Browsing by Author "Lin, Amy"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    IDENTIFICATION OF MICROGLIAL CELLS DURING CHICK EMBRYO BRAIN DEVELOPMENT AND HUMAN XENOGRAFT TUMOR FORMATION
    (University of Delaware, 2020-05) Lin, Amy
    Glioblastoma (GBM) is the most aggressive and invasive human brain tumor, which despite advancement in surgery and immunology, only 10% of patients survive 18 months after diagnosis. The Galileo lab studies GBM, focusing on the role of the protein L1CAM on increasing the motility, proliferation, and invasiveness of GBM cells using in vitro cell tracking and a novel xenograft chick embryo brain tumor model. My work focused on microglial cells, which are resident brain immune system cells that are not well understood. Previous research in mice showed that microglial cells concentrated in areas with greatest cancer cell invasion. It was hypothesized that a difference in microglia will be seen in the chick embryo brain that corresponds with areas of human xenograft tumor growth and invasion of glioma cells. The first aim for my project was to find biomarkers that specifically identify microglial cells in normal chick embryo brain during development. The second aim was to use those markers to examine any potential interaction microglial cells have with xenograft tumors in the chick embryo brain. Chick embryo brains were fixed, frozen, and cut into serial cryostat sections or fixed and vibratome sectioned, followed by immunofluorescent staining using antibodies or lectins that identified microglial cells in other model organisms. The optimal staining protocol required overnight primary antibody/lectin with 0.1% Triton X-100 detergent and 5% normal goat serum in phosphate buffered saline. The antibodies and lectins that I investigated included Isolectin B4, Ricinus Communis Agglutinin I (RCA-I), 3H11, V2E9 and anti-CD45. Several of the antibodies and lectins tested either did not stain cells specifically or exhibited a high background staining. Immunofluorescence analysis revealed RCA-I to stain microglial cells with the most specificity. Anti-CD45 staining reveal microglial cell morphology to progressively change from a compact amoeboid shape (E5) into ramified branched shape (E12 and E15). Instead of locating throughout the brain, microglial cells appeared to be located as clusters in certain areas. In the chick brains with xenograft human tumors, there appeared to be a cluster of amoeboid shaped microglia in the tumor periphery. Further investigations are needed to verify the increased presence of reactive microglial cells and their interaction with tumor cells.
  • Thumbnail Image
    Item
    Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior
    (Journal of Visualized Experiments, 2023-05-26) Pastorino, Nicole G.; Tomatsu, Saori; Lin, Amy; Doerr, Jackson; Waterman, Zachary; Sershen, Krisztina; Ray, Pulak; Rodriguez, Analiz; Galileo, Deni S.
    Summary Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis. Abstract The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 µm) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 µm) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.