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.

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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<p>Breast cancer remains a major cause of cancer-related deaths among women despite several advances in the field due to metastasis with a 5-year survival rate of less than 30% for metastatic breast cancer. Dissemination of tumor cells to metastatic sites begins as early as the primary tumor is diagnosed at just 4mm in size. These cells remain dormant for extended periods of time evading immune surveillance and later turn into therapy resistant metastases resulting in the poor prognosis in breast cancer patients. Hence, there is a <b>critical need </b>to improve our understanding of the metastatic programs in breast cancer and its contributors to develop better therapy options.</p><p>One such contributor is alcohol which is listed as a carcinogen by the National Toxicology Program. Alcohol consumption is a risk factor for several cancers and increases the risk of breast cancer incidence in a dose dependent manner. We have observed in preliminary studies, that alcohol consumption causes increased neutrophil extracellular trap (NET) formation in the lungs and outgrowth of previously dormant cancer cells in mice. Further, NETs increase cancer cell seeding and play a role in metastasis. Hence, we hypothesized that alcohol consumption breaks cancer cell dormancy by activating neutrophils.</p><p>In this study, we have broken cancer cell dormancy and generated a novel cell line, Alcohol-D2.OR, by inducing outgrowth of the dormant D2.OR cells in mice through alcohol consumption. Reinjection of the Alcohol-D2.OR cells, into alcohol-naïve mice results in aggressive outgrowth of the cells suggesting these cells are modified on a genetic level. Indeed, RNA sequencing analysis of the gene expression in the cells showed that these cells have significantly modified gene expression as well as modified morphology and surface protein expression than the parental D2.OR cells. Importantly, from our analysis we have identified a tumor suppressor, SPINK5 which was significantly downregulated in the alcohol line. Further, SPINK5 expression in cancer cells suppressed neutrophil activity in-vitro. Knockdown of SPINK5 in the parental D2.OR line resulted in outgrowth of the cells in-vivo with increased lung NETs highlighting the importance of this gene for maintenance of dormancy by suppression of neutrophil activity.</p><p>Hence, we have successfully identified a gene responsible for dormancy maintenance, SPINK5 which will aid in not only therapeutic intervention but also in identification of breast cancer patients likely to progress to metastasis. Further, the newly established Alcohol-D2.OR cells provide a novel tool to study other initiators of metastasis in breast cancer.</p><p>A common side-effect of most chemotherapeutic treatments is neutropenia, reduced neutrophils in circulation increasing susceptibility to infections. Hence, GM-CSF is often administered to patients to mobilize bone marrow neutrophils. However, neutrophils have been increasingly shown to promote distant metastases. Circulating disseminated cancer cells (DCCs), which are present as early as primary diagnosis, have been shown to activate neutrophils resulting in the release of neutrophil extracellular traps (NETs). These NETs alter the lung architecture providing a suitable environment for the seeding and growth of DCCs promoting lung metastases. One key player in neutrophil activation is spleen tyrosine kinase (SYK), an intracellular non-receptor kinase which is activated by the engagement of b-integrin on the neutrophil surface.</p><p>Using a chemical genetics approach we are able to specifically inhibit SYK in the murine host. Using our transgenic model of specific SYK inhibition as well as the FDA approved SYK inhibitor, fostamatinib, we see similar results of decreased lung metastases compared to controls. We also observed decreased neutrophil viability in-vitro in the presence of fibronectin, an effect that was not seen on plastic highlighting the importance of integrin mediated activity of SYK. We also observe decreased neutrophil and macrophage infiltration into the lungs upon host-specific SYK inhibition. Overall, these findings suggest a paracrine effect of SYK in stromal cells that promotes favorable tumor microenvironment (TME) and its inhibition may be a useful therapeutic option to combat DCCs from forming metastases.</p><p>Hence, through this work we address two mechanisms of neutrophil-mediated breast cancer metastasis and that therapeutic intervention by rescuing SPINK5 expression in cancer cells or inhibition of SYK in the tumor microenvironment can suppress pulmonary metastasis in breast cancer.</p>

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24496号 / 医博第4938号 / 新制||医||1064(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 滝田 順子, 教授 上野 英樹, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Bladder cancer, as one of the most common cancer types and with high recurrence risk, is considered a candidate for novel immunotherapy strategies. An important aspect of the research for immunotherapy drug development for bladder cancer is to study the tumor microenvironment (TME) and it’s immune contexture. Besides tumor-infiltrating lymphocytes (TILs) as the main drivers of anti-tumor response, recent studies revealed the importance of tumor-associated tertiary lymphoid structures (TLSs) and high endothelial venules (HEVs) in the TME. Structures similar to these were found to spontaneously form in the orthotopic MB49 model used for bladder cancer research in our group. The aim of this study was to perform a deeper characterization of the TME in this model, by using immunofluorescent staining and microscopy. Specifically, the co-localization of tumor infiltrating lymphocytes (CD8+ and CD4+ T cells, CD19+ B cells), CD11c+ dendritic cells and HEVs along with CCL21 signaling were analyzed within orthotopic MB49 tumors, with and without immune stimulation. The quantification of cells expressing CD8, CD19 and CD11c immune markers, CCL21 levels, vascular density and numbers of HEVs, showed higher densities within the immune-stimulated tumors, indicating a rapid effect of immune stimulation on increasing immune cell infiltration and vascular density after only 24 hours post CpG therapy. Also, the highest frequency of TILs, CCL21 chemokine and vascular density was located in regions of the tumor border indicating that these regions should be studied further in depth as a potential target for entry of cells to the tumor with immunotherapy or as a model of the tumor microenvironment since tumor cell density is maintained high in these locations.

Systematic characterization of microbes in several tumors including colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) has revealed the presence of multiple species of intracellular bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in co-culture of host epithelial cells and bacteria, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging oncomicrobe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a Gram-negative, anaerobic bacterium that is normally found within the oral cavity. However, its selective enrichment in CRC and PDAC tumors is correlated with poor clinical outcomes. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum infection of host cancer cells induced robust secretion of select cytokines that increased cancer cell migration, impacted cell seeding, and enhanced immune cell recruitment. In PDAC, we uncovered additional cytokines that were secreted from both normal and cancerous pancreatic cell lines upon infection with F. nucleatum that increased cancer cell proliferation and migration via paracrine and autocrine signaling, notably in the absence of immune cell participation. In order to examine the contribution of a hypoxic TME on infection dynamics, we used a multi-omics approach that combined RNA-seq and ChIP-seq of H3K27ac to determine epigenomic and transcriptomic alterations sustained within hypoxic CRC cells upon infection with F. nucleatum. Our findings revealed that F. nucleatum can subvert host cell recognition in hypoxia and can modulate the expression of multiple cancer-related genes to drive malignant transformation. Insights gained from this research will pave the way for future studies on the impact of the tumor microbiome in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer. / Doctor of Philosophy / Colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are the second and third leading causes of cancer death in the United States, respectively. Recent systematic characterization of various tumor types revealed the presence of distinct bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in developing host cell-microbe co-culture models, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging cancer-associated microbe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a microbe commonly found within the oral cavity. However, clinical studies revealed that selective enrichment of F. nucleatum in CRC and PDAC tumors significantly correlated with poor prognosis. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum invasion of host cancer cells induced the secretion of select proteins called cytokines that cells use to signal and communicate with each other. These cytokines directly stimulated the cell migration of host cancer cells which is usually associated with increased cancer aggressiveness. In PDAC, F. nucleatum infection induced the secretion of additional cytokines from both cancer cells and normal cells that, in addition to cell migration, impacted the proliferation of cancer cells, another feature of aggressive cancers. F. nucleatum usually thrives in a low oxygen environment that is prevalent in cancer tissue and hence, we examined how a low oxygen environment can influence infection dynamics using sequencing technologies that probe the genomic constitution within cells. Our findings revealed that F. nucleatum can escape recognition in low oxygen environments and can modulate the expression of multiple cancer-related programs within the cell to drive cancer progression. Insights gained from this research will pave the way for future studies on the impact of the tumor-associated microbes in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer.

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