INCREASED L1CAM EXPRESSION AFFECTS STEM CELL MOTILITY IN VITRO AND STEM CELL BEHAVIOR IN VIVO
Date
2019-05
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Glioblastoma Multiforme (GBM) is a type of aggressive brain cancer whose
high mortality rate is attributed to invasion of tumor cells in the brain beyond where
they can be surgically removed. There is currently no successful treatment for GBM,
which ranks as the most common and most lethal form of brain cancer. While GBM
always invades surrounding brain tissue, it does not metastasize to other organs. The
motility of rare glioblastoma stem cells (GSCs) is thought to drive tumor expansion,
and the abnormal expression of L1CAM, a transmembrane cell adhesion molecule, by
tumor cells is hypothesized to increase GSC motility. Endogenous expression or
exogenous presence of L1CAM previously has been demonstrated by our lab to
increase motility in “established” glioblastoma cell lines (Yang et al., 2009, 2011;
Anderson and Galileo, 2016), but not yet in GSCs.
One objective of my study was to investigate whether or not the effects that
L1CAM has on established glioma cell lines are similar to the effects it has on GSCs.
It is important to define the role that L1 plays in motility of GSCs, as the mechanisms
by which these stem cells drive tumor expansion are not yet understood. The
heterogeneity of GBM is another factor that plays a role in its fatality. Tumor cell
access to different factors has been shown to cause changes in cell morphology and
behavior. In a patient, these changes potentially could result in a more aggressive
phenotype, such as increased invasiveness. One stem cell line (GSC2016-4) grown in
“differentiation media”, which contains 10% FBS (GSC2016-4/HiFBS) but no growth
factors, exhibited an increase in L1 expression by western blot analysis. Another
objective of my study was to determine how this change in L1 expression affected cell
behavior, both in vitro and in vivo.
To determine whether L1CAM affected motility of GSCs, cells were plated
with differing concentrations of L1-producing cells (U-118/L1LE or GSC2016-
4/HiFBS), and in vitro time-lapse SuperScratch assays of these mixtures were
completed in order to track the velocity of cells over a 22-hour time period. The
effects of mixing U-118/1879 or U-118/L1LE cells with different stem cells lines
varied depending on cell type, but mixing GSC2016-4 cells with GSC2016-4/HiFBS
(L1-producing) cells resulted in a clear, linear increase in cell velocity that correlated
with increased presence of L1-producing cells. Tumor morphology and marker
expression also were studied by injecting mixtures of stem cells into a xenograft chick
embryo brain tumor model. Cells expressing fluorescent marker proteins were injected
into the optic tecta of embryonic day (E) 5 chick embryos, and allowed to develop
until E15, when brains were dissected. These brains then were fixed, embedded,
vibratome sectioned, immunostained for different markers, and analyzed by confocal
microscopy. Results indicated that tumor formation follows specific patterns based on
cell type, and that there is variability in stem cell marker expression in vivo.
A main finding of this study is that paracrine L1CAM can modulate the
motility of GSCs. Velocity of GSCs increased with increased exposure to L1 from
nearby L1-producing cells. However, this increase in motility may differ between stem
cell lines as a result of their inherent L1 expression. Secondly, injection of GSC2016-
4/HiFBS cells mixed with GSC2016-4 cells produced tumors similar in morphology to
those that formed from injections of GSC2016-4 cells mixed with U-118/L1LE cells.
This indicates that GSC2016-4 cells grown in “differentiation” media have the ability
to affect cells in a manner similar to an established cell line that has been modified to
exhibit increased L1 expression.
Description
Keywords
Biological sciences, Stem cell motility and behavior, L1CAM