1. Glioblastoma Reprograms Macrophages Gene Expression
Abstract Methods Results
Acknowledgements
Conclusions
Mrunali Patel, Gina M. Rodriguez, Kennedy Adams, Lara Brown, Shefali Das, Jacqueline Fezza, Neshama Fournier, Rohan Paturu, Nicole Serio, Rahil Shah, Ava Weitz, Dr. Salvatore Coniglio
New Jersey Center for Science, Technology & Mathematics;
Group Summer Scholars Research Program, Marianne Gass, and STEM Faculty
Glioblastoma multiforme is one of the most aggressive brain cancers,
with a survival rate of less than 12 months after the diagnosis. The
methods available to manage this cancer include surgery,
chemotherapy, and radiation; however, they do not ensure a complete
removal of metastatic glioblastoma cells. Recent studies show that
microglia, immune cells in the brain, are the key to the metastasis
(spreading) of cancer cells. The cancer cells and microglia
communicate via signaling pathways that ultimately result in altered
gene expression in microglia. This study used quantitative real time
PCR to measure levels of gene expression in microglia treated with
GCM as compared to untreated microglia. The genes studied coded for
a class of proteins called CC chemokines, which are known to play a
role in inflammation and immunity.² Blocking these proteins has been
shown to increase human susceptibility to infection. Upregulation or
downregulation of these genes in microglia could play a role in the
glioblastoma’s ability to metastasize without eliciting an immune
response. The purpose of this experiment was to identify which CC
chemokines in microglia are regulated by glioblastoma cells. Studying
the resulting changes in the behavior of the microglia can lead to a
better understanding of metastasis while opening new possibilities in
the field of therapeutics.
1
2
3 4
Figure 2: Master mix of: SYBR
Green Dye (1), Nuclease Free
Water (2), cDNA (Macrophages
treated with Glioma-Conditioned
Media + Control cDNA) (3), and
various primers (4) [photo
courtesy of directindustry.com]
Figure 3: 20ul
master mix was
added to PCR
reaction tube.
[picture courtesy
of brand.de]
Figure 1: Lyse cells, harvest mRNA, produce
cDNA [photo courtesy of tataa.com]
Figure 4: PCR
tubes were placed
in thermocycler.
[picture courtesy
of bio.rad.com
Figure 5: Illustrates what is
occurring in the thermocycler. (1)
Denaturation (2) Annealing (3)
Melting Curve of Analysis [photo
courtesy of openwetware.org
siRNA can be used to block target genes in microglia that were seen
upregulated in these experiments. An invasion assay can then be
performed using these microglial cells to determine whether blocking
the expression of these genes in microglia affects glioblastoma invasion.
Glioblastoma multiforme is an aggressive form of brain cancer with a
high mortality rate.¹ Previous studies have shown that microglia, a
form of macrophage cell, are critical in the growth and metastasis of
this cancer. In order for the macrophages to enable metastasis, the
glioblastoma cells must first reprogram the macrophages via changes in
gene expression. In this study, we attempted to elucidate which genes in
microglia are regulated by glioblastoma cells. Using quantitative
real-time PCR, the changes in the gene expression between untreated
microglia and microglia treated with glioblastoma conditioned media
(GCM) were quantified by finding the difference in the cycle threshold
between the two samples. This was used to calculate the fold change in
gene expression. In microglia treated with GCM, CCL4 and CCL3
were downregulated while CCL5, CCL6, CCL7, and CCL9 were
upregulated. Future studies will investigate whether blocking these
genes inhibits tumor metastasis.
➔ The expression of the CCL4 and CCL3 genes in macrophages
decreased when treated.
➔ CCL5 and CCL6 genes exhibited slightly greater expression.
➔ The expression of CCL7 and CCL9 increased greatly when the
microglia were treated with GLCM.
➔ CCL2, uPAR, and CCR1 did not express any signal in either the
treated or untreated sample.
Figure 7:
Gene expression in
GCM treated
microglia was
compared to
expression in
untreated samples.
A higher fold
change indicates
increased gene
expression.
Expression
increased in the
CCL7 and CCL9
genes.
Introduction
Future Works
Figure 6: Illustrated here are the primers chosen. They were selected due to their recognized
capabilities to increase or decrease gene expression.
References
¹Survival Statistics for Brain Tumours. (n.d.). Retrieved August 09, 2016.
² Viola, A., & Luster, A. D. (2007, September 17). Chemokines and Their Receptors: Drug Targets
in Immunity and Inflammation. Annual Review of Pharmacology and Toxicology, 177-182.
Retrieved August 9, 2016.