The tumor microenvironment is composed of cellular and stromal components such as tumor cells, mesenchymal cells, immune cells, cancer associated fibroblasts and the supporting extracellular matrix. The tumor microenvironment provides crucial support for growth and progression of tumor cells and affects tumor response to therapeutic interventions. To better understand tumor biology and to develop effective cancer therapeutic agents it is important to develop preclinical platforms that can faithfully recapitulate the tumor microenvironment and the complex interaction between the tumor and its surrounding stromal elements. Drug studies performed in vitro with conventional two-dimensional cancer cell line models do not optimally represent clinical drug response as they lack true tumor heterogeneity and are often performed in static culture conditions lacking stromal tumor components that significantly influence the metabolic activity and proliferation of cells. Recent microfluidic approaches aim to overcome such obstacles with the use of cell lines derived in artificial three-dimensional supportive gels or micro-chambers. However, absence of a true tumor microenvironment and full interstitial flow, leads to less than optimal evaluation of tumor response to drug treatment. Here we report a continuous perfusion microfluidic device coupled with microscopy and image analysis for the assessment of drug effects on intact fresh tumor tissue. We have demonstrated that fine needle aspirate biopsies obtained from patient-derived xenograft models of adenocarcinoma of the lung can successfully be analyzed for their response to ex vivo drug treatment within this biopsy trapping microfluidic device, wherein a protein kinase C inhibitor, staurosporine, was used to assess tumor cell death as a proof of principle. Lastly, we tested the model for its ability to demonstrate similar results found in clinic when using a Wee1 inhibitor on osteosarcoma and an epidermal growth factor receptor inhibitor, Erlotinib, and inhibitors of programmed death 1 receptor and programmed death ligand 1 on lung adenocarcinoma fine needle aspirate biopsies. This approach has the potential to study tumor tissue within its intact microenvironment to better understand tumor response to drug treatments and eventually to choose the most effective drug and drug combination for individual patients in a cost effective and timely manner.

Background: Near-IR fluorescence (NIRF) imaging is becoming a promising approach in preclinical tumor detection and clinical image-guided oncological surgery. While heptamethine cyanine dye IR780 has excellent tumor targeting and imaging potential, its hydrophobic property limits its clinical use. In this study, we developed nanoparticle formulations to facilitate the use of IR780 for fluorescent imaging of malignant brain tumor. Methods: Self-assembled IR780-liposomes and IR780-phospholipid micelles were prepared and their NIRF properties were characterized. The intracellular accumulation of IR780-nanoparticles in glioma cells were determined using confocal microscopy. The in vivo brain tumor targeting and NIRF imaging capacity of IR780-nanoparticles were evaluated using U87MG glioma ectopic and orthotopic xenograft models and a spontaneous glioma mouse model driven by RAS/RTK activation. Results: The loading of IR780 into liposomes or phospholipid micelles was efficient. The particle diameter of IR780-liposomes and IR780-phospholipid micelles were 95 and 26nm, respectively. While stock solutions of each preparation were maintained at ready-to-use condition, the IR780-phospholipid micelles were more stable. In tissue culture cells, IR780-nanoparticles prepared by either method accumulated in mitochondria, however, in animals the IR780-phospholipid micelles showed enhanced intra-tumoral accumulation in U87MG ectopic tumors. Moreover, IR780-phospholipid micelles also showed preferred intracranial tumor accumulation and potent NIRF signal intensity in glioma orthotopic models at a real-time, non-invasive manner. Conclusion: The IR780-phospholipid micelles demonstrated tumor-specific NIRF imaging capacity in glioma preclinical mouse models, providing great potential for clinical imaging and image-guided surgery of brain tumors.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18146号 / 医博第3866号 / 新制||医||1002(附属図書館) / 31004 / 京都大学大学院医学研究科医学専攻 / (主査)教授 鈴木 茂彦, 教授 妻木 範行, 教授 戸口田 淳也 / 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(工学) / 甲第19002号 / 工博第4044号 / 新制||工||1622(附属図書館) / 31953 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 白川 昌宏, 教授 田中 庸裕, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21449号 / 医博第4416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 妹尾 浩, 教授 松原 和夫, 教授 川上 浩司 / 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(生命科学) / 甲第21474号 / 生博第405号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 影山 龍一郎, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Fluorescence microscopy has revolutionized medicine and biological science with its ability to study the behavior and chemical expressions of living cells. Fluorescent probes can label cell components or cells of a particular type. Clinically the impact of fluorescence imaging can be seen in the diagnosis of cancers, AIDS, and other blood related disorders. Although fluorescence imaging devices have been established as a vital tool in medicine, the size, cost, and complexity of fluorescence microscopes limits their use to central laboratories. The work described in this thesis overcomes these limitations by developing a low cost integrated fluorescence microscope so single use fluorescence microscopy assays can be developed. These assays will enable at-home testing, diagnostics in resource limited settings, and improved emergency medicine.

The human cell is complex, with countless processes ongoing in parallel in specialized compartments, the organelles. Cells can be studied in vitro by using immortalized cell lines that represent cells in vivo to a varying degree. Gene expression varies between cell types and an average cell line expresses around 10,000-12,000 genes, as measured with RNA sequencing. These genes encode the cell’s proteome; the full set of proteins that perform functions in the cell. In paper I we show that RNA sequencing is a necessary tool for studying the proteome of the human cell. By studying the proteome, and proteins’ localization in the cell, information can be assembled on how the cell functions. Image-based methods allow for detailed spatial resolution of protein localization as well as enable the study of temporal events. Visualization of a protein can be accomplished by using either a cell line that is transfected to express the protein with a fluorescent tag, or by targeting the protein with an affinity reagent such as an antibody. In paper II we present subcellular data for a majority of the human proteins, showing that there is a high degree of complexity in regard to where proteins localize in the cell. Cellular energy is generated in the mitochondria, an important organelle that is also active in many other different functions. Today approximately only a third of the estimated mitochondrial proteome has been validated experimentally, indicating that there is much more to understand with regard to the functions of the mitochondria. In paper III we explore the mitochondrial proteome, based on the results of paper II. We also present a method for sublocalizing proteins to subcompartments that can be performed in a high-throughput manner. To conclude, this thesis shows that transcriptomics is a useful tool for proteome-wide subcellular localization, and presents high-resolution spatial distribution data for the human cell with a deeper analysis of the mitochondrial proteome. / <p>QC 20170512</p>

The two-photon absorption (2PA) phenomenon has attracted attention from various fields ranging from chemistry and biology to optics and engineering. Two of the common NLO applications in which organic materials have been used are three-dimensional (3D) fluorescence imaging and optical power limiting. Two-photon absorbing materials are, therefore, in great demand to meet the needs of emerging technologies. Organic molecules show great promise to meet this need as they can be customized through molecular engineering, and as the development of two-photon materials that suit practical application intensifies, so does research to meet this need. However, there remains some uncertainty in the particulars of design criteria for molecules with large 2PA cross sections at desired wavelengths, as such research to understand structure-property relationships is matter of significant importance. As a result, the full potential of 2PA materials has not been fully exploited. Several strategies to enhance the magnitude and tune the wavelength of 2PA have been reported for ?-conjugated organic molecules. On this account, we have designed novel fluorophores using the fluorene moiety and modified it to tune the properties of the compounds. Chapter 2 of this dissertation reports the successful application of fluorene-based compounds in photocatalysis; a process that involves the decomposition of organic compounds into environmentally friendly carbon dioxide and water attesting to the photostability of the fluorene moiety. A facile organic nanoparticle preparation method is reported in chapter 3 using the reprecipitation method, whose surface was then modified using a naturally occurring surfactant, Lecithin, and were then successfully used in fluorescence cell imaging. Chapter 4 reports the design and synthesis of a fluorene-based compound using an acceptor, s-indacene-1, 3, 5, 7(2H, 6H)-tetra one, or Janus Dione, a moiety that is relatively new and that has not been fully exploited despite its very attractive features. Owing to the hydrophobicity of this compound, notwithstanding its unprecedented 2PA cross section, it was not applicable in fluorescence cell imaging but provided the tenets for the design of related derivative. This limitation was circumvented in the concluding chapter by tuning the compound's hydrophilicity. The hydrophilic Janus dione probe was then used as envisioned for cell imaging as the dual prerequisites for fluorescence imaging probes; large 2PA cross sections and high fluorescence quantum yields were met.

Treatments for skeletal tissue injuries include surgery and rehabilitation but in adult patients, the healing process is slow and incomplete, and the underlying biological mechanisms are largely unknown. Skeletal tissues contain stem cells responsible for their maintenance and repair, but the identity and location of these stem cells, and what molecular mechanisms regulate their fate decisions remain unclear. To design more effective regenerative therapies for skeletal conditions, understanding the fundamental biology of skeletal stem cells (SSC) in postnatal organisms is required. Our project aims at identifying and characterizing these SSC populations in postnatal murine and human tissues using lineage tracing techniques, combined with multicolor 3D confocal microscopy and computational image analysis, in vitro assays, and single cell transcriptomics. We hypothesized that the postnatal skeleton contains self-renewing and multipotent Sox9+ SSCs that persist in adulthood. We showed that the adult mouse skeleton contains Sox9+ cells self-renewing, multipotent skeletal stem cells (SSCs) with osteogenic and chondrogenic potential. They are located adjacent to the growth plates and in periosteum and persist in adulthood. Transcriptome analysis revealed that these cells express other putative SSCs markers, as well as genes involved in skeletal development, stem cell self-renewal, and fate decision. This data provides testable drug targets to pharmacologically manipulate SSCs fate decisions in situ. In addition, we showed that human tissues contain SSCs similar to murine tissues. This is the first experimental proof of self-renewal in postnatal Sox9+ SSCs in vivo. These findings provide actionable insights for the use of culture-expanded stem cell product for regenerative medicine product or pharmacological targeting of these stem cells in situ. We believe our data will help improve stem-cell based and tissue engineering therapies, increasing success rate of regenerative orthopaedic surgeries while reducing reoccurrence of injuries.