FLOW CYTOMETRY ANALYSIS OF THE DIFFERENTIATION OF GLIOBLASTOMA STEM CELLS USING STAINING PROCEDURES WITH VARIOUS MARKERS
Date
2024-05
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University of Delaware
Abstract
Glioblastoma (GBM) is an incurable and deadly form of brain cancer that spreads quickly
and aggressively throughout the human brain. Since there is no effective way to treat
glioblastoma, the median survival rate is just over a year. The reason for this incurability is
believed to be because Glioblastoma Stem Cells (GSCs) are resistant to radiation and
chemotherapy. GSCs infiltrate the brain tissue and are believed to drive the spread of the tumor.
These cells also migrate from the main tumor mass to escape surgical removal and ultimately
end up causing tumor regrowth.
Currently, the Galileo lab performs experiments in vitro on culture dishes and in vivo in
chick embryo brains. Chick embryos are injected with fluorescently labeled GBM cells and/or
GSCs at embryonic day 5 and then dissected anywhere from embryonic day 10-15. GSCs form
small tumor masses along blood vessels in the chick brain, leading to the appearance that the
cells are invading the brain along blood vessels (Pastorino et al., 2023). The Galileo Lab has
previously demonstrated that a molecule that contributes to tumor motility, proliferation, and
invasiveness is the L1 cell adhesion molecule, also referred to as L1CAM, or just L1 (Yang et
al., 2009; Yang et al., 2011; Mohanan et al., 2012; Pace et al., 2019). Further injection
experiments also revealed that patient derived GSC cell lines (GSC 2015-2 and GSC 2016-4)
formed patterned tumors in vivo when mixed with non-stem GBM cells (U-118/L1LE) that
secrete the L1CAM ectodomain (SFN abstract; Pastorino et al., 2023). The two different GSC
lines interacted differently with the L1-producing GBM cells, where in one the L1-producing
GBM cells formed an outer cortex around the GSC 2015-2 which also formed a ring of L1CAM.
This specific pattern led to the hypothesis that GSCs are induced to express L1CAM when mixed
with U-118/L1LE cells in vitro.
To further validate this hypothesis, I co-cultured GSCs with U-118 cells, immunostained
the GSCs for L1CAM and GFAP, and then analyzed them by flow cytometry to observe whether
expression of these markers varied. These experiments supported that co-culturing U-118/L1LE
with GSC 2015-2 and GSC 2016-4 upregulates the production of L1CAM and GFAP, while also
possibly leading to a way of differentiating cancer cells.
Building on the concept of differentiated GSCs, I propose to further expand upon this
possibility by exploring various mixtures of media and supplements in hopes of inducing
differentiation. Specific supplements that were used here include retinoic acid (RA), B27 with
RA, and N2, which will then be incorporated into a previously confirmed GBM differentiation
media, DMEM+10% FBS (HiFBS). Testing for differentiation included staining the cells for
different GSC markers Nestin, Sox-2, and Integrin 6 and differentiation markers GFAP and
L1CAM and analyzing their expression levels with flow cytometry. Experimental data of percent
positive cells gathered from the GSC 2016-4 cell line were averaged and resulted in the
indication that differentiation tentatively, but not significantly, occurred in DMEM+10% FBS
media through morphological changes and marker expression (only Integrin 6 was significantly
downregulated). To deeper analyze potential differentiation media conditions, histogram
statistics of shifts in mean and peak fluorescence values were applied to each protein marker
cultured in DMEM+10% FBS, DMEM+10% FBS+20M RA, and DMEM+10%
FBS+B27+20M RA providing similar statistics in that neither analysis (mean or peak
fluorescence levels) provided significant differences in protein marker expression. However, this
form of analysis, gave equal potentiality for differentiation in all 3 media conditions. Tentatively,
for GSC 2016-4, DMEM+10% FBS seems to most effectively prompt differentiation.
Due to the varying results found among the two forms of analysis conducted, further
testing and a determination of which analysis is better suited for this research needs to be
performed. The only marker tested that resulted in a significant difference for conditions with
previously said supplements was Integrin 6, which showed reduced expression. It was also
observed that different GSC lines exhibited different marker expressions and effects based on
media conditions. GSC 2015-2 cell line was only analyzed a few times, making conclusions less
certain than for GSC 2016-4.Future experiments are needed, including an increased
concentration of B27 with RA, additional supplements not considered yet, and continued use of
differing histogram statistics to better understand if serum induced differentiation is an effective
method for potentially reducing invasiveness and propagation of GSCs.