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Molecular targets for treatment of glioblastoma multiforme

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Molecular targets for treatment of glioblastoma multiforme

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MOLECULAR TARGETS FOR TREATMENT OF
GLIOBLASTOMA MULTIFORME

Vijaya Pooja Vaikari

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
DECEMBER 2014

Copyright 2014 Vijaya Pooja Vaikari

i

ACKNOWLEDGMENT

I would like offer my sincere gratitude to my advisor Dr. Florence Hofman for
giving me this opportunity to be a part of a laboratory that immensely helped me learn
and develop my research skills. Dr. Hofman made me understand that perfection, hard
work, and patience are the keys to good research. She has been extremely motivating
for me and I am grateful for the invaluable support she has given me. I would also like to
thank Niyati Jhaveri for teaching me all the techniques in detail and helping me design
and carry out my experiments in the best possible manner. I sincerely appreciate all the
support she has offered me over the past few months.
I am extremely grateful to our collaborator, Dr. Jean Shih, who provided
immense guidance to me for my project with her extensive research expertise. I would
also like to express my gratitude to Dr. Thomas Chen, Dr. Axel Schönthal, and Dr. Stanley
Tahara for all their insights and guidance that helped me better develop my project. I
am also very thankful to Dr. Swati Mishra, Dr. Weijun Wang, and Dr.Heeyon Cho for all
their help and guidance throughout.
Working in an environment with extremely enthusiastic people has helped me
develop as a scientist and learn from others and their experience. I would like to thank
members of the Hofman and Shih laboratories - Swati, Christopher, Lucy, Tiffany and
Alex for all their help.
ii

I would also like to thank Dr. Joseph Landolph for serving on my committee.

None of this would have been possible without the support of my family. I am
extremely grateful to my parents, Vijay and Bindu Kuruganti, and my sister, Kiran Vaikari
who are the reasons I have made it this far. They have been the most constant source of
encouragement with all their love, motivation and belief in me that made me strive
harder at each step. I would also like the thanks my grandmother, Seshu Chirravuri, for
all her blessings and good wishes; and Satwik, Ishan and Saumya for their support.

iii

CONTENTS

ACKNOWLEDGMENT ................................................................................................ i
LIST OF FIGURES ....................................................................................................... v
ABSTRACT ............................................................................................................... vii
CHAPTER 1: INTRODUCTION ................................................................................... 1
1.1 Glioblastoma multiforme .............................................................................. 1
1.1.1 Introduction ........................................................................................... 1
1.1.2 Treatment .............................................................................................. 2
1.1.3 Therapeutic targets for GBM treatment................................................ 3
1.1.4 Specific targeting of GBM ...................................................................... 4
CHAPTER 2: MATERIALS AND METHODS ............................................................... 9
2.1 Cell lines ........................................................................................................ 9
2.2 MTT Assay ..................................................................................................... 9
2.3 Cell viability assay ....................................................................................... 10
2.4 Tissue sectioning ......................................................................................... 10
2.5 Immunohistochemistry ............................................................................... 10

iv

CHAPTER 3: RESULTS............................................................................................. 12
PART 1: EFFECT OF INTRATUMORAL ADMINISTRATION OF BEVACIZUMAN ON
ANGIOGENESIS AND STEM CELLS ..................................................................... 12
3.1.1 Purpose of this study ............................................................................... 14
3.1.2 Results ...................................................................................................... 14
PART 2: EFFECT OF AN INFLAMMATORY FACTOR (IF) ON THE IMMUNE
RESPONSE IN GBM ........................................................................................... 19
3.2.1 Purpose of study ...................................................................................... 19
3.2.2 Results ...................................................................................................... 19
3.2.2a To determine immune cell differences in GBM tumors between IF
knockout mice and wild type with GBM ....................................................... 19
3.2.2b To determine immune cell differences in GBM tumors between mice
treated with IF inhibitors and vehicle with GBM .......................................... 26
PART 3: EFFECT OF SEROTONIN ON GBM CELLS .............................................. 31
3.3.1 Effect of serotonin on cytotoxicity on mouse glioma cells ..................... 32
3.3.2 Effect of serotonin on cytotoxicity on human GBM cells ....................... 35
3.4 Conclusion .................................................................................................. 38
CHAPTER 4: DISCUSSION ....................................................................................... 39
BIBILIOGRAPHY ..................................................................................................... 42
v

LIST OF FIGURES

Figure 1- 1: Gioblastoma Multiforme.. .............................................................. 2
Figure 1- 2: Hallmarks of cancer. ....................................................................... 4
Figure 1- 3: Pathways that connect cancer and inflammation .......................... 8
Figure 3- 1: Survival study……………………………………………………………………………13
Figure 3- 2 : SDF-1 staining .............................................................................. 15
Figure 3- 3: SOX-2 staining. .............................................................................. 17
Figure 3- 4: Nestin staining .............................................................................. 17
Figure 3- 5: CD133 staining .............................................................................. 18
Figure 3- 6: Macrophage staining. ................................................................... 20
Figure 3- 7: INOS and Arginase staining. .......................................................... 21
Figure 3- 8: CD11C staining. ............................................................................. 22
Figure 3- 9: Granulocyte staining. .................................................................... 24
Figure 3- 10: B cell staining. ............................................................................. 25
Figure 3- 11: NK cell staining. ........................................................................... 25
Figure 3- 12: Macrophage staining. ................................................................ 27
Figure 3- 13: INOS and arginase staining. ........................................................ 29
Figure 3- 14: CD11C staining. ........................................................................... 30
Figure 3- 15: Effect of serotonin on GBM-24 hour MTT assay. ...................... 32
vi

Figure 3- 16: Effect of serotonin on GBM-48 hour MTT assay. ....................... 33
Figure 3- 17: Effect of serotonin on GBM-72 hour MTT assay. ....................... 34
Figure 3- 18: Effect of serotonin on GBM-24 hour MTT assay. ....................... 35
Figure 3- 19: Effect of serotonin on GBM-48 hour MTT assay. ....................... 36
Figure 3- 20: Effect of serotonin on GBM-72 hour MTT assay ........................ 37

vii

ABSTRACT

In this study we have tried to understand mechanisms by which tumor
suppression of glioblastoma mutliforme (GBM) can be enhanced. To do so we analyzed
the effect of bevacizumab, an inflammatory factor (IF), and the effect of serotonin on
GBM.

Bevacizumab is a humanized monoclonal antibody derivated against vascular
endothelial growth factor (VEGF), a stimulator of angiogenesis. Bevacizumab is the
single most important therapeutic agent for glioblastoma since temozolomide.
Bevacizumab has been shown to increase the progression free survival by 4 months but
is known to have side effects like hypertension, renal failure and deep venous
thrombosis. In our study we determined whether there are differences in angiogenesis
and stem cell population when bevacizumab is administered locally as compared to
systemic administration. Bevacizumab when administered locally improved survival, and
there were more cells secreting stromal derived factor-1 (SDF-1) that plays a role in
angiogenesis in the vehicle and mice treated with bevacizumab systemically. Further, we
found no differences in the number of stem cells between the mice treated with
bevacizumab locally or systemically.

viii

We further analyzed the effect of an inflammatory factor (IF) on the immune
response in GBM. We found that an increase in survival with absence or reduced levels
of IF occurred. This could be a result of increased number of macrophages in IF
knockout mice or mice treated with IF inhibitors.

Serotonin is a neurotransmitter present in the brain. We found that serotonin
can increase cell death in GBM cells as compared to serum-free medium.

1

CHAPTER 1: INTRODUCTION

1.1 Glioblastoma multiforme
1.1.1 Introduction
Tumors that arise from glial cells of the brain are called gliomas. Gliomas
represent approximately 40% of primary brain tumors. The most frequently seen
gliomas in the clinical setting are astrocytomas, oligodendrogliomas, and
oliogoastrocytomas. Astrocytomas are further subdivided by a four-tiered grading
system based on the World Health Organization (WHO) classification, where the most
benign are designated as grade I, and the most malignant and aggressive are assigned
higher grades of III and IV (Louis et al., 2007). Based on the histopathological features
like regions of necrosis, pseudopalisading tumor cells, and increased blood vessels,
glioblastoma mutliforme GBM (grade IV astrocytoma) are the most malignant brain
tumors (Homma et al., 2006). These patients have a median survival of approximately
one year (Ohgaki & Kleihues, 2007) from the time of diagnosis. Glioblastoma multiforme
(GBM) is an extremely invasive, well-vascularized tumor believed to be of astroglial
origin (Davis, Freels, Grutsch, Barlas, & Brem, 1998).

Primary and secondary glioblastoma constitute distinct disease subtypes,
affecting patients of different age and developing through different genetic pathways.
The vast majority of glioblastomas (~90%) develop rapidly de novo in elderly patients,
2

without clinical or histologic evidence of a less malignant precursor lesion (primary
glioblastomas). Secondary glioblastomas progress from low-grade diffuse astrocytomas
or anaplastic astrocytoma (Ohgaki & Kleihues, 2007).

Figure 1- 1: Gioblastoma Multiforme. A) MRI scans of a glioblastoma patient’s
histological appearance of grade IV GBM. B) Histological appearance of low grade
glioma. C) Histological appearance of high grade glioma. (Figure adapted from
www.mir.wustl.edu and Decker, 2003).

1.1.2 Treatment
The object of treatment for GBM is to improve the quality of life and increase
the survival time. Treatment includes surgery, radiation therapy, and chemotherapy.
The first stage of treatment involves surgery, following which patients receive radiation
A B
C
3

along with temozolomide chemotherapy (Nagasawa et al., 2012). Temozolomide works
by causing DNA alkylation and subsequent tumor cytotoxicity as well as sensitizing
tumor cells to radiation(Anton, Baehring, & Mayer, 2012). These treatments only
increase survival for a few months. Treating GBM is difficult because the tumor cells
become resistant to temozolomide (Cho et al., 2012). This causes recurrence of GBM,
which is very aggressive. Most drugs do not cross the drug brain barrier, thus limiting
the treatment options once resistance to temozolomide occurs. Thus, there is an
increasing need for alternate chemotherapeutic treatments to treat GBM.

1.1.3 Therapeutic targets for GBM treatment
Novel therapies are designed to target pathways by blocking oncogenic
mechanisms by either inhibiting downstream signaling pathways that will inhibit tumor
growth and progression, or by altering ligand-receptor interaction to down-regulate
oncogenic effects (Wick et al., 2011). Various enzymes are now known to be involved in
GBM. The most common gene amplifications in GBM were the enzymes having tyrosine
kinase activity, such as epidermal growth factor receptor (EGFR) and platelet-derived
growth factor receptor (PDGFR)α-a promoter of cell cycle progression- cyclin dependent
kinase 4 (CDK4) and suppressors of P53 activity(Bigner et al., 1988),(Hayashi et al.,
1997). The most frequently mutated genes in GBM were found to be Phosphatase and
tensin homolog (PTEN), P53, EGFR, nuclear factor (NF-1), PIK3R1 and PIK3CA—2
components/regulators of the Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)
signaling pathway (Wick et al., 2011). Identification of the genes that are altered in
4

GBM has made it more efficient to design drugs that can target these genes that are
amplified or deleted to reverse their action and slow cancer progression by increasing
specificity.
1.1.4 Specific targeting of GBM
In an excellent review, Weinberg and Hanahan outlined the basic characteristics
that define malignant tumors. These properties include tissue invasion and metastasis,
evasion of apoptosis, sustained angiogenesis, a limitless replication potential,
insensitivity to growth inhibitors, and self-sufficiency in growth signals (Hanahan &
Weinberg, 2011). In addition to these six properties, Mantovani proposed an additional
property of inflammation as a pro-tumor characteristic.

Figure 1- 2: Hallmarks of cancer. (Adapted from Mantovani, 2008)

5

Self- sufficiency in growth signals: To move to an active proliferating state from a
quiescent state normal cells require mitogenic growth signals. Tumor cells show much
reduced dependence on exogenous growth signals for cell proliferation (Hanahan &
Weinberg, 2011) . Cancer cells develop the ability to synthesize growth factors which
are mediated by autocrine stimulations. For instance, GBM cells produce platelet
derived growth factor (PDGF) and Tumor growth factor (TGF α) growth factors known to
stimulate cell proliferation.
Insensitivity to anti-growth signals: In addition to pro-replication growth factors,
there are cytokines that down-regulate or inhibit tumor cell proliferation (REF). The
mechanism of blocking cell proliferation could be by either forcing the cells out of the
proliferative cycle into the quiescent (G0) state or by inducing the cells into post-mitotic
states. These anti-proliferative signals are known mainly to be modulated by
hypophosporylated retinoblastoma protein (pRb). E2F transcription factor controls
progression of cells from G1 to S phase. This transcription factor is modulated by pRb. In
tumors, it was found that this pathway is disrupted rendering cells insensitive to
antigrowth factors (Weinberg, 1995).
Resistance to apoptosis: To maintain a functional cellular homeostasis, it is very
essential that there is a balance between cell proliferation and cell death. In cancer a
resistance to apoptosis is acquired, which disrupts cellular homeostasis. Mutations and
defects in p53 tumor suppressor genes (Levine, 1997), the PI3 kinase –AKT/PKB
pathway (Evan & Littlewood, 1998) and the pTEN tumor suppressor gene (Cantley &
6

Neel, 1999), are commonly found in cancers. These genes serve as important targets in
cancer treatment. This will have implications on cellular death leading to reduced
tumorgenicity.
Limitless replication potential: Normal cells replicate in a finite manner. Resistance
to apoptosis, insensitivity to anti-growth signals, and self-sufficiency in growth signals,
leads to uncontrollable replication of cells. Furthermore, telomerase has been shown to
be involved in limitless replication of tumor cells. Telomerase is known to be up-
regulated in 80% of the cancers, and this helps maintaining the telomeres(Shay &
Bacchetti, 1997) . When telomeres are maintained above a certain threshold, it leads to
unlimited multiplication of the descendant cells (Hanahan & Weinberg, 2011). Novel
anti-telomerase therapies are thus emerging as a potential cancer treatment (Agrawal,
Dang, & Gabrani, 2012)
Tissue invasion and metastasis: When primary tumor cells spread to other tissues
by invasion, they form new colonies and often become unresponsive to cancer
treatment highly (Hanahan & Weinberg, 2011). A critical component of tissue invasion
in malignant tumors is the alteration of specific malignant tumor proteins that are
involved in cell tethering cells to surrounding tissues. These proteins include E-cadherin,
a molecule expressed on epithelial cells that cause homotypic cell-to-cell interaction
(Christofori & Semb, 1999), cell-cell adhesion molecules (CAMs), and intergrins.
Targeting invasion and preventing it is an important avenue of cancer therapy.
Currently, drugs like Contortrostatin that work by disrupting and preventing the binding of
7

integrins to the extracellular matrix, are being studied for their anti-invasive effects on
gliomas (Schmitmeier, Markland, & Chen, n.d.).
Sustained Angiogenesis: Angiogenesis refers to the formation of blood vessels
from pre-existing vessels (Nishida, Yano, Nishida, Kamura, & Kojiro, 2006). Increased
blood vessel formation leads to tumor progression, as blood vessels provide the
necessary nourishments needed for tumor cell replication. In normal physiology the
formation of blood vessels is tightly regulated. One of the most important stimulators
of angiogenesis is VEGF (Vascular endothelial growth factor or vascular permeability factor).
VEGF functions by stimulating endothelial cell proliferation, migration and increasing blood
vessel permeability (Goel & Mercurio, 2013). Anti-VEGF therapeutic drugs have been extensively
studied as a potential therapy for different types of cancer. Two FDA approved anti-VEGF
therapeutics are the humanized anti-VEGF antibody, bevacizumab (Avastin anti-VEGF
antibody) and Sunitanib (Lien & Lowman, 2008).
Inflammation: Activation of oncogenes causes the production of inflammatory
reactions. These reactions can be causes by either an extrinsic diseases such as
inflammatory bowel disease or activated by intrinsic pathway driven by genetic
alterations that leads to the activation of transcription factors like NK-kB and STAT3.
Activation of these transcription factors can stimulate production of chemokines and
cytokines, which can further lead to suppression of the adaptive immune response,
tumor cell migration, stimulation of angiogenesis, and cell proliferation (Mantovani,
2009).

8

Figure 1- 3: Pathways that connect cancer and inflammation (adapted from Mantovani,
2008)

As mentioned, angiogenesis, inflammation, and insensitivity to cell death play a
very important role in tumor progression. Therefore in our study with bevacizumab and
an inflammatory factory, we examined these two hallmarks of cancer.
Further, we also analyzed the effect of a neurotransmitter on cell death, thus targeting
the characteristic insensitivity to growth inhibitors of cancer cells.
9

CHAPTER 2: MATERIALS AND METHODS

2.1 Cell lines
The human glioma cell line, U251 and the mouse glioma cell line, GL26, were
cultured in 10% fetal calf serum (Omega Scientific) in Dulbecco’s Modified Eagle’s Media
(USC Cell Culture Core Facility) supplemented with 100 U/mL penicillin and 0.1 mg/mL
streptom sin ( S ell ulture ore Facilit ) in a humidi ed incubator at C and 5%
CO
2
.
2.2 MTT Assay
Cells were seeded in triplicate at a density of 5 X 10
3
/ 50 μL/well in 96 well
plates in DMEM 10% FCS. After 24 hours, the medium was replaced by DMEM alone.
Three hours after replacing the medium, serotonin (SIGMA Cat. No: H-7752) dissolved in
PBS (USC Cell Culture Core Facility) was added in concentrations of 1 nM, 10 nM, 100
nM and 1000 nM. After 24, 48, and 72 hours of treatment with serotonin, 10 μ L of a 5
mg/ml stock of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye
(Sigma Aldrich, St. Louis, Missouri ) was added to each well and incubated for 4 hours.
The mitochondrial enzymes in viable cells cleaved the tertazolium salts, forming a
colored formazan dye. Following this, 100 μL l sis buffer containing 10 % SDS and 0.01 %
HCl was added to each well. The following day, the absorbance was measured at 490 nm
using a microtiter plate reader (Dynatech MR4000). A plot of serotonin treated versus
untreated control was constructed using percentage viability calculated relative to the
untreated control.
10

2.3 Cell viability assay
Cells were seeded in triplicates at a concentration of 10 x 10
4
cells/mL of DMEM
10% FCS in 6 well plates. After 24 hours, the medium was replaced by DMEM alone.
Three hours after replacing the medium, serotonin dissolved in PBS was added in
concentrations of 1 nM, 10 nM, 100 nM and 1 μM. After 24, 48, and 2 hours of
treatment with serotonin, cells were trypsinized, and the live and dead cells were
counted with trypan blue using a hemacytometer. The percentage of live cells were
calculated and plotted.
2.4 Tissue sectioning
Frozen brain tissues were sectioned using a cr ostat at a temperature of -21 C.
The tissues were embedded on a microtome and fixed using optimum cutting
temperature medium (OCT) (VWR). Sectioning provides the very thin specimens needed
for cell analysis using a microscope. The tissues were sectioned at a thickness of 8 μM
and mounted on superfrost slides (VWR). A Hematoxylin test was done for a section
from each tissue to confirm the presence of tumor areas in the tissue. Sections were
fixed in acetone for 10 mins. Fixation stabilizes and preserves the tissue.
2.5 Immunohistochemistry
Frozen tissue sections were washed with PBS and blocked with Sea Block
(Thermo Scientific, Rockford, IL) for 20 mins. SEA Block buffer is fish serum; it is effective
in stabilizing samples for antibody-binding interactions and minimizes cross-reactions
with mammalian samples. Primary antibodies were kept overnight after blocking.
11

Sections were stained with F4/80 (1:50), CD11c (1:50), CD69 (1:100), iNOS (Abcam,
Cambridge, MA), GR 1 (1:50), Arginase (1:100), CD20 (1:100), Ki67 (1:100) (Santa Cruz
Biotechnology Inc., Santa Cruz), Nestin (1:100), CD133 (1:50) (EMD Millipore,
Massachusetts), SOX-2 (1:100), SDF-1 (1:100). Following this, tissues were washed with
PBS, and the biotinylated secondary antibodies (Vector Laboratories, Berlingame, MA)
were added for 45 min. The secondary antibodies were washed away with PBS, and the
tissues were incubated with avidin-biotin peroxidase complex (ABC kit, Vector
Laboratories) for 30 min and treated with amino-ethylcarbozol substrate for 10 min.
Avidin is a large glycoprotein having affinity for biotin and can be labeled with
peroxidase. Biotin is a low molecular weight vitamin conjugated to the secondary
antibody. Adding ABC elite produces a complex of avidin with biotin and the peroxidase
can be developed by the substrate to produce colored end products. Tissues were then
counterstained with hematoxylin for 2 min; the red precipitate identifies positive
staining. Controls included no primary antibody. Images were photographed at 200X
and analyzed using ImageJ software.

12

CHAPTER 3: RESULTS
PART 1: EFFECT OF INTRATUMORAL ADMINISTRATION OF
BEVACIZUMAN ON ANGIOGENESIS AND STEM CELLS

Bevacizumab, in combination with irinotecan, is currently an FDA approved
treatment for recurrent GBM. Bevacizumab works by binding to soluble VEGF receptors,
thus reducing subsequent signaling processes that leads, to angiogenesis (Ma &
Waxman, 2008). A significant improvement in progression-free survival (PFS) and
overall survival (OS) as compared to historic controls was observed in patients with
recurrent GBM treated with bevacizumab (Nghiemphu et al., 2009). To improve the
effect of bevacizumab, it is currently being administered in combination with Irinotecan,
a topoisomerase-1 inhibitor that prevents unwinding of DNA (Pommier, Leo, Zhang, &
Marchand, 2010). It was found that bevacizumab alone or in combination with
irinotecan posed certain clinical problems like increased risk of renal toxicity, poor
wound healing, deep venous thrombosis, and hypertension (Chamberlain, Lassman, &
Iwamoto, 2010).
To test whether bevacizumab improves survival when administered
intratumorally rather than a systemically, an in vivo experiment was performed by
Dr.Weijun Wang (Department of Neurosurgery, USC) to study the effect of local
administration of bevacizumab and irinotecan as compared to systemic administration.
Human glioma cells U8251 were intracranially implanted into athymic nude mice, and
the mice were grouped as follows- 1) vehicle; 2) IV bevacizumab; 3) IT bevacizumab; 4)
13

IP irinotecan; 5) IV bevacizumab + irinotecan; 6) IT bevacizumab + irinotecan.
Bevacizumab was administered either by IV injection at 10 mg/kg/once every 14 days
(two injections in 28 days), or continuously delivered by direct IT infusion (Alzet micro-
osmotic pump) for 28 days, resulting in a total dose of bevacizumab of 0.8 mg. The
irinotecan was administered IP on days 1–5 and 8–12 at a dose of 12 mg/m2 or 4 mg/kg
(in saline). After treatment was completed (28 days), animals were maintained without
any treatment until they died. Animals treated with vehicle received saline IT using the
Alzet micro-osmotic pump.

Figure 3- 1: Survival study of tumor-bearing mice treated with Intratumoral (IT) or
intravenous (IV) bevacizumab as a monotherapy or in combination with irinotecan (CPT-
11)

Dr. Weijun Wang, unpublished
14

Kaplan Meier survival plotted by Dr.Weijun Wang revealed that Intratumoral
administration significantly (p < 0.002) increased animal survival as compared to
intravenous drug delivery. Survival of combination therapy of IT bevacizumab was also
significantly increased (p < 0.02) as compared to IV bevacizumab in combination with
irinotecan (CPT-11)

3.1.1 Purpose of this study
In our study we hypothesized that intratumoral delivery of bevacizumab alone or
in combination with irinotecan are more effective because they are more effective in
reducing VEGF. Thereby, there is a greater reduction in angiogenesis and stem cell
populations as compared to the systemic delivery of these drugs.

3.1.2 Results
Tissues were stained for SDF-1 Stromal derived factor, also known as C-X-C motif
chemokine 12 (CXCL12), a chemokine. CXCL12 plays a role in angiogenesis by recruiting
endothelial progenitor cells (EPCs) from the bone marrow (Guyon, 2014). CXCL12 also
has a role in tumor metastasis where cancer cells that express the receptor CXCR4 are
attracted to metastasis target tissues that release the ligand, CXCL12 (Müller et al.,
2001). Tissues were stained with SDF at 1:100 concentrations and tumor sections and
the microenvironment was analyzed.
15

We found the cell secreting SDF-1 were present in the tumor microenvironment
of the vehicle and mice treated with intravenous bevacizumab while very few positive
cells were found in intratumorally treated bevacizumab.

Figure 3- 2 : Images represent tumor microenvironment of SDF-1 staining at 200X. A)
Vehicle. B) Tissue section of mice treated with Intravenous bevacizumab C) Tissue
section of mice treated with intratumoral bevacizumab

To determine whether there is a difference in the cancer stems cells between mice
treated with bevacizumab delivered intratumorally or intravenously alone or in
combination with irinotecan, tissues were stained for SOX-2, Nestin and CD133. SOX-2 is
a transcription factor that is essential for maintenance of self-renewal. An increased
level of SOX-2 is associated with tumor cell proliferation and increased infiltrations
(Berezovsky et al., 2014). Nestin is an intermediate filament protein mostly expressed
on nerve cells and is a marker for neural stem cells, proliferation and migration (Jin, Jin,
Jung, Beck, & Kim, 2013). CD133 is a glycoprotein that is known to be expressed in
A.Vehicle B.IV bevacizumab C.IT bevacizumab
16

hematopoietic stem cells, endothelial progenitor cells,
glioblastoma, neuronal and glial stem cells (Sanai, Alvarez-Buylla, & Berger,
2005)(Corbeil et al., 2000)

Vehicle IV bevacizumab IT bevacizumab

-
C
PT
11
+C
PT
11
CPT-11 IV bevacizumab IT bevacizumab

IT Avastin
IV bevacizumab+ CPT-11 IT bevacizumab+CPT-11
A)
B)
17

Figure 3- 3: A) Tumor cell images of tissue sections stained for SOX-2. B) Images
represent stem cells infiltrating into the normal parenchymal cells.

Figure 3- 4: Tumor cell images of tissue sections stained for Nestin (200X)

Vehicle IT bevacizumab IV bevacizumab
- CTP 11
CPT 11
18

Figure 3- 5: Tumor cell images of tissue sections stained for CD133 (200X)

There were no differences in the number of stem cells with bevacizumab (Avastin) as a
monotherapy or in combination with irinotecan (CPT 11) given locally as compared to
the systemic administration of these drugs.

Vehicle IV bevacizumab IT bevacizumab
- CTP 11
CPT 11
19

PART 2: EFFECT OF AN INFLAMMATORY FACTOR (IF) ON
THE IMMUNE RESPONSE IN GBM

Effects of an inflammatory factor (IF) were examined on brain tumor progression
using GBM tumor-bearing mice that were genetically ablated or chemically depleted of
IF. Dr.Weijun Wang documented survival of these mice. The results demonstrated that
IF knockout or inhibition resulted in improved survival of mice with GBM
3.2.1 Purpose of study
Based on these data from Dr.Wang, we hypothesized that an improved survival
in the IF knockout or IF inhibited mice compared to the wild type may be due to the
stimulation of an anti-tumor response. To determine whether this is so, we evaluated
the numbers of macrophages, dendritic cells, granulocytes, B cells and natural killer cells
in the different groups

3.2.2 Results
3.2.2a To determine immune cell differences in GBM tumors between IF knockout mice
and wild type with GBM

Macrophages are extremely important in determining the kind of immune
responses elicited by the tumor environment. Staining for macrophages was done using
20

F4/80, a mouse analogue of human EMR1, which is a transmembrane protein present
on cell surfaces of human and murine macrophages.

Figure 3- 6: Macrophage Staining. Tumor Images from wild type and IF knockout 200 X.
Red precipitations identifies the positive cells. Arrow points to a positive cell A)
Macrophage staining Image in Wild type tissue. B) Macrophage staining Image in IF
knockout tissue. C) ImageJ analysis of typical area of macrophages present in tumors of
wild type and IF knockout

A. Wild type B. IF knockout
0
500
1000
1500
2000
2500
Wild type IF knockout
Area μM
C. Macrophage Staining
p< 0.005
21

The results show a significant (p<0.005) increase in the number of macrophages
in the IF knockout GBM tumor tissue as compared to the wild type.
The macrophages can be M1 polarized (pro-inflammatory/anti-tumorigenic) or
M2 polarized (anti-inflammatory/pro-tumorigenic) (Martinez & Gordon, 2014). To
analyze the type of polarization, tissues were stained for arginase to determine M2
polarized macrophages and for inducible nitric oxide synthase to determine M1
polarized macrophages.

Figure 3- 7: INOS and Arginase staining. This staining are is cytoplasmic. Tumor
image (200 X). A) Wildtype/Arginase staining. B) IF knockout/Arginase staining.
C) Wild type/INOS staining. D) IF knockout/INOS staining.
A.Wild type
B.IF Knockout
C.Wild type
D. IF Knockout
Arginase staining INOS staining
22

The staining results show no significant difference in the number and distribution of M1
or M2 macrophage populations in the IF deleted mice as compared to wild type.
We next examined dendritic cells which are antigen presenting cells using the marker
CD11C.Tissues were stained for CD11C, a transmembrane protein found in high
amounts on the surface of dendritic cells.

Figure 3- 8: CD11C Staining. Tumor images (200 X) in wild type and IF knockout (200
da s’ old). Red precipitation implies positive cells. Arrow points to a positive cell. A)
CD11C staining image in Wild Type. B) CD11C staining image in IF knockout. C) ImageJ
analysis of average area of dendritic cells present in wild type and IF knockout.

A. Wild Type
B. IF knockout
0
100
200
300
400
500
600
700
800
900
Wild type IF knockout
Area μM C. CD11 C Staining
23

There were no significant differences in the number of CD11C positive cells present in
the wild type as compared to the IF knockout groups though there was a trend of
increased CD11C positive cells in the wild type.
To examine the role of granulocytes in the IF knockout compared to wild type,
granulocytes were stained using Gr1, a myloid differentiation marker expressed
predominantly on granulocytes. This antigen is expressed on monocytes in the bone
marrow. The level of antigen expression correlates with granulocyte differentiation and
maturation; the more intense staining is expressed on more mature granulocytes.
The results show that very few positive cells were present in both the wild type
and IF knockout mice. The range of granulocytes in the wild type was great; thus there
was no significant difference observed in the number of granulocytes present between
the wild type (WT) and IF knockout groups although the trend showed more
granulocytes in the WT.

24

Figure 3- 9: Granulocyte Staining. Tumor images (200 X) in wild type and IF knockout
(200 da s’ old). Red precipitation implies positive cells. Arrow points to a positive cell A)
GR-1 staining image in Wild type. B) GR-1 staining image in IF knockout. C) ImageJ
analysis of average area of granulocytes present in wild type and IF knockout

Antibody- forming B cells are shown to regulate tumor growth (Nelson, 2010).
We therefore stained these GBM tumors for B cells using CD20 antibody, a glycosylated
A. Wild type B.IF knockout
0
50
100
150
200
250
300
Wild Type NOBO knockout
Area μM
Granulocyte Staining
25

phosphoprotein present on the surface of B cells. Tumors were analyzed. The results
showed that no B cells were observed in the wild type or knockout. Mice spleens used
as a control were positive.

Figure 3- 10: B cell Staining. Tumor images (200 X) of wild type and IF knockout mice
(200 da s’ old). A) B cell staining image in Wild t pe. B) B cell staining image in IF
knockout. C) Positive control-Mice spleen

Natural killer cells were analyzed in GBM tissues using CD 69.

Figure 3- 11: NK cell Staining. Tumor images (200 X) of wild type and IF knockout mice
(200 da s’ old). A) B cell staining image in Wild t pe. B) B cell staining image in IF
knockout. C) Positive control- Mice spleen

A. Wild type
B. IF knockout C. Spleen
A. Wild type B. IF knockout C. Sleen
26

No natural killer cells were detected in the wild type or IF knockout. Mouse spleen was
used as a positive control.

To summarize, we found a significant increase in the number of macrophages in the IF
knockout as compared to the wild type. There was an increase in the number of
granulocytes and dendritic cells in the wild type as compared to the IF KO though this
was not significant. There were no apparent B cells and NK cells present in either the
wild type or IF KO.

3.2.2b To determine immune cell differences in GBM tumors between mice treated with
IF inhibitors and vehicle with GBM

Staining for macrophages was done on GBM tumor tissues of animals treated with IF
pharmacological inhibitor-1 and pharmacological inhibitor-2 or vehicle.
27

Figure 3- 12: Macrophage staining. Tumor images (200 X ) in treated mice. A) F4/80
staining of vehicle treated mice. B) F4/80 staining of tissue in mice treated with IF
inhibitor 1. C) F4/80 staining of tissues in mice treated with IF inhibitor 2. D)
Macrophage staining analysis using imageJ
A. Vehicle
B. IF inhibitor-1 Treated C. IF inhibitor-2 Treated
0
500
1000
1500
2000
2500
3000
3500
Vehicle IF inhibitors 1 IF inhibitor 2
Area μM
D) Macrophage Staining Analysis
28

IF inhibitor 1 and 2 showed a significant increase (p< 0.005) in the number of
macrophages compared to the vehicle.
To determine the kind of macrophages groups, vehicle and mice treated with IF
inhibitors 1 and 2 tissues were stained for INOS and arginase.

A.Wild type
C.IF inhibitor-1 treated
D.IF inhibitor-1 treated

E. IF inhibitor-2 treated

F. IF inhibitor-2 treated
B. Wild type
Arginase staining INOS staining
29

Figure 3- 13: INOS and arginase staining. These staining are cellular as we all
cytoplasmic stains. Tumor image (200 X). A) Vehicle-Arginase staining B) Vehicle-
INOS staining. C) IF inhibitor -1 treated mice-arginase staining. D) IF inhibitor -1
treated mice- INOS staining. E) IF inhibitor -2 treated mice- Arginase staining. F)
IF inhibitor -2 treated mice- INOS staining.

Analysis of these tumor tissues showed that there were no significant difference
between INOS and arginase staining in the vehicle and IF inhibitors treated mice.

To determine whether there were differences in the number of dendritic cells present in
the mice treated with IF inhibitor 1 and 2 as compared to the vehicles, tumor sections
were stained for CD11C.

30

Figure 3- 14: CD11C staining. Tumor images (200 X) in treated mice. A) CD11C
staining of vehicle. B) CD11C staining of IF inhibitor-1 treated mice. C) CD11C
staining in IF inhibitor-2 treated mice. D) CD11C staining analysis of average area
of CD11C staining using imageJ

A. Vehicle
B. IF inhibtior-1 Treated
C. IF inhibtior-2 Treated
0
100
200
300
400
500
600
Vehicle IF Inhibitor-1 IF inhibitor-2
Area μM
D) CD11C Staining
31

No significant difference was observed between the IF inhibitor 1 and 2 treated mice
and the vehicle.

To summarize, we demonstrated that mice bearing intracranial GBM tumors when
treated with IF inhibitors 1 and 2 showed increased numbers of macrophages as
compared to the vehicle. However these were no significant differences in the numbers
of dendritic cells among these groups.

PART 3: EFFECT OF SEROTONIN ON GBM CELLS

Serotonin is a neurotransmitter present in the brain. Based on the serotonin
receptors present on the cells, serotonin can have a pro-proliferative or anti-
proliferative effect (Liang et al., 2013). We therefore tested glioma cells for their
response with serotonin. Concentrations of serotonin added to Gl26 and U251 cells
were 1nM, 10nM, 100nM and 1 uM for 24, 48 and 72 hours. The cells were seeded in
triplicates in DMEM containing 10% FBS. After 24 hours, the medium was replaced by
serum free DMEM, and after two hours, serotonin was added to the cells. Serum free
medium was used as baseline because there is no serotonin in medium alone.

32

3.3.1 Effect of serotonin on cytotoxicity on mouse glioma cells
24 Hour proliferation Gl26 cells
A. SFM B. 100 nM serotonin

Figure 3- 15: Effect of serotonin on GBM. GL26 cells at different doses were treated
with serotonin for 24 hour. A) GL26 cells in Serum free medium (SFM). B) 100 nM
serotonin added to GL26 cells. C) Summary of data 24 Hours MTT assay of GL26 cells

0
20
40
60
80
100
120
140
SFM 1 nM 10 n M 100 nM 1 um
% of cell survival
C. GL26 24 Hour MTT Assay
33

48 Hour proliferation assay of GL26 cells
A. SFM B. 100 nM serotonin

Figure 3- 16: Effect of serotonin on GBM. GL26 cells at different doses were treated with
serotonin for 48 hour. A) GL26 cells in Serum free medium (SFM). B) 100 nM serotonin
added to GL26 cells. C) Summary of data 48 Hours MTT assay of GL26 cells

0
20
40
60
80
100
120
SFM 1 nM 10 n M 100 nM 1 um
% of cell survival
C. GL26 48 Hour MTT Assay
34

72 Hour proliferation assay of Gl26 cells
A.SFM B. 100 nM serotonin

Figure 3- 17: Effect of serotonin on GBM. GL26 cells at different doses were treated with
serotonin for 72 hour. A) GL26 cells in Serum free medium (SFM). B) 100 nM serotonin
added to GL26 cells. C) Summary of data 72 Hours MTT assay of GL26 cells

These preliminary results revealed that after 24 hours with a serotonin
concentration of 100 nM, there was significant (p<0.001) level of cell death as compared
to serum free medium alone. At 48 hours the number of dead cells with 100 nM
serotonin increased as compared to 24 hour assay but was still significantly (p< 0.01)
less than serum free medium. At 72 hours there was again a significant (p<0.001)
0
20
40
60
80
100
120
SFM 1 nM 10 n M 100 nM 1 um
% cell survival
C) GL26 72 Hour MTT Assay
35

increase in the number of dead cells at a concentration of 100 nM serotonin as copared
to serum free media alone.
3.3.2 Effect of serotonin on cytotoxicity on human GBM cells
24 Hour proliferation assay of U251 cells
A. SFM B. 100 nM 5 HT

Figure 3- 18: Effect of serotonin on GBM. U251 cells at different doses were treated with
serotonin for 24 hour. A) U251 cells in Serum free medium (SFM). B) 100 nM serotonin
added to U251 cells. C) Summary of data 24 Hours MTT assay of U251 cells.

0
20
40
60
80
100
120
140
SFM 1 nM 10 n M 100 nM 1 um
% of cell survival
C) U251 24 Hour MTT Assay
36

48 Hours proliferation assay of U251 cells
A. SFM B. 100 nM 5HT

Figure 3- 19: Effect of serotonin on GBM. U251 cells at different doses were treated with
serotonin for 48 hour. A) U251 cells in Serum free medium (SFM). B) 100 nM serotonin
added to U251 cells. C) Summary of data 48 Hours MTT assay of U251 cells.

0
20
40
60
80
100
120
140
SFM 1 nM 10 n M 100 nM 1 um
% of cell survival
C. U251 48 Hour MTT Assay
37

72 Hour proliferation assay cells
A.SFM B. 100 nM 5H

Figure 3- 20: Effect of serotonin on GBM. U251 cells at different doses were treated with
serotonin for 48 hour. A) U251 cells in Serum free medium (SFM). B) 100 nM serotonin
added to U251 cells. C) Summary of data 48 Hours MTT assay of U251 cells.

In these preliminary results we observed that at 24 hours, there were no
significant difference between cells containing serotinin and serum free medium. At 48
and 72 hours, the number of cells treated with 100 nM serotonin was significantly
(p<0.001) reduced as compared to serum free medium only.

0
50
100
150
SFM 1 nM 10 n M 100 nM 1 um
% cell survival
C. U251 72 Hour MTT Assay
38

3.4 Conclusion

In our study we found that there were increased numbers of cells secreting SFD-
1 in the vehicle and mice treated with bevacizumab systemically as compared to mice
treated locally. There were no significant differences in SOX-2, nestin, or CD133
between these groups.

We also found that a there were a significantly increased number of
macrophages in mice with absence and reduced levels of an inflammatory factor (IF).
There were no difference in the number of dendritic cells and granulocytes, thought
there was an increased trend in the wild type as compared to IF knockout mice.

Serotonin has a pro-cytotoxic effect on human glioma cells. At a concentration of
100 nM serotonin caused the most significant increase in the death of U251 and Gl26
cells as compared to serum-free medium.

39

CHAPTER 4: DISCUSSION

Tumor heterogeneity makes treatment difficult because different cell
populations respond differently to treatment. Identifying these subpopulations should
greatly increase GBM treatment (Altaner, 2008) Small populations of GBM cells also
include cancer stem cells (CSC). CSC can lead to highly aggressive tumors due to
resistance to treatment. GBM cells that consist of CSC markers are known to be more
resistant to temozolomide and radiation. As mentioned earlier, angiogenesis also leads
to increased tumorigenicity. In our study we tried to determine whether an improved
survival with local administration of bevacizumab was a result of decreased
angiogenesis and a decreased number of stem cells. There was an increase in the
number of cells secreting SDF-1 in the vehicle and mice treated with bevacizumab
systemically, suggesting that there may be increased angiogenesis in these groups as
compared to mice treated intratumorally. Further analysis of angiogenesis in these
groups will perhaps reveal more confirmatory results. We did not find any significant
differences in the number of stem cells between bevacizumab administered systemically
or locally alone or in combination with irinotecan.

To understand the mechanism involved in the increased survival of IF knockout
or IF inhibited mice we studied the difference in the immune response between the
knockouts, inhibitor treated groups, and the controls. Our data revealed that there were
significantly increased numbers of macrophages in the knockout and inhibitor-treated
40

mice as compared to the controls. This could be one of the mechanisms that lead to
delayed tumor progression in these groups. Macrophages can be classified as M1 and
M2 based on the immune response they stimulate. Classical or M1 macrophage
activation in response to microbial products or interferon-γ is characterized b : a) high
capacity to present antigen, b) high interleukin-12 (IL-12) and IL-23 production, c)
consequent activation of a polarized type I response and d ) high production of toxic
intermediates [nitric oxide (NO), reactive oxygen intermediates (ROI)]. M1 macrophages
are generally considered potent effector cells which kill microorganisms and tumor cells
and produce high amounts of pro-inflammatory cytokines (Martinez & Gordon, 2014). In
contrast, various signals (e.g. IL-4, IL-13, glucorticoids, IL-10, immunoglobulin
complexes/TLR ligands) induce distinct M2 functions, able to tune inflammatory
responses and adaptive Th2 immunity, scavenge debris, promote angiogenesis, tissue
remodeling and repair (Sica et al., 2008) . The INOS/arginase ratio in our study was
inconclusive. Further investigations based on M1 and M2 markers could give
confirmatory results. Various therapies are targeting the inhibition of M2 macrophages
for treatment as anti-angiogenic factors. Drugs that target immune response and elicit a
pro-inflammatory response are beneficial since this leads to an anti-tumor activity.

Serotonin is known to have an anti-tumorigenic or pro-tumorigenic based on the
receptors present on the cells (Manda, Nishigaki, Mori, & Shimomura, 1988), (Dizeyi et
al., 2004). Our results showed that serotonin had a pro-cytotoxic effect on GBM cells.
Understanding the serotonin receptors present on GBM cells will help develop a more
41

clinically relevant approach in treatment of GBM. One of the hallmarks of cancer is
insensitivity to growth inhibitors (Hanahan & Weinberg, 2011). By understanding the
mechanism by which serotonin causes cell death, we can target this pathway to
overcome the cancer cell’s insensitivit to growth inhibitors. Serotonin can also play a
role in macrophage polarization based on the cell receptors present. For instance
Serotonin modulates macrophage polarization and contributes to the maintenance of
an anti-inflammatory state via 5HT 2B and 5HT7(de las Casas-Engel et al., 2013).
Serotonin inhibits the LPS-induced release of proinflammatory cytokines without
affecting IL-10 production, upregulates the expression of M2 polarization-associated
genes (de las Casas-Engel et al., 2013) where as in presence of a 5HT1A receptor
serotonin causes the activation of inflammatory macrophages (Manéglier et al., 2008).
Thus, serotonin can play a role in proliferation and polarization of macrophages.

42

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Abstract (if available)

Abstract In this study we have tried to understand mechanisms by which tumor suppression of glioblastoma mutliforme (GBM) can be enhanced. To do so we analyzed the effect of bevacizumab, an inflammatory factor (IF), and the effect of serotonin on GBM. ❧ Bevacizumab is a humanized monoclonal antibody derivated against vascular endothelial growth factor (VEGF), a stimulator of angiogenesis. Bevacizumab is the single most important therapeutic agent for glioblastoma since temozolomide. Bevacizumab has been shown to increase the progression free survival by 4 months but is known to have side effects like hypertension, renal failure and deep venous thrombosis. In our study we determined whether there are differences in angiogenesis and stem cell population when bevacizumab is administered locally as compared to systemic administration. Bevacizumab when administered locally improved survival, and there were more cells secreting stromal derived factor-1 (SDF-1) that plays a role in angiogenesis in the vehicle and mice treated with bevacizumab systemically. Further, we found no differences in the number of stem cells between the mice treated with bevacizumab locally or systemically. ❧ We further analyzed the effect of an inflammatory factor (IF) on the immune response in GBM. We found that an increase in survival with absence or reduced levels of IF occurred. This could be a result of increased number of macrophages in IF knockout mice or mice treated with IF inhibitors. ❧ Serotonin is a neurotransmitter present in the brain. We found that serotonin can increase cell death in GBM cells as compared to serum-free medium.

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Asset Metadata

Creator Vaikari, Vijaya Pooja (author)

Core Title Molecular targets for treatment of glioblastoma multiforme

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 10/14/2016

Defense Date 07/29/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag angiogenesis,bevacizumab,Dendritic cells,glioblastoma multiforme,glioblastoma multiforme (GBM) treatment,macrophages M1 and M2,OAI-PMH Harvest,serotonin,temozolomide

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Hofman, Florence M. (committee chair), Landolph, Joseph R., Jr. (committee member), Schönthal, Axel H. (committee member), Shih, Jean C. (committee member)

Creator Email pooja.vaikari@gmail.com,vaikari@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-506490

Unique identifier UC11297830

Identifier etd-VaikariVij-3013.pdf (filename),usctheses-c3-506490 (legacy record id)

Legacy Identifier etd-VaikariVij-3013.pdf

Dmrecord 506490

Document Type Thesis

Format application/pdf (imt)

Rights Vaikari, Vijaya Pooja

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA