Developing chick embryo brain slice cultures to investigate the role of N-cadherin in GSC invasion
| Author(s) | Tomatsu, Saori | |
| Date Accessioned | 2022-10-31T12:16:04Z | |
| Date Available | 2022-10-31T12:16:04Z | |
| Publication Date | 2022 | |
| SWORD Update | 2022-08-10T19:09:49Z | |
| Abstract | Glioblastoma (GBM) is the most aggressive form of brain cancer formed from astrocytes, classified as a grade IV astrocytoma by the World Health Organization (WHO). GBM has been associated with poor prognosis and the medium survival time of a patient with GBM is around 14 months. It is theorized that GBM invasion and relapse is connected to the small population of glioblastoma stem cells (GSCs) that is resistant to treatments including radiotherapy and chemotherapy, and how they interact with non-cancer stem cells. A major molecule known to take part in increasing GBM motility and invasion is the L1 cell adhesion molecule (L1CAM). Cleavage of L1CAM ectodomain by the metalloproteinase ADAM10 can induce cell migration and neuronal growth. Along with L1CAM, the transmembrane protein N-cadherin, is also speculated to increase GBM motility by similar processes as L1CAM involving cleavage of the protein’s ectodomain by ADAM10. GSC and non-GSC invasion can involve several paths including along neuronal axon bundles, migrating diffusely into brain tissue, or along blood vessels. ☐ My goal was to develop an ex vivo brain slice culture system using embryonic chick brain slices to facilitate time-lapse analysis of GBM cell and GSC behavior. Ex vivo brain slice cultures survived best in MEM media (with additives) as shown by Sox-2 and laminin stainings which screened for brain viability and blood vessel conditions, respectively. The implantation method used in this study proved to be a successful method to observe live U-118/L1LE cell migration along blood vessels in the live brain slice cultures via time-lapse under the widefield fluorescent microscope. This was validated with immunostaining and confocal imaging with z-stack analysis. However, less invasion was seen using GSC2016-4, suggesting further investigation with live brain slice cultures and GSC invasion. ☐ A second goal was to determine N-cadherin expression in GSC and GBM cell lines in the Galileo Lab using antibodies against the cytoplasmic domain and ectodomain of N-cadherin. By being able to identify N-cadherin expression in specific cells, further experimentation on how N-cadherin influences GBM motility can be investigated in the future. My second goal was to establish an ex vivo organotypic brain slice culture model to visualize live invasion of GSC and GBM cells, particularly along blood vessels. ☐ Western blot analysis, flow cytometry, and coverslip immunostaining with the antibody against the cytoplasmic domain of N-cadherin showed N-cadherin expression in all GSC and GBM cells tested. N-cadherin expression using the antibodies against the cytoplasmic domain and ectodomain was evident in western blot analysis, fixed and live coverslip staining, and immunostained tumors in brain slices. ☐ My results demonstrate that ex vivo chick embryo brain slice cultures are a suitable system for analyzing invasiveness of GBM cells and GSCs. Furthermore, N-cadherin appears to be widely expressed in GBM cells and GSCs, although further experiments are needed to determine if it is cleaved and released to facilitate autocrine/paracrine stimulation of motility, invasiveness, and/or proliferation similarly to what occurs with L1CAM. | en_US |
| Advisor | Galileo, Deni S. | |
| Degree | M.S. | |
| Department | University of Delaware, Department of Biological Sciences | |
| Unique Identifier | 1349339242 | |
| URL | https://udspace.udel.edu/handle/19716/31547 | |
| Language | en | |
| Publisher | University of Delaware | en_US |
| URI | https://login.udel.idm.oclc.org/login?url=https://www.proquest.com/dissertations-theses/developing-chick-embryo-brain-slice-cultures/docview/2700756512/se-2?accountid=10457 | |
| Keywords | Chick embryo | en_US |
| Keywords | Ex vivo brain slice culture | en_US |
| Keywords | GBM and GSC | en_US |
| Keywords | N-cadherin | en_US |
| Keywords | Time-lapse | en_US |
| Title | Developing chick embryo brain slice cultures to investigate the role of N-cadherin in GSC invasion | en_US |
| Type | Thesis | en_US |