Clinical ototoxicity - permanent hearing loss caused by medications - is estimated to affect millions of patients annually. Two classes of drug are largely to blame: platinum-based chemotherapeutics, primarily cisplatin, and aminoglycoside antibiotics. Development of methods to prevent ototoxicity depends upon an understanding of its mechanisms and may benefit from an understanding of native protective pathways of the inner ear. As the mechanisms behind cisplatin ototoxicity remain unclear, I first sought, and herein report, a refined mouse model of cisplatin ototoxicity which will allow for further in vivo investigation of cisplatin ototoxicity and potential methods for its prevention. This low-dose, multi-cycle model was found to accurately reproduce cisplatin ototoxicity as it has been described clinically and histopathologically. I then used this mouse model of cisplatin ototoxicity to investigate cisplatin pharmacokinetics in the cochlea and their role in driving cisplatin ototoxicity. Cisplatin was found to be retained within the cochlea for months following its administration. This initial finding in mice was extended to cochlear tissue samples from deceased human patients. Analysis of intra-cochlear cisplatin distribution in murine and human tissue identified the stria vascularis region of the cochlea as a promising target for intervention. With the nature of aminoglycoside ototoxicity better understood, I investigated a native inner ear protective pathway which could be leveraged to promote sensory hair cell survival. The improved hair cell survival that has previously been demonstrated as a result of heat stress was found to be mediated by cell-cell communication via extracellular vesicles. Further, hair cell protection against aminoglycosides could be reproduced through the application of exogenous, non-inner ear-derived extracellular vesicles. In sum, these data provide new insight into mechanisms of ototoxicity and details of cellular pathways which can help protect against it.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24840号 / 医博第5008号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田, 恭之, 教授 松田, 道行, 教授 柳田, 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

La progression tumorale dépend d'un dialogue entre les cellules cancéreuses et leur environnement. Parmi les cellules du microenvironnement du mélanome, les adipocytes ont longtemps été ignorés. Pourtant, ces cellules sont le composant majeur de l'hypoderme, la couche la plus profonde de la peau. Ainsi, elles sont proches du mélanome lors de la tumorigenèse et, lorsque la tumeur envahi les couches profondes de la peau, les deux types cellulaires entrent en contact. Il est donc important de comprendre l'impact des adipocytes sur la progression du mélanome, d'autant plus que des études épidémiologiques montrent que l'obésité est un facteur de mauvais pronostic pour ce cancer. Le surpoids et l'obésité sont en hausse constante avec près d'un tiers de la population mondiale affectée, faisant du lien entre l'obésité et le cancer un enjeu de santé publique majeur. Parmi les différents moyens de communication cellulaire, les vésicules extracellulaires (VE) jouent un rôle important dans le cancer. Les VE régulent la communication entre les cellules cancéreuses mais aussi entre les composants du microenvironnement et la tumeur. Les VE sécrétées par les adipocytes sont peu caractérisées et leur rôle sur la progression tumorale reste à élucider. Les VE adipocytaires pourraient être modifiées qualitativement et quantitativement en obésité car différents stress (inflammation, hypoxie...), connus pour modifier les VE, sont retrouvées dans le tissu adipeux d'individus obèses. Dans ce contexte, le premier objectif de ma thèse était de caractériser les VE adipocytaires et déterminer leur impact sur le mélanome dans un contexte normopondéral et d'obésité. Les résultats obtenus montrent que ces VE favorisent la migration et l'invasion des cellules de mélanome. Une analyse protéomique a révélé une signature spécifique dans ces VE, fortement enrichies en protéines du métabolisme des acides gras (AG).[...] / It is now clear that tumor progression is the result of a permanent dialog between cancer cells and the tumor microenvironment (TME). Among the cells found within the melanoma microenvironment, adipocytes had long been ignored. However, adipocytes are the main component of the hypodermis, the deepest skin layer, and are therefore close to melanoma from tumorigenesis and, as the tumor becomes aggressive and invades the deeper skin layers, the two cell types come into contact. Thus, understanding how adipocytes influence melanoma progression is of major importance, especially since epidemiological studies show that obesity is a poor prognosis factor for melanoma. As overweight and obesity are constantly rising and affect around a third of the World's population, the link between obesity and cancer is a major public health issue. Among the different ways in which cells communicate, extracellular vesicles (EV) play a particularly important role in cancer. Moreover, not only can tumor cells communicate with each other through EV, but the cellular components of the TME also use EV to communicate with cancer cells. Adipocyte-derived EV are poorly characterized and their role in tumor progression remains to be determined. In obesity, adipocyte EV may be qualitatively and quantitatively altered since various stresses (inflammation, hypoxia etc.), which are known to modify EV, are found in the adipose tissue of obese individuals. In this context, the first aim of my thesis was to characterize adipocyte EV and their impact on melanoma in lean and obese individuals. The results obtained show that EV secreted by adipocytes promote migration and invasion of melanoma cells. Analysis of their proteome revealed a protein signature specific to adipocyte EV, which was highly associated with fatty acid (FA) metabolism, a metabolic pathway involved in tumor aggressiveness. In melanoma treated with adipocyte EV, fatty acid oxidation (FAO) is increased and FAO inhibitors reverse their pro-invasive effect. Moreover, adipocytes secrete increased numbers of EV in obesity and, using equal numbers of EV from lean or obese subjects, their effect on tumor aggressiveness is increased and remains dependent on FAO. T[...]

Changes in the biomechanical properties of cells accompanying the development of various pathological conditions have been increasingly reported as biomarkers for various diseases and as a predictor of disease progression stages. For instance, cancer cells have been found to be less stiff compared to their healthy counterparts due to the proteomic and lipidomic dysregulations conferred by the underlying pathology. The separation and selective recovery of cells or extracellular vesicles secreted from such cells that have undergone these changes have been suggested to be of diagnostic and prognostic value. This dissertation first describes the implementation of a stiffness-based separation of phosphatidylcholine-based vesicles using a method first introduced based on the research in this work and was dubbed thermally-assisted acoustophoresis, or thermo-acoustophoresis. By tuning the temperature, we achieved the separation of vesicles of the same size, shape, and charge but with different stiffness values. It was observed that at a specific transition point, the acoustic contrast factor of vesicles changed sign from positive to negative. This change was mainly due to change in the compressibility of the vesicles, which is inversely proportional to stiffness. The acoustic contrast temperature (Tϕ), corresponding to the temperature at which the contrast factor switches sign, was determined to be unique to the composition of the vesicles. This unique temperature signature allowed us to develop this separation method of vesicles with distinct membrane stiffness with target outlet purities exceeding 95%. We have further explored the functionality of this method by experimenting with cholesterol-containing vesicles. In cells, the cholesterol content plays a crucial role in determining stiffness. Changes in the cholesterol content in cellular membranes can be an indication of pathological disorders. We evaluated the Tϕ of vesicles at different cholesterol molar ratios (Xchol) and developed a multi-stage lab-on-a-chip method to accomplish for the first time the separation of a three-vesicle mixture. Using Xchol = 0.1, 0.2, and 0.3 vesicles, we obtained efficiencies exceeding 93%. The simplicity, rapidity, and label-free nature of this approach holds promise as a diagnostic and separation tool for cells affected by diseases that affect the stiffness and extracellular vesicles such as exosomes and microvesicles.

Background: Between 2005 and 2015 the number of new cancer cases per year in Canada rose by 29% and this number is projected to increase to 277,000 cases per year. Ionizing radiation is used as therapy in the majority of cancer cases; however, it can have long-term detrimental effects on the hematopoietic system. Recent work from our lab, in a preclinical model of radiation damage, demonstrated that endurance exercise training can enhance hematopoietic recovery, while obesity can impair it. Extracellular vesicles (EVs) are a mode of cellular communication that has been implicated in regulating hematopoiesis acutely following radiation exposure. However, the long-term, radiation-induced changes to EVs, and the role of exercise and/or obesity at modulating marrow EVs remains unknown. Thus, the purpose of this project was to determine the extent to which obesity and exercise influence the regenerative potential of bone marrow-EVs following radiation. Method: Mice were randomly divided into control (n=20; CON) or high fat diet (n=20; HF) groups, then subdivided into exercise-trained (EX, n=10) or sedentary (SED, n=10). Mice underwent whole-body exposure to a 3 Gy dose of gamma-radiation at age 13 weeks of age followed by bone marrow collection at 20 weeks of age. EVs were then isolated from the bone marrow by ExoQuick and ultracentrifugation. A non-irradiated, sedentary, control diet group (n=10) was used to determine the effects of radiation alone. Data was evaluated using repeated-measures three-factor (diet, exercise, time) and two-factor ANOVA. Results: High fat diet-induced changes in body weight and composition and altered food consumption (p<0.05). Isolated EVs measured between 78 and 195 nm and western blot confirmed the presence of EV protein markers Alix, TSG101, and Flotillin. No size difference was observed between the groups. The concentration of EVs in irradiated mice was significantly lower compared to EVs from control mice (p<0.01). Radiation, obesity, exercise, or their combination had no significant effect on hematopoietic stem progenitor cells (HSPC) content in co-culture assays. Conversely, EVs from irradiated mice significantly increased the number of CFU-GEMM, CFU-G, and the TOTAL number of colonies compared to EVs from non-irradiated mice (p<0.01). However, EVs from the CON+SED, CON+EX, HF+SED, and HF+EX groups did not have a significant effect on colony formation. Conclusion: Our findings demonstrate that ionizing radiation can diminish the concentration of bone marrow-EVs and that irradiated bone marrow-EVs can increase the total number of myeloid colonies formed in vitro. These results suggest that radiation induces myelopoiesis via a mechanism that includes EVs; however, exercise and obesity induce their effects via a different mechanism.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22396号 / 薬科博第118号 / 新制||薬科||13(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23225号 / 工博第4869号 / 新制||工||1760(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 秋吉 一成, 教授 跡見 晴幸, 教授 大塚 浩二 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Detection of cancer at the early stages greatly increases the chance for successful treatment and favourable prognosis for patients. However, a liquid-based biopsy has yet to be developed for most cancers. Extracellular vesicles (EVs) are an attractive candidate for early cancer detection since their surface proteome mirrors the cell of origin. Thus, there is a need for the development of reliable probes that can detect cancer derived EVs. In this thesis, the VBS-1 aptamer was developed to selectively bind to triple-negative breast cancer cell line derived EVs. Initially, several EV isolation methods were compared and isolated EVs were validated and characterized. Aptamer clones were developed by Systematic Evolution of Ligands by Exponential Enrichment to EVs isolated by differential ultracentrifugation and their binding was validated by flow cytometry. The binding partner of the selected VBS-1 aptamer was identified by LC-MS/MS to be the transmembrane protein ATP1A1. The presence of an ATP1A1-positive EV population was validated by flow cytometry. The selected aptamer may find further application in biosensors for the detection of EVs as cancer biomarkers in biological fluids.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24586号 / 工博第5092号 / 新制||工||1975(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 秋吉 一成, 教授 大塚 浩二, 教授 田中 一生 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

O câncer colorretal (CCR) é o terceiro tipo de câncer mais comum no mundo. Apesar dos avanços nos tratamentos convencionais, aproximadamente dois terços dos pacientes com CCR são submetidos à cirurgia potencialmente curativa. Entretanto, grande parte desses pacientes evolui mal, apresentando recidivas e/ou metástases. A busca de novos alvos moleculares para a terapia do CCR revelou a proteína celular Prion (PrPC) como um possível candidato. Trabalhos recentes sugerem participação direta ou indireta de PrPC no crescimento de tumores, na formação de metástases, na composição de complexos multiproteicos e na indução de vias de sinalização envolvidas em diversos processos biológicos, como proliferação. Além disso, PrPC foi descrito como um importante modulador do crescimento de tumor colorretal. Resultados prévios mostraram que a interação da proteína PrPC com a proteína HSP70/HSP90 Organizing Protein (HOP) induz proliferação em glioblastomas. HOP é uma proteína predominantemente citoplasmática, podendo também ser secretada associada às vesículas extracelulares. Assim, o presente estudo objetivou avaliar o papel do complexo PrPC-HOP e das vesículas extracelulares no desenvolvimento e progressão dos tumores colorretais. Os nossos resultados mostram que HOP induziu migração e invasão em linhagens de CCR de maneira dependente de PrPC, uma vez que o uso do peptídeo sem atividade que compete pelo sítio ligação de HOP a PrPC inibiu estes processos. Além disso, nossos dados apontaram que o aumento de migração e invasão das células de CCR induzida pela interação PrPC-HOP é mediada pela ativação da via ERK1/2. Os achados in vitro estimularam a avaliação do perfil de expressão de PrPC e HOP por imuno-histoquímica em tecidos de pacientes com diferentes tipos de tumores colorretais. Nossos resultados sugeriram que essas proteínas são importantes no início do desenvolvimento tumoral e na transição de adenomas para adenocarcinomas, não havendo correlação entre a presença de HOP e/ou PrPC com metástase, linfonodos acometidos, estadiamento, sobrevida ou região tumoral versus tecido normal. Em relação ao papel das vesículas extracelulares na progressão dos tumores colorretais, nossos resultados mostraram que linhagens celulares que apresentam padrões parecidos de agressividade tumoral podem ter perfis de secreção de proteínas e vesículas extracelulares bastante diferentes, induzindo, portanto, processos biológicos com intensidades distintas. O meio condicionado e as vesículas extracelulares da linhagem WiDr apresentaram maior potencial de indução de migração quando comparado com a linhagem HCT8. Além disso, a modulação negativa da proteína VPS4, uma das responsáveis pela formação dos corpos multivesiculares, mostrou-se uma abordagem interessante no estudo da secreção de vesículas por células de CCR, uma vez que o dominante negativo de VPS4 promoveu diminuição do cargo proteico e da secreção de vesículas extracelulares, redução da proliferação celular e do efeito indutor do processo de migração na linhagem WiDr. Assim, em conjunto, o presente trabalho indicou que o complexo PrPC-HOP pode ser um bom alvo terapêutico nos processos de migração e invasão em CCR. Ainda, essas proteínas se mostraram importantes nos estágios iniciais da formação dos tumores. A modulação da secreção de vesículas extracelulares pode contribuir para retardar a progressão dos tumores colorretais. / Colorectal cancer (CRC) is the third most common type of cancer in the world. Despite improvements in conventional treatments, approximately two-thirds of CRC patients undergo potentially curative surgery. However, most of these patients evolve poorly, showing recurrence and/or metastasis. Search of new molecular targets for CRC therapy revealed the cellular protein Prion (PrPC) as a putative candidate. Recent studies have shown that PrPC exhibit direct or indirect participation in tumor growth, formation of metastasis, composition of multiprotein complexes and induction of signaling pathways involved in many biological processes such as proliferation. Moreover, PrPC has been described as an important modulator of colorectal tumor growth. Previous findings showed that the interaction between PrPC and its ligand HSP70/90 heat shock organizing protein (HOP) induces gliobastoma proliferation. It is well known that HOP localizes mainly in the cytoplasm but HOP is also secreted associated with extracellular vesicles. In this way, the present study sought to evaluate the role of PrPC-HOP complex and extracellular vesicles in the development and progression of CRC. We demonstrate that HOP induces the migration and invasion of CRC cell lines in a PrPC-dependent manner because the use of HOP peptide, which is able to bind to PrPC, blocking PrPC-HOP complex formation, inhibited the migration and invasion processes. In addition, our data showed that the enhancement of migration and invasion induced by PrPC-HOP interaction is mediated by ERK1/2 pathway activation. These in vitro results lead us to evaluate the PrPC and HOP expression by immunohistochemistry in tissues from patients with different tumor types. Our data showed that these proteins could be important for the initial steps of tumor development, represented by the transition from adenoma to adenocarcinoma. No correlation was found among HOP and/or PrPC expression and metastasis, lymph node involvement, staging, survival or tumor area versus normal tissue. Regarding the role of extracellular vesicles in the progression of colorectal tumors, our results showed that cell lines exhibiting similar aggressive tumor behavior can have a different protein secretion pattern and a distinct profile of extracellular vesicles release, which could induce biological process with different intensities. The conditioned medium and the extracellular vesicles derived from WiDr cell line showed a higher potential to induce migration than HCT8 cell line. Moreover, the negative modulation of VPS4, one of the proteins responsible for multivesicular body formation, showed to be an interesting approach in the study of extracellular vesicles secretion secreted by CRC cells; the negative dominant of VPS4 promoted in the WiDr cell line a reduction in the protein cargo and secretion of the extracellular vesicles, a decrease of cell proliferation and induction of migration process. Therefore, taken together, our data highlights that PrPC-HOP complex can be considered a new therapeutic target in migration and invasion processes of CRC. Moreover, these proteins appeared to be important at onset of tumor formation. The modulation of extracellular vesicles secretion may contribute for delaying the progression of colorectal tumors.