Cancer is a common disease and one out of two gets cancer in Japan. Therefore costs for medical treatment would constantly increase if appropriate diagnosis and therapeutic treatments were not given. Tumorigenesis consists of several biological steps, which include autonomous cell growth, invasion and metastasis, genomic instability caused by genomic and epigenetic aberrations in cancer. So far basic research to reveal the mechanism of each step successfully identified the genes responsible for tumorigenesis (oncogenes) and molecular targeted therapies against some types of cancers have been developed. Efficacious drugs against many other cancers, however, remain to be developed. Generally, tumor cells are thought to evolve against external stress such as low oxygen, low nutrition and internal stress such as DNA damage by X-ray irradiation. Namely tumorigenesis can be regarded as adaptation to stressful conditions. In order to understand the mechanism of tumorogenesis as “adaptation to stress”, we have been developing in vitro and in vivo experimental systems to identify and analyze the functions of genes that regulate tumor growth, invasion and metastasis under stressful conditions. These systems include reconstitution of functional protein complex using biochemically-purified proteins, single cell analysis, animal models of tumor cell growth and metastasis. We not only perform independent research themes but also share experimental procedures to constitute the “stress biology of tumor” in this project.
In vivo growth and metastasis of tumors are thought to be strictly regulated by micro environments including low nutrition, low pH or low oxygen. To understand the mechanism for the tumor cells to overcome such environmental stress, we still need appropriate cell lines and assay systems. We are going to develop methods to purify mammary stem cells and establish appropriate immortalized cell lines, retrovirus-mediated high efficient gene transfer techniques and methods to assess growth and invasion of tumor cells in vivo and then integrate those methods into a experimental system to evaluate the function of genes.
Aim of this team is to understand the molecular mechanisms of DNA repair pathways, especially for DNA double strand break (DSB) repair by homologous recombination. DSBs are induced by ionizing radiation (IR), DNA crosslinkers, and replication errors. Homologous recombination (HR) is an accurate DSB repair pathway in mitosis, and the mitotic recombination reaction is preferred to occur between sister chromatids. HR also functions in meiosis, and the meiotic recombination occurs between homologous chromosomes. The meiotic HR ensures the correct chromosome segregation at meiosis I. Therefore, both mitotic and meiotic homologous recombination pathways play important roles in chromosome maintenance, and may suppress tumorigenesis.
Short wavelength light such as UV and blue light induce the generation of ROS in dermal and retinal cells. ROS not only give serious stress of DNA damages to induce skin cancer but also play important roles in light-dependent signal transduction pathways for modulation of immune responses and apoptosis, though the pathways are far less understood. To reveal novel ROS- or light-dependent signaling pathways, it is important to establish the cellular system in which we easily detect the light-dependent responses. We are developing a model system using human dermal cells, avian pineal cells, and fish cultured cells, along with molecular and functional characterization of candidate blue light or ROS receptors.
For analyzing metastasis ability of tumor cell, assay systems for cell migration both in two dimensional and three dimensional cultures were originally developed. For a start, migration of fibroblast, which was known to lead infiltration of cancer cell, was analyzed. Directional migration of single fibroblast solely by the topography of the micropatterned substrates was achieved without applying any bio signaling factors, so as to exclude their unidentified side effect. Furthermore, fibroblasts were embedded and three dimensionally cultured within a type I collagen gel, with applying flow of the culture medium continuously and slowly, and time course behavior of migration was examined. In next step, migration of cancer cell will be assayed, correlating with metastasis ability.
Single-cell analysis has attracted a great deal of attention as the technology that can reveal the heterogeneity of individual cells in living organisms. Understanding the complexity and heterogeneity of cellular behavior requires a technique capable of tracing the temporal behavior of cells at a single-cell resolution for minutes, hours, and even days. However, the lack of such technology has made it extremely difficult to continuously observe the behavior of suspension cells at the single-cell level. Recently, we introduced a highly efficient cell entrapment platform whereby blood cells were successfully entrapped onto 10,000 microcavity structures. Because the cells are arrayed on the addressable microcavity array, thousands of individual cells, arranged in a spatially defined pattern, can be cultivated and imaged by using microscopy. Since non-adherent cells are retained at fixed position for a long time, measurements can be made at multiple time points for the same set of single cells.
Therefore, this technology shows great potential as an inexpensive and simple tool for the continuous long-term single-cell observation of cellular behavior.