IDENTIFICATION OF MICROGLIAL CELLS DURING CHICK EMBRYO BRAIN DEVELOPMENT AND HUMAN XENOGRAFT TUMOR FORMATION
Files
Date
2020-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
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.
Description
Keywords
biological sciences, microglial cells, human xenograft tumor formation