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Targeting glioma cancer stem cells for the treatment of glioblastoma multiforme

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Targeting glioma cancer stem cells for the treatment of glioblastoma multiforme

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Targeting Glioma Cancer Stem Cells for the Treatment of
Glioblastoma multiforme

by
Niyati Jhaveri

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC MOLECULAR AND CELLULAR BIOLOGY)

December 2015

Copyright 2015 Niyati Jhaveri
ii

“Much to learn you still have…my old padawan.”
Yoda (2002)

iii

DEDICATION
I dedicate this dissertation to my family for their unconditional love and
encouragement. To my father Sandip Jhaveri, my mother Bela Jhaveri and my
sister Aditi, thank you for supporting me throughout this journey and beyond!
iv

ACKNOWLEDGMENTS
This work has been made possible with the support of many people.
Firstly, I would like to thank my mentor Dr. Florence Hofman for her constant
guidance and encouragement throughout my time in graduate school. I came in
with little research experience and Dr. Hofman was very patient and supportive.
She is an eternal optimist and I am very grateful to her for always encouraging
me to continue trying. I owe a large part of my confidence and scientific prowess
to her mentorship and my experience in her laboratory has been truly enriching. I
would also like to thank Dr. Thomas Chen. Dr. Chen leads the USC Glioma
Research Group and was instrumental in initiating the NEO212 project. Having a
clinical co-mentor like him has really enhanced the translational aspect of this
study and I am excited about the implications for patients and future directions for
this novel compound. I wish him all the success and am grateful to have had the
opportunity to be a part of this study.
Additionally, I would also like to thank my committee members Dr. Pradip
Roy-Burman, Dr. Louis Dubeau and Dr. Young Hong for their valuable input
during my qualifying exam and annual research appraisals. Their critique has
really improved this dissertation. I would also like to express my gratitude to
members of the Glioma Research Group – Dr. Axel Schönthal, Dr. Stanley
Tahara and Dr. Josh Neman-Ebrahim for providing critical suggestions through
the course of this study.
v

I am especially grateful to Dr. Fabienne Agasse. Dr. Agasse isolated the
glioma stem cells and opened up a whole new avenue of research for the lab. I
learned a lot of the specialized stem cell techniques from her and I am very
thankful to her for her guidance and friendship. We have had a lot of volunteers,
research technicians and postdocs in the lab over the years that are great friends
and have been an incredible source of emotional support for me. Thank you –
Shering, Heeyeon, Marcela, Alex, Kim, Raquel, Tiago, Rachel, Pooja, Mickey,
Shayane, Natasha, David and Michelle.
I would also like to acknowledge all the people that have provided
valuable technical assistance for this project. I would like to thank Dr. Weijun
Wang for help with intracranial implantations, Dr. Don Armstrong and Dr.
Raphael Zidovetski for microarray data analysis, Michelle McVeigh at the USC
Liver Center for help with confocal microscopy, Lora Barsky and Bernadette
Masinsin for assistance with flow cytometry and Ryan Park and Tautis Skorka at
the USC Molecular Imaging Center for their endless support with bioluminescent
imaging and analysis. I would also like to thank Lisa Doumak for all her
administrative support and making the graduate school experience a lot of fun.
Finally, I would like to thank my family in Los Angeles – Surbhi, Malini,
Divya, Ayesha, Lindita and Ahanya for all the laughter and fun times that kept me
sane. I could not have made it this far without you. And to my family, thank you
for being my inspiration and strength.
vi

TABLE OF CONTENTS

Dedication iii
Acknowledgments iv
List of Tables viii
List of Figures ix
Abbreviations xii
Abstract xvii
Chapter I: Introduction 1
- Glioblastoma multiforme 1
- Glioma Cancer Stem Cells 6
- Temozolomide 14
- Glioma Cancer Stem Cells and Resistance 16
- Perillyl Alcohol 19

Chapter II: Characterization and Establishment of Glioma Cancer
Stem Cells from Human GBM resections 23
- Introduction 23
- Materials and Methods 26
- Results 31
- Significance and Purpose of Study 41

vii

Chapter III: Temozolomide-perillyl alcohol (NEO212), a Novel
Conjugate Targeting Proneural and Mesenchymal
Glioma Cancer Stem Cells 46
- Abstract 46
- Introduction 48
- Materials and Methods 51
- Results 56
- Discussion 73

Chapter IV: Discussion and Conclusion 79
- Overall Significance of the Study 79
- Clinical Implications 80
- Limitations of the Study and Future Directions 82
- Proposal for Pilot Project – Investigating the
Interactions Between Glioma Cancer Stem Cells
and Endothelial Cells in GBM Progression 86
- Concluding Remarks 108

Bibliography 110

viii

LIST OF TABLES
Table 2-1: Summary of GSC characteristics
Table 3-1: IC50 values for TMZ and NEO212 in GSCs

ix

LIST OF FIGURES
Figure 1-1: GBM incidence
Figure 1-2: GBM pathology
Figure 1-3: Molecular classification of GBM
Figure 1-4: Models of tumor heterogeneity
Figure 1-5: Origin of glioma cancer stem cells
Figure 1-6: Isolation and culture of glioma stem cells
Figure 1-7: Mechanism of temozolomide action and role of MGMT
Figure 1-8: Perillyl alcohol
Figure 2-1: Growth pattern of GSCs
Figure 2-2: Expression of stem cell markers in GSCs
Figure 2-3: In vivo tumor initiating capacity of GSCs
Figure 2-4: Characteristics of GSC-derived tumors
Figure 2-5: Classification of GSCs based on gene expression
Figure 2-6: Microvessel density in GSC-derived tumors
Figure 2-7: Classification of GSCs based on Lottaz’s gene
signature
x

Figure 2-8. NEO212
Figure 3-1: NEO212 cytotoxicity in GSCs
Figure 3-2: Long-term NEO212 cytotoxicity in GSCs
Figure 3-3: NEO212 cytotoxicity in normal neural stem cells
Figure 3-4: Role of MGMT in TMZ and NEO212 cytotoxicity
Figure 3-5: Mechanism of NEO212 cytotoxicity
Figure 3-6: In vivo efficacy of NEO212 in an intracranial GSC-
derived tumor model
Figure 4-1: NEO212 cytotoxicity in tumor endothelial cells
Figure 4-2: Endothelial-mesenchymal transition
Figure 4-3: Comparison of vasculature in normal human brain and
GBM
Figure 4-4: Localization of GSCs relative to the vasculature
Figure 4-5: Interactions between glioma stem cells and endothelial
cells
Figure 4-6: Increased α-SMA expression in co-cultures of USC02
and BEC
xi

Figure 4-7: Comparison of contact co-cultures and conditioned
medium for induction of EndMT
Figure 4-8: Decreased α-SMA expression with inhibition of Notch
signaling
Figure 4-9: Increased α-SMA expression in co-cultures of proneural
USC04 and BEC
Figure 4-10: NEO212 is a CSC-directed therapy targeting glioma
stem cell self-renewal and chemoresistance

xii

ABBREVIATIONS

BBB: Blood Brain Barrier
BCA: Bicinchoninic Acid
BEC: Brain Endothelial Cell
BMI-1: B cell-specific Moloney murine leukemia virus integration site 1
BMP7: Bone Morphogenetic Protein-7
BSA: Bovine Serum Albumin
CAF: Cancer-associated Fibroblasts
CDKN2A: Cyclin-dependent Kinase Inhibitor 2A
CM: Conditioned Medium
CSC: Cancer Stem Cell
DG: Dentate Gyrus
DMSO: Dimethyl Sulfoxide
EC: Endothelial Cell
ECM: Extracellular Matrix
EDTA: Ethylenediaminetetraaceticacid
xiii

EGF: Epidermal Growth Factor
EGFR: Epidermal Growth Factor Receptor
EMT: Epithelial-Mesenchymal Transition
EndMT: Endothelial-Mesenchymal Transition
ER: Endoplasmic Reticulum
ET-1: Endothelin-1
FBS: Fetal Bovine Serum
FDA: Food and Drug Administration
FGF-2/bFGF: Fibroblast Growth Factor-2/Basic Fibroblast Growth Factor
FSP1: Fibroblast-specific Protein 1
GBM: Glioblastoma Multiforme
GFAP: Glial Fibrillary Acidic Protein
GFP: Green Fluorescent Protein
GSC: Glioma Cancer Stem Cell
HDGF: Hepatoma-derived Growth Factor
IC: Inhibitory Concentration
xiv

IDH1: Isocitrate Dehydrogenase 1
IHC: Immunohistochemical
MGMT: O6-methylguanine-DNA Methyltransferase
MMP: Matrix Metalloproteinase
MMR: Mismatch Repair
MTIC: Methyltriazen-1-yl imidazole-4-carboxamide
MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
NF1: Neurofibromin 1
NOD-SCID: Non-obese Diabetic-Severe Combined Immunodeficient
NSC: Neural Stem Cell
O6-BG: O6-Benzylguanine
PARP: Poly-ADP Ribose Polymerase
PBS: Phosphate Buffered Saline
PDGFRA: Platelet-derived Growth Factor Receptor A
PDGFβ: Platelet-derived Growth Factor β
PE: Phycoerythrin
xv

PLG: Poly-lactide-co-glycolide
POH: Perillyl Alcohol
PTEN: Phosphatase and Tensin Homolog
RBC: Red Blood Cells
RTK: Receptor Tyrosine Kinase
SDF-1: Stromal-derived Factor 1
SDS: Sodium Dodecyl Sulfate
SFA: Sphere Forming Assay
SHH: Sonic Hedgehog
SMA: Smooth Muscle Actin
SOX2: Sex Determining Region Y Box-2
SP: Side Population
SVZ: Subventricular Zone
TAM: Tumor-associated Macrophage
TCGA: The Cancer Genome Atlas
TGFβ: Transforming Growth Factor β
xvi

TMZ: Temozolomide
Tregs: Regulatory T Cells
TuBEC: Tumor-associated Brain Endothelial Cell
VEGF: Vascular Endothelial Growth Factor

xvii

ABSTRACT

Glioblastoma multiforme (GBM) is the most common malignant brain
tumor characterized by high vascularity and invasion of tumor cells into the
surrounding brain parenchyma. Despite surgery, radiation and chemotherapy
with temozolomide (TMZ), a DNA alkylating agent, the prognosis for GBM
remains poor with a median survival time of 12-15 months. Tumor recurrence is a
major cause for mortality in GBM and glioma cancer stem cells (GSCs) have
been implicated as the source of origin and tumor recurrence. TMZ, the current
standard-of-care does not affect the GSCs and therefore, therapies directed
towards this critical drug resistant cell population are sorely needed.
In this study, we have identified two subtypes of GSCs differing in their
growth characteristics, marker expression, genetic profile and tumorigenic
potential. The proneural and mesenchymal patient-derived GSCs together
represent approximately 80% of human GBM. Thus, these GSCs recapitulate the
heterogeneity of human tumors and provide a clinically representative model to
evaluate novel therapeutic strategies. Elimination of GBM will require
chemotherapies that target both subtypes of GSCs particularly the clinically
aggressive mesenchymal subtype.
The significant finding of this study is the efficacy of a novel chemotherapy
NEO212, a conjugate of TMZ and perillyl alcohol (POH), a drug shown to have
moderate effects against resistant GBM. We found that NEO212 is cytotoxic to
xviii

both proneural and mesenchymal GSCs in vitro and in an orthotopic xenograft
model. NEO212 specifically affects GSCs through targeting two critical properties
of cancer stem cells – self-renewal and chemoresistance. NEO212 decreases
the expression of key self-renewal factors Bmi-1 (in proneural GSCs) and Sox2
(in mesenchymal GSCs) and induces extensive DNA damage independent of
MGMT, an important protein involved in DNA repair and drug resistance.
Our results showing the efficacy of NEO212 against both proneural and
mesenchymal GSCs make it a promising new therapy for GBM. NEO212 can be
effective as a first line therapy to prevent tumor recurrence or can be
administered following drug resistance to eliminate GSCs and delay tumor
recurrence. Thus, NEO212 has significant clinical implications for the treatment
of GBM.

1

CHAPTER I
INTRODUCTION
1.1 GLIOBLASTOMA MULTIFORME
Glioblastoma multiforme (GBM) is the most aggressive brain tumor
accounting for over 40% of primary brain and central nervous system
malignancies (Figure 1-1)(Ostrom et al., 2013). ‘Gliomas” are used to describe
tumors having histological features similar to astrocytes, oligodendrocytes or
ependymal cells – the glial cell populations found in the brain. However, the cell
of origin of these tumors is still unclear. Gliomas are further classified by grade
based on degree of malignancy which is a critical factor in determining choice of
treatment and regimen (Louis et al., 2007). According to the World Health
Organization, astrocytomas are classified into 4 grades: grade I are benign
pilocytic astrocytomas with low proliferation, grade II are infiltrative astrocytomas
with cytological atypia, grade III are anaplastic astrocytomas and grade IV are
GBM (Louis et al., 2007). Primary GBM tumors occur de novo and represent over
90% of all cases whereas secondary GBM accounting for less than 10% of
tumors arise from pre-existing anaplastic or low grade astrocytomas (The Cancer
Genome Atlas (TCGA) Research Network, 2008).

2

Figure 1-1. GBM incidence. GBM accounts for 45.2% of malignant primary
brain and central nervous system tumors (Figure adapted from Ostrom et al.,
2013).

Glioblastoma multiforme, as the name suggests is highly heterogeneous.
Macroscopically, it is characterized by regions of necrosis and hemorrhage
(Figure 1-2A). Microvascular proliferation (Figure 1-2B), pseudopalisading
necrosis (Figure 1-2C) and infiltration into the surrounding brain parenchyma are
distinguishing hallmarks of GBM (Holland, 2000; Wippold et al., 2006). Scherer
described the characteristic pattern of glioma invasion along white matter tracts
or blood vessels which is referred to as secondary structures of Scherer (Figure
1-2D) (Holland, 2000). In some GBM cases, the entire brain is diffusely infiltrated
with tumor cells, a condition known as gliomatosis cerebri (Holland, 2000).
3

Figure 1-2. GBM pathology. (A) Gross pathology of GBM showing areas of
necrosis and hemmorhage (dotted circle). The invasive nature of GBM is also
seen in this image with a primary tumor that has crossed the midline and formed
a secondary site of tumor growth (circle) (Figure adapted from WebPath: The
Internet Pathology Library). (B) Immunostaining for CD31 (red arrow), a marker
for blood vessels showing abnormal tumor vasculature in GBM. (C)
Pseudopalisading necrosis characterized by tumor cells (black arrows) lined up
in rows around a central area of necrosis (asterisk) (Figure adapted from Wippold
et al., 2006). (D) Distinctive pattern of tumor cell invasion along white matter
tracts or vasculature described as secondary structures of Scherer (black arrows)
(Figure adapted from Frontal Cortex, Inc. website).

The first step in GBM treatment is surgical debulking of the tumor but
owing to its infiltrative nature, surgery alone is not sufficient. Following surgery,
*
A. B.

C. D.

4

radiation and chemotherapy with temozolomide (TMZ), a DNA alkylating agent is
the current standard of care (Johnson et al., 2012). However, the median survival
of patients is less than 2 years due to drug resistance attributed to a cancer stem
cell population (Beier et al., 2011; Chen et al., 2012; Stupp et al., 2009). Thus,
treatment for GBM remains a significant challenge and there is an imperative
need to find novel and effective therapeutic strategies.
1.1.1 MOLECULAR CLASSIFICATION OF GBM
Cancer is a genetic disease and The Cancer Genome Atlas (TCGA) has
sequenced over 200 GBM specimens to obtain a comprehensive view of all the
genetic alterations driving tumor pathogenesis. The TCGA data indicates three
core pathways that are frequently disrupted in GBM – (1) Receptor tyrosine
kinase (RTK)/Ras/PI3K signaling, (2) p53 signaling and (3) Rb signaling (The
Cancer Genome Atlas (TCGA) Research Network, 2008). Verhaak et al. have
expanded on the TCGA data to identify four molecular subtypes of GBM based
on abnormalities in gene expression – classical, mesenchymal, proneural and
neural (Figure 1-3) (Verhaak et al., 2010). This designation is based on the
salient features of genes characterizing each subtype. The classical subtype is
distinguished by epidermal growth factor receptor (EGFR) amplification but lack
of TP53 mutations (Verhaak et al., 2010). The mesenchymal subtype is
characterized by deletions in neurofibromin 1 (NF1), high expression of
mesenchymal markers such as MET and CD44 and increased necrosis and
5

inflammation (Verhaak et al., 2010). Platelet-derived growth factor receptor A
(PDGFRA) amplifications, point mutations in isocitrate dehydrogenase 1 (IDH1)
and TP53 mutations are frequently observed in the proneural subtype. Proneural
tumors also have high expression of oligodendrocytic development genes such
as OLIG2 and proneural development genes such as SOX, DCX and DLL3
(Verhaak et al., 2010). The neural subtype expresses neuron markers such as
GABRA1 and SLC12A5. Normal brain tissue classifies under the neural subtype
(Verhaak et al., 2010). Secondary GBMs are found more in the proneural group
which has younger patients and is associated with longer survival (Phillips et al.,
2006; Verhaak et al., 2010).
Gene expression-based molecular classification of GBM provides valuable
information about glioma pathogenesis as well as for designing effective targeted
therapeutic strategies. It is superior to the histological-based classification which
is highly subjective. Most tumors are heterogeneous and cannot be placed into
one group based on morphological criteria. In a number of cases, the histological
diagnosis and clinical course do not correspond (Louis et al., 2001). A new
molecular classification system based on the TCGA data is being devised
currently and will be more predictive of patient/treatment outcome in the future.
6

Figure 1-3. Molecular classification of GBM. Gene expression analysis of over
100 GBM tumors indicating stratification into four subtypes – proneural, neural,
classical and mesenchymal based on mutations and alterations in gene
expression (Figure reproduced from Verhaak et al., 2010).

1.2 GLIOMA CANCER STEM CELLS
Two models of tumor development have been proposed: (1) the stochastic
model suggests that tumors are highly heterogeneous and all tumor cells have
the potential to give rise to tumors through clonal evolution driven by mutations
but this occurs at a low frequency (2) the hierarchical/cancer stem cell model
suggests that only a small subpopulation within tumors called cancer stem cells
can initiate and sustain tumor progression (Figure 1-4) (Reya et al., 2001).
7

Figure 1-4. Models of tumor heterogeneity. Two models have been proposed
to explain the initiation and development of tumors. The stochastic model posits
that all tumor cells have a low probability of initiating tumors whereas the
hierarchical model suggests that only a small subpopulation of cancer stem cells
(CSCs) within the tumor can initiate and propagate tumors. (Figure adapted from
Reya et al., 2001).

The cancer stem cell hypothesis has been proven in the development of
many cancers most notably acute myeloid leukemia and breast cancer (Al-Hajj et
al., 2003; Bonnet et al., 1997; Collins et al., 2005). Recently, several groups have
isolated cells with stem cell-like properties from human brain tumors (Galli et al.,
2004; Ignatova et al., 2002; Singh et al., 2004; Yuan et al., 2004), evidence
supporting the hierarchical model for GBM. Like neural stem cells (NSCs), glioma
cancer stem cells (GSCs) show limitless self-renewal potential and multipotent
differentiation (Galli et al., 2004; Singh et al., 2004; Yuan et al., 2004). GSCs
express many neural stem cell markers such as CD133, a cell surface
Stochastic model Hierarchical/CSC model
8

transmembrane protein and Nestin, an intermediate filament protein (Yuan et al.,
2004). GSCs also express Sox2, a transcription factor involved in self-renewal
(Gangemi et al., 2009). A defining characteristic of GSCs that distinguishes them
from NSCs is the ability of GSCs (also called tumor initiating cells) to give rise to
tumors in immune compromised mice at low cell numbers; NSCs do not form
tumors. Moreover, GSCs are also characterized by karyotypic or genetic
alterations that are not present in NSCs (Vescovi et al., 2006). GSC-derived
tumors recapitulate the invasive nature of human GBM (Galli et al., 2004; Singh
et al., 2004; Yuan et al., 2004). Moreover stem cells can be isolated from these
tumors and can be serially transplanted to give rise to secondary tumors,
demonstrating in vivo self-renewal potential (Galli et al., 2004; Singh et al., 2004).
GSCs are believed to be the source of origin and tumor recurrence in
glioblastoma (Chen et al., 2012; Lathia et al., 2011). Lathia et al. co-injected
differentially labelled GSCs and non-GSCs isolated from a recurrent human GBM
tumor orthotopically into a murine brain. When implanted in the same
microenvironment, only the GSC fraction grew and produced tumors (with
neurological symptoms observed around days 37-42 post implantation), even
though the GSC population represented only 10% of the injected cells (Lathia et
al., 2011). The non-GSC population did not form tumors; thus the GSC
containing population rather than the non-stem cell population are responsible for
tumor initiation and growth. GSCs have also been implicated in tumor recurrence
following drug resistance. Chen et al. have shown that in a spontaneous murine
9

glioma model with 100% penetrance when temozolomide treatment is
discontinued, the first cell population to undergo proliferation and lead to tumor
regrowth is the nestin-positive stem cell population (Chen et al., 2012). Moreover,
CD133 positive-GSCs are enriched in recurrent GBM (Huang et al., 2008; Liu et
al., 2006; Pallini et al., 2011). GSCs isolated from recurrent tumors form more
aggressive and invasive tumors in athymic mice compared to GSCs isolated from
primary tumors derived from the same patient (Huang et al., 2008). These data
strongly suggest that GSCs contribute to tumor regrowth from minimal residual
disease post-surgery.
GSCs are found in close association with vascular niches which regulate
their self-renewal and tumorigenicity (Calabrese et al., 2007; Galan-Moya et al.,
2011; Infanger et al., 2013; Lathia et al., 2011; Zhu et al., 2011). Other studies
have found GSCs to be highly enriched in the hypoxic core of the tumor than the
periphery (Piccirillo et al., 2009; Pistollato et al., 2010). Hypoxia supports GSC
self-renewal, proliferation and tumorigenicity (Heddleston et al., 2009). Moreover,
hypoxia can induce non-stem cells to acquire GSC characteristics and increased
tumorigenic potential (Heddleston et al., 2009).
1.2.1 ORIGIN OF GLIOMA CANCER STEM CELLS
Cancer is a result of mutagenic events that provide a growth or survival
advantage to cells. These mutagenic events occur over a long time period,
10

supporting stem cells that proliferate indefinitely as the targets for transformation
and source of tumor compared to quiescent terminally differentiated cells.
Seminal studies in the early 1990s by Reynold and Weiss identified
multipotent neural stem cells in the adult murine brain (Reynolds et al., 1992).
Adult neurogenesis was also described in humans with neural stem cells found
mainly in the subventricular zone (SVZ) of the lateral ventricles or the dentate
gyrus (DG) of hippocampus (Lie et al., 2004; Vescovi et al., 2006). Neural stem
cells have the capacity to self-renew indefinitely and give rise through
differentiation to all three cell lineages found in the central nervous system -
astrocytes, oligodendrocytes and neurons (Vescovi et al., 2006).
Studies in mice suggest that GSCs may arise from neoplastic
transformation of NSCs (Figure 1-5). NSCs and GSCs share many pathways that
regulate self-renewal and mutations in these pathways cause tumors (Pardal et
al., 2003). Activation of Ras and Akt or overexpression of platelet-derived growth
factor β (PDGFβ) in Nestin-positive neural progenitors increases tumor incidence
in mice (Dai et al., 2001; Holland et al., 2000). Moreover, inactivation of tumor
suppressors - p53, PTEN and NF1 in NSCs induces astrocytomas (Alcantara
Llaguno et al., 2009). However, overexpression of EGFR and loss of CDKN2A in
mature astrocytes also leads to glioma tumors (Bachoo et al., 2002). Thus, GSCs
may also arise by acquisition of stem cell-like properties by dedifferentiation of
mature tumor cells (Figure 1-5).
11

Figure 1-5. Origin of glioma cancer stem cells. Glioma cancer stem cells
(GSCs) may arise from oncogenic transformation of neural stem cells (NSCs) or
by dedifferentiation of progenitor or mature tumor cells. (Figure adapted from
Goffart et al., 2013).

1.2.2 ISOLATION AND CULTURE OF GSCS
A selective culture system consisting of serum-free medium with
epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) has been
used for isolation and enrichment of stem cells (Figure 1-6) (Galli et al., 2004;
Yuan et al., 2004). Under these conditions, most differentiated cells will die after
several passages whereas stem cells can undergo indefinite self-renewal as
De
differentiation
NSC GSC
Transformation
Self-
renewal
Self-
renewal
Differentiated
tumor cell
GBM
Neural progenitor Glioma progenitor
Neuron
Oligodendrocyte

Astrocyte
Transformation
Transformation
De
differentiation
12

neurospheres and can be serially passaged. The percent of stem cells varies
with the stage of culture, the highest being during dissociating and re-passaging.
However, transient amplifying precursor cells can also form neurospheres and be
passaged a limited number of times. Furthermore, neurospheres may arise from
cell aggregation rather than self-renewal.

Figure 1-6. Isolation and culture of glioma stem cells. GSCs are isolated from
brain tumors using the neurosphere assay. Brain tumors are homogenized and
enzymatically dissociated. Cells are cultured in serum-free medium with
epidermal growth factor (EGF) and fibroblast growth factor (FGF-2) which allows
preferential selection of stem cells. Stem cells self-renew forming neurospheres
which can be dissociated into single cells and generate secondary neurospheres.
Stem cells can also be induced to differentiate into astrocytes, oligodendrocytes
or neurons following growth factor withdrawal and addition of fetal bovine serum
(FBS) (Figure adapted from Vescovi et al., 2006).

GSCs can also be isolated based on expression of markers such as
CD133, L1CAM or SSEA-1/CD15 (Bao et al., 2008; Singh et al., 2004; Son et al.,
Serum free medium
+ EGF + FGF-2
Serum free medium
+ EGF + FGF-2
+ FBS
13

2009). L1CAM is a neural cell adhesion molecule that is involved in axon
guidance and neuronal migration and differentiation (Bao et al., 2008). CD15, an
extracellular matrix-associated carbohydrate expressed on embryonic stem cells
and neural progenitors is important for cell adhesion (Capela et al., 2006).
CD133, a cell surface transmembrane protein is the most widely used GSC
marker. Studies have shown that only CD133-positive cells have tumorigenic
potential in non-obese diabetic, severe combined immunodeficient (NOD-SCID)
mice (Singh et al., 2004). As few as 100 CD133-positive cells formed tumors
whereas 10
5
CD133-negative cells did not show any tumor formation up to 12
weeks (Singh et al., 2004). The percentage of CD133-positive cells within GBMs
varied between 19-29% (Singh et al., 2004). Moreover, CD133-positive cells
could be serially transplanted in mice, evidence of self-renewal in vivo (Singh et
al., 2004). Subsequent studies, however, have found that CD133-negative cells
also have stem cell-like properties and can give rise to tumors. (Lottaz et al.,
2010; Wang et al., 2008). In addition, CD133 expression may vary depending on
environmental factors such as hypoxia and cellular stress causing mitochondrial
dysfunction (Griguer et al., 2008). These studies have put into question the
reliability of CD133 as a GSC marker and it remains a controversial and highly
debated topic in GSC research.
GSCs have also been isolated based on expression of ATP-binding
cassette transporters such as ABCG2 which efflux Hoechst 33342 dye resulting
in an unlabeled side population (SP) of enriched stem cells (Chua et al., 2008;
14

Hirschmann-Jax et al., 2004). However, not all stem cells are found within the SP
and cells other than stem cells are also present in the side population. Moreover,
Hoechst staining decreases clonogenic potential which may give spurious
results. Regardless of the method used to isolate GSCs, it is important to validate
the presence of GSCs by functional assays for drug resistance and tumorigenic
potential.
1.3 TEMOZOLOMIDE
The current standard of care for GBM is surgical resection followed by
radiation and chemotherapy with temozolomide. Radiotherapy consists of daily
fractioned focal irradiation of 2 Gy administered 5 days a week for 6 weeks
totaling 60 Gy (Stupp et al., 2009). Conventionally, temozolomide is given orally
at 75 mg/m
2
of body surface area daily along with radiation followed by six cycles
of 150-200 mg/m
2
for 5 days of a 28 day cycle (Stupp et al., 2009). A critical
phase III trial conducted by the European Organization for Research and
Treatment of Cancer (EORTC) and National Cancer Institute of Canada Clinical
Trials Group (NCIC) demonstrated that addition of temozolomide to radiotherapy
increased patient survival from 12.1 months to 14.6 months (Stupp et al., 2009)
and this is currently the accepted regimen for newly diagnosed GBM.
Temozolomide is an imidazotetrazine derivative prodrug that undergoes
spontaneous metabolism to methyltriazen-1-yl imidazole-4-carboxamide (MTIC)
at alkaline pH (Jarvis et al., 2002). MTIC then undergoes degradation to form a
15

highly reactive methyl diazonium cationic species that alkylates DNA. DNA
methylation occurs more frequently at the N7 position of guanine and N3 position
of adenine. O
6
methylation of guanine represents only 5% of the lesions induced
by TMZ but is the most cytotoxic because O
6
-methylguanine mispairs with
thymine instead of cytosine during replication initiating mismatch repair (MMR)
(Figure 1-7) (Fu et al., 2012; Jarvis et al., 2002). The MMR system removes the
mispaired thymine but is unable to remove the methylated moiety eventually
leading to repeated futile cycles of mismatch repair, double-stranded DNA
breaks, replication fork arrest and cell death by apoptosis (Jarvis et al., 2002).
A major consideration in GBM therapy is the blood brain barrier (BBB).
The BBB is comprised of endothelial cells, pericytes and astrocytes in close
association to prevent the entry of toxic substances into the brain. BBB
permeability is an essential characteristic for drugs designed for the treatment of
GBM. Temozolomide is lipophilic and can easily cross the BBB (Jarvis et al.,
2002). Furthermore, it is 100% orally bioavailable and has an elimination half-life
of less than 2 hours in plasma (Jarvis et al., 2002). However, a major dose
limiting factor is the toxicity associated with temozolomide particularly
myelosuppression (Jarvis et al., 2002). Additionally, although temozolomide has
increased median survival of GBM patients to over a year, eventually tumors
develop resistance and recur. Therefore, there is an urgent need to develop
novel and effective therapies for the treatment of temozolomide-resistant GBM.
16

1.4 GLIOMA CANCER STEM CELLS AND RESISTANCE
Chemoresistance of glioma cancer stem cells has been attributed as the
cause of tumor recurrence. Indeed, several studies have shown that GSCs are
resistant to temozolomide in vitro (Beier et al., 2008; Beier et al., 2012; Blough et
al., 2010; Chamberlain et al., 2011; Eramo et al., 2006; Liu et al., 2006).
Temozolomide induces reversible G2/M cell cycle arrest and inhibits proliferation
but most GSCs are able to recover, resume proliferation and maintain their
tumorigenic potential (Mihaliak et al., 2010). In accordance with this, GSCs are
found to be enriched and more aggressive in recurrent GBM tumors (Huang et
al., 2008; Liu et al., 2006).
1.4.1 O
6
-METHYL GUANINE-DNA METHYL TRANSFERASE (MGMT)
Several mechanisms of drug resistance have been reported.
Overexpression of O
6
-methylguanine-DNA methyltransferase (MGMT) is seen in
approximately 40% of gliomas and is a major mechanism for resistance to TMZ
(Johannessen et al., 2012). MGMT transfers the methyl group on O
6
guanine to
a cysteine residue within its active site and is irreversibly inactivated in the
process (Figure 1-7). MGMT expression is regulated by epigenetic silencing
through methylation of its promoter. Patients with methylated MGMT promoter
survive longer with temozolomide treatment than patients expressing MGMT
(Hegi et al., 2005). GSCs have varying degrees of sensitivity to temozolomide in
vitro. GSCs isolated from the core of the tumor have high CD133 and MGMT
17

expression and are more resistant to temozolomide than GSCs isolated from the
tumor periphery (Pistollato et al., 2010). MGMT-negative GSCs can be
eliminated in vitro with an alternating dosing regimen of 7 days on temozolomide
followed by 7 days off (Beier et al., 2012). This treatment protocol also inhibits
growth of MGMT-positive GSCs to some extent but requires significantly higher
concentrations which are clinically unachievable due to toxic side effects. O
6
-
benzyl guanine (O6-BG), a pseudosubstrate for MGMT, has been shown to
increase sensitivity to TMZ in GSCs in vitro but has met with limited success in
the clinic (Beier et al., 2008; Quinn et al., 2009).

TMZ
G
C
Replication
G
T
G
MMR
Replication
Apoptosis
MGMT
Survival
MGMT
G
C
18

Figure 1-7. Mechanism of temozolomide action and role of MGMT.
Temozolomide (TMZ), an alkylating agent methylates DNA at the O
6
position of
guanine. O
6
methyl guanine mispairs with thymine during replication inducing the
mismatch repair system (MMR). MMR is unable to remove the methyl group from
guanine resulting in repeated unsuccessful attempts ultimately leading to DNA
double strand breaks and apoptosis. MGMT removes the methyl group from
guanine and restores the DNA to its original state, thus protecting against TMZ
cytotoxicity (Figure adapted from Fu et al., 2012).

1.4.2 OTHER MECHANISMS OF RESISTANCE
Additional mechanisms of resistance reported in tumors involving DNA
repair pathways include a defective mismatch repair system and overexpression
of proteins involved in the base excision repair pathway which fixes N7 methyl
guanine and N3 methyl adenine lesions (Agnihotri et al., 2012; Cahill et al., 2008;
Trivedi et al., 2005; Yip et al., 2009). These mechanisms remain to be explored
in GSCs.
Moreover, overexpression of drug efflux pumps belonging to the ATP-
binding cassette transporter family including p-glycoprotein or ABCG2 may also
play a role in drug resistance by decreasing therapeutic concentrations of drug
within the tumor (Chua et al., 2008; Schaich et al., 2009). Studies have shown
that temozolomide treatment increases the ‘side population’ cells expressing high
ABCG2 (Chua et al., 2008). However, temozolomide has not been shown to be a
substrate for ABCG2. Moreover, knockdown of ABCG2 does not alter
temozolomide sensitivity (Chua et al., 2008).
19

The Sonic hedgehog (SHH) and Notch signaling pathways have been
shown to be important for GSC self-renewal and tumorigenicity (Clement et al.,
2007; Fan et al., 2010). Recent studies have shown that these two pathways are
also involved in TMZ resistance (Ulasov et al., 2011). Temozolomide treatment of
GSCs increases expression of NOTCH1 and GLI1, critical mediators of the Notch
and SHH signaling pathway respectively (Ulasov et al., 2011). Pharmacological
inhibition of these pathways in combination with temozolomide significantly
enhances cell death (Ulasov et al., 2011).
GSCs are also resistant to other chemotherapeutic drugs such as
etoposide, cisplatin, campothecin, doxorubicin and paclitaxel (Chamberlain et al.,
2011; Eramo et al., 2006). GSCs are particularly resistant to radiation due to an
effective DNA damage repair response (Bao et al., 2006). Moreover, Notch
signaling in GSCs promotes self-renewal and protects against radiation (Wang et
al., 2010).
Extrinsic factors such as hypoxia and the vascular niche may also
contribute to GSC resistance to chemotherapy. Moreover, the blood brain barrier
limits the availability of chemotherapies to the tumor.
1.5 PERILLYL ALCOHOL
Perillyl alcohol (POH) is a naturally occurring monocyclic terpene found in
lavender oil, peppermint, celery and several other plants (Figure 1-8) (Chen et
al., 2015). It is derived from limonene through hydroxylation by cytochrome P450
20

enzymes (Duetz et al., 2003). POH is commonly found in household cleaning
products, cosmetics and food additives (Chen et al., 2015). Moreover, in vivo
studies in animal models of pancreatic, breast, liver and brain cancer showed
significant anti-tumor effects (Cho et al., 2012; Lebedeva et al., 2008; Mills et al.,
1995; Yuri et al., 2004). Clinical trials with POH, however, have met with limited
success because of lack of efficacy, intolerable gastrointestinal toxicity and
patient non-compliance due to the unpleasant taste associated with oral
administration of POH (Chen et al., 2015). In Brazil, POH has been tested in
clinical trials for recurrent malignant glioma via inhalational intranasal delivery (da
Fonseca et al., 2008). Intranasal delivery avoids first-pass hepatic metabolism
and allows for increased and quick access to the tumor site in a non-invasive
manner (Turker et al., 2004). It may also bypass the blood brain barrier by entry
through the olfactory or trigeminal nerve pathway (Chen et al., 2015). In a Phase
I/II study, intranasal administration of POH was well tolerated and increased
progression-free survival in patients with recurrent malignant glioma (da Fonseca
et al., 2008). Moreover, POH was more effective in patients with secondary
GBMs arising from preexisting tumors and patients with deep midline or
subcortical tumors (da Fonseca et al., 2011). After 4 years of continuous POH
treatment, 19% of patients were in remission without any observable
hematological, hepatic, renal or lung toxicity (da Fonseca et al., 2013) compared
to an overall survival of 12.1% at 4 years with the standard Stupp protocol (Stupp
et al., 2009).
21

Figure 1-8. Perillyl alcohol. Perillyl alcohol (POH) is a monoterpene shown to
be effective against recurrent GBM in clinical trials in Brazil (Figure adapted from
Cho et al., 2012).

Previous studies from our lab have shown that in an intracranial murine
model of human glioma, intranasal administration of POH significantly increased
survival without any apparent toxicity to normal organs (Cho et al., 2012). The
mechanism of action is not fully understood. Moreover, the effect of POH on
glioma stem cells has not been investigated. In vitro studies in tumor cells have
shown that POH induces G1 arrest, endoplasmic reticulum (ER) stress and
apoptosis (Cho et al., 2012; Clark, 2006; Shi et al., 2002). Inhibition of the
oncogenic Ras signaling pathway by inhibition of posttranslational isoprenylation
has also been reported but is controversial (Chaudhary et al., 2009; da Fonseca
et al., 2008). We have also shown that POH inhibits glioma cell invasion and may
have an effect on tumor angiogenesis by decreasing the production of pro-
angiogenic cytokines such as vascular endothelial growth factor (VEGF) and
interleukin-8 (IL-8) (Cho et al., 2012). The United States Food and Drug
22

Administration (FDA) has recently approved clinical trials for intranasal
administration of NEO100, a purified form of POH, in patients with recurrent
glioblastoma (Chen et al., 2015).

23

CHAPTER II
CHARACTERIZATION AND ESTABLISHMENT OF GLIOMA CANCER STEM
CELLS FROM HUMAN GBM RESECTIONS

INTRODUCTION
There is strong evidence supporting the hierarchical model of tumor
propagation for glioblastoma multiforme with the isolation of glioma cancer stem
cells by several groups (Galli et al., 2004; Ignatova et al., 2002; Singh et al.,
2004; Yuan et al., 2004). Glioma stem cells have the cardinal cancer stem cell
characteristics of unlimited self-renewal and tumor initiation (Lathia et al., 2011).
GSCs can also undergo multi-lineage differentiation although they show aberrant
patterns co-expressing multiple markers of differentiation such as glial fibrillary
acidic protein (GFAP) and class III beta tubulin (Tuj1) (Lee et al., 2006). In
addition, GSCs are critical in tumor recurrence due to resistance (Chen et al.,
2012). Radiation and temozolomide target the bulk of the tumor comprised of
actively proliferating cells but GSCs are resistant in part due to activated DNA
repair mechanisms (Bao et al., 2006; Beier et al., 2011). Residual GSCs
eventually cause tumor recurrence which is associated with the poor survival of
GBM patients.
GSCs can be identified on the basis of expression of markers such as
CD133, Nestin and Sox2; however, the reliability of these markers is
controversial. CD133/Prominin-1 is a penta-transmembrane glycoprotein with
24

unknown function expressed in fetal neural stem cells (Holmberg Olausson et al.,
2014). CD133-positive cells isolated from GBM specimens have properties of
NSCs but are also highly tumorigenic, indicating that CD133 also labels a
population of glioma cancer stem cells (Singh et al., 2004). Nestin is an
intermediate filament protein also found in NSCs and GSCs (Singh et al., 2004;
Wiese et al., 2004; Yuan et al., 2004). Nestin expression is lost upon
differentiation (Wiese et al., 2004). Sex determining region Y box-2 (Sox2), a key
transcription factor belonging to the high mobility group domain protein family is
essential for induction of pluripotency in human somatic cells along with Oct4,
Klf4 and c-Myc (Takahashi et al., 2007). Sox2 is also important in self-renewal
and tumorigenicity of GSCs (Gangemi et al., 2009). Recently, c-Met has
emerged as a functional marker for a subset of GSCs. Cells expressing high c-
Met show long term self-renewal in vitro and faster tumor growth in vivo than
cells with low levels of c-Met (De Bacco et al., 2012).
GSCs with different characteristics have been described based on gene
expression patterns (Gunther et al., 2008; Lottaz et al., 2010). Lottaz et al. have
characterized two subtypes of GSCs – type I expressing CD133 and
corresponding to the proneural subtype and type II lacking CD133 expression
and corresponding to the mesenchymal GBM subtype described by Verhaak et
al. (Lottaz et al., 2010). Furthermore, GSCs may have different phenotypic and
genetic profiles based on their location within tumors. GSCs isolated from the
core of the tumor grow as neurospheres and have higher clonogenic potential
25

and tumorigenicity compared to GSCs at the tumor periphery (Piccirillo et al.,
2009). Pistollato et al. have described a three layer concentric model with more
immature GSCs expressing CD133 located within the hypoxic core of the tumor
(Pistollato et al., 2010). These core GSCs also express MGMT and are resistant
to temozolomide in comparison to cells located at the vascularized periphery of
the tumor (Pistollato et al., 2010).
Since GSCs are the critical population involved in tumor initiation and
recurrence, these chemoresistant cells are an important target for GBM therapy
and strategies directed towards eliminating GSCs would significantly improve
GBM prognosis. Moreover, tumors derived from GSCs faithfully mimic the
phenotypic heterogeneity and invasiveness of human GBM tumors (Lee et al.,
2006; Pollard et al., 2009; Wakimoto et al., 2012); this makes GSCs an excellent
and reliable model for in vitro and in vivo analyses to investigate clinically
relevant therapeutics. To this end, we sought to isolate and establish GSC
cultures from human GBM surgical resections.
Two GSCs – USC02 and USC04 were isolated from human GBM and
were able to self-renew up to 20 passages (thus far) in serum-free medium.
GSCs differed in growth pattern and stem cell marker expression. USC02 was
semi-adherent and CD133 negative whereas USC04 grew as neurospheres and
had a subpopulation of CD133 positive cells. Both GSCs were able to initiate
invasive tumors in immune compromised mice. Microarray analysis revealed that
26

these GSCs belong to two distinct subtypes; USC02 had a mesenchymal
signature and USC04 was proneural. Thus, we have established two distinct
GSC cultures which show characteristics of glioma stem cells mainly self-renewal
and tumorigenicity. These GSCs provide a representative model to evaluate
novel chemotherapies.

MATERIALS AND METHODS
Isolation of glioma stem cells and differentiated primary tumor cells
Freshly resected GBM tissues were obtained and processed in
accordance with the University of Southern California (USC) Institutional Review
Board guidelines. Tissues were rinsed in sterile phosphate buffered saline (PBS),
cut into small pieces and homogenized with a cell douncer. The cells were
centrifuged at 3500 rpm for 5 minutes and the pellet resuspended in trypsin-
EDTA in PBS dissociation buffer (1:1). After digestion for 20 minutes at 37°C,
trypsin activity was neutralized with fetal bovine serum (FBS) (Omega Scientific,
Inc., Tarzana, CA). Red blood cells (RBCs) were eliminated by treatment with
RBC lysis buffer (155 mM ammonium chloride, 11 mM sodium bicarbonate, 68
µM EDTA, pH 7.4) for 5 minutes at room temperature. Next, the mixture was
centrifuged again and the final pellet resuspended in cancer stem cell culture
(CSC) medium containing DMEM F12 medium (Life Technologies, Grand Island,
NY) supplemented with 1% penicillin-streptomycin, 1% B-27 (Life Technologies)
and 20 ng/ml EGF and FGF-2 (Peprotech, Rocky Hill, NJ). This solution was
27

passed through a 70 µm cell strainer to get rid of tissue debris and plated on a 10
cm petri dish. Cultures were observed routinely and replenished with new
medium once a week. For passaging and expanding sphere cultures, spheres
were dissociated into single cells with PBS dissociation buffer and cells were
resuspended in CSC culture medium. For isolation of primary tumor cells, tissue
was rinsed in PBS, cut into small pieces and homogenized. The homogenate
was centrifuged and the cell pellet was resuspended in DMEM medium
(Mediatech, Inc., Manassas, VA) with 1% penicillin-streptomycin and 10% FBS.
Immunofluorescence staining
Glioma stem cells were seeded onto glass coverslips precoated with
Matrigel (BD Biosciences, San Jose, CA). After 24 hours allowing for cell
adherence, cells were rinsed with PBS, permeabilized and blocked with 3%
bovine serum albumin (BSA), 0.3 % Triton-X in PBS for 30 minutes at room
temperature. Primary antibodies Sox2 (Santa Cruz Biotechnology, Dallas, TX;
sc-17320 - 1:200) and Nestin (EMD Millipore, Billerica, MA; MAB5326 - 1:200)
were added overnight at 4°C. The next day, cells were washed with PBS and
incubated with appropriate Alexafluor secondary antitbodies (Life Technologies)
at recommended dilutions for 1 hour at room temperature. Hoechst was added to
the secondary antibody solution as a counterstain. Cells were subsequently
washed and mounted with DAKO mounting medium (Vector Laboratories,
28

Burlingame, CA). Immunofluorescence images were taken using Zeiss LSM 710
confocal scanning microscope.
Flow cytometry
Glioma stem cells were dissociated into single cells with PBS dissociation
buffer. For CD133 analysis, 10
5
single cells were resuspended in 80 µL PBS
buffer containing 0.5 % BSA and 2 mmol/L EDTA. FcR blocking reagent and
CD133/2 (293C3)-PE antibody (Miltenyi Biotec, San Diego, CA; 130-098-046 –
1:11) were added according to manufacturer’s recommendations. Normal mouse
IgG1-PE antibody (Santa Cruz Biotechnology; sc-2866) was used as isotype
control. Cells were incubated at 4°C in the dark. After 30 minutes, cells were
centrifuged, washed with buffer solution and resuspended in 400 µl buffer
containing 20 µl BD Viaprobe solution (BD Biosciences,) to gate out dead cells
for flow cytometry. For c-Met analysis, 10
5
cells were washed and blocked with
incubation buffer containing 0.5% BSA in PBS for 10 minutes. c-Met antibody
(Cell Signaling, Danvers, MA; 5631 - 1:50) was added and cells incubated for 1
hour on ice. CD68 (Santa Cruz Biotechnology; sc-20060) was used as isotype
control. After two washes with buffer, cells were incubated with donkey anti-
mouse Alexafluor 488 antibody (Life Technologies; A21202 – 1:50) for 30
minutes on ice, rinsed and resuspended in PBS with BD Viaprobe (BD
Biosciences) for flow analysis. Analysis was performed on a CyAn ADP analyzer.
At least 1,000 live events were calculated.
29

In vivo experiments
All animal protocols were approved by the USC Institutional Animal Care
and Use Committee and strictly adhered to. Four to six week male NOD-SCID
mice (Harlan Laboratories, Indianapolis, IN) were implanted intracranially with
1000 GSCs or primary human glioma cells as described previously (Virrey et al.,
2009). Briefly, animals were held in a stereotactic frame, a paramedian incision
was made and a 1.5 mm bur hole was drilled 1 mm anterior to the coronal suture
on the right hemisphere and 2 mm lateral from the midline. Cells were injected
(1000 cells in 5 µL PBS) via a Hamilton syringe into the right frontal lobe and the
incision was sutured with a silk thread. Mice were monitored for signs of tumor
growth such as significant weight loss, hunched back, immobility, ataxia or
seizures. At time of euthanasia, brains were harvested, frozen in optimal cutting
temperature (O.C.T.) compound (VWR, Visalia, CA) and sectioned for analysis.
Immunohistochemistry and microvessel density analysis
Tissue sections were fixed in acetone, washed with PBS and blocked with
SeaBlock (Thermo Scientific, Rockford, IL) for 20 minutes. Subsequently,
sections were incubated with antibodies to Ki67 (DAKO, Carpinteria, CA; M7240
– 1:100), CD133 (EMD Millipore; MAB4399 – 1:100), Sox2 (Santa Cruz
Biotechnology, Dallas, TX; sc-17320 – 1:150), Nestin (EMD Millipore, Billerica,
MA; MAB5326 – 1:200), c-Met (Cell Signaling, Danvers, MA; 4560 – 1:100),
MAP2 (Abcam, Cambridge, MA; ab5392 – 1:1000) and CD31 (BD Biosciences,
30

San Jose, CA; BD550274 – 1:50) overnight at room temperature. The following
day, sections were rinsed with PBS and incubated with corresponding
biotinylated secondary antibodies at recommended concentrations (Vector
Laboratories) for 45 minutes. Tissues were washed, treated with avidin-biotin
peroxidase complex for 30 minutes followed by incubation with amino-ethyl
carbazol for 1-5 minutes. Hematoxylin was the counterstain. Red color denotes
positive staining. For microvessel density analysis, 5-10 random fields per tumor
tissue were imaged. At least 3 mice per group were analyzed. Microvessel
density was quantified using ImageJ software (NIH).
Microarray analysis and GSC classification
Total RNA was extracted using RNeasy Plus Mini Kits (Qiagen, Valencia,
CA) and initial RNA quality was assayed using an Agilent 2100 BioAnalyzer.
Gene expression was examined using HuGene 1.0 ST arrays utilizing
Affymetrix's standard protocols. Probe pair intensities were extracted using R ("R
Core Team. R: A language and environment for statistical computing. R
Foundation for Statistical Computing, Vienna, Austria.," 2013) and the affy
package (Gautier et al., 2004), and were normalized using the MvA kernel
density method (Yang et al., 2002). For assignment to proneural and
mesenchymal types, Hu133 expression values from classification of banked
GBM samples to Proneural (and Neural) and Mesenchymal (and Classical) were
obtained from Verhaak et al. (Verhaak et al., 2010), and matched to HuGene 1.0
31

ST probes. Hierarchical clustering using Euclidean distance was performed on
centered and scaled expression values of matched probes. For assignment to
type I and type II, gene signatures were obtained from Lottaz et al. Probe pairs
from Hu133 were matched to HuGene 1.0 ST using the best match according to
Affymetrix’s published comparison document and centered and scaled to have
average expression 0 and standard deviation 1 across all samples studied.
Heatmaps were plotted using heatmap.2 ("http://CRAN.R-
project.org/package=gplots,") to indicate this assignment.
RESULTS
Glioma cancer stem cells isolated from human GBM express stem cell
markers
Two GSCs isolated from surgically resected human GBM tissue, identified
as USC02 and USC04 showed continuous self-renewal in serum-free medium
supplemented with epidermal growth factor (EGF) and basic fibroblast growth
factor (bFGF/FGF-2). USC02 grew as a semi-adherent culture whereas USC04
grew as free floating neurospheres (Figure 2-1). Immunofluorescence analysis
showed that USC02 cells expressed the neural stem cell markers Sox2 (Figure
2-2A, top left panel) and Nestin (Figure 2-2A, top right panel). USC04 also
expressed Nestin (Figure 2-2A, bottom right panel), but had no detectable Sox2
expression (Figure 2-2A, bottom left panel). We next analyzed the GSCs for
CD133 expression, a recognized glioma stem cell surface marker (Singh et al.,
32

2004). Flow cytometry revealed that USC04 had 32.8% CD133 positive cells,
whereas USC02 was CD133 negative (Figure 2-2B, left panel). GSCs were also
tested for c-Met, a tyrosine kinase receptor for hepatocyte growth factor (HGF)
shown to be a functional marker for GSCs (De Bacco et al., 2012). Both cultures
expressed c-Met positive cells: USC02 - 49.24%; and USC04 - 42.43% (Figure
2-2B, right panel).

Figure 2-1. Growth pattern of GSCs. USC02 show semi-adherent growth (left
panel) and USC04 grows as non-adherent neurospheres in serum-free medium
(right panel). Bar represents 100 µm.

USC04 USC02
Sox 2 Nestin
Nestin Sox 2
A.
33

Figure 2-2. Expression of stem cell markers in GSCs. (A) Expression of stem
cell markers Sox2 (red nuclear staining, left panel) and Nestin (red, right panel) in
GSCs by immunofluorescence staining. Bar represents 20 µm (B) Expression of
CD133 (red, left panel) and c-Met (red, right panel) in GSCs relative to isotype
controls (black) by flow cytometry. Percent CD133 and c-Met positive cells are
expressed.

Glioma cancer stem cells form invasive tumors
A key characteristic of cancer stem cells is their ability to initiate tumors at
low cell numbers. To investigate in vivo tumorigenicity of GSCs, we implanted
1000 cells intracranially into NOD-SCID mice. For comparison, we implanted
1000 differentiated primary tumor cells. Only the mice implanted with USC02 or
USC04 cells formed tumors (Figure 2-3). Mice implanted with differentiated
CD133 c-Met
0.03
32.8
49.24
42.43
B.
34

primary tumor cells showed no signs of tumor development up to 160 days when
the experiment was terminated. Immunohistochemical (IHC) analysis of the
tumors derived from GSCs showed that USC02 retained Sox2 and Nestin
expression in vivo (Figure 2-4A, B), however USC04 was negative. Both USC02
and USC04-derived tumors had c-Met and CD133 positive cells (Figure 2-4C, D).
These results confirmed that USC02 and USC04 are two different tumor-initiating
stem cell populations expressing unique patterns of GSC markers. Tumors
derived from GSCs recapitulate the characteristics of patient tumors (Figure 2-
4A-D). Ki67 staining of GSC-derived tumors in mice (Figure 2-4E) also
demonstrated a highly invasive pattern, reflective of human gliomas.

Figure 2-3. In vivo tumor initiating capacity of GSCs. Kaplan Meier curve
demonstrating in vivo tumorigenic potential of 1000 intracranially implanted
GSCs USC02 (red, squares) and USC04 (blue, triangles) in comparison with
differentiated (diff.) human GBM tumor cells (black, circles) in NOD-SCID mice.

35

Figure 2-4. Characteristics of GSC-derived tumors. Expression of (A) Sox2
(B) Nestin (C) c-Met and (D) CD133 in USC02, USC04 and human GBM tumors
c-Met
USC02 USC04 Human GBM
Sox2 Nestin
A.

B.

C.

D.

CD133
E. F.
USC02 USC04
Ki67 Ki67
36

by immunohistochemistry. Scale bar represents 50 µm. (E) Ki67 staining in
USC02 (left) and USC04 (right) tumors. Red denotes positive staining. Arrows
indicate invasive nature of GSC tumors. Scale bar represents 100 µm.

Gene expression-based classification of glioma stem cells identifies two
distinct subtypes
Verhaak et al. classified GBMs into proneural, neural, mesenchymal and
classical subtypes. This classification may reflect different cells of origin and is
also important clinically because of differences in response to treatment (Phillips
et al., 2006; Verhaak et al., 2010). GSCs corresponding to the proneural and
mesenchymal subtypes have thus far been identified (Lottaz et al., 2010). To
identify the subtypes of the GSCs used in these studies, an 840 gene signature,
established by Verhaak, was analyzed. Microarray analysis identified USC02 as
having a mesenchymal gene signature and USC04 as having a proneural
signature (Figure 2-5A). IHC analysis of GSC tumors demonstrated that USC02
tumors had high CD44 (mesenchymal marker) expression (Figure 2-5B), and
USC04 had high DLL3 (proneural marker) expression (Figure 2-5C), validating
the microarray data. USC04 tumors also expressed MAP2, a neuronal marker
(Figure 2-5D, right panel). In accordance with their mesenchymal nature, USC02
also formed highly vascular tumors compared to USC04 (Figure 2-6).

37

A.
Proneural
Mesenchymal
USC02
USC04
38

Figure 2-5. Classification of GSCs based on gene expression. (A)
Classification of USC02 (yellow) and USC04 (blue) into mesenchymal (green)
and proneural (orange) subtypes respectively based on Verhaak’s 840 gene
signature. Immunohistochemical analysis of USC02 (left panel) and USC04 (right
panel) derived tumors showing (B) expression of CD44 (mesenchymal marker),
(C) DLL3 (proneural marker) and (D) MAP2 (neuronal marker). Red denotes
positive staining. Scale bar represents 50 µm.

B.

C.

D.

USC02 USC04
CD44 DLL3 MAP2
39

Figure 2-6. Microvessel density in GSC-derived tumors. (A) CD31 staining of
blood vessels in USC02 (left) and USC04 (right) tumors. Red denotes positive
staining. Scale bar represents 50 µm. (B) Quantification of microvessel density by
ImageJ analysis.

Lottaz et al. have shown two clusters of GSCs – type I and type II based
on differential gene expression patterns (Lottaz et al., 2010). Based on the Lottaz
gene signature, USC02 was classified as type II whereas USC04 was classified
A.

B.
USC02 USC04
CD31
40

as type I (Figure 2-7). These results further demonstrated the unique properties
of these two GSCs, and the diverse gene signatures present in human gliomas.

Type I Type II
41

Figure 2-7. Classification of GSCs based on Lottaz’s gene signature.
Assignment of USC02 and USC04 as type II and type I respectively based on
Lottaz’s gene signature.

SIGNIFICANCE AND PURPOSE OF STUDY
We have established two glioma stem cell cultures with distinct phenotypic
and gene expression profiles, representing at least in part, the heterogeneity
found in human GBM tumors (Table 2-1). These GSCs will serve as an important
tool for the study of GBM biology and for the evaluation of novel preclinical
therapeutic strategies directed towards GSCs which are the source of tumor
recurrence.
Several groups have identified unique molecular classes of GBM tumors
bearing different genetic signatures (Phillips et al., 2006; Verhaak et al., 2010).
USC02 has a mesenchymal gene profile whereas USC04 has a proneural
signature (Figure 2-5A). Studies have shown that mesenchymal tumors are
aggressive and associated with poor survival compared to proneural tumors
(Phillips et al., 2006; Verhaak et al., 2010). Indeed, our studies showed that the
mesenchymal USC02 formed more aggressive and vascular tumors than the
proneural USC04 (Figure 2-3, 2-6). A recent study comparing matched primary
and recurrent tumors from the same patients showed that in approximately 25%
of cases, tumors shifted to a mesenchymal phenotype upon recurrence. This
involves upregulation of genes such as YKL40, CD44 and Vimentin (VIM)
regulating invasion, corresponding to the aggressive behavior of recurrent
42

gliomas. The tumors derived from USC02 or USC04 retain their mesenchymal
and proneural gene signature respectively; therefore we did not observe any
class switching. Proneural and mesenchymal GBM tumors account for 80% of all
tumors, thus we have two distinct GSCs which represent a large majority of GBM
tumors and will be a valuable resource for studying the pathogenesis of GBM.
Table 2-1. Summary of GSC characteristics

CD133 is a popular marker for GSCs (Singh et al., 2004). However,
several groups have found that CD133 negative cells also show all the
phenotypic and functional characteristics of GSCs (Lottaz et al., 2010; Wang et
al., 2008). We have found that our GSCs differ in CD133 expression in vitro with
USC02 lacking CD133 expression and a subpopulation of cells within USC04

USC02 USC04
Self-renewal in serum-free medium
 
Growth pattern

Semi-adherent

Non-adherent,
growth as
neurospheres
Expression of stem cell markers

Sox 2 +
Nestin +
CD133 –
c-Met +
Sox 2 –
Nestin +
CD133 +
c-Met +
In vivo tumorigenicity

Highly vascular

Less vascular
Gene signature

Mesenchymal,
Type II
Proneural,
Type I
43

expressing CD133 (Figure 2-2B). However, tumors from both USC02 and USC04
have CD133-positive cells (Figure 2-4D) suggesting that CD133-negative cells
can give rise to CD133-positive tumors. The CD133-positive cells in the USC02
could also represent normal neural stem cells migrating towards the tumor. The
different pattern of CD133 expression in the two GSCs may also reflect different
cells of origin or location within tumors. CD133 is expressed by fetal neural stem
cells but is absent in adult neural stem cells (Lottaz et al., 2010). Therefore,
USC02 may be derived from transformation of adult neural stem cells into GSCs
whereas USC04 may originate from a fetal neural stem cell. Additionally, GSCs
expressing CD133 are enriched in the core of the tumor (Pistollato et al., 2010);
thus USC04 may have been derived from the core of the tumor while USC02
may be at the tumor periphery.
Traditionally, glioma cell lines passaged indefinitely in serum containing
medium have been ubiquitously used as models to understand the pathobiology
of GBM and in preclinical studies to investigate novel therapies. However, this
approach has not been shown to have significant predictive value in terms of
clinical efficacy mainly because cell lines are not an accurate representation of
human tumors. Serum cultured cells accumulate de novo mutations over
extensive passages as artifacts of culture and progressively lose characteristics
of the parental tumor (Li et al., 2008). Genes involved in cell cycle and replication
are significantly upregulated in glioma cell lines, an effect of the in vitro selection
pressure on these cells (Li et al., 2008). Tumors derived from cell lines also bear
44

little resemblance to primary tumors (Lee et al., 2006). While a large majority of
cells within glioma cell lines are actively cycling, only about 7-14% cells within
human tumors are highly proliferative (Li et al., 2008). This may explain why
chemotherapies targeting proliferating cells are effective in vitro and in cell line-
based murine models but fail to translate to success in clinical trials.
On the other hand, numerous studies have shown that GSCs isolated
from primary human tumors retain the phenotype and genetic profile of the tumor
they are derived from (Lee et al., 2006; Pollard et al., 2009; Wakimoto et al.,
2012). GSCs - USC02 and USC04 form tumors that recapitulate the
distinguishing histological features of human GBM including pleomorphism and
invasion into the normal brain parenchyma (Figure 2-4A-E). Thus, these GSCs
represent a more clinically relevant system for studying the pathogenesis of GBM
and for screening therapies that would be potentially effective in the clinic.
Moreover, a GSC-derived tumor model preserving the heterogeneity and
genotype of the patient-specific tumor has great value for the design and
validation of personalized cancer therapy.
The purpose of this study is to utilize the two established patient-derived
GSC cultures as a representative model to evaluate new and effective strategies
for GBM treatment. GSCs have been implicated as the source of origin and
tumor recurrence in GBM (Chen et al., 2012; Lathia et al., 2011). GSCs are
resistant to radiation and temozolomide (Bao et al., 2006; Beier et al., 2011), the
45

current standard of care and hence there is a critical need to find new GSC-
directed chemotherapeutics to improve the dismal prognosis of GBM. In this
study, we evaluated a novel chemotherapy, NEO212 (Figure 2-8), against
proneural and mesenchymal GSCs. Specifically, we investigated (1) the effects
of NEO212 on GSCs in vitro (2) the mechanisms of GSC cytotoxicity and (3) the
efficacy of NEO212 in an intracranial proneural and mesenchymal GSC-derived
tumor model. Overall, these findings have significant implications for the
treatment of GBM tumors and are of great clinical importance.

Figure 2-8. NEO212. NEO212 is a novel chemical entity which is a chemical
conjugate of temozolomide (red) and perillyl alcohol (blue) via a carbamate bond.

46

CHAPTER III

TEMOZOLOMIDE-PERILLYL ALCOHOL (NEO212), A NOVEL CONJUGATE
TARGETING PRONEURAL AND MESENCHYMAL GLIOMA CANCER STEM
CELLS

ABSTRACT
Glioblastoma multiforme (GBM) is a highly invasive grade IV astrocytoma
accounting for approximately half of all gliomas. The standard-of-care is surgery,
radiation and chemotherapy with temozolomide (TMZ), a DNA alkylating agent.
Unfortunately the median survival of GBM patients is between 12-15 months.
The poor prognosis is due to tumor recurrence which is attributed to
chemoresistant glioma cancer stem cells (GSCs).
A novel compound, NEO212, the chemical conjugate of two drugs: TMZ
and perillyl alcohol (POH) has recently been developed. In the present study we
tested the effects of NEO212 on two unique cultures of patient-derived GSCs:
one exhibiting proneural characteristics; and the other expressing the
mesenchymal gene signature. Together, these GSCs represent 80% of human
GBM tumors.
Our findings show that NEO212 is over 10 fold more cytotoxic to GSCs
than either of its parent compounds alone or in combination. Furthermore,
NEO212 is effective against both subtypes of GSCs, including the more
aggressive mesenchymal subtype. NEO212 is effective irrespective of the status
47

of MGMT, a critical DNA repair protein involved in chemoresistance. The
mechanism of NEO212 cytotoxicity is through downregulation of key proteins
involved in self-renewal, double strand DNA breaks and subsequent apoptosis.
In vivo studies show that NEO212 significantly delays tumor growth of both
proneural and mesenchymal derived-stem cell tumors. Patient-derived GSCs
and tumors derived from these are highly reflective of the heterogeneity and
invasiveness of human GBM and are a more clinically relevant model than cell
lines. The efficacy of NEO212 against both proneural and mesenchymal GSCs
indicates that NEO212 has great potential as a promising new therapy for GBM
by targeting GSCs, the source of tumor recurrence.

48

INTRODUCTION

Glioblastoma multiforme (GBM) is the most malignant and aggressive
glioma. GBM treatment involves surgical resection followed by concomitant
radiation and chemotherapy with temozolomide (TMZ). Unfortunately, most
patients do not survive beyond two years due to tumor recurrence. Recent
evidence implicates chemoresistant glioma cancer stem cells (GSCs) as the
source of the tumor recurrence (Chen et al., 2012).
The cancer stem cell hypothesis has been proven in the development of
many tumors, most notably acute myeloid leukemia and breast cancer (Al-Hajj et
al., 2003; Bonnet et al., 1997; Lapidot et al., 1994). More recently, cancer stem
cells have been isolated from glioblastoma by several groups (Galli et al., 2004;
Singh et al., 2004; Yuan et al., 2004). GSCs exhibit characteristics similar to
normal neural stem cells such as capacity for self-renewal, differentiation along
multiple lineages and expression of neural stem cell markers such as CD133,
Sox2 and Nestin (Gangemi et al., 2009; Vescovi et al., 2006). Low numbers of
GSCs are able to initiate tumors in immune compromised mice, a defining
characteristic of cancer stem cells (Singh et al., 2004). GSCs have been shown
to be resistant to temozolomide in vitro (Blough et al., 2010). Moreover, a recent
study by Chen et al. demonstrated that a Nestin-positive glioma stem cell-like
population is the first to proliferate and repopulate the tumor when TMZ treatment
is discontinued in a spontaneous murine model of glioma (Chen et al., 2012).
Gene expression profiling studies have revealed proneural GSCs associated with
49

longer patient survival and mesenchymal GSCs associated with aggressive
tumors and shorter survival (Lottaz et al., 2010; Verhaak et al., 2010). GSCs are
thus an important target in designing and evaluating drugs for GBM. Targeting
this critical drug resistant population is essential in preventing tumor recurrence
and improving patient prognosis.
Two critical proteins involved in GSC self-renewal and tumor formation are
Bmi-1 and Sox2. Bmi-1 (B cell-specific Moloney murine leukemia virus
integration site 1), an oncogene and member of the polycomb family of proteins,
is a transcriptional repressor that regulates expression of p16
Ink4a
and p14
Arf
by
chromatin modifications (Park et al., 2004; Siddique et al., 2012). In the absence
of p16, the cyclin-Cdk complex can phosphorylate Rb thereby activating
transcription of genes involved in cell cycle progression. Moreover, in the
absence of p14, MDM2 causes p53 degradation which in turn inhibits cell cycle
arrest and apoptosis (Park et al., 2004). Thus Bmi-1 promotes proliferation and
cell survival. Bmi-1 is expressed in >90% of gliomas compared to normal brain
and correlates with tumor grade and poor survival (Li et al., 2010). Bmi-1 is
enriched in CD133-positive glioma stem cells and mediates GSC self-renewal
and tumorigenic potential in vivo (Abdouh et al., 2009). Moreover, Bmi-1 has also
been implicated in DNA repair and chemoresistance (Ginjala et al., 2011; Li et
al., 2010). Sox2, a critical transcription factor of the high mobility group domain
protein family is overexpressed in GBM and is important for GSC self-renewal
and tumorigenicity (Annovazzi et al., 2011; Gangemi et al., 2009). Sox2 activates
50

key pathways such as IL-8 signaling involved in GBM progression (Berezovsky et
al., 2014) and antagonizes tumor suppressive pathways such as Hippo signaling
(Basu-Roy et al., 2015). Thus, Bmi-1 and Sox2 are critical GSC targets.
We have previously shown that perillyl alcohol (POH), a naturally
occurring monoterpene, has significant anti-tumor activity against TMZ-resistant
tumor cells in vitro and in an in vivo model (Cho et al., 2012). We have since
developed a novel chemical entity, the conjugate of TMZ and POH, NEO212,
which is significantly cytotoxic to both TMZ-sensitive and TMZ-resistant tumor
cell lines in vitro. Moreover, in vivo studies have demonstrated that NEO212
crosses the blood brain barrier, and is effective in delaying tumor growth of
temozolomide-resistant tumors (Cho et al., 2014). NEO212 is also effective
against breast cancer and melanoma (Chen et al., 2014; Chen et al., 2015).
In this study, we tested the effects of NEO212 on GSCs expressing two
different genetic signatures, the proneural and mesenchymal glioma stem cells.
NEO212 was significantly more cytotoxic than either TMZ or POH alone, or the
mixture of TMZ and POH. NEO212 was an effective cytotoxic agent against both
proneural and mesenchymal types of GSCs, and was independent of O
6
-methyl
guanine methyl transferase (MGMT), a DNA repair protein and mediator of TMZ
resistance. In the in vivo orthotopic mouse model, NEO212 significantly
increased survival of animals with either proneural or mesenchymal GSC-derived
tumors. The mechanism of NEO212 cytotoxicity in both GSC populations was
51

through double strand DNA breaks, activation of the DNA damage response
pathway and subsequent apoptosis. Moreover, NEO212 decreased stem cell
self-renewal by downregulation of Bmi-1 in the proneural population and Sox2 in
the mesenchymal GSC population.

MATERIALS AND METHODS

Chemicals
Temozolomide (Merck, Whitehouse Station, NJ), perillyl alcohol (POH)
(NEO100) (NeOnc Technologies, Inc., Woodland Hills, CA), NEO212 (NeOnc
Technologies, Inc.) and O
6
-benzylguanine (Sigma Aldrich, St. Louis, MO) were
prepared as stock solutions in DMSO for all in vitro studies.
MTT cytotoxicity assay
Glioma stem cells were seeded in 96 well plates in 50 µL CSC medium
containing BSA (Sigma Aldrich) equivalent to 10% FBS to mimic the protein
concentration found in human plasma. The next day, drugs were added in a
volume of 50 µL/well at the concentrations indicated. After 72 hours, the MTT
assay was performed as described previously (Kardosh et al., 2005). 10 µL 3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) dye was added
and the plate was incubated for 4 hours at 37°C. Cells were lysed by addition of
50 µL lysis buffer containing 10% sodium dodecyl sulfate (SDS) in 0.01 M
hydrochloric acid. After overnight incubation, the absorbance was measured at
52

490 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). Percent
cell viability was calculated relative to untreated control. Each condition was
plated in triplicate; experiments were repeated thrice.
Sphere/Colony forming assay
For sphere forming assay, GSCs were dissociated and 3000 single cells
were seeded in a 24 well plate in CSC medium with BSA. Cells were treated with
drugs for 4 days, and then primary spheres were counted. Primary spheres were
subsequently dissociated and reseeded at 3000 cells/well. For conditions with
less than 3000 cells, all the remaining cells were added. Cells were incubated for
an additional 7 days without drug after which the secondary spheres were
counted. Each treatment condition was set up in triplicate and experiments
repeated thrice. For colony forming assay, 5000 cells were seeded in CSC
medium with BSA and treated for 4 days. Subsequently, cells were centrifuged
and resuspended in CSC medium containing 10% FBS to promote cell
adherence. After 7 days, cells were stained with 1% methylene blue; intensity
was measured using Odyssey LI-COR scanner. For O
6
BG experiments, cells
were pre-treated with 50 µmol/L MGMT inhibitor O
6
benzyl guanine (O6-BG) for 2
hours followed by treatment with TMZ or NEO212. After 48 hours, cells were
retreated with O6-BG at 10µmol/L.

53

SVZ and DG stem cell isolation and sphere forming assay
Neural stem cells from the subventricular zone (SVZ) and dentate gyrus
(DG) of 1-3 day old C57BL/6 donor mice were isolated as described previously
(Bernardino et al., 2008). Spheres were dissociated into single cells using PBS
dissociation buffer and sphere forming assay was performed as described above.
Cells were incubated with NEO212 and primary spheres were enumerated after
4 days. Each treatment condition was set up in triplicate.
Immunohistochemistry for MGMT
Frozen tissue sections were fixed in 10% neutral buffered formalin for 10
minutes followed by antigen recovery (Neman et al., 2014). Briefly, sections were
placed in 10 mmol/L sodium citrate buffer (pH 6.0) for 30 min at 80-85°C and
permeabilized with 0.25% Triton-X for 18 min. Non-specific binding was blocked
with SeaBlock for 20 minutes followed by incubation with anti-MGMT antibody
(Abcam; ab39253 – 1:50). The following day, sections were rinsed with PBS and
incubated with biotinylated anti-mouse secondary antibody (Vector Laboratories;
1:100) for 45 minutes. Tissues were washed, treated with avidin-biotin
peroxidase complex for 30 minutes followed by incubation with amino-ethyl
carbazol for 5 minutes. Hematoxylin was the counterstain. Positive cells were
identified by red nuclear staining.

54

Immunofluorescence staining for pH2AX foci
USC02 cells were treated with drugs at indicated concentrations for 24
hours and cytocentrifuge cell preparations were fixed in acetone. Subsequently,
immunofluorescence staining was performed as described earlier with rabbit anti-
pH2AX (Cell Signaling; 9947 - 1:1000) overnight at room temperature.
Immunofluorescence images were taken using Zeiss LSM 710 confocal scanning
microscope. At least five representative images were taken per condition at high
power objective. Total fluorescent intensity was calculated using ImageJ and
normalized to cell number. Two independent experiments were performed.
Single cell gel electrophoresis (Comet) assay
USC04 GSCs were treated with drugs at indicated concentrations for 24
hours. Subsequently, the single cell gel electrophoresis (Comet) assay was
performed according to the manufacturer’s protocol (Trevigen, Gaithersburg,
MD). Briefly 1 x 10
5
cells/ml were mixed with low melting agarose at a ratio of
1:10. 50 µL of this solution was spread on a coated Comet slide and allowed to
gel at 4°C. Cells were lysed and treated with alkali solution to unwind and
denature DNA. Subsequently, cells were subjected to electrophoresis, fixed and
allowed to dry overnight. Comets were visualized with the SYBr Gold staining
solution. At least 5 representative images were taken for each condition using a
fluorescent microscope. The number of cells with comet tails was enumerated.

55

Western blot
Total cell lysates were prepared by incubating with
radioimmunoprecipitation assay buffer for 30 minutes. Protein concentration was
determined using the BCA assay (Thermo Scientific) according to the
manufacturer’s recommendations. Fifty microgram of total protein was loaded,
and gels were run using the Biorad Mini protean gel apparatus. Proteins were
transferred to a PVDF membrane using the Biorad Semi-dry Transblot Transfer
system. Membranes were incubated with primary antibodies overnight at 4°C.
MGMT (Thermo Scientific; PA5-17278), c-Met (Cell Signaling; 4560), PARP (Cell
Signaling; 9532), Actin (Santa Cruz Biotechnology; sc-130656) and antibodies to
DNA damage response proteins p-H2AX, p-p53 and p-Chk2 (Cell Signaling;
9947) were used according to manufacturers’ recommendations. Corresponding
HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) were used.
The reaction was developed using Supersignal west chemiluminescent substrate
(Thermo Scientific) and bands detected with the LAS-4000 Gel Imager.
In vivo experiments
All animal protocols were approved by the USC Institutional Animal Care
and Use Committee. Four to six week male NOD-SCID mice (Harlan
Laboratories, Indianapolis, IN) were implanted intracranially with 5000 USC02 or
USC04 GSCs as described earlier. Seven days post implantation, mice were
randomly divided and treated subcutaneously with (1) Vehicle – 45% ethanol/
56

45% glycerol and 10% DMSO solution or (2) NEO212 – 25 mg//kg in vehicle
solution. Treatment was given in a cycle of 10 days on/5 days off for 9 cycles for
USC02 and 6 cycles for USC04. Dosage and treatment of NEO212 were
determined based on previous experiments (Chen et al., 2014).
For the USC02 in vivo experiment, firefly luciferase positive USC02 cells
were used to monitor tumor progression. Mice were anesthetized with 2%
isoflurane in oxygen, injected with 50 mg/kg D-Luciferin Firefly, potassium salt
(Caliper Life Sciences, Alameda, CA) intravenously and imaged after 1.5 minutes
using the IVIS Spectrum (Xenogen Corp., Alameda, CA); images were analyzed
using the Living Image 4.3 software (Perkin Elmer, Alameda, CA).
Statistical analysis
Student’s t test was used to evaluate statistical significance. For survival
analysis, Log rank test was used to determine statistical significance. p values
less than 0.05 were considered significant.
RESULTS
NEO212 is cytotoxic and decreases self-renewal of TMZ-resistant glioma
stem cells in vitro
We had previously shown that NEO212 is significantly cytotoxic for a
broad range of TMZ-resistant glioma cell lines (Cho et al., 2014). To test the
effects of NEO212 on GSC viability, GSCs were treated with TMZ, POH, TMZ +
57

POH or NEO212 for 72 hours in the MTT assay. NEO212 was found to be
significantly more cytotoxic than either TMZ or POH alone (Figure 3-1A, B), and
more potent than the combination of TMZ and POH, suggesting that NEO212
has unique mechanisms of action. The IC50 for NEO212 in GSCs ranged from 8-
43 µmol/L, approximately 7-40 fold lower than the IC50 for TMZ (Table 3-1).
POH, previously shown to be effective against drug-resistant GBM, was effective
only in millimolar doses. These data confirmed that NEO212 was significantly
more effective than TMZ.

A.

*
***
USC02
58

B.

Figure 3-1. NEO212 cytotoxicity in GSCs. 72 hour MTT cytotoxicity assay with
TMZ (black, circles), POH (red, squares), TMZ + POH (blue, triangles) and
NEO212 (green, inverted triangles) in (A) USC02 and (B) USC04 GSCs. The
data are expressed as percent cell survival relative to untreated control.
Conditions were performed in triplicate. Data is represented from three
independent experiments. Black dotted line indicates IC50. *p<0.05, **p<0.005,
***p<0.0005.

Table 3-1. IC50 values for TMZ and NEO212 in GSCs

Values are obtained from three independent experiments, based on 72 hour MTT
cytotoxicity assay.

IC50 (µmol/L)
Fold
difference

TMZ TMZ-POH

USC02 317 ± 42 43 ± 9 7
USC04 323 ± 61 8 ± 2 40
*
***
**
USC04
59

To test the long-term effects of NEO212 on GSC self-renewal, the serial
sphere forming assay (SFA) was performed on proneural GSCs (USC04).
Neurospheres were dissociated into single cells and treated with TMZ or
NEO212. Primary spheres were counted on day 4, dissociated and replated for
an additional 7 days. At this stage secondary spheres, arising from viable stem
cells in the primary sphere, were counted. The results showed that USC04 was
resistant to TMZ treatment, and survived high TMZ concentrations (100 µmol/L).
By contrast, no spheres remained after treatment with 10 µmol/L NEO212,
indicating that NEO212 exhibited a greater than 10 fold more potent response
compared to treatment with TMZ (Figure 3-2A). Cultures were maintained for an
additional 45 days, and still no spheres were observed in the NEO212-treated
group indicating that NEO212 effectively inhibited USC04 GSC self-renewal
(Figure 3-2B). Consistent with these findings, NEO212 also decreased
expression of Bmi-1, an important factor for GSC self-renewal (Figure 3-2C).
Since USC02 is a semi-adherent culture, we were able to perform the colony
forming assay (CFA). Results indicated that for the mesenchymal USC02
population, NEO212 was also over 10 fold more potent than TMZ in inhibiting
proliferation (Figure 3-2D). Moreover, NEO212 decreased expression of Sox2, a
key protein regulating GSC proliferation more effectively than TMZ (Figure 3-2E).

60

***
A.

B.

Control
NEO212 10 µmol/L
TMZ 10 µmol/L
Control
NEO212 10 µmol/L
TMZ 10 µmol/L
Day 11 Day 45
61

C.

D.

***
*
Bmi-1
Actin
(µmol/L)
USC04
62

E.

Figure 3-2. Long-term NEO212 cytotoxicity in GSCs. (A) Long-term sphere
forming assay for NEO212 and TMZ. USC04 were dissociated into single cells
and treated with different concentrations of TMZ and NEO212. After 4 days,
primary spheres were counted and plotted relative to untreated control (solid
black bars). Primary spheres were further dissociated into single cells and
replated for an additional 7 days without drug. Secondary spheres (striped bars)
were counted and plotted relative to untreated. Data is expressed from three
independent experiments. ***p<0.0005 (B) Images of USC04 secondary spheres
on day 11 (left panel) and day 45 (right panel) post drug treatment. Bars
represent 100 µm. (C) Expression of Bmi-1 relative to Actin in USC04 treated
with different concentrations of NEO212 or TMZ for 72 hours. (D) Colony forming
assay with USC02 treated with different concentrations of TMZ and NEO212 for
4 days and subsequently incubated for 7 days. Conditions were performed in
triplicate. Data is expressed from five independent experiments. *p<0.05,
***p<0.0005 (E) Expression of Sox2 relative to Actin in USC02 treated with
increasing concentrations of NEO212 or TMZ for 72 hours.

NEO212 selectively targets GSCs over normal neural stem cells
Selectivity of chemotherapy for tumors is critical to avoid non-specific side
effects. To this end, NEO212 was examined for its effects on normal neural stem
cells in the sphere forming assay. Normal stem cells were isolated from the
Sox2
Actin
(µmol/L)
USC02
63

subventricular zone (SVZ) and dentate gyrus (DG) of new born mice. GSCs and
NSCs were incubated with NEO212 for 4 days and primary spheres were
enumerated. These data showed that NEO212 preferentially targets glioma stem
cells as compared to normal neural stem cells (Figure 3-3). This selectivity
provides a therapeutic window to target GSCs without affecting the normal neural
stem cell pool.

Figure 3-3. NEO212 cytotoxicity in normal neural stem cells. Sphere forming
assay comparing NEO212 cytotoxicity in USC04 human GSCs (striped bars) with
murine neural stem cells from subventricular zone (mSVZ, solid black bars) and
dentate gyrus (mDG, checkered bars). Cells were dissociated into single cells
and treated with NEO212. After 4 days, primary spheres were counted and
plotted relative to untreated control. Data is expressed from two independent
experiments.

64

NEO212 cytotoxicity is independent of MGMT
O
6
-methyl guanine methyl transferase (MGMT) is a key protein mediating
TMZ resistance in GBM (Johannessen et al., 2012). USC02 cells expressed
MGMT whereas USC04 had no detectable MGMT expression (Figure 3-4A).
Moreover, USC02-derived tumors retained MGMT expression in vivo whereas
USC04-derived tumors remained MGMT negative (Figure 3-4B). To determine
the role of MGMT in TMZ resistance in GSCs, we pre-treated USC02 cells with
the MGMT inhibitor O
6
-benzyl guanine followed by treatment with TMZ or
NEO212, in the CFA. Inhibition of MGMT partially sensitized USC02 to TMZ but
there was no significant change in response of USC02 to NEO212 (Figure 3-4C)
suggesting that there are additional mechanisms of resistance to TMZ other than
MGMT in these cells and that NEO212 is cytotoxic irrespective of the presence of
MGMT. Moreover, NEO212 reduced MGMT expression in USC02 more
effectively than TMZ at equimolar concentrations (Figure 3-4D).

A. B.

MGMT
Actin
MGMT
USC02 USC04
65

Figure 3-4. Role of MGMT in TMZ and NEO212 cytotoxicity. (A) Expression of
MGMT protein in GSCs relative to Actin by Western blot. (B) Immunostaining for
MGMT (red nuclear staining) in USC02 and USC04-derived tumor sections.
Scale bar represents 50 µm. (C) Colony forming assay with USC02 treated with
TMZ or NEO212 alone (solid black bars) and in combination with MGMT inhibitor
O
6
-benzylguanine (striped bars). Data is represented from two independent
experiments. *p<0.05, ns – not significant (D) Expression of MGMT in USC02
treated with increasing concentrations of NEO212 or TMZ.

MGMT
Actin
(µmol/L)
D.
C.
ns
ns
ns
*
*
*
66

NEO212 induces DNA damage
To identify the mechanisms of NEO212-mediated cell death, we
investigated the effects of NEO212 on DNA damage. GSCs were treated with
NEO212 and DNA damage analyzed by the single cell gel electrophoresis
(Comet) assay. In this assay, cells with damaged DNA when lysed and subjected
to an electrophoretic field form characteristic comet tail structures (Collins, 2004).
The results showed that 100% of cells treated with NEO212 at 50 µmol/L had
comet tails, indicative of DNA damage; by contrast only 63% of cells treated with
TMZ (50 µmol/L) exhibited similarly damaged cells (Figure 3-5A). Another marker
for DNA damage is pH2AX which binds to and marks sites of double strand DNA
breaks (Kuo et al., 2008). USC02 cells were treated with NEO212 or TMZ for 24
hours, and then stained for pH2AX. The results showed that NEO212 induced a
dose dependent increase in the number of pH2AX foci. Quantifying pH2AX
fluorescence intensity indicated that at equimolar concentrations, NEO212
caused 5 fold more double strand breaks than TMZ (Figure 3-5B).
Phosphorylation of H2AX was also examined 72 hours post-drug treatment.
Western blot results demonstrated that at equimolar doses, pH2AX expression
was higher with NEO212 compared to TMZ in both USC02 and USC04 (Figure
3-5C, D).
To further investigate the potential mechanisms of cell death mediated by
NEO212, activation of the DNA damage response (DDR) was analyzed by
67

examining the phosphorylation of checkpoint protein Chk2, which can cause cell
cycle arrest and eventually induce apoptosis (Zhou et al., 2004). GSCs were
treated with NEO212 for 72 hours. NEO212 increased phosphorylation of Chk2
in both USC02 and USC04, which correlated with increased apoptosis as
determined by poly-ADP ribose polymerase (PARP) cleavage (Figure 3-5C, D);
such effects were not observable even with TMZ at 10 fold higher concentrations.
These results show that NEO212 is a potent DNA-damaging agent.

68

A.
Control NEO212 TMZ
***
69

B.
NEO212 TMZ
50µmol/L 50µmol/L
NEO212 TMZ
10µmol/L 10µmol/L
Control
***
70

Figure 3-5. Mechanism of NEO212 cytotoxicity. (A) Comet assay images at
low magnification and high magnification (inset) of USC04 cells treated with TMZ
or NEO212 for 24 hours. Scale bar represents 100µm. Quantification of percent
total cells with Comet tails. ***p<0.0005. (B) Immunofluorescence staining for
pH2AX (green foci) in USC02 treated with TMZ or NEO212 for 24 hours. Bar
USC04
p-H2AX
PARP
Cl. PARP
p-Chk2
Actin
(µmol/L)
USC02
p-H2AX
PARP
Cl. PARP
p-Chk2
Actin
(µmol/L)
C.
D.
71

represents 20 µm. Quantification of fluorescent intensity using ImageJ software.
Intensity was normalized to cell number. Data are expressed from two
independent experiments. ***p<0.0005. Western blot for DNA damage response
and apoptosis proteins with (C) USC04 and (D) USC02 treated with TMZ or
NEO212 for 72 hours.

NEO212 prolongs survival of mice bearing proneural and mesenchymal
GSC-derived tumors
To determine whether NEO212 is effective on GSCs in vivo, GSCs were
implanted intracranially into NOD-SCID mice. Seven days post GSC
implantation, mice were randomly divided into vehicle and NEO212 treatment
groups. Kaplan-Meier survival analysis showed that NEO212 significantly
prolonged survival of mice bearing USC04 proneural tumors (p<0.005) (Figure 3-
6A). The median survival of vehicle-treated mice was 61 days compared to 158
days for NEO212-treated mice, representing a 159% increase in survival with
NEO212. Histological examination of the brains from 2 out of 6 NEO212-treated
mice exhibited no visible tumor formation.
NEO212 also significantly increased survival of mice with USC02-derived
MGMT-positive mesenchymal tumors (p<0.05) (Figure 3-6B) without any
significant differences in body weight (Figure 3-6C). The median survival of
USC02 tumor bearing mice treated with vehicle was 65 days and treated with
NEO212 was 140 days. Bioluminescence imaging showed a delay in tumor
progression with NEO212 (Figure 3-6D). At day 121 post implantation, 2 out of 5
72

mice showed negligible tumor signal. These results indicated that NEO212 is
effective against both proneural and mesenchymal-derived GSC tumors in vivo.

A.

B.

USC04 Proneural tumor

USC02 Mesenchymal tumor
**
*
73

C.

D.

Day 7
(1st day
of Tx)

Day 30

Day 78

Day 121
Vehicle NEO212
74

Figure 3-6. In vivo efficacy of NEO212 in an intracranial GSC-derived tumor
model. GSCs were implanted intracranially into NOD-SCID mice and divided into
Vehicle (black, square) and NEO212 (red, circle) treatment groups. NEO212 was
administered subcutaneously in a 10 day on/5 day off cycle starting 7 days after
implantation. Kaplan-Meier survival analysis of mice bearing (A) proneural
USC04-derived tumors and (B) mesenchymal USC02-derived tumors. n denotes
number of mice in each group. **p< 0.005 for USC04 and *p< 0.05 for USC02
using Log Rank Test. (C) Body weight measurements (in gms) of USC02
derived-tumor bearing mice treated with Vehicle (black, square) or NEO212 (red,
circle). (D) Bioluminescent images of USC02-firefly luciferase bearing mice
treated with vehicle or NEO212 at day 7(first day of treatment(Tx)), 30, 78 and
121 post implantation.

DISCUSSION
Our study demonstrates the therapeutic advantage of the unique drug
NEO212 which targets two unique patient-derived glioma stem cell
subpopulations.
The current FDA-approved chemotherapeutic agent, temozolomide,
eliminates proliferating tumor cells but is not very effective in targeting the glioma
stem cell population. At clinically achievable doses, the maximum concentration
of TMZ in the plasma is 50 µmol/L and 5 µmol/L in the cerebrospinal fluid (Brada
et al., 1999; Ostermann et al., 2004). At these concentrations, we have shown
that the bulk of glioma stem cells are not affected by TMZ. However, NEO212 at
equivalent concentrations exhibited over 95% cytotoxicity (Figure 3-2A, D).
Perillyl alcohol (POH) is a monoterpene that showed promising results in Phase
II trials for recurrent resistant GBM but is required to be administered four times
daily intranasally (da Fonseca et al., 2011). NEO212, the conjugate of TMZ and
75

POH is greater than 10 fold more potent than its parent compounds, and not
likely to require these high doses. From the results with combination of O6-BG
and the intensity of cytotoxic activity in vitro and in vivo, NEO212 is likely to be
acting through different pathways.
The mechanism of NEO212 cytotoxicity is under investigation. Bmi-1 and
Sox2 have been shown to be expressed by GSCs and mediate self-renewal and
GSC tumorigenicity (Abdouh et al., 2009; Gangemi et al., 2009). NEO212
decreased expression of Bmi-1 in the proneural USC04 GSCs (Figure 3-2C) and
Sox2 in the mesenchymal USC02 GSCs (Figure 3-2E) whereas TMZ had only a
minimal effect on expression of these critical proteins. Therefore, by targeting
Bmi-1 and Sox2, NEO212 inhibits self-renewal, a key characteristic of GSCs.
Both Bmi-1 and Sox2 are regulated by the Hedgehog-Gli signaling pathway (Liu
et al., 2006; Santini et al., 2014). The effect of NEO212 on Hedgehog-Gli
signaling and the mechanism by which NEO212 affects Bmi-1 and Sox2
expression remains to be investigated.
Another mechanism of NEO212 is through DNA damage and subsequent
apoptosis. TMZ is a DNA methylating agent, known to induce methylation of O
6

guanine, which mispairs with thymine during replication. This leads to futile
cycles of mismatch repair which eventually causes double strand breaks,
replication fork collapse and cell death (Fu et al., 2012). However, TMZ does not
induce significant DNA damage in glioma stem cells (Figure 3-5). NEO212 is a
76

more potent DNA damaging agent than TMZ. At equimolar concentrations,
NEO212 induced 5 fold more DNA damage as measured by immunostaining for
pH2AX, a marker for DNA double strand breaks (Figure 3-5B). Longer exposure
to NEO212 showed an increase in DNA damage-associated proteins and
apoptosis (Figure 3-5C, D). Glioma stem cells have been shown to activate DNA
repair mechanisms (Bao et al., 2006) which may protect these cells from the
extent of DNA damage induced by TMZ. Alternatively, NEO212 may have
induced more effective and persistent DNA damage which was beyond the cell’s
repair capacity making NEO212 a more potent cytotoxic agent. We have
previously shown that NEO212 has increased stability in vitro compared to TMZ
(Chen et al., 2014). NEO212 retained 50% cytotoxic potency after 4 hours of
incubation in medium containing cells whereas TMZ lost all cytotoxic activity after
4 hours (Chen et al., 2014). In addition, the conjugation of POH, a highly
lipophilic drug, to TMZ may have enhanced entry of NEO212 into cells making
this novel agent an effective and efficient cytotoxic agent. Further studies
analyzing the contribution of the POH entity in NEO212 are essential to
deciphering the mechanism of NEO212 and are currently under investigation.
Several groups have identified unique molecular classes of GBM tumors
bearing different gene expression profiles. GBM tumor specimens have been
classified into four subtypes (Verhaak et al., 2010), with 80% of all GBM tumors
represented by the proneural and mesenchymal tumors (Phillips et al., 2006).
Mesenchymal tumors are aggressive and associated with poorer survival
77

compared to proneural tumors (Verhaak et al., 2010). Moreover, MGMT
overexpression is detected in approximately 40% of gliomas and correlates with
TMZ resistance (Johannessen et al., 2012). These studies show that USC02 is
MGMT positive (Figure 3-4A, B) and is relatively more resistant to TMZ
compared to the MGMT negative USC04 (Figure 3-2A, D). Based on studies in
tissues, it appears that approximately 50% of the USC02 GSCs are MGMT
positive (Figure 3-4B). NEO212 is cytotoxic for this mesenchymal population,
most likely because this agent is cytotoxic for both proneural and mesenchymal
glioma stem cell subtypes irrespective of their MGMT status (Figure 3-2A, D; 3-
4C). These results suggest that NEO212 can be a valuable therapeutic tool for a
broad range of GBM tumors.
A major concern with any chemotherapy is the eradication of normal stem
cells with treatment. This was examined using NEO212 on normal murine neural
stem cells. Data showed that NEO212 is preferentially cytotoxic for glioma stem
cells as compared to normal non-tumor neural stem cells derived from the
subventricular zone and dentate gyrus (Figure 3-3). This selectivity provides a
therapeutic window to target GSCs without affecting the normal neural stem cell
pool. Preserving the normal neural stem cell population is especially important
with recent data showing that NSCs migrate towards tumors and secrete factors
such as bone morphogenetic protein-7 (BMP7) that induce GSC differentiation
and suppress GSC self-renewal and tumor growth (Chirasani et al., 2010). The
mechanism of NEO212 selectivity is not well understood but may correlate with
78

differences in proliferation and inherent genetic instability of GSCs. Moreover, the
NSCs used in this study were derived from mice; it will be important to evaluate
the effects of NEO212 on human NSCs.
The blood brain barrier is an important consideration for GBM therapy. In
the intracranial xenograft model of proneural or mesenchymal GSC-derived
tumors, NEO212, administered subcutaneously delayed tumor progression and
prolonged survival (Figure 3-6). In the mesenchymal GSC model, on day 7 post
implantation, all mice exhibited low levels of bioluminescence. However by day
121, while 2 out of 5 NEO212-treated animals were active and showed minimal
tumor signal, no vehicle-treated animals were alive (Figure 3-6D). This observed
variability is not well understood. In the proneural GSC tumor bearing animals, 2
out of 6 mice showed no signs of tumor development after 6 cycles of NEO212
treatment suggesting that NEO212 may have eliminated the tumor-initiating
population (Figure 3-6A). These data show that although tumors derived from
both GSC populations are susceptible to NEO212, the proneural tumor is
somewhat more sensitive to this agent. This information can be useful in
identifying appropriate chemotherapy dosages and protocols.
In conclusion, the results presented here have significant implications for
the treatment of GBM. In vitro studies demonstrate that NEO212 is a superior
cytotoxic agent to TMZ and is cytotoxic for GSCs with proneural and
mesenchymal signatures. Furthermore, NEO212 inhibits GSC self-renewal and is
79

cytotoxic independent of MGMT, a key DNA repair protein. Thus NEO212 has
great potential as a novel therapeutic agent for GBM derived from different
glioma stem cell subpopulations, thereby preventing or delaying tumor
recurrence.

80

CHAPTER IV

DISCUSSION AND CONCLUSION
OVERALL SIGNIFICANCE OF THE STUDY
Cancer is considered to be a ‘disease of unregulated self-renewal’ and
glioma cancer stem cells are implicated as the source of origin and recurrence in
GBM (Chen et al., 2012; Lathia et al., 2011; Reya et al., 2001). GSCs are
resistant to radiation and the current standard of care chemotherapy
temozolomide (Bao et al., 2006; Beier et al., 2011). Moreover, GSCs have been
shown to modulate tumor angiogenesis by secretion of pro-angiogenic factors
such as vascular endothelial growth factor (VEGF) and hepatoma-derived growth
factor (HDGF) (Bao et al., 2006; Thirant et al., 2012). Furthermore, recent studies
have shown that GSCs can directly contribute to the tumor vasculature by
transdifferentiation into endothelial cells and pericytes, the cell populations which
constitute blood vessels (Cheng et al., 2013; Guichet et al., 2014; Ricci-Vitiani et
al., 2010; Wang et al., 2010). Owing to their critical role in tumor growth and
progression, GSCs are an important target for preventing tumor recurrence and
improving the prognosis of GBM patients.
A significant finding of our study is that GSCs are not a homogenous
population. We have described two diverse GSC populations with differential
growth characteristics, expression of stem cell markers and genetic profiles. The
two GSCs also differ in their level of chemoresistance and response to
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temozolomide with the mesenchymal USC02 being more resistant than the
proneural USC04. The resistance of USC02 can be partially attributed to the
presence of MGMT. However other mechanisms of resistance exist and remain
to be elucidated.
Using the patient-derived GSCs as a representative model for human
GBM, we found that a novel conjugate chemotherapy NEO212 is highly effective
in eliminating both proneural and mesenchymal GSCs. The mechanisms of cell
death may be downregulation of self-renewal pathways and extensive DNA
double strand breaks and damage eventually resulting in apoptosis. NEO212
also crosses the blood brain barrier, a major hurdle in GBM therapy and prolongs
survival of mice bearing proneural and mesenchymal GSC-derived tumors.
CLINICAL IMPLICATIONS
Our study has added a new chemotherapy to our armamentarium against
the most malignant and invasive brain tumor glioblastoma multiforme.
Temozolomide, the current chemotherapy targets actively proliferating tumor
cells but GSCs are resistant to TMZ resulting in tumor recurrence usually within a
year from diagnosis. Tumor recurrence is associated with the dismal clinical
prognosis of GBM patients with a five year survival rate of less than 10%. Since
the development of TMZ in the 1990s, extensive research and clinical trials have
been conducted to find a more effective therapy including tyrosine kinase
inhibitors such as Gefitinib and immunotherapeutic approaches but TMZ still
82

remains the standard of care. Dendritic cell-based vaccines and adoptive transfer
of tumor-reactive autologous cytotoxic T lymphocytes are being developed as
strategies to activate the immune response against glioma but the safety and
efficacy of these treatments remain to be investigated (Okada et al., 2009). The
history of GBM research shows that while most drugs show promising results in
preclinical studies, they fail to be effective in the clinic. A major reason for this
discrepancy is the cell line models used in preclinical studies which are a poor
representation of human tumors. Cell lines are very unstable and accumulate de
novo mutations over extensive in vitro passaging resulting in significant
divergence from the original tumors. GSCs derived from patient samples and
propagated in serum-free medium are very similar genetically and phenotypically
to their parental tumors. The two GSCs we have established are used at low
passage numbers and are highly reflective of the heterogeneity and invasiveness
of human GBM. Therefore, these patient-derived GSCs are a superior and more
clinically relevant model compared to cell lines for investigating new therapeutic
strategies.
In vitro and in vivo studies with the two heterogeneous GSC populations
have shown that NEO212 is effective against both proneural and mesenchymal
subtypes. Clinically, the mesenchymal subtype is highly vascular and associated
with a poor prognosis compared to the proneural subtype. Our results
demonstrating that NEO212 was cytotoxic to the mesenchymal USC02 and
delayed tumor progression of mice bearing mesenchymal tumors within clinically
83

relevant doses has significant implications for GBM therapy. Proneural and
mesenchymal tumors constitute 80% of human GBM; thus the efficacy of
NEO212 against both subtypes is very promising. Moreover, MGMT is expressed
in over 40% of human gliomas and correlates with resistance to temozolomide.
The MGMT-independent mechanism of NEO212 makes it a valuable therapeutic
strategy for a broad range of temozolomide-resistant tumors.
NEO212 is able to cross the blood brain barrier and has selective
cytotoxicity for GSCs and tumor cells over normal neural stem cells, astrocytes
and endothelial cells. Thus, we have compelling evidence to believe that
NEO212 is a promising new therapy for the treatment of GBM tumors both as a
first line therapy to prevent recurrence and for prolonging survival after
established temozolomide resistance.
LIMITATIONS OF THE STUDY AND FUTURE DIRECTIONS
Though isolation and culture of cells in serum-free medium enriches for
the glioma stem cell fraction, our in vitro GSC cultures contain a mixed
population of cells at varying stages of differentiation. The bulk of the population
may be progenitor cells and the true stem cell fraction may be a small subset of
the entire population as is seen in tumors; thus results from mixed cultures may
be more reflective of the progenitor cells. Until more specific markers for glioma
stem cells are identified or a panel of multiple markers is used for GSC isolation,
we are limited in our technical abilities to detect GSC-specific chemotherapies.
84

Moreover, considering the heterogeneity of GBM there might be different
markers for different subtypes of GSCs, thereby providing another level of
complexity to the identification of GSCs. Single cell analysis may help to a certain
extent to investigate GSC-specific drug effects but these studies are highly
complex and cumbersome (Laughney et al., 2014).
The patient-derived glioma cancer stem cells we have established provide
a valuable model system to investigate the potential of novel chemotherapies in
vitro and in xenograft murine models; however, our model is still not truly
representative of human tumors. GBM is a highly angiogenic tumor and GSCs
have been found in close association with the vasculature (Calabrese et al.,
2007; Lathia et al., 2011). Cross talk between GSCs and endothelial cells (ECs)
regulates tumor growth, angiogenesis and invasion (Bao et al., 2006; Folkins et
al., 2009; Infanger et al., 2013; Zhu et al., 2011). Though our orthotopic in vivo
model provides the murine brain microenvironment, it may not reflect specific
interactions between GSCs and the vasculature in humans. IL-8 secretion by
endothelial cells upregulates IL-8 receptors (CXCR1 and CXCR2) in GSCs in
vitro; this signaling mediates increased GSC growth and chemotactic migration
towards endothelial cell conditioned medium (Infanger et al., 2013). IL-8 is
absent in mice and therefore murine models fail to recapitulate this critical
signaling pathway in GSC-EC interaction. Co-implantation of human brain
endothelial cells with GSCs can overcome this limitation. Indeed, studies have
shown that mice implanted subcutaneously with GSCs and hCMEC, a human
85

immortalized brain endothelial cell line within poly (lactide-co-glycolide) (PLG)
based polymeric scaffolds showed accelerated tumor growth (Infanger et al.,
2013). IHC analysis of these tumors revealed pseudopalisading necrosis and
microvascular hyperplasia, characteristic features of GBM (Infanger et al., 2013).
Previous studies from our laboratory have shown that glioma tumor-associated
brain endothelial cells (TuBECs) secrete high levels of IL-8 compared to normal
brain endothelial cells (BECs) (Charalambous et al., 2005). Co-implantation of
TuBEC with GSCs in an orthotopic murine model would more accurately
reproduce the microenvironment of human GBM. Analyzing the efficacy of
NEO212 in such a model would give us more representative results of the
potential of this drug in the clinical setting. Preliminary in vitro data show that
TuBEC are resistant to NEO212 (Figure 4-1). In light of the importance of TuBEC
as a component of the vascular niche for GSCs, combination of anti-angiogenic
therapies targeting TuBEC along with NEO212 targeting GSCs might be an
effective strategy to combat GBM.

86

0 20 40 60 80 100
0
20
40
60
80
100
120
TuBEC
U251
NEO212 ( M)
Percent survival (%)

Figure 4-1. NEO212 cytotoxicity in tumor endothelial cells. 72 hour MTT
cytotoxicity assay with NEO212 in tumor-associated brain endothelial cells
(TuBEC) (red, squares) and U251 glioma cell line (black, triangles). The data are
expressed as percent cell survival relative to untreated control. Conditions were
set up in triplicates. Data is represented from one experiment.

Another major shortcoming of the in vivo model used in our studies is the
lack of a functional immune system. NOD-SCID mice lack B and T lymphocytes
which play a crucial role in the adaptive immune response. Overcoming immune
surveillance by activation of immunosuppressive mechanisms including
regulatory T cells (Tregs) and tumor associated macrophages (TAMs) is a very
important hallmark of glioma growth and progression. Xenograft models using
immune compromised mice fail to reproduce this aspect of tumor development
(Perng et al., 2015). Several genetically engineered mouse models of glioma
have been created including a mouse model with conditional inactivation of tumor
87

suppressors – Nf1, p53 and Pten, frequently mutated in human GBM (Alcantara
Llaguno et al., 2009). This model develops spontaneous gliomas with 100%
penetrance and reproduces the initiation and progression of tumors in humans.
Studies with NEO212 in genetic immunocompetent mouse models would be very
important to further understand the clinical significance of NEO212.
PROPOSAL FOR PILOT PROJECT – INVESTIGATING THE INTERACTIONS
BETWEEN GLIOMA CANCER STEM CELLS AND ENDOTHELIAL CELLS IN
GBM PROGRESSION
(1) Introduction
Endothelial-mesenchymal transition
Epithelial-mesenchymal transition (EMT) is a reversible biological process
which enables a polarized epithelial cell to acquire phenotypic and functional
characteristics of an invasive mesenchymal cell through a series of biochemical
changes (Kalluri et al., 2009). EMT can be divided into 3 subtypes: (1) type I
which occurs during embryo formation and organogenesis, (2) type II which is
involved in wound healing, tissue repair and fibrosis following injury and
inflammation and (3) type III which mediates invasion and metastatic spread in
cancer (Kalluri et al., 2009). A process similar to EMT has been described for
endothelial cells (ECs) lining blood vessels called endothelial-mesenchymal
transition (EndMT) (Potenta et al., 2008). EndMT is the progressive loss of
endothelial markers such as CD31 and intercellular junction proteins (ZO-1,
88

Claudin-5) and transition towards a mesenchymal phenotype characterized by
high expression of α- smooth muscle actin ( α-SMA) or fibroblast specific protein 1
(FSP1) and increased migration and invasion (Figure 4-2) (Potenta et al., 2008).

Figure 4-2. Endothelial-mesenchymal transition. EndMT is characterized as
the progressive loss of endothelial features (CD31 expression, tight junction
proteins such as VE-cadherin, ZO-1 and Claudin-5) and gain of mesenchymal
features such as α-SMA expression and increased migratory and invasive
properties.

EndMT has been shown to be important during cardiac development but
also in cancer as the source of cancer-associated fibroblasts (CAFs), which
promote angiogenesis and tumor progression. Up to 40% of CAFs in the tumor
microenvironment are derived from EndMT (Zeisberg et al., 2007). Moreover
EndMT has been implicated in metastasis. The metastatic cascade including
degradation of local extracellular matrix (ECM), intravasation of tumor cells into
the vasculature and subsequent extravasation and seeding of tumors at distant
Endothelial
phenotype
Intermediate
Mesenchymal
phenotype
89

sites is responsible for the poor prognosis of most cancers. Though, GBMs do
not metastasize outside the central nervous system, there is significant local
infiltration of tumor cells in the brain. In some GBM cases, the entire brain is
diffusely infiltrated with tumor cells, a condition known as gliomatosis cerebri
(Holland, 2000). In addition, a majority of brain tumors represent metastasis of
primary lung or breast tumors. In response to transforming growth factor β
(TGFβ) secreted by tumor cells, rat brain endothelial cells have been shown to
undergo EndMT which mediates increased adhesion of tumor cells to endothelial
cells and increased transendothelial migration (Krizbai et al., 2015). Besides the
TGFβ signaling pathway, other pathways that are involved in EMT are also
shown to be important for EndMT including BMP and Notch signaling (Medici et
al., 2010; Noseda et al., 2004). However, more studies investigating the
mechanisms inducing EndMT are needed.
Tumor vasculature in GBM
The vasculature is a highly dynamic, tissue specific organ. It plays a
critical role in transport of oxygen and nutrients, removal of cellular waste and
regulation of blood flow. The normal vasculature is organized as a hierarchy of
arteries, arterioles, capillaries, venules and veins. In capillaries, the blood vessels
are lined with a thin layer of tightly packed endothelial cells surrounded by
pericytes; in larger vessels, arterioles and arteries, endothelial cells are
associated with smooth muscle cells (Aird, 2012). The basement membrane,
90

secreted by ECs, is the extracellular matrix responsible for structural support,
stability and anti-coagulation status (Aird, 2012). Brain ECs are unique in that
these cells are directly associated with astrocytes and express tight junction
proteins, resulting in the formation of the blood–brain barrier (Jain et al., 2007;
Obermeier et al., 2013).
In sharp contrast to normal blood vessels, the tumor vasculature,
particularly in GBM, is highly proliferative resulting in abnormal blood vessel
structures (Carmeliet et al., 2000; Jain et al., 2007). These tumor blood vessels
lack the organization and regulation found in normal vessels. Morphologically,
tumor vessels are tortuous, exhibiting dead ends leading to hypoxic regions
(Figure 4-3). Furthermore these tumor blood vessels exhibit shunting from
arterioles to venules thereby causing unstable blood vessel structures and often
resulting in hemorrhaging (Jain et al., 2007). Tumor EC overexpress vascular
endothelial growth factor (VEGF) receptors, therefore an environment of high
VEGF will cause increased endothelial cell proliferation, migration and blood
vessel permeability (Jain et al., 2007). Permeability changes are associated with
increased edema routinely observed in GBM (Jain et al., 2007). The tumor
vasculature is also characterized as demonstrating a compromised blood brain
barrier (Jain et al., 2007).

91

Figure 4-3. Comparison of vasculature in normal human brain and GBM.
Immunostaining for CD31, an endothelial cell marker in normal human brain
tissue (left panel) and GBM tissue (right panel) demonstrating tortuous, abnormal
blood vessels in tumors compared to normal brain. Red denotes positive
staining. Scale bar: 0.1 mm

In our approach to understanding the interactions between the tumor
vasculature and the tumor environment, we have previously isolated tumor
endothelial cells from glioma tissue and characterized these cells. We found that
the tumor-associated brain endothelial cells (TuBECs) exhibited distinct
morphological and functional differences compared to normal brain endothelial
cells (BECs) isolated from non-tumor epilepsy specimens (Charalambous et al.,
2006; Charalambous et al., 2005). TuBECs are large, flat, veil-like cells as
compared to normal BECs, which are small and plump in appearance. TuBECs
have lower proliferation rates, increased migration and enhanced invasion
properties (Charalambous et al., 2006). These tumor-associated brain ECs
produce high amounts of pro-angiogenic growth factors such as VEGF, IL-8 and
endothelin-1 (ET-1) (Charalambous et al., 2006). A subpopulation of the tumor-
92

associated brain EC also express α-smooth muscle actin, a mesenchymal
marker (Charalambous et al., 2006).
Glioma stem cells and interaction with the vasculature
GSCs have been detected close to vascular niches which regulate their
self-renewal and tumorigenicity (Figure 4-4). Calabrese et al. found
Nestin+/CD133+ GSCs in close association with CD34+ endothelial cells in
human GBM tissues (Calabrese et al., 2007). In vitro studies show that co-culture
of human brain microvascular ECs with GSCs promotes GSC self-renewal. One
of the major pathways involved is the Notch signaling pathway (Zhu et al., 2011).
ECs have also been shown to secrete factors that maintain GSC self-renewal
and survival through activation of the mTOR signaling pathway (Galan-Moya et
al., 2011). Conditioned medium from endothelial cells can rescue GSCs from
apoptosis and autophagy induced by growth factor deprivation (Galan-Moya et
al., 2014). Moreover, the Ang1/Tie2 pathway has been found to mediate
adhesion of GSCs to ECs and promote invasion and chemoresistance (Liu et al.,
2010).

93

Figure 4-4. Localization of GSCs relative to the vasculature. Projection
micrographs showing GFP-labeled GSCs (green) in close association with the
vasculature visualized with fluorescent dextran dye (red). (Figure reproduced
from Lathia et al., 2011).

GSCs in turn can also influence ECs by paracrine interactions and
secretion of soluble factors to stimulate tumor angiogenesis. In vitro studies
revealed that conditioned medium from GSCs contain approximately 10–20 fold
higher levels of VEGF than medium from non GSCs; these high levels of VEGF
promote human microvascular EC migration and tube formation (Bao et al.,
2006). GSCs may also play a role in recruiting endothelial progenitor cells from
the bone marrow and contributing to vasculogenesis by secretion of stromal-
derived factor 1 (SDF-1) (Folkins et al., 2009). A summary of the interactions
between GSCs and ECs is shown in Figure 4-5.
94

Figure 4-5. Interactions between glioma stem cells (GSC) and endothelial
cells (EC). (A) EC effects on GSC: EC produce JAG1 and DLL4 which bind to
Notch 1/2
JAG1
Tie2
DLL4
CXCR1/2
IL-8
Ang1
GSC
EC
mTOR
A.
SELF-RENEWAL
TUMOR GROWTH
MIGRATION
TUMOR GROWTH
ADHESION
INVASION
CHEMORESISTANCE
EC EFFECTS ON GSC
CD133+
VEGF
SDF-1
Endothelial
progenitor cells
VEGF
independent
mechanism
Notch
GSC
VEGF
CD105+
CD31+
HDGF
CD133+
/
CD144+
B.
MIGRATION
ANGIOGENESIS
VASCULOGENESIS
TRANSDIFFERENTIATION
GSC EFFECTS ON EC
95

Notch receptors on GSC and promote GSC self-renewal and tumor growth. EC
secrete IL-8 which stimulates migration of GSC to EC and GSC tumor growth in
vivo. Ang1 produced by EC binds to Tie2 on GSC and mediates adhesion,
invasion and chemoresistance. EC also secrete factors that maintain GSC self-
renewal and survival through activation of mTOR pathway. (B) GSC effects on
EC: GSC produce VEGF and HDGF which promotes EC migration and
angiogenesis. SDF-1 produced by GSC induces recruitment of endothelial
progenitor cells from the bone marrow to the tumor and vasculogenesis. GSC
can also differentiate into endothelial cells in a two-step process mediated by
Notch and VEGF signaling or by VEGF independent mechanisms. (Figure
courtesy: Rachel Rosenstein-Sisson).

(2) Hypothesis
Since glioma stem cells are found in close association with endothelial
cells in GBM and since tumor-associated brain endothelial cells (TuBEC) display
mesenchymal features such as α-SMA expression and enhanced migration and
invasion, we hypothesize that GSCs interact with ECs to induce endothelial-
mesenchymal transition in GBM which mediates increased GSC invasion and
tumor progression.
(3) Preliminary data
Co-culture of mesenchymal GSC USC02 with BEC increases α-SMA
expression
To analyze if glioma stem cells induce endothelial-mesenchymal
transition, mesenchymal USC02 GSCs were co-cultured with normal BECs in
vitro. USC02 were seeded on collagen coated coverslips on day 1 in CSC
medium supplemented with 10% FBS to promote cell attachment. BECs were
96

added on to USC02 on day 2 and medium was replaced with EC medium. For
comparison, USC02 and BEC were cultured alone in EC medium. After 72 hours,
coverslips were fixed and cells stained with antibody to α-SMA, a mesenchymal
marker. Our data demonstrated that USC02 and BEC alone had very few α-SMA
positive cells. However, co-culture of USC02 with BEC significantly upregulated
α-SMA expression as demonstrated by increased α-SMA staining (Figure 4-6A).
To investigate the kinetics of α-SMA upregulation, USC02 and BEC were
co-cultured and cells fixed at different time points. IHC results showed that α-
SMA expression increased with time with high expression at 120 hours post co-
culture (Figure 4-6B). Furthermore, to determine that α-SMA upregulation occurs
in BECs, BEC were labeled with green fluorescent protein (GFP) and sorted to
enrich for GFP positive cells. GFP-BECs were co-cultured with USC02 for 120
hours followed by staining for α-SMA. Immunofluorescence analysis
demonstrated that α-SMA was expressed by GFP-positive BECs, evidence
supporting EndMT (Figure 4-6C).

A.

USC02 BEC USC02+BEC
α-SMA
97

Figure 4-6. Increased α-SMA expression in co-cultures of USC02 and BEC.
(A) USC02 were cultured in CSC 10% FBS medium. After 24 hours, BEC were
B.

C.
24 hour 48 hour

72 hour 120 hour
α-SMA
GFP-BEC
DAPI
α-SMA
98

added to USC02 and medium replaced with EC medium. USC02 and BEC
cultured alone in EC medium served as controls. Representative images of
immunostaining for α-SMA (red) in USC02 (left panel), BEC (middle panel) and
USC02+BEC co-culture (right panel) conditions is shown. Scale bar represents
50 µm. (B) Immunostaining for α-SMA in USC02+BEC co-cultures after 24, 28,
72 and 120 hours. Scale bar represents 100 µm. (C) Immunostaining for α-SMA
in USC02+GFP-BEC co-cultures after 120 hours. Bar represents 50 µm.

Conditioned medium from USC02 does not induce EndMT
To determine if soluble factors secreted by GSCs may be involved in
EndMT, conditioned medium (CM) was collected from USC02 and BEC cultured
alone as well as from USC02+BEC co-cultures. CM was added to BEC alone at
a ratio of 1:1 CM:EC medium. CM was collected every 24 hours and added to
BEC; the experimental set-up is described in Figure 4-7A. After 120 hours, cells
were fixed and analyzed for α-SMA expression. IHC results showed that co-
culture and physical interaction of USC02 and BEC is required for α-SMA
upregulation and EndMT (Figure 4-7B). Conditioned medium from USC02, BEC
or USC02+BEC cultures does not induce EndMT in BEC (Figure 4-7C).

Seed USC02 Seed BEC Collect 50% CM, add 50% fresh
medium to plate

Seed BEC Add 50% CM to existing
50% medium
Fix and stain
for α-SMA
D1 D2 D3 D4 D5 D6 D7
A.
99

Figure 4-7. Comparison of contact co-cultures and conditioned medium for
induction of EndMT. (A) Experimental set-up. USC02 were cultured on day 1
(D1). After 24 hours, BEC were added to USC02. USC02 alone and BEC alone
cultures served as controls. Conditioned medium (CM) from all these conditions
was collected every 24 hours up to day 6. In parallel on day 2, BEC were seeded
on coverslips in EC medium. After 24 hours, 50% conditioned medium from the
B.

C.
USC02 BEC USC02+BEC
α-SMA
BEC in EC medium BEC in USC02 CM

BEC in BEC CM BEC in USC02+BEC CM
α-SMA
100

USC02, BEC and USC02+BEC cultures collected fresh on days 3-6 was added
to BEC alone. EC medium served as control. On day 7, all cultures were fixed
and stained for α-SMA. (B) Immunostaining for α-SMA in USC02, BEC and
USC02+BEC 120 hour contact cultures. Scale bar represents 100 µm. (C)
Immunostaining for α-SMA in BEC exposed to CM from USC02, BEC or
USC02+BEC contact cultures after 120 hours. EC medium served as control.
Red denotes positive staining. Scale bar represents 100 µm.

Notch signaling may mediate EndMT induction by GSCs
The Notch pathway involves Notch receptors and ligands, cell surface
transmembrane proteins which mediate important cellular functions such as
proliferation and differentiation through direct cell-cell contact (Kopan et al.,
2009). Activation of the Notch signaling pathway has been shown to mediate
EndMT in endocardial cushion formation (Noseda et al., 2004). To determine if
the Notch pathway is involved in EndMT induction by GSCs, we pretreated BEC
with 10 µM DAPT, an inhibitor of γ-secretase which inhibits Notch signaling. BEC
were then co-cultured with USC02 and fresh DAPT was added to the medium.
DAPT was added every 48 hours to maintain Notch inhibition. After 72 hours or
120 hours, cells were fixed and immunostained for α-SMA. These results showed
that Notch inhibition decreased α-SMA positive cells, indicating that Notch may
be involved in USC02-mediated induction of EndMT in BEC (Figure 4-8).

101

Figure 4-8. Decreased α-SMA expression with inhibition of Notch signaling.
BEC were pre-treated with DAPT at 10 µM for 2 hours followed by co-culture with
USC02. Fresh 10 µM DAPT was also added at the time of co-culture and every
48 hours thereafter. After 72 hours or 120 hours, cells were fixed with acetone.
Representative images of immunostaining for α-SMA (red) in USC02+BEC co-
cultures at 72 hours (upper panel) and 120 hours (lower panel) in control and
DAPT treated cells is shown. Scale bar represents 100 µm.

Co-culture of proneural USC04 with BEC also increases α-SMA expression
Co-culture of mesenchymal USC02 GSCs with BEC induced EndMT as
demonstrated by increased expression of α-SMA, a mesenchymal marker
(Figure 4-6A). To determine if EndMT is specific to mesenchymal GSCs or is a
general GSC-EC interaction phenomenon, we co-cultured proneural USC04
Control + DAPT (10 μM)
α-SMA
102

GSCs with BEC. Immunostaining for α-SMA showed that after 120 hours,
USC04+BEC co-cultures also show increased α-SMA expression (Figure 4-9).

Figure 4-9. Increased α-SMA expression in co-cultures of proneural USC04
and BEC. USC04 were cultured in CSC 10% FBS medium. After 72 hours, BEC
were added to USC04 and medium replaced with EC medium. USC04 and BEC
cultured alone in EC medium served as controls. Representative images of
immunostaining for α-SMA (red) in USC04 (left panel), BEC (middle panel) and
USC04+BEC co-culture (right panel) conditions at 120 hours post co-culture is
shown. Scale bar represents 50 µm.

(4) Proposed studies
Preliminary data showed that both proneural and mesenchymal GSCs
induce α-SMA expression in BECs, an indication of the process of endothelial-
mesenchymal transition. Induction of EndMT only occurs when GSCs are in
physical contact with BECs; conditioned medium from GSCs does not cause
increased expression of α-SMA in BECs. The mechanism and functional
implications of EndMT by GSCs is not known. The following experiments are
proposed to further elucidate the mechanism and significance of EndMT in GBM.

USC04 BEC USC04+BEC
α-SMA
103

(i) Confirming EndMT in BECs in vitro and demonstrating EndMT in clinical
samples of GBM
We have shown that co-culture of GSCs with BEC induces expression of
α-SMA, an important marker for EndMT and myofibroblasts (Figure 4-6A, 4-9).
However, relying on only one marker is insufficient. We propose to assay a panel
of endothelial markers such as VE-cadherin, claudin-5, ZO-1, Tie1/2, CD31 and
CD105 along with mesenchymal markers such as FSP1, vimentin, fibronectin,
vitronectin and type I collagen by PCR analysis. Since DNA from GSCs will
interfere with the results, we propose to differentially label GSCs and ECs. After
co-culture, ECs can be subsequently sorted from GSCs and the enriched ECs
evaluated for additional EndMT markers by PCR. Based on our hypothesis, we
expect to see a decrease in endothelial markers and upregulation of
mesenchymal markers. Additionally, we propose to analyze the same panel of
markers in normal and human GBM tissue. Laser dissection microscopy can be
used to isolate the vasculature from normal and GBM brain sections followed by
PCR for endothelial and mesenchymal markers. We predict that the GBM
vasculature will have high expression of mesenchymal markers.
These studies will provide further proof for the occurrence of EndMT in the
GBM vasculature. Moreover, the demonstration of EndMT in human GBM
samples is critical to extend our in vitro findings to clinical significance.

104

(ii) Identifying the mechanism underlying EndMT by GSCs in BECs
Since preliminary data showed that EndMT by GSCs requires physical
interaction between GSCs and ECs, the Notch pathway was investigated as a
potential mechanism. Inhibition of Notch signaling by DAPT decreased EndMT
as evidenced by reduced staining for α-SMA (Figure 4-8). We propose to follow
up on these initial findings with further mechanistic studies. GSCs and BECs can
be analyzed for the expression of Notch ligands (Jagged 1/2 and Delta like
ligands) and receptors (Notch 1-4) respectively. Moreover activation of the Notch
signaling pathway can be analyzed in BECs following co-culture with GSCs. This
can be investigated by measuring expression of transcriptional factors such as
Hes and Hey, downstream regulators of Notch activation by western blots. In
addition to DAPT, Notch ligands produced by GSCs can be knocked down by
siRNA approaches followed by co-culture of GSCs with BECs. Addition of
recombinant ligands to rescue inhibition of EndMT can definitively confirm the
role of Notch signaling in EndMT of BECs by GSCs.
Additional cell contact-dependent mechanisms such as integrin β1
signaling has been shown to be involved in EndMT in diabetic nephropathy (Shi
et al., 2015). These mechanisms should also be investigated in our system.
These experiments delineating the specific proteins and pathways
underlying EndMT in BECs is essential for identifying novel targets for the design
of drugs targeting GBM.
105

(iii) Determining the functional significance of EndMT in GBM
EndMT has been shown to be the source of cancer-associated fibroblasts,
an important component of the tumor stroma that is involved in tumor
progression and angiogenesis (Zeisberg et al., 2007). Moreover, EndMT has also
been shown to mediate tumor cell adhesion to the vasculature and
transendothelial migration to promote metastasis (Krizbai et al., 2015). To
determine the function of EndMT in GBM, we propose to investigate the
migration and invasion of BEC cultured alone or sorted after co-culture with
GSCs in Boyden chamber assays. Moreover, proteins such as matrix
metalloproteinase 2 (MMP-2) and MMP-9 which are involved in cell migration
and invasion can also be analyzed. We hypothesize that BECs co-cultured with
GSCs will have increased migration and invasion as well as high levels of MMP-2
and MMP-9. To analyze the effects of EndMT on angiogenesis, BECs cultured
alone or BECs that have undergone EndMT by interaction with GSCs can also
be subjected to the tubule formation assay, a measure of in vitro angiogenic
potential.
Moreover, we have shown that the tumor vasculature in GBM is resistant
to chemotherapy (Virrey et al., 2009). The role of EndMT in mediating
chemoresistance can be explored. Also, the effects of mesenchymal BECs on
GSCs can be investigated. GSCs cultured with mesenchymal BEC can be
analyzed for self-renewal and proliferation. The effects of mesenchymal BEC on
106

GSC sensitivity to drugs including TMZ and NEO212 can also be investigated to
determine the role of the vasculature in chemoresistance.
Information from these studies will be crucial to understanding the
pathogenesis of GBM and for developing novel strategies to target GSCs, the
source of origin and tumor recurrence.
(iv) In vivo co-implantation of GSCs and BECs and investigating
therapeutic strategies to inhibit EndMT
To investigate therapeutic strategies targeting EndMT, it is essential to
have an in vivo model that recapitulates EndMT. GSCs can be implanted alone
or along with mesenchymal BECs into the brains of NOD-SCID mice. Luciferase
labeling of GSCs can enable us to monitor tumor progression. Survival can be
used as an end point for this study. In addition, after sacrifice, tumors can be
analyzed for GSC markers such as CD133, Sox2 and Nestin. Invasion can be
investigated by immunostaining for markers such as MMP-2, MMP-9 and FSP1.
We predict that mice co-implanted with GSCs and mesenchymal BEC will have
shorter survival and highly invasive tumors.
This in vivo model can also be used for analyzing the efficacy of novel
therapies. Based on our findings of the mechanism inducing EndMT in vitro, we
can test different drugs such as DAPT in combination with NEO212. We expect
that a combination of NEO212 targeting GSCs and DAPT inhibiting EndMT will
lead to delayed tumor progression and longer survival.
107

(5) Overall significance
EndMT has been demonstrated in melanoma and pancreatic tumor
models (Zeisberg et al., 2007). EndMT has been implicated as the cause of the
plasticity and heterogeneity of the tumor vasculature in a spontaneous mammary
tumor model (Xiao et al., 2015). However, to our knowledge, this study is the first
demonstration of EndMT in glioblastoma multiforme. GSCs are found near
vascular niches in GBM tumors. Though, there are many studies about the
effects of the vasculature on GSC self-renewal and tumorigenesis, very little is
known about the effects of GSCs on the vasculature. This pilot project aims to
further our understanding about the dynamic cross talk between GSCs and ECs
by studying EndMT, an essential process during tumor progression and invasion.
GBMs are one the most vascularized and invasive tumors. Moreover, the
tumor vasculature is resistant to temozolomide. This study will investigate the
role of EndMT in chemoresistance. Furthermore, by understanding the
mechanism triggering EndMT in GBM, this study will provide new insights into
gliomagenesis and provide novel targets for the development of drugs targeting
GBM. Most preclinical studies with GBM use cell lines to model the human
disease in mice. These models fail to recapitulate many crucial aspects of GBM
including invasion and heterogeneity. Most importantly, they fail to take into
consideration the critical role of the tumor microenvironment especially the tumor
vasculature. The co-implantation model we propose will directly address the
interaction between GSCs and ECs isolated from human specimens and is a
108

more clinically representative model. This model will be valuable for preclinical
screening strategies to better predict clinical response of GBM to novel
therapeutics.
In summary, the interactions between glioma stem cells and the tumor
vasculature is a key driving force for tumor growth and progression. Knowledge
of the specific mechanisms regulating these interactions will provide a better
understanding of GBM pathogenesis and will provide new perspectives for the
development of anti-GBM therapies.

109

CONCLUDING REMARKS
In conclusion, the significant finding from this dissertation is the
identification of the novel chemotherapy NEO212 targeting glioma cancer stem
cells, the source of origin and tumor recurrence in GBM. GSCs have two key
properties – self-renewal and chemoresistance that are critical in their
contribution to tumor initiation and progression. TMZ targets proliferating
differentiated tumor cells but spares the GSCs which are responsible for tumor
recurrence. NEO212, on the other hand downregulates self-renewal pathways -
Bmi-1 and Sox2 and induces extensive DNA damage independent of cellular
DNA repair and chemoresistance mechanisms such as MGMT. In this way,
NEO212 targets GSCs by a dual effect on both self-renewal and
chemoresistance. NEO212 affects both proneural and mesenchymal GSCs
representing approximately 80% of human GBM tumors. Moreover, we have also
previously demonstrated that NEO212 is cytotoxic to glioma tumor cells (Cho et
al., 2014). Thus NEO212 has significant clinical value and provides a new
direction in the treatment of GBM. In the future, tumor and GSC-directed
treatments such as NEO212 administered in combination with anti-angiogenic
drugs targeting the tumor vasculature have great potential to significantly alter
the course of GBM progression.

110

Figure 4-10. NEO212 is a CSC-directed therapy targeting glioma stem cell
self-renewal and chemoresistance. TMZ (Temozolomide) only targets
proliferating differentiated tumor cells. The glioma cancer stem cells (CSC) are
not affected by TMZ, thereby contributing to tumor recurrence. NEO212
decreases CSC self-renewal and induces DNA damage independent of MGMT.
Thus, NEO212 effectively targets the CSC population and can potentially prevent
tumor recurrence. Previous studies have shown that NEO212 also targets glioma
tumor cells (Cho et al., 2014).
TMZ NEO212
Differentiated tumor cells Glioma cancer stem
cell
DNA damage,
Independent of
MGMT
Chemo-
resistance
Sox2
Bmi-1
Self-renewal
111

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Abstract (if available)

Abstract Glioblastoma multiforme (GBM) is the most common malignant brain tumor characterized by high vascularity and invasion of tumor cells into the surrounding brain parenchyma. Despite surgery, radiation and chemotherapy with temozolomide (TMZ), a DNA alkylating agent, the prognosis for GBM remains poor with a median survival time of 12-15 months. Tumor recurrence is a major cause for mortality in GBM and glioma cancer stem cells (GSCs) have been implicated as the source of origin and tumor recurrence. TMZ, the current standard-of-care does not affect the GSCs and therefore, therapies directed towards this critical drug resistant cell population are sorely needed. ❧ In this study, we have identified two subtypes of GSCs differing in their growth characteristics, marker expression, genetic profile and tumorigenic potential. The proneural and mesenchymal patient-derived GSCs together represent approximately 80% of human GBM. Thus, these GSCs recapitulate the heterogeneity of human tumors and provide a clinically representative model to evaluate novel therapeutic strategies. Elimination of GBM will require chemotherapies that target both subtypes of GSCs particularly the clinically aggressive mesenchymal subtype. ❧ The significant finding of this study is the efficacy of a novel chemotherapy NEO212, a conjugate of TMZ and perillyl alcohol (POH), a drug shown to have moderate effects against resistant GBM. We found that NEO212 is cytotoxic to both proneural and mesenchymal GSCs in vitro and in an orthotopic xenograft model. NEO212 specifically affects GSCs through targeting two critical properties of cancer stem cells—self-renewal and chemoresistance. NEO212 decreases the expression of key self-renewal factors Bmi-1 (in proneural GSCs) and Sox2 (in mesenchymal GSCs) and induces extensive DNA damage independent of MGMT, an important protein involved in DNA repair and drug resistance. ❧ Our results showing the efficacy of NEO212 against both proneural and mesenchymal GSCs make it a promising new therapy for GBM. NEO212 can be effective as a first line therapy to prevent tumor recurrence or can be administered following drug resistance to eliminate GSCs and delay tumor recurrence. Thus, NEO212 has significant clinical implications for the treatment of GBM.

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Asset Metadata

Creator Jhaveri, Niyati (author)

Core Title Targeting glioma cancer stem cells for the treatment of glioblastoma multiforme

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 04/22/2016

Defense Date 10/13/2015

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

Tag glioblastoma,glioma cancer stem cells,novel chemotherapy,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Dubeau, Louis (committee chair), Chen, Thomas C. (committee member), Hofman, Florence M. (committee member), Hong, Young (committee member)

Creator Email niyati.s.jhaveri@gmail.com,nsjhaver@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-192550

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