High-grade serous ovarian cancer (HG-SOC) is the most lethal gynecologic malignancy. Although most respond to initial therapy, the vast majority of patients eventually recur and die of their disease. Understanding intra-tumor cellular heterogeneity and inter-patient variability is necessary to effectively cure HG-SOC. The work described in this thesis should help to speed the progress of ovarian cancer research in several ways. First, I generated a robust xenograft model that recapitulates the cellular heterogeneity of HG-SOC. In addition, I performed gene expression profiling on a subset of xenografts and showed that they recapitulate the inter-patient diversity of this disease. Second, I applied this model to pre-clinical testing of a folate-targeted imaging agent and showed that it can identify metastatic studding by PET/CT and fluorescence imaging. Using my xenograft model, I investigated the properties of tumor-initiating cells (TIC) and demonstrated that TIC in HG-SOC are rare. Furthermore, although CD133 marks most TIC, heterogeneity in the phenotype is observed within individual tumors and between different patients. Finally, I used a transformative technology, CyTOF, to develop a novel pipeline for prioritization of candidate TIC markers, as well as for characterization of cellular heterogeneity in primary HG-SOC samples.

High-grade serous ovarian cancer (HG-SOC) is the most lethal gynecologic malignancy. Although most respond to initial therapy, the vast majority of patients eventually recur and die of their disease. Understanding intra-tumor cellular heterogeneity and inter-patient variability is necessary to effectively cure HG-SOC. The work described in this thesis should help to speed the progress of ovarian cancer research in several ways. First, I generated a robust xenograft model that recapitulates the cellular heterogeneity of HG-SOC. In addition, I performed gene expression profiling on a subset of xenografts and showed that they recapitulate the inter-patient diversity of this disease. Second, I applied this model to pre-clinical testing of a folate-targeted imaging agent and showed that it can identify metastatic studding by PET/CT and fluorescence imaging. Using my xenograft model, I investigated the properties of tumor-initiating cells (TIC) and demonstrated that TIC in HG-SOC are rare. Furthermore, although CD133 marks most TIC, heterogeneity in the phenotype is observed within individual tumors and between different patients. Finally, I used a transformative technology, CyTOF, to develop a novel pipeline for prioritization of candidate TIC markers, as well as for characterization of cellular heterogeneity in primary HG-SOC samples.

La vaccination est l'un des plus grands progrès réalisés en santé publique. Toutefois, malgré de nombreuses connaissances sur le système immunitaire, de nombreux pans d’ombre empêchent la conception de vaccins contre des pathogènes complexes. Pour pallier ce problème, une meilleure compréhension des modes d'action des vaccins est requise. En particulier, la plupart des vaccins nécessitent plusieurs immunisations pour induire une mémoire immunitaire adaptative au long terme, mais l'impact du délai entre primo-vaccination, induisant une mémoire primaire, et rappel(s) la restimulant pour générer une mémoire secondaire, est peu défini. De plus, la réponse immunitaire innée, induite à chaque immunisation et façonnant l'immunité adaptative, reste peu caractérisée dans ce contexte vaccinal. En vaccinant des macaques cynomolgus avec le virus de la vaccine modifiée Ankara, selon un schéma de primo-vaccination suivie d’un rappel homologue à deux mois, et en utilisant la cytométrie de masse couplée à des analyses bio-informatiques, nous avons caractérisé la réponse innée induite par chaque immunisation. Les réponses innées diffèrent entre primo-vaccination et rappel, avec induction par la primo-vaccination d’une modification phénotypique tardive des cellules innées, suggérant une meilleure capacité à répondre au rappel. De surcroît, la réduction à deux semaines du délai entre primo-vaccination et rappel abroge la mobilisation de ces cellules innées phénotypiquement modifiées et altère la qualité de la réponse humorale. En définitive, en plus de la réponse innée précoce, ce projet a mis en évidence l'induction par la primo-vaccination d'un vraisemblable entraînement inné tardif, un concept émergent traduisant la capacité de mémorisation des cellules innées via des modifications épigénétiques. Ce vraisemblable entraînement, non seulement des monocytes et cellules tueuses naturelles, mais aussi des cellules dendritiques et surprenamment des neutrophiles, est corrélé à la qualité de la mémoire immunitaire adaptative, de manière hautement dépendante du délai entre primo-vaccination et rappel. Ces résultats contribuent à ouvrir la voie vers l’optimisation rationnelle des futurs vaccins, via l'optimisation des calendriers vaccinaux et la valorisation de l'entraînement inné. / Vaccination is one of the best achievements made in public health. However, designing vaccines against complex pathogens is currently challenging. The immune system is indeed uncompletely characterized, despite large amount of accumulated knowledges. A better understanding of vaccine-induced immunity is then required to optimize vaccine design. In particular, while most vaccines require several immunizations to induce a long-lasting adaptive immune memory, little is known on the impact of the delay beween the prime inducing a primary memory and the boost restimulating it to induce a secondary memory. Also, the innate immunity induced by each immunization and shaping the adaptative immunity is poorly characterized in this vaccine context.We studied the innate immune responses in cynomolgus macaques immunized with the modified vaccinia virus Ankara, following an homologous prime-boost vaccination at two months apart. We applied mass cytometry and bioinformatic analyses to characterize the innate response induced by each immunization. We showed that prime and boost vaccination triggered distinct innate responses. Actually, prime induced late phenotypic modifications of innate cells. These phenotypic changes suggest a stronger ability to react to the boost. Moreover, reducing the delay between prime and boost to two weeks impeded the mobilization of these phenotypically modified innate cells, and qualitatively altered humoral response.In conclusion, beyond the early innate responses, these results highlight the late induction by the prime of "likely trained" innate cells. This emerging concept corresponds to the ability of innate cells to display memory features based on epigenetic modifications. This "likely training" occured not only on monocytes and NK cells, but also on dendritic cells and strikingly on neutrophils. It was deeply connected with adaptive immune memory establishment, in a prime-boost delay dependant fashion. These findings contribute to pave the way towards to the rationale design of future vaccines, via vaccine schedule optimization and harnessment of innate training.

No description available.

Mass spectrometry (MS) as an analytical approach could provide comprehensive identification and quantitation of the biomolecules (proteins, peptides, nucleic acids, lipids) in a cell, tissue, or organism, from biomarker discovery to prediction of response to therapy or intervention. Inductively coupled plasma mass spectrometry (ICP-MS), can determine the elemental composition of materials and has been used for below ppt levels (ppq) and better in some cases (transuranics and non-metals) to detect metals and other elements in water, soil, and air or blood and urine samples. Mass cytometry is an implementation of ICP-MS to single-cell analysis; it is based on metal isotope-tagged antibodies to quantify these bioconjugates. Imaging mass cytometry (IMC), a commercially available immunohistochemistry laser ablation-inductively coupled plasma-time-of-flight-mass spectrometry (LA-ICP-TOF-MS) system, was designed for molecular biomarkers imaging in the tissue sections (e.g., brain) through metal-tagged antibodies (typically, lanthanides). This thesis highlights the contributions of ICP-TOF-MS-based approaches towards advanced developments of mass cytometry (CyTOF) and discusses its biomedical applications for investigating neurodegenerative diseases while comparing it to other imaging modalities such as PET, MRI, Allen brain, etc. In conclusion, CyTOF, as a high-dimensional imaging tool, provides information on many clinical applications, such as hematopoiesis, transplantation, cancer, and autoimmunity.

Introduction: Le mélanome est le type de cancer de la peau le plus fréquent et les métastases du système nerveux central en sont une complication fréquente et grave. Les cellules de mélanome interagissent avec une grande variété de types de cellules dans le microenvironnement tumoral (MET), ce qui peut entraîner des effets pro- ou antitumoral. Plusieurs voies immunosuppressives ont été récemment découvertes comme des cibles médicamenteuses prometteuses, notamment la voie de l'adénosine. L'adénosine extracellulaire s'accumule dans le MET suite à l'hydrolyse de l'ATP par les ectonucléotidases CD39 et CD73. Les principaux régulateurs de la voie de l'adénosine sont CD39, CD73, et les récepteurs A2a et A2b. Matériel et Méthodes: Pour caractériser spatialement le MET des métastases cérébrales du mélanome (MCM), nous avons quantifié l'expression de 35 marqueurs protéiques à l'aide du time of flight (CyTOF) Imaging Mass Cytometry (IMC) dans 21 MCM, et segmenté et classé plus de 130 000 cellules. Ensuite, pour évaluer les effets du ciblage du récepteur A2b et du CD73 dans la voie de l'adénosine sur le développement du mélanome, nous avons utilisé les tests de prolifération IncuCyte et MTS pour évaluer la prolifération des cellules de mélanome. Résultats: Dans notre ensemble de données, les caractéristiques immunitaires du MET étaient hétérogènes dans tous les échantillons et le type de cellule le plus courant après les cellules cancéreuses du mélanome était les macrophages dérivés de la moelle osseuse (MDMO). Les échantillons à propagation leptoméningée avaient significativement moins de neutrophiles, de MDMO de type M1, d'autres cellules T et plus de cellules cancéreuses dans leur microenvironnement. Nous avons observé que la stimulation du récepteur A2b a un effet antiprolifératif sur les cellules cancéreuses du mélanome. Conclusion: Cette recherche met en évidence le rôle du MET dans la progression du mélanome et l'importance du MET comme base pour le développement de nouvelles thérapies pour les patients atteints de cancer. / Background: Melanoma is the most frequent type of skin cancer and metastasis to the central nervous system is a common and serious complication of it. Melanoma cells interact with a wide variety of cell types in the tumor microenvironment (TME) which can lead to tumor-promoting or tumor suppressive effects. Several immunosuppressive pathways have emerged as promising drug targets, including the adenosine pathway. The extracellular adenosine accumulates in the TME as the result of ATP hydrolysis by the ectonucleotidases CD39 and CD73. Key regulators of the adenosine pathway are CD39, CD73, A2a and A2b receptor. Methods: To spatially characterize the TME of melanoma brain metastases (MBM), we quantified the expression of 35 protein markers using time of flight (CyTOF) Imaging Mass Cytometry (IMC) in 21 MBMs, and segmented and classified over 130 000 cells. Then, to evaluate the effects of targeting the A2b receptor and CD73 in the adenosine pathway on the development of melanoma, we used the IncuCyte and MTS proliferation assays to assess the proliferation of melanoma cells. Results: In our dataset, the immune landscape of the TME was heterogeneous across all samples and the most common cell type after melanoma cancer cells were bone marrow derived macrophages (BMDM). Samples with leptomeningeal spread had significantly less neutrophils, M1-like BMDM, T other cells and more cancer cells in their microenvironment. We observed that stimulation of the A2b receptor has an antiproliferative effect on melanoma cancer cells. Conclusion: This research highlights the role of the TME in the progression of melanoma and the importance of the TME as grounds for development of new therapies for cancer patients.