICB) has revolutionized therapeutic approaches in the field of medical oncology and has largely contributed to the fact that immunotherapy is now being regarded as the fourth pillar of cancer treatment alongside surgery, radiotherapy and chemotherapy. Despite encouraging results from clinical trials using ICB, most patients ultimately relapse or succumb to their disease. Therefore, the field of immunotherapeutic resistance research is rapidly expanding. Many strategies to improve ICB responses have been undertaken, including: 1) the search for novel, actionable targets in the immune tumor microenvironment (TME) and; 2) therapeutic combination studies where an ICB backbone is combined with different, synergistic treatment modalities. Each of the studies presented in this research thesis embraces one of these strategies. In our first study, we show that WISP1 represents a promising TME target in multiple solid tumor types by demonstrating its association with prognostic and pro-inflammatory variables, as well as to a complex epigenetic program termed Epithelial-Mesenchymal Transition (EMT). Furthermore, we show that increased WISP1 expression is associated to primary resistance to ICB, particularly when EMT-related signatures are found concomitantly. In our second study, we used in vivo mouse models of KRAS-mutant Non-Small Cell Lung Cancer (NSCLC) to test different therapeutic combinations of targeted therapies (i.e., MEK inhibitor) and ICB. We found that the addition of anti-CTLA-4 ICB to MEK inhibitor AZD6244 (selumetinib) and anti-PD-L1 ICB increases survival, and that these benefits are associated with the downregulation of EMT-related markers. Therefore, there exists a common link between these studies, which relies on the significance of EMT as a detrimental factor within the TME and its association with ICB resistance. Moreover, we show for the first time that the benefits of ICB combination therapy can be associated to the downregulation of EMT markers in vivo. Consequently, we discuss how our results may constitute the basis for future work aiming at reducing EMT-mediated therapeutic resistance, as well as the translational relevance of our pre-clinical results through the development of a clinical trial.

Background Primary radiochemotherapy with photons is the standard treatment for locally advanced-stage non-small cell lung cancer (NSCLC) patients. Acute radiation-induced side effects such as oesophagitis and radiation pneumonitis limit patients’ quality of life, and the latter can be potentially life-threatening. Due to its distinct physical characteristics, proton therapy enables better sparing of normal tissues, which is supposed to translate into a reduction of radiation-induced side effects. Methods/design This is a single-centre, prospective, randomised controlled, phase II clinical trial to compare photon to proton radiotherapy up to 66 Gy (RBE) with concomitant standard chemotherapy in patients with locally advanced-stage NSCLC. Patients will be allocated in a 1:1 ratio to photon or proton therapy, and treatment will be delivered slightly accelerated with six fractions of 2 Gy (RBE) per week. Discussion The overall aim of the study is to show a decrease of early and intermediate radiation-induced toxicity using proton therapy. For the primary endpoint of the study we postulate a decrease of radiation-induced side effects (oesophagitis and pneumonitis grade II or higher) from 39 to 12%. Secondary endpoints are locoregional and distant failure, overall survival and late side effects. Trial registration Registered at ClinicalTrials.gov with Identifier NCT02731001 on 1 April 2016.

Indiana University-Purdue University Indianapolis (IUPUI) / All platinum (Pt)-based chemotherapeutics exert their efficacy primarily via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. Repair and tolerance of Pt-DNA lesions by nucleotide excision repair and homologous recombination (HR) can substantially reduce the effectiveness of the Pt therapy. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt treatment and increase clinical efficacy. Replication Protein A (RPA) plays essential roles in both NER and HR, along with its role in DNA replication and DNA damage checkpoint activation. Each of these functions requires RPA binding to single-stranded DNA (ssDNA). We synthesized structural analogs of our previously reported RPA inhibitor TDRL-505, determined the structure activity relationships and evaluated their efficacy in tissue culture models of epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC). These data led us to the identification of TDRL-551, which exhibited a greater than 2-fold increase in in vitro and cellular activity. TDRL-551 showed synergy with Pt in tissue culture models of EOC and in vivo efficacy, as a single agent and in combination with platinum, in a NSCLC xenograft model. These data demonstrate the utility of RPA inhibition in EOC and NSCLC and the potential in developing novel anticancer therapeutics that target RPA-DNA interactions.