Study On The Role Of Circulating Cancer Cells And TGF-β In Fibroblast Differentiation By New Fluorescent Microscopy

Part I:The study of the metastatic potential of prostate cancer cells and the role of macrophages in circulating prostate cancer cells by in vivo flow cytometryMetastasis is a very complicated multi-step process and accounts for the low survival rate of the cancerous patients. To metastasize, a cancer cell must break away from its tumor and invade either the circulatory or lymphatic system, which will carry it to a new location, and establish itself in the new site. Once in the blood stream, the cancer cells now have access to every portion of the body. In this study, we have utilized an emerging technique, namely in vivo flow cytometry (IVFC) to study the relationship between metastatic potential and depletion kinetics of circulating prostate cancer cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labeled cells in vivo. Interestingly, more invasive PC-3prostate cancer cells are depleted faster from the circulation than LNCaP cells. More than70%PC-3cells are depleted within the first hour after tail vein injection. In comparison, less than30%LNCaP cells are depleted within the first hour. In vivo confocal microscopy imaging shows the cell numbers of DiD-labeled PC-3cells spreading in the bone marrow areas within mouse skull are considerably higher than those of LNCaP cells at1h and5h after injection. The differences in depletion kinetics might provide insights into early metastasis processes of prostate cancer.Macrophages appear to be directly involved in tumor progression and metastasis. However, the role of macrophages in affecting cancer metastasis has not been fully elucidated. Here, we have used the in vivo flow cytometer to study the depletion kinetics of circulating prostate cancer cells in mice and how depletion of macrophages by the liposome-encapsulated clodronate affects the depletion kinetics. Our results show different depletion kinetics of PC-3cells between macrophage-deficient group and the control group. The number of circulating tumor cells (CTCs) in macrophage-deficient group decreases in a slower manner compared to the control mice group. The differences in depletion kinetics indicate that the absence of macrophages facilitates the stay of prostate cancer cells in circulation. In addition, our imaging data suggest that macrophages might be able to arrest, phagocytose and digest PC-3cells. Therefore, the phagocytosis may mainly contribute to the depletion kinetic differences. The developed methods here would be useful to study the relationship between macrophages and tumor metastasis in small animal cancer model. Part II:Using two-photon fluorescence microscopy and bio-image informatics to study the TGF-β signaling in fibroblasts inside porous collagen scaffoldsIn order to learn the new advanced fluorescence microscopy imaging techniques, the author was sponsored by the China Scholarship Council and went to the laboratory of Prof. So in Massachusetts Institute of Technology to carry out a scientific research for18months since November,2010.Cells sense and respond to their environment via signal transduction pathways, cascades of proteins and small molecules. While the vast majority of existing knowledge about cell signaling is based on long-established biochemical and genetic methods, the increasing complexity of biological systems under study requires novel approaches to infer cellular interactions and speed-up discovery. Two-photon fluorescence microscopy, which has the advantages of high resolution, deeper tissue penetration and less photo-damage, has been wildly used in the field of live cell and tissue imaging, especially focusing on long-term three-dimensional cell imaging. Bio-image informatics methodologies combine genetic manipulation of cells, large scale imaging, image processing and statistics to study complex signal transduction pathways in cells to elucidate physiological processes and pharmaceutical interactions. So far, bio-image informatics has been applied in studies of cells cultured on2D surfaces in standard culture dishes.Fibroblasts play an important role in wound healing and fibrosis disease. TGF-β isoforms regulate myofibroblast differentiation and therefore affect significantly both the shape of the cells as well as cell-matrix interactions. TGF-β-induced signaling in wound contraction and scar formation affect significantly the outcome of wound healing in injured organs. Porous collagen scaffolds are currently used as tissue extracellular matrix (ECM) analogs and have been applied successfully clinically to induce regeneration in injured organs. In this study, stable protein expression interference of each component of TGF-β/SMAD pathway in fibroblasts was achieved by transfection with a shRNA lentivirus. We used a custom-made16-channel spectral multi-photon microscope, combined with a new bio-image informatics framework, which is suitable for3D cell-matrix system, to study the TGF-β signaling in fibroblasts inside porous collagen scaffolds. The integrated system outputs of imaging-based single-cell morphometrics and cell-matrix interaction metrics obtained by high-content3D two-photon imaging, as well as the biochemical validation assays, were finally processed and statistical analyzed, which makes several conclusions could be inferred:Compared with the isoform TGF-P3, TGF-β1makes a more important role in mediating myofibroblast differentiation; SMADl and SMAD3play key roles in TGF-β1mediated myofibroblast differentiation; SMAD1and SMAD3were excited through different way in TGF-β1mediated myofibroblast differentiation. All these inferences here were consistent with the literatures whose results were obtained by biochemical or genetic methods. Hence, the integrated framework of two-photon fluorescence microscopy and bio-image informatics we established could be well used to study the cell signaling in a3D cell-matrix system, which optimizes the experimental design and creates an opportunity to accelerate the pace of knowledge discovery.