Characterization and evaluation of L1CAMs effect on glioblastoma stem cells in vitro and in vivo
Date
2020
Authors
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Publisher
University of Delaware
Abstract
Glioblastoma (GBM) is a grade 4 astrocytoma classified by its extreme invasiveness into the adjacent brain tissue. It is considered the most common and deadly form of brain cancer in adults, with a medium survival time of 15 months, which has not changed in decades. Many recent experiments have pointed towards the existence of glioblastoma stem cells (GSCs) within these tumors. This cell population has been predicted to be the main cause of malignancy; however, the mechanisms underlying GSC invasiveness are not well understood. The Galileo lab previously has shown that the L1 cell adhesion molecule (L1CAM) was able to increase overall GBM cell motility and proliferation through autocrine/paracrine stimulation in vitro. My project objective was to address the question of whether GSCs derived from patient surgical specimens also were equally affected by L1CAM. The focus was on characterizing GSC lines for stem cell marker expression, analyzing responsiveness to exogenous L1CAM, and determining how manipulating L1CAM expression levels affect cell motility both in vivo and in vitro. GSC characterization for seven glioblastoma or stem cell “markers” revealed that the cells isolated with the Failsafe adherent culture method were consistently positive for SOX2 and Nestin with varying positivity levels for OCT3/4, integrin a6, L1CAM, L1cyto, and CD133 by flow cytometry. Western blot analysis supported the expression of SOX2, Nestin, L1CAM, and L1cyto. Exogenously added L1CAM, as L1-Fc, to GSC cultures did not significantly affect cellular motility in scratch assays over a 22-hour time frame. Attenuated (shL1) GSCs showed complete eradication of L1CAM expression post-infection. Overexpressed (L1LE) GSCs have yet to be analyzed due to their slow growth rate. Motility assays of modified GSC lines showed unexpected results, as the control vector and the attenuated vector had similar average velocities that were both significantly lower than that of the uninfected GSC line. In addition, to test the in vivo impact of L1CAM in GSCs, modified and labeled GSCs were injected into chick embryo brains at E5 by themselves. Tumors were allowed to form for 9 days prior to dissection and fixation of the brains. Attenuated L1CAM GSCs in vivo did not form tumors noticeably different than those of the modified control GSCs. Additionally, both GSC 2016-4/1879 and GSC 2016-4/shL1 lines resulted in denser tumor masses with fewer invading cells compared to the uninfected GSC 2016-4 line. This study determined that the patient-derived GSCs expressed well-accepted stem cell markers, such as SOX2 and Nestin, while also expressing various levels of several other classical stem cell markers. Additionally, each GSC line showed fundamentally different motility potential, tumor formation, and invasive behavior both in vivo and in vitro. Not only can the GSC lines established in the Galileo lab be used to study patient-specific characteristics of the clinical disease, the in vitro and in vivo methods also are suitable for this.
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Keywords
Chick embryo, Glioblastoma, L1 cell adhesion molecule, Migration