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Interleukin-11: a study of its effects in glioblastoma multiforme

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Interleukin-11: a study of its effects in glioblastoma multiforme

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INTERLEUKIN-11: A STUDY OF ITS EFFECTS IN GLIOBLASTOMA MULTIFORME

by

Zhi Liu

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

December 2010

Copyright 2010 Zhi Liu

ii
DEDICATION
I would like to dedicate this work to my parents, who gave me so much love and
care and strengthened me no matter where I was. Through their support, I learnt to
appreciate life and always live it in a positive way.

iii
ACKNOWLEDGEMENTS
I would like to bring my gratitude to my PI Dr. Florence Hofman, who opened the
door of science to me and showed me how fascinating it was. Her guidance, help and
patience inspired me to work hard and do my best. I express my appreciation for my
committee members, Dr. Zoltan Tokes, Dr. Yves De Clerck, and Dr. Thomas Chen, for
their advice and support. I thank the members of the glioma research group, especially
Dr. Axel Schönthal, Dr. Stan Louie and Dr. Stan Tahara for their valuable opinions on my
research. I bring my appreciation to my previous and current lab members, particularly
Ms. Ligaya Pen, who taught me solid lab skills and always kept me company; Dr. Jenilyn
Virrey, who was encouraging me, sharing her precious experiences and my dear friend,
together we had a great time; Dr. Fabienne Agasse, Dr. Heeyeon Cho, Ms. Niyati Jhaveri
and Mr. Christopher Stapleton. I also appreciate the help from Ms. Anne Rice and Ms.
Lisa Doumak. And last but not least, I want to thank all my friends I had made during my
study in USC, with who graduate study life became so fantastic!
iv
TABLE OF CONTENTS
Dedication ii
Acknowledgements iii
List of Figures v
Abbreviations vi
Abstract viii
Chapter I – Introduction 1
- Angiogenesis and the Tumor Vasculature 1
- Glioblastoma Multiforme 10
- Interleukin-11 16
- Purpose of Study 22

Chapter II – Materials and Methods 24
- Histological Studies 24
- Functional Studies 29
Chapter III – Results and Conclusion 33
- Experimental Results 33
- Conclusion 49

Chapter IV – Discussion 51

Bibliography 58
v
LIST OF FIGURES
Figure 1: The structure of an artery 2
Figure 2: The angiogenic process 4
Figure 3: Models of tumor angiogenesis 7
Figure 4: Normal and tumor vasculatures 9
Figure 5: GBM tissue sections 12
Figure 6: Morphologies of TuBEC and BEC 15
Figure 7: IL-11Rα-gp130 signaling pathway 20
Figure 8: BEC, TuBEC and glioma cells expressed IL-11 34
Figure 9: IL-11 expression levels in normal brain and GBM tissues 37
Figure 10: BEC and TuBEC expressed IL-11Rα in vitro and in vivo 40
Figure 11: Effects of IL-11 on BEC and TuBEC migration 44
Figure 12: Effects of IL-11 on U87 cell migration and invasion 45
Figure 13: Effects of IL-11 on cell proliferation of BEC 46
Figure 14: Effects of IL-11 on cell proliferation of TuBEC 47
Figure 15: Effects of IL-11 on cell proliferation of U87 cells 48
Figure 16: IL-11 in the normal brain and GBM environment 49
vi
ABBREVIATIONS
Ang-1: Angiopoietin-1
Ang-2: Angiopoietin-2
BBB: Blood-Brain-Barrier
BEC: Brain-associated Endothelial Cells
bFGF: basic Fibroblast Growth Factor
BrdU: Bromodeoxyuridine
di-acet-LDL: deacetylated Low-Density Lipoprotein
ECM: Extracellular Matrix
EGF: Epidermal Growth Factor
Epo: Erythropoietin
ET-1: Endothelin-1
GBM: Glioblastoma Multiforme
GFAP: Glial Fibrillary Acidic Protein
GM-CSF: Granulocyte-Macrophage Colony-Stimulating Factor
gp130: glycoprotein 130
HDMEC: Human Dermal Microvascular Endothelial Cells
HIF: Hypoxia-Inducible Factors
HIMEC: Human Intestinal Microvascular Endothelial Cells
HUVEC: Human Umbilical Vein Endothelial Cells
IFN-γ: Interferon-γ
vii

IL-1β: Interleukin-1β
IL-6: Interleukin-6
IL-8: Interleukin-8
IL-11: Interleukin-11
IL-11Rα: IL-11 receptor alpha
MGMT: Methylguanyl Methyltransferase
MMP: Metalloproteinase
MRP: Multidrug resistance protein
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
PDGF: Platelet-Derived Growth Factor
pRB: Retinoblastoma Protein
rhIL-11: recombinant human IL-11
SCF: Stem Cell Factor
TGF-β: Transforming Growth Factor-β
TNF-α: Tumor Necrosis Factor-α
TPO: Thrombopoietin
TuBEC: Tumor-associated Brain Endothelial Cells
VE-Cadherin: Vascular Endothelial-Cadherin
VEGF: Vascular Endothelial Growth Factor
vWF: von Willebrand Factor
viii
ABSTRACT
Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) cytokine family,
which utilizes a common membrane glycoprotein 130 (gp130) for signal transduction.
Previously it had been shown IL-11 was an anti-inflammatory cytokine, stimulated
megakaryocyte diffentiation and was used for treatment of chemotherapy-induced
thrombocytopenia. Recently more attentions have been paid to its function in cancer.
There is growing evidence showing IL-11 plays a role in tumor progression in many types
of cancer. In our investigation, we studied the expression of IL-11 and its function on
tumor angiogenesis and tumor progression, using glioblastoma multiforme (GBM), the
most malignant, highly vascular and invasive brain tumor. Our studies show that IL-11 is
present abundantly in the GBM tumor tissue as compared to the normal brain, and IL-11
has multiple effects. First, IL-11 stimulates normal and tumor-associated brain
endothelial cells to migrate, and has no effect on cell proliferation. This suggests that IL-
11 may play a role in promoting angiogenesis as a pro-angiogenic factor. Second, it
enhances glioma cell migration and invasion without affecting cell number, indicating
that IL-11 may be involved in glioma tumor invasion and progression. In addition, IL-11
receptor α (IL-11Rα), the binding receptor of IL-11, is expressed by tumor cells in GBM
tissues while not expressed by normal astrocytes in the normal brain specimens. In
summary, our studies suggest novel functions of IL-11 in GBM tumor. These findings are
of great importance since they provide meaningful evidence for us to understand GBM
ix
better and potentially provide new therapeutic target for the treatment of this fatal
disease.
1
CHAPTER I – INTRODUCTION
1.1. ANGIOGENESIS AND THE TUMOR VASCULATURE
1.1.1. Overview of angiogenesis
Angiogenesis is the physiological process involving the formation of new blood
vessels from the pre-existing vessels. It is not only a vital process under normal
physiological circumstances, such as growth, development and wound healing, but also
a fundamental step in tumor progression.
Blood vessels are variable in morphologies and functions. Typically, the structure
of larger blood vessels, such as arteries, arterioles, veins and venules, can be divided
into three layers, from inside to outside: tunica intima, tunica media, and tunica
adventitia. Tunica intima is formed by a monolayer of endothelial cells, surrounded by a
thin layer of subendothelial connective tissue interlaced with internal elastic lamina (the
elastic bands). The subendothelial connective tissue secretes collagen and laminin,
which provide support to the endothelial cells and maintain the lumen integrity. Tunica
media is the thickest layer of the blood vessel. It is rich in smooth muscle cells,
connective tissue and polysaccharide substances. They function together to control the
constriction of the vessels. Tunica adventitia is made entirely by the fibroblast
connective tissue. It secretes collagen and contains nerves and nutrient capillaries called
vasa vasorum for the vascular walls. Figure 1 shows a typical blood vessel structure.
However, capillaries, the smallest blood vessels, are made solely by a single layer of
endothelial cells. There are three types of capillaries, continuous, fenestrated and
2
sinusoidal. The continuous capillaries, mainly found in muscle, lung, skin and brain, are
those endothelial cells provide an uninterrupted lining, the tight junction, which only
allows small molecules like water and ions to diffuse through, while blocks large
molecules passage between cells. The large molecule transportation requires specific
transporters. In the brain, this kind of capillary is highly specialized, and it is a major
constituent of the blood-brain-barrier (BBB). The fenestrated capillaries have pores in
the endothelial cells that allow small molecules and limited amounts of protein to
diffuse. These capillaries can be found in tissues such as pancreas, intestines and
endocrine glands. The sinusoidal capillaries are discontinuous endothelial cells and have
larger openings in the endothelium to allow red and white blood cells and various serum
proteins to pass. This structure helps exchange between the blood and tissues, and can
be found in livers, spleens, and bone marrows (Gartner, et al., 2001).

Figure 1. The structure of an artery. The figure indicates three layers (tunica intima,
tunica media, and tunica adventitia) of a blood vessel. (Picture adapted from
www.adam.com)
3
Angiogenic process involves in four well-characterized stages (Bergers, et al.,
2003). In the first stage, angiogenesis is initiated. The current mature and inactive blood
vessels become dilated, and the pericytes detach from the blood vessels. The blood
vessels become activated. In the second stage, the activated endothelial cells begin to
secrete enzymes and degrade the extracellular matrix, and this provides space for
endothelial cells to escape from the original blood vessel walls. In the third stage, the
activated endothelial cells proliferate and migrate into the surrounding extracellular
matrix. Under the guide of mural cells, such as pericytes, endothelial cells migrate in
tandem, connect to each other with integrins, and form solid sprouts connecting the
neighboring vessels. The last stage is also known as the maturation stage. After the
lumen formation, the newly formed vessels secrete extracellular matrix, and the mural
cells including the pericytes and smooth muscle cells reattach to the blood vessels and
provide support.
4

Figure 2. The angiogenic process. Normal angiogenic process involves four well-
characterized stages, which include (A) blood vessels destabilization, (B) endothelial
cells proliferation and migration, (C) lumens formation, (D) new vessels perfusion and
maturation. Each stage is tightly regulated by growth factors (Figure adapted from
Adams, et al., 2007).
Under normal physiological circumstances, such as wound healing, the
angiogenic process is determined by the balance of pro- and anti-angiogenic factors.
When pro-angiogenic factors are overexpressed, the pre-existing blood vessels become
destabilized, and the angiogenic process is triggered, resulting in new blood vessels
formation. When in reverse, the anti-angiogenic factors become dominant, angiogenic
process is inhibited and no new blood vessel formation. Each step of angiogenesis is
tightly regulated by multiple growth factors. During the initiation of angiogenesis,
endothelial cells are typically activated by hypoxia, ischemia or reduced blood flow, and
5
this step is mediated by hypoxia-inducible factors (HIF-1, 2). HIF-1 can activate the
transcription of several pro-angiogenic factor genes, especially vascular endothelial
growth factor (VEGF) and angiopoietin-2 (Ang-2) (Semenza, et al., 2003 and Daly, et al.,
2006), which together enhance vascular remodeling and blood vessel sprouting. VEGF is
a major contributor to angiogenesis, and it has diverse functions including but not
limited to: stimulates vessel permeability, acts as a survival factor of endothelial cells
and promotes their proliferation and migration, and induces enzyme production for
extracellular matrix degradation. Ang-2 is required in blood vessel maturation. Growth
factors that modulate endothelial cell proliferation and migration include VEGF, basic
fibroblast growth factor (bFGF), epidermal growth factor (EGF) and interleukin-8 (IL-8).
During the extracellular matrix degradation, metalloproteinases (MMPs) play
important roles, especially MMP2 and MMP9. MMPs can be provided by endothelial
cells, as well as other nonmalignant, infiltrating inflammatory cells such as neutrophils
and macrophages. While the MMPs are being released and digesting the extracellular
matrix, more pro-angiogenic factors originally trapped in the extracellular matrix are
released and promote blood vessel formation. As the blood vessel formation is nearly
completed, extracellular matrix undergoes further degradation and generates fragments
with bioactivities that inhibit angiogenesis. These fragments include endostatin,
tumstatin, canstatin and arrestin. Growth factors involve in the maturation stage
includes angiopoietin-1 (Ang-1), transforming growth factor-β (TGF-β), and platelet-
derived growth factor (PDGF). Ang-1 production is stimulated by the blood flow, and it
6
maintains the stability of blood vessels. Together with VEGF, it can also increase the
number and circumference of blood vessels. Low dose TGF-β acts pro-angiogenically,
while in high dose it acts anti-angiogenically. In low dose it up-regulates angiogenic
factors and proteases, and in high dose it inhibits endothelial cells growth, promotes
basement membrane reformation and stimulates smooth muscle cell differentiation and
recruitment to the blood vessel. PDGF recruits pericytes and smooth muscle cells to
stabilize nascent blood vessels.
1.1.2. Tumor angiogenesis
Angiogenesis is necessary for tumor progression. Tumor growth requires
vasculature to supply oxygen, nutrients, and clear out metabolic wastes. Angiogenesis is
critical in tumor survival and helps the tumor to overcome size limitation and become
malignant. There are two models of tumor angiogenesis, the avascular tumor initiation
model and the host-vessel co-option tumor growth model (Yancopoulos, et al., 2000). In
the first model, tumor initiates as a small avascular mass, which corresponds to small
and occult lesions of no more than 1–2 mm in diameter. These lesions stay dormant by
reaching a steady state between proliferation and apoptosis (Bergers, et al., 2003).
Hypoxia and MMP-9 trigger the “angiogenic switch”, inducing release of VEGF and
proteases; angiogenesis is switched on, and tumor growth enters vascular phase. In the
vascular phase, tumor growth can overcome size limitation and even become metastatic.
In the second model, tumor cells can initially grow along existing host blood vessels, and
thus can start off as a vascular small tumor. In response to the tumor, the host vessels
7
produce high level of Ang-2, resulting in vascular regression, and the tumor undergoes a
secondary avascular and hypoxia. The hypoxic condition triggers angiogenesis and the
tumor grows exponentially (Yancopoulos, et al., 2000).
In contrast with normal physiological process of angiogenesis, tumor
angiogenesis has lost the appropriate control of pro-angiogenic factors, such as VEGF,
TGF-β, bFGF, PDGF and ET-1, and anti-angiogenic factors, for example thrombospondin-
1, Ang-1, and Endostatin. Tumor cells constitutively produce and secrete pro-angiogenic
factors (Papetti, et al., 2002) and hypoxia continuously activates the transcription of
pro-angiogenic genes (Semenza, et al., 2003), resulting in constantly activated
angiogenic process. Tumor blood vessels fail to become quiescent and grow
continuously.

Figure 3. Models of tumor angiogenesis. (A) Model of avascular tumor initiation. (B)
Host vessel co-option tumor growth model (Picture adapted from Yancopoulos, et al.,
2000).
8
1.1.3. Comparison of the normal and tumor vasculature
The blood vessels in normal and tumor microvasculature are significantly
different. There are morphological and functional differences, which provide clear-cut
evidences to distinguish normal and tumor vasculature.
Normal vasculature offers a highly organized blood vessel network system, and
the arterioles, venules and capillaries are distinguishable. In the vessel, endothelial cells
line in tandem, connect each other through adhesion molecules, gap junctions and
desmosomes, and form the inner layer of the vessel lumen. The lumen is wrapped
around with pericytes and smooth muscle cells, to ensure the integrity of the blood
vessel without dilation or permeability, allowing the blood flow to be maintained in a
regular rate. By coating with mural cells, endothelial cells are quiescent and hardly
proliferate. Normal vasculature has intact basement membrane. This well-established
structural system enables normal vasculature to supply sufficient oxygen and nutrients,
sense and respond to the conditions in the extracellular environment.
In comparison to the normal vasculature, the tumor vasculature is highly chaotic.
It is not organized into definitive arterioles, venules and capillaries, but rather shares
chaotic features of all of them (Bergers, et al., 2003). The tumor vessels are
discontinuous, dilated, tortuous, and can have dead ends. Endothelial cells in the tumor
vessels tend to have abnormal shapes, grow on top of each other and project into the
lumen, resulting in sluggish blood flow (Nagy, et al., 2009). Integrins and adhesion
molecule expression are altered in tumor endothelial cells (Brooks, et al., 1994 and
9
Erdreich-Epstein, et al., 2000). Furthermore, pericytes and basement membrane form
loose connection with the blood vessel, and VEGF is abnormally elevated in the tumor
vasculature, causing the tumor endothelium continuously activated, the permeability is
increased and the vessel becomes leaky and hemorrhagic (Gerstner, et al., 2009).

Figure 4. Normal and tumor vasculatures. (A) Normal vasculature: simple, organized
arrangement of arterioles, capillaries, and venules. (B) Tumor vasculature: disorganized,
arterioles, capillaries, and venules are not identifiable. (C) Normal vasculature: pericytes
(red, arrowheads, anti-α-SMA staining) tightly wrap around endothelial cells (green,
anti-CD31 staining). (D) Tumor vasculature: pericytes (red, arrowheads, anti-α-SMA
staining) loosely attach to blood vessels (green, anti-CD31 staining). (E) Tumor
vasculature: blood vessels (green, anti-CD31 staining) are covered by an abnormally
loose, fragmented and multilayered basement membrane (red, arrowheads, anti-type IV
collagen staining) (Figure adapted and modified from McDonald, et al., 2003).

10
1.2. GLIOBLASTOMA MULTIFORME
1.2.1. Introduction
Glioblastoma Multiforme (GBM) is the grade IV astrocytoma, the most common
and most malignant primary brain tumor in human. The term “glioma” indicates the
tumor is originated from glial cells, such as astrocytes or oligodendrocytes, which
support neurotransmission and insulate axons to accelerate the electrical
communication (Allen, et al., 2009). According to the World Health Organization, there
are three grades of diffuse astrocytic tumors divided according to their malignancies:
astrocytoma (grade II), anaplastic astrocytoma (grade III), and GBM (grade IV). GBM
accounts for 52% of all glial tumors and occurs at a frequency of 5 cases per 100 000
people (McCarthy, et al., 2005). The average survival for patients with GBM, although
individually variable, merely lasts for an average of 14 months after diagnosis under the
current standard care (Van Meir, et al., 2010).
GBM is a very challenging disease to treat. The current standard treatment for
GBM is to start with maximal safe surgical resection followed by radiotherapy and
adjuvant temozolomide therapy (Clarke, et al., 2010). More recently, the targeting
therapy is finding its way to the clinical practice, such as bevacizumab, an antibody
targeting to the vascular endothelial growth factor (VEGF). Although the radiotherapy
technique has been improved during the past five years and the temozolomide
combination treatment has showed some effects in increasing patient survival, the
overall effect is still limited (Van Meir, et al., 2010). It has been acknowledged the tumor
11
has the ability to overcome radiation-induced injury by aberrant or amplified growth
and survival signaling pathways (Noda, et al., 2009). Temozolomide-induced tumor cell
injury can be repaired by DNA repairing enzymes, including methylguanyl
methyltransferase (MGMT), and temozolomide has no effects on killing glioblastoma
stem cells (Fu, et al., 2009). Meanwhile, GBM tends to develop chemoresistance over
time, so it provides another challenge for finding a cure. Bevacizumab has been
observed to cause a conversion of the tumor phenotype to a more invasive one,
resulting in a rapid recurrence and more malignant type by the time the treatment
failed (Paez-Ribes, et al., 2009 and Tuettenberg, et al., 2009). In all, improvement of the
treatment is highly required.
The invasion of tumor cells into healthy brain tissue is a distinguishing feature of
primary gliomas and contributes to the failure of currently available treatments (surgery,
radiotherapy and chemotherapy) (Bellail, et al., 2004). The border between the normal
brain cells and the primary tumor cells is ill-defined, as the tumor zone continuously
transitions to the adjacent normal area (Van Meir, et al., 2010). Glioma cells have the
unique characteristic of individual or groups of cells detaching from the major tumor
mass and traveling significant distances (Van Meir, et al., 2010). Understanding the
mechanism of the glioma cell invasion becomes critical since it plays a role in glioma
progression and may be the cause of the failure of current treatment due to tumor
recurrence.
12

Figure 5. GBM tissue sections. (A) Histological sections show typical GBM character:
Dense cellularity, striking polymorphism and neoplastic cells formed necrosis area in the
center (arrow). (B) The cells of a GBM can infiltrate widely, particularly along white
matter tracts, and even through the cerebrospinal fluid (Figure adapted from website:
www.pathconsultddx.com and www.pathology.umn.edu/clinical/).
1.2.2. Brain tumor angiogenesis
GBM is a highly vascular brain tumor. It relies on blood vessels for survival and
requires angiogenesis for tumor progression (Jain, et al., 2007). Angiogenesis in GBM is
complex and involves multiple dynamic processes. Recent studies have shown that the
GBM tumor generates new blood vessels through several simultaneously happened
mechanisms (Jain, et al., 2007). One of them is the classical “sprouting angiogenesis”
process. Hypoxia stimulates the secretion of growth factors, such as VEGF, the
endothelial cells in the vicinity are activated, and the angiogenic process is initiated.
Another mechanism is the host vessel co-option tumor growth model, which is GBM can
initially grow by “co-opting” pre-existing normal blood vessels. Glioma cells migrate
along existing, normal blood vessels and destabilize them, causing vessel regression,
reduced perfusion, hypoxia, and necrosis (Holash, et al., 1999). In response, growth
13
factors and cytokines are secreted and angiogenic processes are triggered (Zagzag, et al.,
2000). Another less understood mechanism of new blood vessel formation in GBM
involves in vasculogenesis, by which bone marrow derived endothelial precursor cells in
the circulation are recruited to the brain tumors, and directly incorporated into the
brain tumor vasculature (Chi, et al., 2009). These mechanisms highly interact with each
other and provide GBM more possibilities to gain blood vessel supply.
1.2.3. Brain tumor vasculature
Blood vessels in the brain are highly specialized and different from other parts of
the body. They are typically consisted of endothelial cells formed tubules surrounded by
pericytes and astrocytes. Together, they create a unique environment, the blood-brain-
barrier (BBB), which selectively restricts the exchange of molecules between the
intracerebral and extracerebral circulatory systems (Jain, et al., 2007). Tight junction
between endothelial cells enables BBB to allow free diffusion of small molecules, such
as O
2
, hormones and CO
2
, while restrict the diffusion of large molecules or
microorganisms, such as bacteria. Endothelial cells formed BBB contains a variety of
active transporters, including P-Glycoprotein and multidrug resistance proteins (MRPs),
the drug pumps (Schinkel, et al., 1999 and Deeken, et al., 2007), which continuously
exclude the drug from the brain parenchyma, and eventually cause the failure of
chemotherapy.
In the brain tumor, the BBB integrity is lost both structurally and functionally
when the tumor exceeds certain size (Deeken, et al., 2007). Structurally, the alterations
14
include a compromised tight junction structure and increased perivascular space
(Liebner, et al., 2000), resulting in high permeability and leakage in the tumor
vasculature. Functionally, there are altered expression levels of P-Glycoprotein and
MRPs on endothelial cells (Haga, et al., 2001). The alteration of drug-exclusion
transporter levels can lead to chemo-resistance of the tumor. Besides, blood vessels in
the brain tumor are significantly larger in diameters and thicker in basement
membranes than those in the normal brain (Bullitt, et al., 2005). In addition, in GBM, the
blood vessel density has been elevated, which becomes a distinguishing feature of GBM
compared to the other low-grade astrocytomas and the normal brains (Bergers, et al.,
2003).
1.2.4. Normal and GBM-associated brain endothelial cells
In both normal and tumor vasculature of the brain, endothelial cells are critical
players. In order to study their characteristics in angiogenesis and anti-angiogenic
therapy in the brain, we isolated and characterized the primary cultured nonmalignant
and GBM-associated brain endothelial cells. Nonmalignant brain endothelial cells (BEC)
are isolated from normal human brain tissues of trauma patients or tissues from
unaffected, cortical regions of patients with epilepsy. Tumor-associated brain
endothelial cells (TuBEC) are isolated from previous untreated, GBM patient tissues.
15

Figure 6. Morphologies of TuBEC and BEC. (A) TuBEC shows larger and veil-like
structure. Cells have small distinct nuclei and abundant cytoplasms. (B) BEC shows
smaller and plump look. Cells have large nuclei and limited cytoplasms (Figure adapted
from Charalambous et al., 2005).
TuBECs have shown different morphology, phenotypic characteristics, and
functional properties comparing with BEC (Charalambous, et al., 2006). In subconfluent
culture, TuBECs are larger and have veil-like structure. They are flat adherent cells with
small distinct nuclei and abundant cytoplasms. In contrast, BECs are smaller and have
plump look, and they exhibit large nuclei with limited cytoplasms (Charalambous, et al.,
2005). Regarding to the phenotype, both BECs and TuBECs are positive for endothelial
cell markers CD31, CD105 and von Willebrand Factor (vWF), while TuBECs exhibit lower
expression of vascular endothelial cadherin (VE-Cadherin) than BEC. VE-Cadherin is a
tight junction protein involves in the BBB integrity. Therefore, the reduced expression of
this tight junction protein may contribute to the leakage of tumor blood vessels (Davies,
et al., 2002). Functional differences are also obvious between these two cells. TuBECs
show characteristics of low proliferation rate and replicative senescence, as well as
insensitive to growth factors, such as VEGF and endothelin-1 (ET-1), and
16
chemoresistance to cytotoxic drugs, such as celecoxib and CPT-11. However, TuBECs
migrate faster than BECs, and show increased growth factor production ability, such as
VEGF, ET-1, IL-8 and PDGF (Charalambous, et al., 2005 and Charalambous, et al., 2007).
These growth factors may act in autocrine fashion that continuously activate TuBECs
and contribute to the cytotoxic protection and the enhanced migration effects.
1.3. INTERLEUKIN-11
1.3.1. Introduction
IL-11 is a member of the interleukin-6 (IL-6) cytokine family, utilizing a
membrane glycoprotein 130 (gp130) as a critical component for the signal transduction.
First isolated in 1990 from bone marrow-derived stromal cells, now it has been shown
to be expressed by many tissues, including brain, spinal cord neurons, gut, colon, breast,
and testis (Hanavadi, et al., 2006, Yoshizaki, et al., 2006, Schwertschlag, et al., 1999 and
Du, et al., 1997). IL-11 is a pleiotropic cytokine with multiple biological effects.
1.3.2. Hematopoietic effects of IL-11
1.3.2.1. Hematopoietic effects of IL-11 on progenitor cells
IL-11 functions together with other growth factors to stimulate hematopoiesis in
multiple stages. These growth factors include IL-3, IL-4, IL-7, IL-12, IL-13, stem cell factor
(SCF), flt3 ligand, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Du,
et al., 1997). IL-11 works synergistically with these growth factors. When combining with
different growth factors or working at a different concentration, IL-11 can either
stimulate the proliferation of primitive stem cells, pluiripotential and committed
17
progenitor cells, or increase the differentiation of primitive stem cells into progenitor
cells (Du, et al., 1997). For example, IL-11 works synergistically with stem cell factor (SCF)
to stimulate murine hematopoietic progenitor cell proliferation (Ariyama, et al., 1995),
and in combination with other cytokines in the hematopoietic microenvironment, IL-11
increases stem cell differentiation into pluripotential progenitor cells (Du, et al., 1995).
1.3.2.2. Hematopoietic effects of IL-11 on megakaryocytopoiesis and
thrombocytopoiesis
Megakaryocytopoiesis is the process by which bone marrow progenitor cells
develop into megakaryocytes, which in turn undergo thrombocytopoiesis to produce
thrombocytes (platelets) required for normal hemostasis. A single megakaryocyte can
give rise to thousands of thrombocytes (platelets). IL-11 acts synergistically with IL-3,
thrombopoietin (TPO), or SCF (Du, et al., 1997) to stimulate various stages of
megakaryocytopoiesis and thrombocytopoiesis in human bone marrow cells.
1.3.2.3. Hematopoietic effects of IL-11 on erythropoiesis and myelopoiesis
IL-11 alone or in combination with other cytokines can act other roles in the
hematopoietic microenvironment. When combining with IL-3, SCF, or erythropoietin
(Epo), IL-11 can stimulate bone marrow cells or fetal liver cells to undergo erythropoiesis
(Du, et al., 1997), the process of producing red blood cells (erythrocytes). It has also
been shown IL-11 modulates the differentiation and maturation of myeloid progenitor
cells in long term human marrow cultures (Keller, et al., 1993).

18
1.3.3. Non-hematopoietic effects of IL-11
1.3.3.1. IL-11 effects on epithelial cells
Recombinant human IL-11 (rhIL-11) inhibits intestinal epithelial cell proliferation
in vitro (Peterson, et al., 1996), through the mechanism of delaying the cell entering S
phase in the cell cycle, and suppressing retinoblastoma protein (pRB) phosphorylation
(Peterson, et al., 1996). The transient cell cycle arrest is a possible mechanism by which
rhIL-11 protects intestinal epithelial cells from damage induced by chemotherapy or
radiation therapy (Peterson, et al., 1996).
1.3.3.2. Anti-inflammatory effects
IL-11 displays anti-inflammation in a variety of animal models of acute and
chronic inflammation, such as mucositis, inflammatory bowel disease and autoimmune
joint disease. This anti-inflammatory effect is partially achieved by attenuation of pro-
inflammatory cytokines production, such as tumor necrosis factor-α (TNF-α),
interleukin-1β (IL-1β) and interferon-γ (IFN-γ), from activated macrophages (Trepicchio,
et al., 1998). IL-11 suppresses gene expression of these cytokines on the activated
macrophages by blocking nuclear translocation of NF-κB, a transcription factor, and
increasing in the cytoplasmic levels of the NF-κB inhibitory proteins (Leng, et al., 1997
and Trepicchio, et al., 1997).
Recombinant human IL-11 (rhIL-11) has been studied for clinical pharmacological
disposition. IL-11 has been shown effective in inflammatory bowel disease in animal
models, as well as inducing remission in a subset of patients with mild to moderate
19
Crohn's disease (CD) (Herrlinger, et al., 2006). However, the treatment seems to be
insufficient in maintaining the remission over three months (Herrlinger, et al., 2006).
1.3.4. IL-11 in cancers
Recombinant human IL-11, commercially known as Neumega, is an FDA
approved drug used in supportive cancer therapy (Kaye, et al., 1998). It was used to
treat chemotherapy-induced thrombocytopenia, a syndrome of reduced thrombocytes
(platelets) following chemotherapy, and therefore reducing the need of platelet
transfusion.
Over time, IL-11 has been addressed more of its functions in cancer. IL-11 has
been found overexpressed in many cancers, including breast cancer (Hanavadi, et al.,
2006), colorectal carcinoma (Yoshizaki, et al., 2006), gastric carcinoma (Howlett, et al.,
2009a), osteosarcoma (Lewis, et al., 2009), choriocarcinoma (Suman, et al., 2009) and
prostate cancer (Campbell, et al., 2001b). By far, researchers have shown IL-11 can up-
regulate the invasive activity of cancer cells, such as gastric carcinoma cells (Nakayama,
et al., 2007), choriocarcinoma cells (Suman, et al., 2009) and colorectal carcinoma cells
(Yoshizaki, et al., 2006). However, the function of IL-11 in tumor progression and tumor
malignancy is still not clear and requires more attention.
1.3.5. IL-11 receptor alpha (IL-11Rα)
The IL-11 receptor complex consists of a ligand binding α chain (IL-11Rα) and a
signal transducing β chain (gp 130). IL-11Rα chain consists of an extracellular domain
containing two potential N-linked glycosylation sites, a transmembrane domain and a
20
cytoplasmic tail (Hilton, et al., 1994, Van Leuven et al., 1996, and Schwertschlag, et al.,
1999). To transduce a signal, IL-11 binding to the IL-11Rα chain alone is insufficient due
to the low binding affinity, and gp130 is required for transducing the signal since co-
expression of IL-11Rα chain and gp130 will increase the binding affinity (Hilton, et al.,
1994).

Figure 7. IL-11Rα-gp130 signaling pathway. Upon binding to the ligand, IL-11Rα
transduces the signal to gp130, and three downstream signaling pathways can be
activated: JAK/STAT pathway, SHP-2/ ERK/ MAPK pathway and the PI3K/AKT pathway
(Figure adapted and modified from Howlett, et al., 2009b).
1.3.6. IL-11 signaling pathways
As an IL-6 cytokine family member, IL-11 utilizes gp130 as a critical component
for signal transduction (Taga, et al., 1997). Following gp130 receptor activation, three
21
alternate signaling pathways can be activated (Howlett, et al., 2009b), the JAK/STAT
pathway (Peters, et al., 1998), SHP-2/ ERK/ MAPK pathway (Kamimura, et al., 2003) and
the PI3K/AKT pathway (Ernst, et al., 2004). In most cell types, IL-11 mediates its effects
through JAK/STAT pathway (Heinrich, et al., 2003), with STAT-3 being the predominant
signaling mediator.
STAT-3 is of special interest in the context of malignant cellular properties
because aberrant STAT-3 activity is directly linked with oncogenesis (Bowman, et al.,
2000). It has been demonstrated that STAT-3 is constitutively activated in many primary
tumors, including breast, head and neck, prostate, skin, ovaries, lung, brain, pancreas
and kidney (Bromberg, et al., 2002). Furthermore, inappropriate STAT-3 activation is
likely contributing to the migratory/invasive activities (Paiva, et al., 2007 and Birner, et
al., 2010), and constitutive phosphorylation of STAT-3 results in increasing cell
proliferation, angiogenesis, inflammation, inhibition of both immunocyte and epithelial
cell apoptosis in many cancer types (Howlett, et al., 2009b).
22
1.4. PURPOSE OF STUDY
In previous study, we examined multiple genes expression in BEC and TuBEC
using microarray technique, and results showed IL-11 mRNA level was different in these
two cells. It triggered our investigation into the expression of IL-11 in normal and GBM
brain tissues, as well as the function of IL-11 in brain tumor angiogenesis. This study
focuses on the effects of IL-11 on endothelial cells and whether it can serve as a pro-
angiogenic factor.
A hallmark of GBM is a highly invasive cellular phenotype. The invasion of tumor
cells into the healthy tissue plays a central role in tumor progression and contributes to
the failure of current therapies. Therefore, understanding the mechanism of tumor cell
invasion becomes critical. Based on our study that IL-11 is expressed differently in GBM
and normal brain tissues, we expanded the work to determine the role of IL-11 in tumor
progression, in terms of tumor cell invasion. This study also explores the function of IL-
11 in GBM tumor invasion, in order to introduce a potential therapeutic target to
suppress tumor cell invasion.
In summary, the general purpose of this study is to understand the role of IL-11
in brain tumor angiogenesis and tumor progression. More specifically, the goals of this
study are: (a) to determine IL-11 and its receptor expression level in GBM brain and
normal brain tissues, as well as in endothelial cells and tumor cells, (b) to explore the
function of IL-11 in brain tumor angiogenesis, including endothelial cell proliferation and
migration, and (c) to study the effects of IL-11 on GBM tumor cell invasion. These
23
findings will be of great significance since they provide meaningful evidence for us to
understand GBM better and give us insights to develop new therapeutic methods for
this fatal disease.
24
CHAPTER II – MATERIALS AND METHODS
2.1. HISTOLOGICAL STUDIES
2.1.1. Tissue samples, cell cultures and reagents
GBM tissues were received after surgery from patients diagnosed with stage IV
Glioblastoma Multiforme previously untreated, and normal brain tissues were obtained
from trauma or epileptic patients. Tissues were obtained and handled under the
approval of Keck School of Medicine, University of Southern California Institutional
Review Board. Tissues were quickly frozen in O.C.T. compound (Tissue-Tek, Torrance, CA)
on dry ice and cryosectioned into 10 µm and placed on slides. Then the tissues were
fixed in acetone for 10 minutes and stored in -20
o
C freezer.
Endothelial cells were isolated from these samples and characterized. Briefly,
freshly received tissue was washed three times with RPMI medium containing 2% FCS
(Atlanta Biologicals, Lawrenceville, GA) and 1% penicillin and streptomycin (RPMI 2%
FCS). The tissue was then cut into small pieces in RPMI 2% FCS medium. Fresh medium
was added to the tissue mixture and transferred to a centrifugation tube, and equal
volume of 30% dextran was added to the centrifugation tube and well mixed to reach
the final concentration of 15% dextran. The tissue mixture was centrifuged for 10
minutes at 10,000 rpm in 4
o
C. This step pulled down the brain microvessels according to
their density. The microvessel pellet was resuspended in 1 mg/mL collagenase-dispase in
RPMI 2% FCS and incubated in 37
o
C water bath with a shaker for 1 hour. Subsequently,
10 mL of RPMI 2% FCS was added to the cells and centrifuged at 1200 rpm for 5 minutes.
25
The pellet was then resuspended in RPMI 2% FCS and centrifuged again. The final pellet
was resuspended in endothelial cell culture medium (RPMI medium supplemented with
100 ng/mL endothelial cell growth supplement (Upstate Biotechnologies, Rochester, NY),
2 mM L-glutamine (GIBCO, Grand Island, NY), 10 mM HEPES (GIBCO, Grand Island, NY),
24 mM sodium bicarbonate (GIBCO, Grand Island, NY), 1 vial of heparin sodium salt
(Sigma-Aldrich, St. Louis, MO), 1% penicillin/streptomycin, and 10% FCS). Cells were
seeded on 1% gelatin coated tissue culture flask (BD, Franklin Lakes, NJ). Medium was
changed every 3 or 4 days until the cells became 80% confluent. Endothelial cells were
then purified from the cellular mixture by selecting cells which absorb the deacetylated
low-density lipoprotein (di-acet-LDL), following with cell sorting. Subsequently, the
population of the endothelial cells was confirmed by immunocytochemistry that the
cells were positive for endothelial cell markers: CD31, vWF, CD105, and negative for
astrocyte cell marker glial fibrillary acidic protein (GFAP), precursor endothelial cell
marker CD34, and macrophage/microglia marker CD11b. Cells between passage 4 and
passage 6 were used for the experiments.
Endothelial cells were cultured in 1% gelatin coated flasks with endothelial cell
growth medium, while the growth factors in the culture medium were removed when
performing the experiments. Glioma cell lines were cultured in DMEM medium
containing 10% FCS and 1% penicillin/streptomycin. The experiments were performed in
the same culture medium unless specially indicated.
26
Recombinant human IL-11 was purchased from Millipore (Bedford, MA) and
reconstituted following the manufacture’s instruction. IL-11 ELISA kit was purchased
from R&D Systems (Minneapolis, MN). Digital pictures were taken using the microscopes
in USC core facilities.
2.1.2. ELISA
6X10
4
cells were planted in RPMI 10% FCS (for endothelial cells) or DMEM 10%
FCS (for glioma cell lines) in 6-well-plate in triplicate overnight for settle down. The next
day, medium was changed and cells were incubated for 48 hours. After the incubation,
medium was collected and centrifuged by 10,000 rpm for 10 minutes. The top portion of
the cell supernatants were collected, filtered through 0.2 µm filter and measured for the
IL-11 concentrations by the commercially available ELISA kit (R&D Systems, Minneapolis,
MN), as instructed by the manufacture’s protocol.
2.1.3. Western Blot
Whole cell lysates were quantified by the BCA Protein Assay Kit (Thermo
Scientific, Waltham, MA), and equal amount of protein (70 µg) was separated using the
SDS-PAGE gel and electrotransfered to 0.2 µm nitrocellulose membranes. The
membranes were then blocked with SEA Block (25 mL SEA Block + 25 mL PBS + 25 µL
Tween 20) and probed with anti-IL-11Rα monoclonal antibody (Santa Cruz Biotechnology,
Santa Cruz, CA, 1:250 dilution) and anti-GAPDH polyclonal antibody (Santa Cruz
Biotechnology, Santa Cruz, CA, 1:5,000 dilution) overnight. Membranes were then
incubated with fluorescent-conjugated secondary antibodies (Thermo Scientific,
27
Waltham, MA, 1:5000 or 1:15,000 dilution) for 45 minutes, and the protein bands were
detected with Odyssey Infrared Imaging (LI-COR Biosciences, Lincoln, NE).
2.1.4. Immunocytochemistry/Immunohistochemistry
To prepare the samples, cells were cytocentrifuged on the slides (2.5X10
4

cells/slides), and fixed with acetone for 10 minutes. And the frozen tissues were
sectioned for 10 µm thickness and fixed with acetone for 10 minutes. The samples were
stored in -20
o
C for prolonged use. For the staining, samples were taken from stock and
air-dried for 10 minutes, rehydrated with PBS for 5 minutes, and blocked with SEA Block
(25 mL SEA Block +25 mL PBS + 25 µL Tween 20) for 20 minutes. Then the samples were
incubated overnight with primary goat anti-IL-11 polyclonal antibody (Santa Cruz
Biotechnology, Santa Cruz, CA, 1:100 dilution), mouse anti-IL-11Rα (Santa Cruz
Biotechnology, Santa Cruz, CA, 1: 100 dilution) polyclonal antibody. Samples were then
incubated with biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA,
1:300 dilution), or biotinylated horse anti-goat IgG (Vector Laboratories, Burlingame, CA,
1:300 dilution) for 45 minutes. Next, the samples were incubated with ABC Elite (Vector
Laboratories, Burlingame, CA) for 30 minutes, followed by AEC substrate (2.12 mL ddH
2
O
+ 25 µL 1M Acetic Acid + 100 µL 1M Sodium Acetate + 12.5 µL H
2
O
2
+ 125 µL AEC) for 10
minutes. Samples were then counterstained for hematoxylin and mounted. Pictures
were taken using Olympus microscope in USC core facility under 20X magnification.
28
2.1.5. Immunofluorescent staining
Samples were prepared, dried, rehydrated and blocked the same method with
immunocytochemistry/immunohistochemistry staining. Subsequently, samples were
incubated with two primary antibodies (no cross reactions in the secondary antibodies)
overnight. The primary antibodies used were: goat anti-CD31 polyclonal antibody (Santa
Cruz Biotechnology, Santa Cruz, CA, 1:100 dilution), rabbit anti-von Willabrand Factor
polyclonal antibody (Dako Corporation, Carpinteria, CA, 1:1000 dilution), rabbit anti-
GFAP polyclonal antibody (Millipore, 1:5000 dilution), goat anti-IL-11 polyclonal antibody
(Santa Cruz Biotechnology, Santa Cruz, CA, 1:100 dilution) and mouse anti-IL-11Rα
(Santa Cruz Biotechnology, Santa Cruz, CA, 1: 100 dilution) polyclonal antibody. Samples
were then incubated with double secondary antibodies (no cross reaction) for 1 hour.
The antibodies were: Alexa Fluor® 594 donkey anti-goat IgG (Invitrogen, Carlsbad, CA,
1:200 dilution), Alexa Fluor® 488 donkey anti-rabbit IgG (Invitrogen, Carlsbad, CA, 1:200
dilution), Alexa Fluor® 594 donkey anti-rabbit IgG (Invitrogen, Carlsbad, CA, 1:200
dilution) and Alexa Fluor® 488 donkey anti-mouse IgG (Invitrogen, Carlsbad, CA, 1:200
dilution). The samples were then stained for nucleus with Hoechst (1:500 dilution) dye
and mounted with Immunofluorescent mounting medium (Dako Corporation,
Carpinteria, CA). Samples were analysed under immunofluorescent microscope and
confocal microscope in USC core facility.
29
2.2. FUNCTIONAL STUDIES
2.2.1. Endothelial cell and tumor cell migration assay
Migration assays were performed using BD Pharmingen (Franklin Lakes, NJ) cell
migration control chambers. Briefly, for endothelial cell migration, 8 µm pore, 6.5 mm
diameter polyethylene terephthalate filters were coated with 0.5% gelatin by emerging
both top (200 µL) and bottom (800 µL) sides of the chamber filters into the gelatin for 2
hours, and air dried in room temperature overnight. 4X10
4
brain endothelial cells were
resuspended in RPMI 10% FCS (100 µL) and seeded on top of the filter of each chamber
overnight for attachment in 37
o
C, 5% CO
2
incubator. Chemoattractant was prepared the
following day and placed in the bottom chamber (600 µL per well). RPMI 10% FCS
without IL-11 was used to test the baseline of migration, and 1, 10, 100 ng/mL IL-11
diluted in RPMI 10% FCS were added into experimental groups. Endogro medium were
used as the positive control. The filters with endothelial cells were then transferred to
the chemoattractant containing wells and allowed to migrate for 8 hours in 37
o
C, 5% CO
2

incubator.
And for the glioma cell migration, 2X10
4
cells (100 µL) were resuspended in
DMEM 10% FCS, seeded on top of the chamber filters without gelatin coating, and
allowed to attach overnight. Chambers were then transferred to 24-well-plate wells
containing chemoattractant. DMEM 1% FCS without IL-11 was used to test the baseline
of tumor cell migration, and 1, 10, 100 ng/mL IL-11 diluted in DMEM 1% FCS were
experimental groups. DMEM 10% FCS containing 50 ng/mL EGF and 50 ng/mL FGF was
30
used as the positive control. The transwell plate was then incubated in 37
o
C, 5% CO
2

incubator for 4 hours to allow cells to migrate.
At the end of the migration experiment, medium was removed from the top of
filters and the non-migrated cells on the top side of the filters were erased with wet Q-
tips. And the migrated cells on the bottom side of the filters were then fixed and stained
with Diff-Quick staining solution (Dade Behring, Inc., Newark, DE), and the filters were
air-dried overnight. Then the membranes of the filters were cut and placed on the glass
slides and mounted with emersion oil. The migrated cells through the membranes were
counted under light microscope. For each membrane, 10 fields were counted under the
20X objective. Groups were plated in duplicate. And experiments were repeated 3 times.
2.2.2. Tumor cell invasion assay
Matrigel invasion chambers (BD, Franklin Lakes, NJ) were allowed to come to
room temperature, and rehydrated by emerging both top (500 µL) and bottom (500 µL)
chambers into warm DMEM 10% FCS for two hours in 37
o
C, 5% CO
2
incubator. And
chemoattractant was prepared as: DMEM 10% FCS without IL-11 was used as the control
to determine the base line of tumor cell invasion. Medium containing 1, 10, 100 ng/mL
IL-11 was used as the experimental groups. Medium containing EGF 50 ng/mL and FGF
50 ng/mL was used as the positive control. After rehydration, chambers were transferred
into 24-well-plate containing chemoattractant (750 µL per well). Then 5X10
4
tumor cells
resuspended in 500 µL DMEM 10% FCS were seeded immediately into the chambers.
And then the transwell plate was incubated in 37
o
C, 5% CO
2
incubator for 24 hours. After
31
completion of the incubation, medium in the top chambers was removed and the non-
invaded cells on the top side of the chamber filter membrane were erased with wet Q-
tips. The cells on the bottom side of the membrane were then fixed and stained with
Diff-Quick staining solution (Dade Behring, Inc., Newark, DE). Cells that invaded through
the membrane were then counted under the 20X objective. Ten fields were counted for
each membrane. The experimental groups were plated in duplicate and the assay was
repeated 3 times.
2.2.3. MTT cell proliferation assay
Cells were seeded triplicate in 96-well plates (5X10
3
cells in 50 µL per well)
overnight before treatment. Cells were then treated with IL-11 by adding 50 µL medium
containing IL-11 to reach the final concentrations of 0, 1, 10, 100 ng/mL. And the cells
were also treated with 100 µM DMC in serum free medium as a negative control. Then
the cells were incubated for 24 and 48 hours. At the end of the experiment, 10 µL 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye (125 mg/25 mL PBS)
was added to each well and incubated for 4 hours. And then the MTT lysis buffer (10%
SDS + 0.1% HCl) were added to each well (100 µL), and the plates were incubated
overnight in 37
o
C, 5% CO
2
incubator for color development. The intensity of the color
was measured by spectrophotometer under 490 nm and proportioned to the cell
viabilities.
32
2.2.4. Bromodeoxyuridine (BrdU) cell proliferation assay
Cells were seeded triplicate in 96-well-plates (5X10
3
cells in 50 µL per well) in
RPMI 10% FCS overnight before IL-11 treatment. The second day, 50 µL RPMI 10% FCS
medium containing IL-11 was added to each well to reach the final concentrations of 0, 1,
10, 100 ng/mL. And the cells were also changed into fresh Endogro medium as the
positive control group. 4 hours before completion the 24 or 48 hours IL-11 treatment,
BrdU was added to the cell culture, and the cells were fixed upon completion of the
treatment. Anti-BrdU-POD antibody was used (Roche, Basel, Switzerland, 1:100 dilution)
to detect the amount of dye absorbed, and after 90 minutes incubation, substrate
solution was added to develop the color. The color development was stopped by adding
1M H
2
SO
4
(stop solution). And within 5 minutes upon adding the stop solution, the color
intensity was measured at 450 nm and proportioned to the cell proliferation rate.
2.2.5. Statistical analysis
Values were presented as the mean +/- SEM. Statistical significance was
evaluated using the Student’s two-tailed t-test or one-way analysis of variance followed
by Dunnett’s multiple comparison test. A P<0.05 was considered statistically significant.
33
CHAPTER III – RESULTS AND CONCLUSION
3.1. EXPERIMENTAL RESULTS
3.1.1. Normal and tumor-associated brain endothelial cells produce IL-11.
In order to determine whether normal and glioma-associated brain endothelial
cells produced IL-11, these cells were isolated, prepared as cytocentrifuge preparations
and stained with anti-IL-11 antibody using immunocytochemistry. Over 80% of both
populations were stained for IL-11 (Figure 8A). The glioma tumor cell line U251 was also
tested for IL-11, and found to produce IL-11 in greater than 80% of the cells (Figure 8A).
These data indicated that both normal and tumor-associated brain endothelial cells and
the glioma cell line expressed IL-11.
To determine whether IL-11 was secreted by brain endothelial cells and tumor
cell lines, we isolated the supernatants derived from these cells in culture and measured
the amount of IL-11 using ELISA technique. We observed the range of secreted IL-11 in
normal brain endothelial cells was 210 pg/mL to 350 pg/mL, in tumor-associated
endothelial cells was 150 pg/mL to 230 pg/mL, and in tumor cell lines was 560 pg/mL to
2410 pg/mL (Figure 8B). All of them were significantly higher than IL-11 detected in the
cell culture medium. These data showed IL-11 was secreted from the cell into the cell
culture environment and present in a soluble form.

34
Figure 8. BEC, TuBEC and glioma cells expressed IL-11. (A) Anti-IL-11 staining on
cytocentrifuge cell preparations showed that these cells expressed IL-11. Negative
controls were cells stained without the primary antibody (not shown). This staining was
performed on 3 different BEC and 3 different TuBEC specimens. U251 staining was
performed 3 times. (B) ELISA analysis of supernatants from 2 BEC samples, 2 TuBEC
samples and glioma cell lines (U87, U251) demonstrates that all these cells secrete IL-11.
Statistical significance (*P<0.05, **P<0.01) was calculated by comparing supernatants
from each cell type to RPMI 10% FCS. The data represent the mean +/- SEM of triplicate
samples for endothelial cell supernatants, and duplicate samples for tumor cell
supernatants and RPMI 10% FCS. The graph represents data from one experiment.
35
Figure 8, continued

36
3.1.2. IL-11 is more highly expressed in human GBM tissue as compared to normal
brain tissue.
To investigate whether IL-11 was expressed in vivo in human disease, normal and
GBM brain tissues were stained with anti-IL-11 antibody. Three samples of GBM and
three samples of normal human brain tissues were analyzed. Figure 9 showed that IL-11
staining was more intense in GBM brain tissues as compared to normal brain tissues.
This indicated that in GBM, IL-11 was up-regulated compared to the normal brain.
As we showed above, normal and tumor-associated brain endothelial cell
cultures expressed IL-11 in vitro. To study the expression of IL-11 in normal and tumor
vasculatures in vivo, we performed double-staining experiments using anti-CD105, an
endothelial cell marker, and anti-IL-11 antibody. Figure 9B showed that IL-11 and CD105
staining were co-localized in both normal and GBM blood vessels, suggesting that both
normal and tumor vasculatures expressed IL-11.
37
Figure 9. IL-11 expression levels in normal brain and GBM tissues. (A) Both
immunohistochemical and immunofluorescent staining showed that GBM tissues
expressed higher levels of IL-11 compared to normal human brain tissues (Red: IL-11,
Green: CD105, Blue: Hoechst). (B) Confocal staining showed that the tumor vasculature
and normal brain blood vessels expressed IL-11 (Red: IL-11, Green: CD105, Blue:
Hoechst). These photomicrographs were representative of 3 normal brain and 3 GBM
tissue specimens.
38
Figure 9, continued

39
3.1.3. Normal and tumor-associated brain endothelial cells express IL-11Rα in vitro and
in vivo.
To determine whether IL-11Rα was expressed in normal and tumor-associated
brain endothelial cells, these cells were stained with anti-IL-11Rα antibody. As shown in
Figure 10A, over 80% of the cells in both normal and tumor-associated brain endothelial
cells stained positively. In addition, cell lysates were harvested and analyzed for IL-11Rα
expression using western blot. As seen in Figure 10B, clear bands of anti-IL-11Rα protein
were detected. These data indicated that brain endothelial cells expressed IL-11Rα. To
confirm that this receptor was expressed in vivo, normal and GBM human brain tissue
were double stained with the endothelial cell marker von Willebrand Factor (vWF) and
IL-11Rα. Figure 10C showed that vWF and IL-11Rα co-localized in the blood vessels of
GBM and normal brain blood vessels. Thus, normal and tumor-associated brain
endothelial cells expressed IL-11Rα.

40
Figure 10. BEC and TuBEC expressed IL-11Rα in vitro and in vivo. IL-11Rα was expressed
by tumor cells in GBM but not astrocytes in normal brain tissue. (A) Cytocentrifuge cell
preparations of normal and tumor-associated brain endothelial cells were stained with
anti-IL-11Rα antibody. Over 80% of the cells were positive for IL-11Rα. Negative controls
were cells stained without the primary antibody (not shown). These data were
representative of 3 normal and 3 GBM endothelial cell specimens. (B) Western blot was
performed to determine IL-11Rα expression on 3 BEC samples, 3 TuBEC samples and 3
glioma cell lines (U87, U251 and LN229). The blot showed positive bands of anti-IL-11Rα
protein. This experiment was performed once. (C) Both the tumor vasculature and
normal brain vasculature expressed IL-11Rα in vivo (Green: vWF, Red: IL-11Rα, Blue:
Hoechst). These staining studies were representative of 3 normal brain and 3 GBM tissue
specimens. (D) Tumor cells in vivo expressed IL-11Rα, while astrocytes in normal brain
were negative for IL-11Rα (Green: IL-11Rα, Red: GFAP , Blue: Hoechst). The staining data
are representative of 3 normal and 3 GBM tissue specimens.
41
Figure 10, continued

42
Figure 10, continued
43
3.1.4. IL-11Rα is expressed by tumor cells in GBM tissue but not by normal astrocytes.
Immunostaining of GBM tissues revealed that cells other than those associated
with the vasculature expressed IL-11Rα (Figure 10C). We further investigated the
identity of those cell types. As observed in Figure 10C, we found that tumor cells also
express IL-11Rα. To confirm this we double stained the GBM and normal tissues with
antibodies to GFAP and IL-11Rα. The results showed (Figure 10D) that GFAP positive
cells co-localized with IL-11Rα in GBM tissue, but not in normal brain. These data
indicated that tumor cells expressed IL-11Rα in vivo, while normal astrocytes did not
express IL-11Rα in vivo.
3.1.5. Brain endothelial cells migration is up-regulated by IL-11.
The effects of IL-11 on brain endothelial cells migration were determined using
the cell migration assay. Endothelial cells were seeded on the upper side of the top
chambers, and IL-11 was added to the bottom chambers at three concentrations (1, 10,
100 ng/mL). Cells were then allowed to migrate for 8 hours, and then the migrated cells
were stained and counted. The results showed that IL-11 stimulated both normal and
tumor-associated brain endothelial cells as compared to untreated cells in a dose-
dependent manner (P<0.05) (Figure 11A, 11B).
44

Figure 11. Effects of IL-11 on BEC and TuBEC migration. (A) BEC and (B) TuBEC were
stimulated with IL-11 (1, 10, 100 ng/mL). Statistical significance (*P<0.05, **P<0.01) was
calculated by comparing treated groups to the untreated control group. The migration
data is expressed as the mean +/- SEM of duplicate samples. These data were
representative of 3 normal and 3 GBM endothelial cell specimens.
3.1.6. IL-11 stimulates tumor cell migration and invasion.
To investigate whether IL-11 affected glioma cell line migration, we performed
cell migration assays using the U87 glioma cell line. U87 cells were seeded on the upper
side of the migration chambers and treated with IL-11 (0, 1, 10, 100 ng/mL) in the
bottom chamber. After 4 hours, the number of tumor cell which migrated through the
filter was quantified. The data (Figure 12A) showed that IL-11 increased U87 cells
migration compared to vehicle treated cells, in a dose-dependent manner.
To determine whether IL-11 affected glioma cell invasion, we performed invasion
assays with U87 cells. As shown in Figure 12B, IL-11 up-regulated U87 cell invasion in a
dose-dependent manner.
45

Figure 12. Effects of IL-11 on U87 cell migration and invasion. U87 cells were stimulated
with IL-11 (1, 10, 100 ng/mL) and analyzed for (A) migration and (B) invasion. Statistical
significance (*P<0.05, **P<0.01) was calculated by comparing treated groups to the
untreated control group. These data represent the mean +/- SEM of duplicate samples.
U87 migration and invasion experiments were performed 3 times.
3.1.7. IL-11 does not affect brain endothelial or glioma cell proliferation.
To determine whether IL-11 increased cell proliferation in BEC and TuBEC, we
analyzed the effects of IL-11 on these cells using the MTT assay. The results showed that
IL-11 did not affect normal brain endothelial cell proliferation during 24 (Figure 13A) or
48 hours (Figure 13B), or tumor-associated brain endothelial cell proliferation during 24
(Figure 14A) or 48 hours (Figure 14B). To confirm these data, BrdU incorporation assays
were also performed. Cells were incubated with BrdU 4 hours before completion of the
24 or 48 hours IL-11 treatment. The results (Figure 13C, 13D, 14C, 14D) showed that
there was no significant difference in incorporation of BrdU with IL-11 treatment
compared to vehicle treated cells. Thus, IL-11 induced brain endothelial cells migration,
without affecting cell proliferation.
46

Figure 13. Effects of IL-11 on cell proliferation of BEC. BEC were plated (5X10
3
cells per
well) and treated with increasing concentrations of IL-11 (0, 1, 10, 100ng/mL) for 24 or
48 hours. After (A) 24 hours or (B) 48 hours treatment, MTT assay was performed, and
cell viability was calculated. Cell proliferations were also confirmed by the BrdU
incorporation assay at (C) 24 hours or (D) 48 hours of IL-11 treatment. Statistical
significance (*P<0.05, **P<0.01) was calculated by comparing treated groups to the
untreated control group. These data represent the mean +/- SEM of triplicate samples.
These results are representative of 2 normal endothelial cell specimens.
47

Figure 14. Effects of IL-11 on cell proliferation of TuBEC. TuBEC were plated (5X10
3
cells
per well) and treated with increasing concentrations of IL-11 (0, 1, 10, 100ng/mL) for 24
or 48 hours. After (A) 24 hours or (B) 48 hours treatment, MTT assay was performed,
and cell viability was calculated. Cell proliferations were also confirmed by the BrdU
incorporation assay at (C) 24 hours or (D) 48 hours of IL-11 treatment. Statistical
significance (*P<0.05, **P<0.01) was calculated by comparing treated groups to the
untreated control group. These data represent the mean +/- SEM of triplicate samples.
These results are representative of 2 GBM endothelial cell specimens.
Experiments were performed to determine whether IL-11 stimulated tumor cell
proliferation. The results showed that IL-11 did not stimulate tumor cell proliferation at
doses effective for cell migration (Figure 15). These data indicated that IL-11 played a
48
role in stimulating glioma cell migration as a chemoattractant, without affecting cell
proliferation.

Figure 15. Effects of IL-11 on cell proliferation of U87 cells. Cells were seeded (5X10
3

cells per well) and treated with increasing concentrations of IL-11 (0, 1, 10, 100ng/mL)
for 24 or 48 hours. After (A) 24 hours or (B) 48 hours treatment, MTT assay was
performed, and cell viability was calculated. Cell proliferations were also confirmed by
the BrdU incorporation assay at (C) 24 hours or (D) 48 hours IL-11 treatment. Statistical
significance (*P<0.05, **P<0.01) was calculated by comparing treated groups to the
untreated control group. These data represent the mean +/- SEM of triplicate samples.
These experiments were performed twice.
49
3.2. CONCLUSION

Figure 16. IL-11 in the normal brain and GBM environment. (A) In the normal brain, IL-
11 is secreted by endothelial cells. The role of IL-11 in the normal brain is not clear. (B) In
GBM, IL-11 is produced by both endothelial cells and tumor cells. Large amounts of IL-11
are present in the tumor environment. IL-11 promotes tumor growth by increasing
angiogenesis, and causing tumor cell migration and invasion.
50
In summary, in the normal brain (Figure 16A), IL-11 is produced by endothelial
cells, and IL-11 can increase normal brain endothelial cell migration. The role of IL-11 in
the normal brain function is not clear. In GBM (Figure 16B), tumor cells produce large
amounts of IL-11. This level of IL-11 may be sufficient to enhance angiogenesis in tumors,
thereby increasing tumor growth. IL-11 in GBM may also increase tumor cell migration
and invasion, and therefore causing the tumor size expansion. Thus, IL-11 enhances
tumor growth by increasing angiogenesis, and causing tumor cell migration and invasion.

51
CHAPTER IV – DISCUSSION
The main objective of this paper was to explore the function of IL-11 presented
in the GBM tumor microenvironment, and its effects on tumor-host interaction, in terms
of brain tumor angiogenesis. We first investigated whether IL-11 and IL-11Rα were
expressed in glioma cells as well as normal and tumor-associated brain endothelial cells,
and we provided evidence of IL-11 and its receptor were expressed by glioma cells and
brain endothelial cells both in vitro and in vivo. We then studied the effects of IL-11 on
endothelial cells migration, tumor cells migration and invasion, and we showed that IL-
11 stimulated endothelial cells and tumor cells migration as well as tumor cells invasion.
Subsequently, in order to determine whether the stimulation of migration and invasion
were due to cell number increase, we investigated the effects of IL-11 on cell
proliferation, and we provided evidence and showed that IL-11 did not affect cell
numbers. This evidence supported the understanding that IL-11 in the GBM tumor
microenvironment promoted tumor cells mobility, invasiveness and stimulated normal
and tumor-associated brain endothelial cells migration.
It has been widely recognized that IL-11 and its binding receptor IL-11Rα are
involved in a wide range of human cancers, including endometrial carcinoma (Yap, et al.,
2010), prostate carcinoma (Campbell, et al., 2001b), breast cancer (Hanavadi, et al.,
2006), gastric cancer (Howlett, et al., 2009a), and colorectal carcinoma (Yoshizaki, et al.,
2006). In glioma, it was previously reported that U87 and U373 glioblastoma cell lines
expressed IL-11 in vitro (Murphy, et al., 1995), which is consistent with what we had
52
shown in our glioma cell lines. However, little has been shown in vivo, especially in GBM.
We showed that in GBM patient samples IL-11 was highly expressed, while in normal
brain tissues IL-11 exhibited very low level. Similar observations had been reported in
the endometrial tumor epithelial cells, IL-11 was elevated from grade 1 to grade 3
compared to endometrial epithelium from tumor free patients (Yap, et al., 2010). In
addition, IL-11 was significantly increased in gastric cancer patient samples compared to
disease-free human tissue samples (Howlett, et al., 2009a and Howlett, et al., 2009b).
Our studies also showed that IL-11Rα was expressed by glioma cells in vitro and
in vivo, while normal astrocytes in normal brain tissues did not express IL-11Rα protein.
In the previous studies, researchers had reported that IL-11Rα was expressed by primary
ovarian carcinoma (Campbell, et al., 2001a), breast cancer (Hanavadi, et al., 2006), and
prostate cancer (Zurita, et al., 2004 and Campbell, et al., 2001b). In prostate cancer, it
was shown that the high-grade prostatic intraepithelial neoplasia and the invasive
carcinoma exhibited significantly frequent and prominent staining for IL-11Rα, while the
epithelial cells of the nonmalignant samples displayed weak staining for IL-11Rα
(Campbell, et al., 2001b). In human osteosarcoma, all tested primary tumor samples
exhibited moderate-to-high-intensity staining for IL-11Rα, and all the tested pulmonary
metastases were positive for IL-11Rα expression, while normal, control lung tissues were
negative (Lewis, et al., 2009). Similarly, in breast cancer, the expressions of both IL-11
and IL-11Rα were distinctively higher in tumor samples compared to normal tissues
(Hanavadi, et al., 2006). However, very little had been done to investigate IL-11Rα
53
expression in GBM, our results reported for the first time that glioma cell lines expressed
IL-11Rα in vitro and in vivo, and GBM tumor tissues exhibited higher IL-11Rα level
compared to normal brain. This suggested that IL-11 and IL-11Rα might play a crucial
role in tumor development and progression, and they might serve as potential
candidates of the markers in GBM diagnosis and progression. It also raised the possibility
of therapeutic target designing.
We also found normal and tumor-associated brain endothelial cells expressed IL-
11 and IL-11Rα in vitro and in vivo. A previous report had shown that in cultured human
umbilical vein endothelial cells (HUVEC) and human intestinal microvascular endothelial
cells (HIMEC), IL-11 mRNA was detected (Nilsen, et al., 1998). HUVEC was also known to
express IL-11Rα (Mahboubi et al., 2000). And in endometrial cancer, researchers had
shown that tumor vasculatures in grade 3 endometrial tumors expressed IL-11 and IL-
11Rα (Yap, et al., 2010), as well as in primary human osteosarcoma, more than half of
the tumor blood vessels showed moderate-to-high intensity staining (Lewis, et al., 2009).
These examples indicated that endothelial cells expressed IL-11 and IL-11Rα in both cell
cultures and tumor samples. And IL-11Rα could be served as a candidate of the
therapeutic target of anti-angiogenic treatment (Lewis, et al., 2009). However, there is
not enough published evidence showing IL-11 and IL-11Rα expression in normal
vasculatures in vivo.
This study is the first report of the effect of IL-11 on brain endothelial cells
migration. IL-11 has been reported to induce rapid phosphorylation of gp130, STAT1,
54
STAT-3, p42/p44 MAPKs in HUVEC, another endothelial cell type, and cause a
cytoprotective effects during immune-mediated injury (Mahboubi, et al., 2000). Besides,
IL-11 was reported to induce survivin, an antiapoptotic protein, in HUVEC through STAT-
3 pathway (Mahboubi, et al., 2001). It was shown that activating STAT-3 signaling
pathway in human dermal microvascular endothelial cells (HDMEC) was critical in
HDMEC migration and tubule formation (Yahata, et al., 2003 and Kortylewski, et al.,
2005). When STAT-3 was blocked in HDMEC, cell migration and tubule formation were
inhibited (Yahata, et al., 2003 and Kortylewski, et al., 2005). Taken together with our
results, we propose that IL-11 can activate brain endothelial cell migration through STAT-
3 signaling pathway. However, more studies are needed to confirm this. Furthermore,
VEGF as a pro-angiogenic factor is also known to activate STAT-3 signaling pathway to
promote angiogenesis (Yahata, et al., 2003, Kortylewski, et al., 2005 and Bartoli, et al.,
2003). It has not been investigated whether there is a synergistic pro-angiogenic effect
of VEGF combined with IL-11.
Our data also demonstrate for the first time IL-11 induces glioma cell invasion
and migration. IL-11 and IL-11Rα are co-expressed by glioma cell lines U87 and U251.
This is in accordance with what was reported previously, IL-11 and IL-11Rα were co-
expressed in colorectal adenocarcinoma (Yoshizaki, et al., 2006) and gastric carcinoma
cell lines (Nakayama, et al., 2007). Exogenous IL-11 up-regulated invasion and
proliferation of human colorectal carcinoma cells (Yoshizaki, et al., 2006), and it up-
regulated invasion but not proliferation on gastric carcinoma cells (Nakayama, et al.,
55
2007), both were mediated through PI3K and MAPK pathways. In addition, there was
publication showing that IL-11 could activate the STAT-3 signaling pathway in
choriocarcinoma cells and increase their invasion (Suman, et al., 2009). IL-11 had also
been shown to significantly increase migration in the breast cancer cell line MDA-MB-
231 (Arihiro, et al., 2000). However, little had been done to study the mechanism of the
effects of IL-11 on glioma cells invasion and migration, including the signaling pathways
involved. IL-11 was known to utilize gp130 for signal transduction, a common
characteristic shared by IL-6 family of cytokines (Taga, et al., 1997). Upon binding to the
gp130 receptor, STAT-3, MEK/MAPK, and PI3K/AKT signaling pathways could be activated
(Alonzi, et al., 2001). These three pathways were the most commonly emphasized
invasion-related signaling pathways (Liu, et al., 2010). Thus, further investigation of the
mechanisms of glioma cell invasion and migration induced by IL-11 can be initiated by
testing the components in these pathways.
It was shown in our results that IL-11 did not stimulate brain endothelial cells or
glioma cells proliferation. Previous reports had indicated that IL-11 could stimulate cell
growth in human colorectal carcinoma cells (Yoshizaki, et al., 2006), and multiple
myeloma cells (Tsimanis, et al., 2004), while IL-11 did not affect cell proliferation in
human gastric carcinoma cells (Nakayama, et al., 2007), Lewis lung carcinoma cells
(Saitoh, et al., 2002), and trophoblast cells (Paiva, et al., 2007). We suggested the effect
of IL-11 on cell proliferation was cell type dependent.
56
By developing a peptide mimic of IL-11 that specifically targets IL-11Rα, it may
yield anti-tumor and anti-angiogenic effects (Lewis, et al., 2009). We can conjugate a
therapeutic region to this peptide, and use vector-mediated delivery approaches
induced transcytosis to have the peptide across the BBB (Newton, et al., 2006). We
propose once the peptide reaches its target, it ceases the signal transduction induced by
IL-11, thereby blocks the functional effects of IL-11 on endothelial cell migration, tumor
cell migration and invasion. As both of the tumor-associated brain endothelial cell and
tumor cell express IL-11Rα, this peptide can target not only the tumors but also their
vasculatures. One aspect that needs to be taken into consideration is that normal brain
endothelial cells also express IL-11Rα, so the targeting to endothelial cells may not be
specific to the tumor-associated ones. This aspect may be conquered by regional
delivery of this therapeutic peptide. By injection of the peptide into the tumor area, it
will diminish its effects on the normal vessels.
In the future study, in order to understand whether the binding of IL-11 to IL-
11Rα is necessary in mediating the cell migration and invasion, we can block IL-11Rα by
using neutralizing antibody, or knock down IL-11Rα expression by using IL-11Rα specific
siRNA, and compare with the untreated control cells of their migration and invasion rate
in response to IL-11. We propose there will be lower migration or invasion rate if IL-11Rα
is blocked or knocked down. And to study whether the disruption of IL-11Rα signaling
can delay the tumor growth, we can use intracranial animal model. By injecting IL-11Rα
specific siRNA treated glioma cells and the untreated glioma cells into the animals, we
57
monitor the tumor growth through bioluminescent animal imaging. We expect to see
slower tumor progression in the IL-11Rα specific siRNA treated cell group. With these
future experiments, we will be able to understand the role of IL-11 and its receptor in
GBM tumor progression.

58
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Abstract (if available)

Abstract Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) cytokine family, which utilizes a common membrane glycoprotein 130 (gp130) for signal transduction. Previously it had been shown IL-11 was an anti-inflammatory cytokine, stimulated megakaryocyte diffentiation and was used for treatment of chemotherapy-induced thrombocytopenia. Recently more attentions have been paid to its function in cancer. There is growing evidence showing IL-11 plays a role in tumor progression in many types of cancer. In our investigation, we studied the expression of IL-11 and its function on tumor angiogenesis and tumor progression, using glioblastoma multiforme (GBM), the most malignant, highly vascular and invasive brain tumor. Our studies show that IL-11 is present abundantly in the GBM tumor tissue as compared to the normal brain, and IL-11 has multiple effects. First, IL-11 stimulates normal and tumor-associated brain endothelial cells to migrate, and has no effect on cell proliferation. This suggests that IL-11 may play a role in promoting angiogenesis as a pro-angiogenic factor. Second, it enhances glioma cell migration and invasion without affecting cell number, indicating that IL-11 may be involved in glioma tumor invasion and progression. In addition, IL-11 receptor alpha, the binding receptor of IL-11, is expressed by tumor cells in GBM tissues while not expressed by astrocytes in the normal brain specimens. In summary, our studies suggest novel functions of IL-11 in GBM tumor. These findings are of great importance since they provide meaningful evidence for us to understand GBM better and potentially provide new therapeutic target for the treatment of this fatal disease.

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The role of tumor angiogenesis in glioblastoma multiforme and implications for anticancer therapy

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Molecular targets for treatment of glioblastoma multiforme

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Quantitative evaluation of the effectiveness of an integrin antagonist, vicrostatin, in cell migration

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Analysis of human brain endothelial cells versus human tumor associated brain endothelial cell tight junction and adherens junction expression differences in glioblastoma multiforme

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Examining perillyl alcochol in glioblastoma treatment and relating its effect to endoplasmic reticulum stress response to further advance its usage in combination treatment with dimethyl-celecoxib

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Targeting glioma cancer stem cells for the treatment of glioblastoma multiforme

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Kaposi's sarcoma associated herpes-virus induces cellular proxi expression to modulate host gene expression that benefits viral infection and oncogenesis

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Bone marrow derived mesenchymal stem cells promote survival and drug resistance in tumor cells

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Blockade of CXCR2 as a novel approach for cancer chemotherapy

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Studies on the role of a novel protein, TMEM 56 in tumorigenic growth for MCF-7 cells

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Ethanol induced modulation of microglial P2X7 receptor expression and its role in neuroinflammation

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The role of CD1d transmembrane and cytoplasmic tail domain in CD1d trafficking pathway

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Bone marrow-derived mesenchymal stromal cells in the tumor microenvironment of neuroblastoma

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The differential effects of selective COX-2 inhibitors on cell proliferation and induced ER stress in glioblastoma and pancreatic carcinoma cell lines

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Investigating the role of SASH1 gene located on chromosome 6 in ovarian cancer

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The noncanonical role of telomerase in prostate cancer cells: exploring a non-telomeric signaling role for telomerase protein (TERT) in a cancer cell line

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The role of survivin in drug resistant pediatric acute lymphoblastic leukemia

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The role of ErbB signaling in dendritic cells during inflammatory bowel disease

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Characterization of a fragment in secreted Hsp90α with potential therapeutic benefits in wound healing and cancer

Asset Metadata

Creator Liu, Zhi (author)

Core Title Interleukin-11: a study of its effects in glioblastoma multiforme

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2010-12

Publication Date 11/22/2010

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

Tag angiogenesis,brain endothelial cells,glioblastoma multiforme,interleukin-11,invasion,migration,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), DeClerck, Yves A. (committee member), Hofman, Florence M. (committee member)

Creator Email zhiliu.usc@gmail.com,zhiliu@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3551

Unique identifier UC1302508

Identifier etd-Liu-4192 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-409725 (legacy record id),usctheses-m3551 (legacy record id)

Legacy Identifier etd-Liu-4192.pdf

Dmrecord 409725

Document Type Thesis

Rights Liu, Zhi

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu