BACKGROUND: Breast cancer is the most common cancer type in women, with 276,480 women diagnosed with breast cancer in the United States in 2020.1 Death from breast cancer is usually caused by metastasis to distant sites, rather than from the primary tumor itself.2 Regional spread of breast cancer to the tumor draining lymph nodes (TDLN) often precedes further dissemination to the rest of the body, and as such is an important prognostic tool during cancer diagnosis and staging.3 During tumor growth, multiple immune cells and stromal cells in both the primary tumor microenvironment (pTME) and the TDLN undergo changes that promote tumor growth, metastasis and immune evasion in the TDLN and to the rest of the body. Among breast cancer subtypes, triple negative breast cancer (TNBC) is one of the most aggressive, and is most likely to be resistant to traditional chemotherapies, has the worst 5-year survival percentage, regardless of stage.4 Additionally, human growth factor receptor-2 (Her2) positive breast cancers are aggressive breast cancers as well, and Her2+ breast cancers are most likely to metastasize to the TDLN.5 PROBLEM: Little is known about how breast cancer cells induce genetic or transcriptomic changes in immune cells and stromal cells in the lymph node microenvironment and pTME as cancer cells metastasize to the TDLN. OBJECTIVE: To elucidate changes observed in immune and stromal cells in the TDLN via single cell sequencing, and to quantify changes in exhaustion status of lymphocytes in the metastatic TDLN microenvironment that may be causal of lymph node metastasis. METHODS: With a bioinformatics approach on single cell RNA sequencing, and with flow cytometry, we will measure changes in the transcriptomes of cells in the primary tumor microenvironment and the TDLN and compare changes in the primary site, tumor draining lymph node, and a normal lymph node. We measure changes in the expression of canonical exhaustion markers and inhibitory receptors: Tim-3, Lag-3, Tigit, CTLA-4, PD-1, and CD160 in lymphocytes in the metastatic TDLN and nonmetastatic spleens in Balb/c mice with flow cytometry. Additionally, we measured changes in myeloid and lymphoid populations in the spleens and lymph nodes with flow cytometry. RESULTS: B cell accumulation was observed in both the single cell RNA sequencing analysis and via flow cytometry in both the metastatic TDLN itself and in the spleens of mice with metastasis to the TDLN. Increased RNA of exhaustion markers in CD8+ T cells was observed in the primary tumor samples. Additionally, a trend of increased Natural Killer cells, B cells, naive and memory CD4+ and CD8+ T cells expressing canonical exhaustion markers was observed in both the metastatic TDLNs and the spleens of mice with cancer, indicating systemic immune suppression may occur as TNBCs and Her2+ breast cancers metastasize to the TDLN. / 2022-11-24T00:00:00Z

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La leucémie lymphoïde chronique (LLC) est une hémopathie maligne caractérisée par l’accumulation, dans le sang et les organes lymphoïdes secondaires, de lymphocytes B matures résistants à l’apoptose. Le microenvironnement tumoral de la LLC au sein des organes lymphoïdes secondaires, et notamment les « Nurse-Like Cells » (NLCs), joue un rôle majeur dans la promotion de la survie et de la prolifération des cellules leucémiques. Au cours de cette étude, la surexpression du récepteur de la neurotensine NTSR2, un récepteur couplé aux protéines G, a été caractérisée. Il est activé de façon constitutive dans les cellules leucémiques circulantes, et son activation dépend de son interaction avec le récepteur à activité tyrosine kinase TrkB suite à la liaison de son ligand, le BDNF, tous deux également surexprimés. L’activation de NTSR2-TrkB par le BDNF entraine une signalisation de survie par les voies Src et Akt, aboutissant à la surexpression des protéines anti-apoptotiques Bcl-2 et Bcl-XL. L’inhibition du récepteur NTSR2 dans ces cellules fait diminuer leur viabilité. En présence des NLCs, les expressions de NTSR2, TrkB, BDNF, et de la sortiline, protéine de transport des neurotrophines et des récepteurs Trk, est accentuée. Les NLCs produisent elle-même du BDNF, activent la voie de signalisation Src, et leur rôle protecteur des cellules leucémiques est BDNF-dépendant. L’ensemble de ces travaux met en évidence un rôle capital de NTSR2-TrkB-BDNF dans la signalisation de survie des cellules leucémiques circulantes et au sein du microenvironnement tumoral de la LLC, et pourrait ainsi constituer une nouvelle cible thérapeutique potentielle. / Chronic lymphocytic leukemia (CLL) is a malignant hemopathy characterized by the accumulation of apoptosis resistant mature B lymphocytes in peripheral blood and secondary lymphoid organs. In these secondary lymphoid organs, the tumor microenvironment, notably Nurse-like Cells (NLCs), plays a major role in leukemic cells survival and proliferation promotion. In this study, an overexpression of neurotensin receptor NTSR2, a G-protein coupled receptor, was identified. NTSR2 is constitutively activated in circulating leukemic cells and its activation depends on its interaction with tyrosine kinase activity receptor TrkB upon binding of its ligand, BDNF. Activation of NTSR2-TrkB by BDNF induces survival signaling by Src and Akt pathways, and in term anti-apoptotic proteins Bcl-2 and Bcl-XL overexpression. Inhibition of NTSR2 in those cells impacts their viability. In the presence of NLCs, expressions of NTSR2, TrkB, BDNF, and sortilin, a neurotrophin and Trk receptor transport regulator, are enhanced. NLCs produce BDNF, stimulate Src activation, and their protective role on leukemic cells is BDNF-dependent. Taken together, this study highlights a key role of NTSR2-TrkB-BDNF in leukemic cells survival signaling, both in the circulation or in the tumor microenvironment, and might thus constitute a potential new therapeutic target.

Orientador: Maria Aparecida Custodio Domingues / Resumo: Mieloma Múltiplo é uma neoplasia maligna de células plasmocitárias, cujas repercussões clínicas de interação da célula tumoral com seu microambiente e com o hospedeiro podem causar danos irreversíveis e progressivos ao doente. Estratégias terapeuticas têm tentado reunir antídotos às várias linhas de atuação da célula tumoral. Objetivou-se avaliar características clíncas e possíveis interações com o microambiente da medula óssea. Foram colhidos dados clínicos, reavaliado material histológico e confeccionado bloco de TMA com grupos de pacientes ao diagnóstico, na primeira recaída e após transplante autólogo de medula óssea. A análise estatística compreendeu descrição dos dados de distribuições de frequência para as variáveis qualitativas e calculadas as médias, desvios padrão, valores mínimo e máximo e mediana para as variáveis quantitativas. O teste de Qui-quadrado de Pearson foi empregado para variáveis qualitativas. Gráficos do tipo mosaico apresentaram os cruzamentos das variáveis discretas e técnica FAMD para verificação da contribuição das variáveis qualitativas e quantitativas simultaneamente. As curvas de sobrevida foram obtidas usando a metodologia de Kaplan & Meier e a estatística de Breslow foi empregada para testar a diferença entre as curvas observadas. Os intervalos de confiança para as curvas e testes foram feitos com nível de significância de 5%. As análises e figuras foram elaboradas com o software R (R Core Team, 2017). Concluímos que pacientes com condições c... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Multiple Myeloma is a malignant neoplasm of plasma cells whose clinical repercussions of tumor cell interaction with its microenvironment and host may cause irreversible and progressive damage to the patient. Therapeutic strategies have attempted to gather antidotes to the various lines of action of the tumor cell. This study aimed to evaluate clinical characteristics and possible interactions with the bone marrow microenvironment. Clinical data were collected, histological material was reevaluated and an AMT block was made with patient groups at diagnosis, in the first relapse and after autologous bone marrow transplantation. Statistical analysis included description of frequency distribution data for qualitative variables and calculated means, standard deviations, minimum and maximum values and median for quantitative variables. Pearson's chi-square test was used for qualitative variables. Mosaic graphs showed the intersections of discrete variables and FAMD technique to verify the contribution of qualitative and quantitative variables simultaneously. Survival curves were obtained using the Kaplan & Meier methodology and the Breslow statistic was employed to test the difference between the observed curves. Confidence intervals for curves and tests were made at a significance level of 5%. The analyzes and figures were elaborated with the R software (R Core Team, 2017).We conclude that patients with clinical conditions for more aggressive initial therapies may impact their su... (Complete abstract click electronic access below) / Doutor

Since their discovery, natural killer cells (NK) cells have been implicated as important players in cancer immunosurveillance. In recent years, researchers have taken advantage of this role by developing NK cell-based immunotherapies in the fight against cancer. While these treatments have been moderately successful against hematological malignancy, they are less effective against solid cancers. This lack of success partially results from the immunosuppressive effects of the tumor microenvironment (TME). While tumors use myriad processes to evade the immune system, the avid consumption of nutrients common to NK and cancer cell metabolism and the production of toxic waste products can have significant deleterious effects on NK cell anti-tumor function. However, it may be possible to avoid some of this tumor-induced inhibition of NK cell anti-tumor function by manipulating NK cell metabolism and/or environmental conditions. Recent studies have revealed that different activation regimens can affect the metabolic dependencies of different NK cell subsets. Furthermore, studies have identified potential targets in the TME that can make the environment less hostile for infiltrating NK cells. By considering the interrelationship of NK cell metabolism and function—especially in the TME—this thesis illuminates potential strategies to modulate immunometabolic suppression. Despite the promising work already done, many gaps in the knowledge of NK cell metabolism remain. Future work will need to investigate the specific molecular mechanisms linking metabolism and function, the role of tissue-resident NK cells in cancer immunosurveillance, and the influences of chronic disease and altered systemic metabolism on NK cell anti-tumor activity.

Directed cancer cell migration induced by the environmental signals is a critical process in cancer metastasis. Cancer cells are exposed to complex chemical and mechanical signals stimulating directed migration in the tumor microenvironment, where the physical nature is highly complex. It is still barely understood how cells sense and process the complex environmental signals through the complex intercellular signaling networks to execute the cell responses. This study explores the migratory response of cancer cells under a single and combined signal. The driving hypothesis is that the cell innate capability constraints the signal stimulations physically in inducing directed cell migration. We assess the hypothesis by engineering the microenvironment in the microfluidic platform, exposing a single or combined signal environment. The combined signal environment is established by 1) two different chemoattractants (TGF-β1 and EGF) and 2) the convection-driven signal environment (TGF-β1 and interstitial flow). The results show that the performance of cancer cell directed migration is physically constrained when the environmental stimulation meets the cell’s innate physical limit. We illustrate the results in a physical and quantitative manner. This approach provides a novel insight to understand the cellular process and eventually enables to predict the cellular response under the complex environmental signals. <br>

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17978号 / 医博第3842号 / 新制||医||1001(附属図書館) / 80822 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長澤 丘司, 教授 河本 宏, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20230号 / 医博第4189号 / 新制||医||1019(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 野田 亮, 教授 武田 俊一, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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