Medulloblastoma (MB) is the most common malignant pediatric brain tumour. Of its four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4), Group 3 MB patients hold the worst clinical prognosis due to their high incidence of tumour recurrence and metastasis to the spinal leptomeninges. Relapsed Group 3 MB patients, particularly those with MYC amplification (known as Group 3𝛾), carry a mortality rate of nearly 100%, given the paucity of effective therapeutic options currently available. The most common cause of mortality amongst these patients is leptomeningeal metastasis, yet this metastatic disease is poorly characterized. Our understanding of MB tumour recurrence and leptomeningeal metastasis is further encumbered by the rare clinical opportunities at which specimens may be collected from relapsed patients. We were able to circumvent this obstacle by establishing a patient-derived xenograft mouse-adapted therapy model, which mimics the treatments administered in the clinic and in turn, recapitulates both local and metastatic human MB recurrence. This model system enabled the collection of valuable, patient-derived Group 3𝛾 MB tumour cells from the spinal leptomeninges at relapse. It provides an opportunity for chemical screening, with the aim of identifying small molecule inhibitors capable of eradicating these cells. Existing studies on MB leptomeningeal dissemination at diagnosis suggest that effective treatments may target signalling proteins, such as phosphatidylinositol 3-kinases and histone deacetylases (HDACs). Therefore, I hypothesized that selective kinase and HDAC inhibitors would pose as effective therapies against Group 3𝛾 MB metastatic recurrences. In this thesis, I conducted a high-throughput screen of 640 kinase inhibitors and robust testing of novel HDAC6-selective inhibitors against these treatment-refractory, metastatic cells. Here, I showed that metastatic recurrences of Group 3𝛾 MB are therapeutically vulnerable to selective inhibitors of checkpoint kinase 1 (CHK1), platelet-derived growth factor receptor beta (PDGFRβ), and HDAC6. Functional studies demonstrated that these inhibitors selectively target the aggressive, migratory Group 3𝛾 MB cells, while sparing healthy neural stem cells. They also showed effective blood-brain-barrier penetration in silico and in vitro, while significantly reducing MB tumour cell properties that are often associated with treatment failure. Taken together, my thesis highlights specific inhibitors of CHK1, PDGFRβ, and HDAC6 that effectively target MB tumour cells that fuel treatment-refractory leptomeningeal metastases. With additional preclinical validation, these compounds may serve as potent therapeutic options to extend survival and improve the quality of life for patients that are ostensibly limited to palliation. / Thesis / Master of Science (MSc) / Medulloblastoma is an aggressive brain cancer in children. While current standard treatment improves patient survival, 30-40% of all medulloblastoma patients relapse at local (brain) or metastatic (spine) sites. Medulloblastoma metastatic recurrences remain poorly understood, yet they result in an alarmingly high mortality rate amongst patients due to inadequate treatment options currently available. Specific molecular targets are common in both medulloblastoma and metastatic cancer research. These targets are particularly important in governing cell signalling pathways that regulate tumour growth and migration. Therefore, treatment against these targets may be effective at preventing medulloblastoma metastatic recurrences. As the collection of local and metastatic tumour samples at patient relapse are rare, the Singh lab developed an animal model that mimics human medulloblastoma recurrence. In this thesis, recurrent medulloblastoma metastases were isolated from our established animal model and tested against compounds that inhibit the specific molecular targets previously implicated in medulloblastoma and other metastatic tumours. We identified potent compounds that effectively target these metastatic cells. Next, we determined which compounds spared healthy cells and were able to penetrate the brain, given our future objective of targeting these MB cells from their source to ultimately prevent metastasis. The identified compounds substantially reduced the ability of these cells to divide. With further investigation, these compounds may pose as effective therapeutic agents to treat human medulloblastoma patients with metastatic recurrences.

abstract: Achieving effective drug concentrations within the central nervous system (CNS) remains one of the greatest challenges for the treatment of brain tumors. The presence of the blood-brain barrier and blood-spinal cord barrier severely restricts the blood-to-CNS entry of nearly all systemically administered therapeutics, often leading to the development of peripheral toxicities before a treatment benefit is observed. To circumvent systemic barriers, intrathecal (IT) injection of therapeutics directly into the cerebrospinal fluid (CSF) surrounding the brain and spinal cord has been used as an alternative administration route; however, its widespread translation to the clinic has been hindered by poor drug pharmacokinetics (PK), including rapid clearance, inadequate distribution, as well as toxicity. One strategy to overcome the limitations of free drug PK and improve drug efficacy is to encapsulate drug within nanoparticles (NP), which solubilize hydrophobic molecules for sustained release in physiological environments. In this thesis, we will develop NP delivery strategies for brain tumor therapy in two model systems: glioblastoma (GBM), the most common and deadly malignant primary brain tumor, and medulloblastoma, the most common pediatric brain tumor. In the first research chapter, we developed 120 nm poly(lactic acid-co-glycolic acid) NPs encapsulating the chemotherapy, camptothecin, for intravenous delivery to GBM. NP encapsulation of camptothecin was shown to reduce the drug’s toxicity and enable effective delivery to orthotopic GBM. To build off the success of intravenous NP, the second research chapter explored the utility of 100 nm PEGylated NPs for use with IT administration. Using in vivo imaging and ex vivo tissue slices, we found the NPs were rapidly transported by the convective forces of the CSF along the entire neuraxis and were retained for over 3 weeks. Based on their wide spread delivery and prolonged circulation, we examine the ability of the NPs to localize with tumor lesions in a leptomeningeal metastasis (LM) model of medulloblastoma. NPs administered to LM bearing mice were shown to penetrate into LM mets seeded within the meninges around the brain. These data show the potential to translate our success with intravenous NPs for GBM to improve IT chemotherapy delivery to LM. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2018

L’apparition de la technique CellSearch® a permis d’obtenir la sensibilité et la spécificité suffisantes et de détecter les CTCs en ciblant les marqueurs spécifiques dans le sang périphérique. Elle permet la numération et l’étude morphologique des CTCs qui est largement utilisée et validée. Nous décrivons une adaptation de la méthode CellSearch® pour détecter les cellules tumorale chez les LM (métastases leptoméningées) patients atteints de cancers du sein, du poumon et mélanomes, qui semble atteindre une sensibilité améliorée en comparaison avec la cytologie conventionnelle. Nous présentons également un cas clinique pour la détection de cellules tumorales dans l’ascite et du sang chez un patient avec le cancer de l’oesophage métastatique. De plus, la détection des cellules tumorales dans le redon chez les patients subis une chirurgie de la tête et du cou a été également réalisée. En utilisant cette méthode, les résultats sont non seulement quatitatifs, mais aussi quantitatifs avec des images numériques de chaque cellule, et des résultats séquentiels ont été étudiés chez certains patients atteints de cancer du sein, de cancer du poumon et de mélanome. Les données ont montré des changements dynamiques des nombres de cellules tumorales détectées dans le LCR, mais leurs corrélations avec la réponse au traitement ou la progression de la maladie ont besoin des études supplémentaires plus contrôlées avec une grande cohorte de patients. La mise en évidence de cette application serait importante en clinique pour le diagnostic, le pronostic et le traitement des patients atteints de cancer avec des métastases aux niveaux du SNC, du péritoine / The introduction of CellSearch® technology allows to give sufficient sensitivity and specificity and to detect CTCs targeting specific markers in peripheral blood. The enumeration and morphological study of CTCs are widely used and validated. We described an adaptation of the CellSearch® method to detect tumor cells in LM (leptomeningeal metastases) patients with breast cancer, lung cancer and melanoma, which appeared to achieve an improved sensitivity in comparison with conventional cytology. We also presented a case report for the detection of tumor cells in the ascites and blood of a patient with metastatic oesophageal cancer. Furthermore, the detection of tumor cells in aspirative drains after neck dissectionin from the patients undergoing surgery for head and neck cancer was also performed. Using this method, the results were not only quatitative but also quantitative with digital images of each cell, and sequential results were studied in some patients with breast cancer, lung cancer and melanoma. The data showed dynamic changes of the numbers of tumor cells detected in CSF, but their correlation with the response to treatment or disease progression need additional more controlled studies with a large cohort of patients. The application would be important for the clinical diagnosis, prognosis and treatment of cancer patients with CNS metastases and peritoneal metastases

<p>    Collateral connections in the brain, also known as Leptomeningeal Anastomoses, are connections between blood vessels originating from different arteries. Despite limited knowledge, they are suggested as an important contributor to cerebral stroke recovery that allows additional blood flow through the affected area. However, few databases and algorithms exist for this specific task of locating them. </p> <p>    In this paper, a MATLAB program is developed to find these connections and detect strokes to replace manual labeling by professionals.  The limited data available for this study are 23 2D microscopy images of mice cerebral vascular structures highlighted by dyes. In the images, strokes are shown to diminish the pixel count of vessels below 80\% compared to the healthy brain. Stroke classification error is greatly reduced by narrowing the scope from comparing the entire hemisphere to one smaller region.</p> <p>    A novel way of finding collateral connections is utilizing connected components. Connected components organize all adjacent pixels into a group. All collateral connections can be found on the border of two neighboring arterial flow regions, and belong to the same group of connected components with the arterial source from each side. </p> <p>    Along with finding collateral connections, a newly created coordinate system allows regions to be defined relative to the brain landmarks, based on the brain's center, orientation, and scale.</p> <p>    The method newly proposed in this paper combines stroke detection, brain coordinate system extraction, and collateral connection detection in stroke-affected mouse brains using only image processing techniques.  This allows a simpler, more explainable result on limited data than other techniques such as supervised machine learning.  In addition, the new method does not require ground truth and high image count for training. This automated process was successfully interpreted by medical experts, which allows for further research into automating collateral connection detection in 3D.</p>

Nous avons abordé dans l’ensemble de nos travaux deux paramètres importants pour l’immunomonitoring des patients atteints d’un cancer : les cellules tumorales circulantes (CTC) comme un indicateur de la « charge antigénique tumorale » et la réponse immune lymphocytaire T spécifique d’antigènes associés aux tumeurs (AAT). Nous avons évalué d’abord la valeur diagnostique et pronostique des CTC dans les cancers des voies aérodigestives supérieures (« HNSCC » en anglais) par une revue systématique et meta-analyse de la littérature. Les preuves actuelles identifient le test de détection de CTC comme un test extrêmement spécifique, mais de faible sensibilité dans les HNSCC. En outre, la présence de CTC indique une DFS (« disease free survival ») inférieure. Nous rapportons également pour la première fois un cas rare d’énumération extrêmement élevée de CTC détectées par le système CellSearch® chez un patient présentant un carcinome épidermoïde de la cavité buccale en utilisant. Le nombre absolu de CTC pourrait donc prédire une phase particulière de développement du cancer ainsi qu'une mauvaise survie, contribuant potentiellement à une prise en charge médicale personnalisée. De plus, nous décrivons une adaptation de la méthode CellSearch® qui nous avons développée pour détecter les cellules tumorales dans le liquide céphalo-rachidien de patients atteints de méningites carcinomateuses. Cette nouvelle approche permet une sensibilité nettement améliorée en comparaison avec la cytologie conventionnelle. La technologie CellSearch®, appliquée à des volumes limités des échantillons et permettant une augmentation du temps pré-analytique, pourrait ainsi avoir un grand intérêt dans le diagnostic de métastases leptoméningées chez les patients atteints d’un cancer d’origine épithéliale. Par une évaluation concomitante des CTC et des réponses lymphocytaires spécifiques aux AAT chez 24 patients avec HNSCC, nous avons trouvé que les CTC pourraient être un indicateur indépendant de la charge tumorale immunogène. L'absence de CTC, la présence de lymphocytes T spécifiques aux AAT, ou la combinaison de ceux-ci, étaient tous des paramètres montrant une tendance pour une meilleure survie globale ou une survie sans maladie. L’amplitude et les signatures fonctionnelles des lymphocytes T spécifiques aux AAT chez les patients atteints de HNSCC étaient associées à la présence de CTC. Ces résultats suggèrent qu’une évaluation concomitante de ces deux paramètres pourrait être plus informative sur le pronostic et potentiellement sur l’impact des traitements (notamment dans la perspective d’un traitement par des « immune checkpoints ») / We have evaluated herein two important parameters in the immunomonitoring of cancer patients: circulating tumor cells (CTC) as an indicator of “tumoral antigenic load” and tumor-associated antigens (TAA) specific T-cells. We firstly evaluated the diagnostic and prognostic value of CTC in Head and Neck Squamous Cell Carcinoma (HNSCC) by a systematic review and meta-analysis. We came to the conclusion that current evidence identifies the CTC detection test as an extremely specific but low sensitive test in HNSCC. In addition, the presence of CTC indicates a worse disease-free disease (DFS). Also, we report for the first time a rare case of extremely high enumeration of circulating tumor cells detected in a patient with squamous cell carcinoma of the oral cavity using the CellSearch® system. The absolute number of CTC could therefore predict a particular phase of cancer development as well as a poor survival, potentially contributing to personalized health. In addition, we describe an adaptation of the CellSearch® method that we have developed for detecting tumor cells in the cerebrospinal fluid of patients with carcinomatous meningitis. This new approach reaches a significantly improved sensitivity compared to conventional cytology. CellSearch® technology, applied to limited sample volumes and allowing an increased pre-analytical time, may be of great interest in the diagnosis of leptomeningeal metastases in patients with epithelial cancer. By a concomitant evaluation of CTC and TAA-specific lymphocyte responses in 24 HNSCC patients, we describe that CTC could be an independent indicator of immunogenic tumor burden. The absence of CTC, the presence of TAA-specific T-cells, or the combination of these, were all parameters showing a trend for a better overall survival or DFS. The amplitude and functional signatures of TAA-specific T-lymphocytes in patients with HNSCC were associated with the presence of CTC. These results suggest that a concomitant evaluation of these two parameters may be more pertinent for prognosis assessment as well as for treatment impact, especially in “checkpoint-inhibitors” new immunotherapies