abstract: Cell-cell interactions in a microenvironment under stress conditions play a critical role in pathogenesis and pre-malignant progression. Hypoxia is a central factor in carcinogenesis, which induces selective pressure in this process. Understanding the role of intercellular communications and cellular adaptation to hypoxia can help discover new cancer biosignatures and more effective diagnostic and therapeutic strategies. This dissertation presents a study on transcriptomic and metabolic profiling of pre-malignant progression of Barrett's esophagus. It encompasses two methodology developments and experimental findings of two related studies. To integrate phenotype and genotype measurements, a minimally invasive method was developed for selectively retrieving single adherent cells from cell cultures. Selected single cells can be harvested by a combination of mechanical force and biochemical treatment after phenotype measurements and used for end-point assays. Furthermore, a method was developed for analyzing expression levels of ten genes in individual mammalian cells with high sensitivity and reproducibility without the need of pre-amplifying cDNA. It is inexpensive and compatible with most of commercially available RT-qPCR systems, which warrants a wide applicability of the method to gene expression analysis in single cells. In the first study, the effect of intercellular interactions was investigated between normal esophageal epithelial and dysplastic Barrett's esophagus cells on gene expression levels and cellular functions. As a result, gene expression levels in dysplastic cells were found to be affected to a significantly larger extent than in the normal esophageal epithelial cells. These differentially expressed genes are enriched in cellular movement, TGFβ and EGF signaling networks. Heterotypic interactions between normal and dysplastic cells can change cellular motility and inhibit proliferation in both normal and dysplastic cells. In the second study, alterations in gene transcription levels and metabolic phenotypes between hypoxia-adapted cells and age-matched normoxic controls representing four different stages of pre-malignant progression in Barrett's esophagus were investigated. Through differential gene expression analysis and mitochondrial membrane potential measurements, evidence of clonal evolution induced by hypoxia selection pressure in metaplastic and high-grade dysplastic cells was found. These discoveries on cell-cell interactions and hypoxia adaptations provide a deeper insight into the dynamic evolutionary process in pre-malignant progression of Barrett's esophagus. / Dissertation/Thesis / Ph.D. Biological Design 2014

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23155号 / 工博第4799号 / 新制||工||1750(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 横川 隆司, 教授 井上 康博, 教授 中部 主敬 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Heterogeneity within tumors has long been studied as a potential confounding factor for effective therapies, with recent studies pointing to heterogeneity resulting in distinct clonal subtypes, each with varying degrees of fitness and metastatic potential. Studies of heterogeneity have previously been limited to microscopy observations, immunohistochemistry, and flow cytometry. Recently, however, it has become possible to examine heterogeneity at a previously unexplored level: the transcriptome of individual cells. Osteosarcomas have been known to be highly heterogeneous, so we have selected osteosarcoma as our primary tumor to study as a proof-of-concept. Additionally, we have elected to create a murine patient derived xenograft (PDX) model from a primary osteosarcoma tumor and examine differences between the primary tumor and resulting xenograft at the single-cell level. Through this, we hope to better understand tumor heterogeneity and add to the current discussion in the scientific community regarding the relevance of PDX models for testing promising new therapies and personalized medicine. Through our examination of single-cell heterogeneity in osteosarcomas, we have confirmed the extensive heterogeneity previously reported, but this time at the level of mRNA. The osteosarcomas were so hetereogeneous that our resulting dataset of over 1,000 cells still did not have enough resolution to generate highly differentiated and separate groupings of cells. Upon examining inter-tumor heterogeneity, we observed the cells from different tumors to generally cluster separately. However, there were certain populations of cells from all tumors that clustered together. We also generated a PDX model and sequenced the resulting tumor, observing markedly reduced heterogeneity as compared to the original primary tumor. Importantly, the cells from the PDX model clustered within the larger group of cells from the original tumor, lending credence to the theory of clonal selection. This work presents evidence of extensive intra- and inter-tumor heterogeneity at the mRNA level within osteosarcoma tumors. This heterogeneity requires further single cell sampling to shed light on the biology of tumor diversity. Further, this heterogeneity is significantly reduced in a generated murine PDX model. This difference should serve as a potential warning about additional factors to take into account when evaluating therapies in PDX models, and suggests that further studies examining cause and effect of this observed heterogeneity are warranted.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13142号 / 論医博第2142号 / 新制||医||1026(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 伊佐 正, 教授 野田 亮, 教授 岩田 想 / 学位規則第4条第2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21453号 / 医博第4420号 / 新制||医||1032(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 渡邉 大, 教授 浅野 雅秀, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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