Glioma stem cells
Evidence supporting the cancer stem cell hypothesis
for gliomas and technical approaches to investigate
glioma stem cells
Marloes van den Nieuwendijk
Writing assignment
Neuroscience and Cognition | Experimental and Clinical Neuroscience track
Student number 3501981
Supervised by professor dr. E.M. Hol
March 2014
PREFACE
This writing assignment for the master Neuroscience and Cognition is the result of five weeks of
intensive literature search about glioma stem cells and their capacity to form and propagate gliomas.
The first chapter of this writing assignment provides an overview of the characteristics of gliomas. A
detailed overview the characteristics of glioma stem cells is provided in the second chapter. Here,
also the possible cell types from which glioma stem cells might arise are summarized. The evidence
supporting the view that gliomas arise from glioma stem cells is summarized in chapter three and the
available in vitro and in vivo models for glioma stem cell research are addressed in chapter four.
Finally, the remaining questions concerning the cell of origin of gliomas are identified in the fifth
chapter.
I would like to thank prof. dr. E.M. Hol for her enthusiasm for the subject and the instructive
feedback.
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ABSTRACT
Gliomas are the most common primary brain tumors and especially high grade gliomas have a bad
prognosis. Treatment comprises surgery, radiotherapy and chemotherapy, but this approach often
fails because tumor cells become resistant to therapy and it is extremely difficult to remove all tumor
cells. Understanding the cell of origin of gliomas might be the only way to develop more effective
treatment strategies. The main hypothesis concerning the glioma cell of origin is that gliomas arise
from glioma stem cells, which are capable of self-renewal and generate a heterogeneous tumor
containing both stem cells and cells which lack the capacity to self-renew. It remains unclear if neural
stem cells give rise to glioma stem cells or whether glioma stem cells derive from committed neural
progenitors or from differentiated neurons or astrocytes. The main questions that is discussed in this
writing assignment is which recent experimental findings are in line with the hypothesis that gliomas
arise from glioma stem cells. Furthermore, the question which in vitro and in vivo techniques can be
used to study glioma stem cells will be addressed. Finally, the remaining questions in the field of
glioma stem cell research are discussed.
LAYMEN ABSTRACT
Gliomas are brain tumors which contain glial cells. Glial cells have several functions in the brain,
including providing support to brain cells. In the Netherlands, about 1000 people are diagnosed with
glioma every year. Glioma symptoms include headache, memory problems, personality changes and
seizures and have a strong impact on the patients’ life. The majority of glioma patients suffers from
an incurable form. Patients diagnosed with the most aggressive form survive on average for 10
months after diagnosis. It is thought that the formation of gliomas might be caused by stem cells.
Stem cells are capable of self-renewal, which means that they can divide and thereby form new stem
cells and other cells, which are not stem cells. A second important feature of stem cells is that they
can divide into different cell types. It is thought that the formation of gliomas is caused by specific,
mutated stem cells, which are called glioma stem cells. These cells comprise only a small part of the
tumor, but they can make a tumor mass by high levels of cell division. Over time they acquire more
mutations and can for example become resistant to chemotherapy and radiotherapy. The current
glioma treatment, which can only extend the patients’ life with a couple of months, tries to destroy
the entire tumor. The identification of glioma stem cells indicates that new treatment should be
targeting the glioma stem cells, because if they remain present in the brain after therapy, the tumor
will regrow. However, there are still a lot of questions about the existence and characteristics of
glioma stem cells. Thus, to develop better treatment options for gliomas, it is crucial to understand
the characteristics of glioma stem cells and know how gliomas are formed. The main question which
is addressed in this writing assignment is what evidence supports the idea that glioma stem cells can
cause the formation of gliomas. Furthermore, the question how glioma stem cells can be studied will
be discussed. Finally, the remaining questions in the field of glioma research will be discussed.
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ABBREVIATIONS
Ara-C
BBB
BrdU
CD15
CD133
Cnp
CSC
DG
DNA
EGF
EGFR
FGF
GCL
GFAP
GFP
GIC
GSC
LGR5
LIF
MAP2
MGMT
miR-200c
NOD-SCID
NSC
OPC
PC
PCR
PDGF-B
SGL
shRNA
Ssh
SVZ
WHO
ZEB1
cytosine-β-D-arabinofuranoside
blood-brain barrier
bromodeoxyuridine
cluster of differentiation-15
cluster of differentiation-133
2’,3’-cyclic nucleotide 3’-phosphodiesterase
cancer stem cell
dentate gyrus
deoxyribonucleic acid
epidermal growth factor
epidermal growth factor receptor
fibroblast growth factor
granule cell layer
glial fibrillary acidic protein
green fluorescent protein
glioma initiating cell
glioma stem cell
leucine-rich repeat containing G protein-coupled receptor 5
leukemia inhibitory factor
microtubule associated protein 2
O6-methylguanine DNA methyltransferase
micro ribonucleic acid-200c
non-obese diabetic, severe combined immunodeficient
neural stem cell
oligodendrocyte precursor cell
progenitor cell
polymerase chain reaction
platelet-derived growth factor B
subgranular layer
short hairpin ribonucleic acid
sonic hedgehog
subventricular zone
World Health Organization
zinc finger E-box binding homeobox 1
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INDEX
Preface................................................................................................................................................. 2
Abstract ............................................................................................................................................... 3
Laymen abstract .................................................................................................................................. 3
Abbreviations ...................................................................................................................................... 4
Index .................................................................................................................................................... 5
1.
Gliomas ............................................................................................................................................ 6
2.
The cancer stem cell hypothesis for gliomas .................................................................................. 8
2.1 The cancer stem cell hypothesis ............................................................................................... 8
2.2 Glioma stem cells..................................................................................................................... 10
2.3 Cell of origin of glioma stem cells ............................................................................................ 11
3.
Evidence supporting the hypothesis that gliomas arise from glioma stem cells .......................... 14
4.
Research techniques to investigate glioma stem cells .................................................................. 16
4.1 In vitro models for human gliomas.......................................................................................... 16
4.2 In vivo models for human gliomas........................................................................................... 17
5.
Remaining questions and conclusion ............................................................................................ 18
References ......................................................................................................................................... 20
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1.
GLIOMAS
Gliomas, which are highly heterogeneous brain tumors composed of glial cells, are the most common
primary brain tumors. They are categorized according to the type of glial cell found in the tumor.
These categories are astrocytomas, which contain astrocytes, oligodendrogliomas, which consist of
oligodendrocytes, and ependymomas, which contain ependymal cells1. Mixed tumors consisting of
both astrocytes and oligodendrocytes, which are called oligoastrocytomas, or tumors containing both
glial cells and neurons, including gangliogliomas and gangliocytomas, are also known2. In the
Netherlands, the glioma incidence remains stable with an incidence of 4 or 6 per 100.000 personyears for females and males respectively3. Astrocytomas are the most common glial tumors, with an
average incidence of 4.8 cases per 100.000 people per year in Europe4. The majority of the
astrocytoma patients suffers from glioblastoma5. Oligodendrogliomas and ependymomas have a
lower incidence, with 0.4 and 0.2 cases per 100.000 people per year 4.
Based on histopathological characteristics which can be observed with light microscopy, gliomas are
divided into four grades in the classification of the World Health Organization (WHO) (Table 1). The
WHO grade predicts the biological behavior of the tumor and subsequently relates to the preferred
treatment options and clinical outcome. WHO grades I and II are considered benign gliomas, while
grades III and IV are malignant gliomas2. Since astrocytomas have the highest incidence of all
gliomas4, the histological abnormalities found in these tumors are summarized here. Grade I pilocytic
astrocytomas show low levels of proliferation and contain bipolar cells which have dense glial
fibrillary acidic protein (GFAP) positive protrusions. Astrocytomas containing GFAP meshworks and
cells with aberrant nuclei are classified as grade II astrocytomas, including diffuse astrocytomas.
Grade III anaplastic astrocytomas show aberrant nuclei, high levels of mitotic activity and anaplasia.
Grade IV glioblastomas also show necrosis and/or microvascular proliferation, next to aberrant
nuclei, high levels of mitotic activity and anaplasia1,2.
Table 1. WHO classification of gliomas. Names of tumors (left column) are followed by the cell
type(s) which are found in the tumor (middle column) and the WHO grade (right column). Adapted
from Louis et al. 2007
Tumor:
Subependymal giant cell astrocytoma
Pilocytic astrocytoma
Pilomyxoid astrocytoma
Diffuse astrocytoma
Pleomorphic xanthoastrocytoma
Anaplastic astrocytoma
Glioblastoma
Giant cell glioblastoma
Gliosarcoma
Oligodendroglioma
Anaplastic oligodendroglioma
Oligoastrocytoma
Cell type(s) of tumor:
Astrocytes
Astrocytes
Astrocytes
Astrocytes
Astrocytes
Astrocytes
Astrocytes
Astrocytes
Astrocytes
WHO grade:
I
I
II
II
II
III
IV
IV
IV
Oligodendrocytes
Oligodendrocytes
II
III
Astrocytes and oligodendrocytes
II
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Anaplastic oligoastrocytoma
Astrocytes and oligodendrocytes
III
Subependymoma
Myxopapillary ependymoma
Ependymoma
Anaplastic ependymoma
Ependymal cells
Ependymal cells
Ependymal cells
Ependymal cells
I
I
II
III
However, the microscopic abnormalities observed in biopsy samples only provide information about
a small part of the glioma. Because of the heterogeneous nature of gliomas, grading based on biopsy
samples is frequently not correct when compared to the surgically resected tumor and as a
consequence patients do not receive optimal treatment and get a wrong prognosis6. Currently, it is
becoming clear that molecular subtyping of gliomas might be a more powerful system to grade these
tumors and to determine how they should be treated. With this approach, it is possible to distinguish
different patients groups based on molecular markers even within a group of patients with identical
histopathological classification. Thereby it can be assessed for individual patients which treatment
they should receive7. O6-methylguanine DNA methyltransferase (MGMT) promoter methylation is
one of the identified molecular markers for gliomas. The MGMT protein is involved in
deoxyribonucleic acid (DNA) repair by removing alkylgroups from the DNA. Hypermethylation of the
MGMT promoter causes silencing of the MGMT gene and thereby inhibits the removal of
alkylgroups. As a consequence, the glioma cells are more sensitive to DNA damage induced by
alkylating agents such as temozolomide. Thus, MGMT promoter methylation status, which can be
assessed with methylation-specific polymerase chain reaction (PCR), can be used as a marker to
predict the response to alkylating chemotherapy in primary gliomas8. The MGMT promoter
methylation status is homogeneous throughout the glioma and thus a single biopsy sample is
sufficient to determine if a patients would benefit from additional therapy with alkylating agents9.
On a macroscopic level, grade I gliomas are frequently not infiltrative, whereas grade II, III and IV
gliomas often infiltrate surrounding tissue. Chances of recurrence for grade II gliomas are high and a
subset of grade II gliomas transforms into higher grade gliomas. Grade IV gliomas are characterized
by possible metastases. Pre- and postoperative disease progression is fast and grade IV gliomas are
often fatal2. Glial tumors in the CNS and pineal gland have a lower average five-year survival rate
than non-glial tumors in the CNS and pineal gland. Highest five-year survival is seen in ependymoma
patients, with an average of 74%, followed by oligodendroglioma patients with 55%. Five-year
survival of astrocytoma patients is not more than 15%, because of the high incidence of the
aggressive grade IV glioblastoma4. Glioblastoma patients have an average survival of 10 months after
diagnosis. Treatment extends survival to an average of 16 months10.
Standard treatment for malignant gliomas is a multimodal approach combining surgical resection,
radiotherapy and chemotherapy. Nevertheless, this approach only extends survival with several
months because of several complicating factors. Surgical resection of the entire tumor is complicated
by the location and the invasive nature of gliomas, and thus often not all tumor cells are removed11.
Furthermore, glioma cells can become resistant against radiotherapy by activating DNA repair
mechanisms which thereby block the effect of radiotherapy12. A third complicating factor is the
blood-brain barrier (BBB), which prevents most systemic chemotherapeutic agents from entering the
glioma11. A promising approach to target gliomas is by using viruses. The idea of using of viruses in
treatment of brain tumors is not new, however this method still needs to be improved. Two different
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approaches are investigated for their use in glioma treatment. The first are the viral vectors which
are used to deliver specific genes with anti-tumor activity to the glioma cells. Secondly, research
focuses on the application of oncolytic viruses, which specifically infect and destroy glioma cells13.
Despite the effort in improving current therapies, it is generally believed that a specific therapy for
gliomas can only be developed when the cells from which gliomas originate are identified14 so that
these cells can be specifically targeted. The cell type which is held responsible for glioma formation
and survival, which is the glioma stem cell, is discussed in the next chapter.
2.
THE CANCER STEM CELL HYPOTHESIS FOR GLIOMAS
2.1 THE CANCER STEM CELL HYPOTHESIS
According to the cancer stem cell hypothesis, cancer cells in general are hierarchically organized and
only a small subpopulation of cancer cells, the cancer stem cells (CSCs), is mitotically active for an
indefinite period of time and has the ability to self-renew. Thereby, CSC maintain and expand the
stem cell population and give rise to a heterogeneous population of cancer cells in which the
remaining majority of cancer cells is not capable of self-renewal and has limited dividing potential
(Fig. 1A)15,16. An evolutional view of the cancer stem cell hypothesis is that, as a consequence of
natural selection and genomic instability, the hierarchy of tumor cells changes over time and new
clones with different mutations
appear. Eventually, the majority
of these cells will have selfrenewing potential independent
of their niche (Fig. 1B)17. The
question is if the cancer stem cell
hypothesis also applies to
gliomas. The term CSC is used for
cancer in general, whereas the
CSCs in gliomas are called glioma
stem cells (GSCs). Thus, GSCs are
also characterized by their
Fig 1. Overview of the classical and evolutional view of the cancer
capacity for self-renewal and the
stem cell hypothesis. (A) Classical view in which only the CSCs are
ability
to
create
a
capable of maintaining and expanding the stem cell population and
heterogeneous tumor of which
differentiation into multiple cell types. (B) Evolutional view proposing
the majority of cells is not
that the hierarchy inside a tumor changes and causes stem cells with
18
new mutations to appear. Adapted from Chen et al, 2012.
capable of self-renewal .
GSCs are often confused with glioma initiating cells (GICs). As described above, GSCs are
characterized by the capacity for self-renewal and the potential to give rise to heterogeneous
progeny including both stem cells and cells without stem-like properties. GSCs are tumorigenic and
can initiate the formation of a recurrent glioma. However, GSCs are not necessarily the cells that
initiate the formation of a primary glioma. The cells that initiate the formation of gliomas, which are
called GICs, are the cells that undergo the initial tumorigenic transformation and thereby give rise to
a glioma containing a GSC population19. If the cancer stem cell hypothesis applies to gliomas, GSCs
might arise from three possible GICs which are the adult neural stem cells (NSCs), restricted
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progenitor cells (PCs) or mature neural cells16,20,21. NSCs might transform into GSCs by acquiring
cancerous mutations which enable them to survive and self-renew in a different niche, to proliferate
in the absence of a niche, to make the cells in the niche proliferate to expand the niche for the
mutant stem cells, or possibly to differentiate but maintain the capacity for proliferation and
reacquire the capacity for self-renewal. Also the niche itself might change and thereby allow only the
survival of stem cells with specific mutations that can later make the cells cancerous16. Mechanisms
which are involved in self-renewal are already activated in NSCs, thus it is hypothesized that less
steps, and thus less mutations, are required for these cells to become GSCs than for mature, fully
differentiated neural cells. A second reason why NSCs might be the target for cancerous
transformation is that stem cells are capable of self-renewal over a long time, whereas most mature
cells have a short lifespan. This makes NSCs more prone to acquire multiple mutations15. However,
another possibility is that GSCs derive from restricted PCs which acquire the capacity for selfrenewal, which is a hallmark of stem cells, and become immortal through mutations16. A third
possible cell of origin of GSCs are mature neural cells which acquire mutations and thereby
dedifferentiate into a more stem-like cell capable of long-term self-renewal22. It is suggested that
mature neural cells need more mutations than NSCs to activate the self-renewal mechanisms15.
The main opposite view of the cancer stem cell hypothesis is the clonal evolution model, which
implies that a tumor initially consists of equivalent tumor cells of which the majority has self-renewal
capacities (Fig. 2). Over time, genetic and epigenetic events will change these cells and the clones
with advantageous mutations will rule out other clones, thus selecting for clones with advantageous
genetic alterations. These mutations will also cause the heterogeneity which is observed in many
cancers. This model shares the feature of selection with the evolutional view of the cancer stem cell
hypothesis17,20.
Fig. 2. Schematic overview of the clonal evolution model, which
proposes that cancer arises from equivalent cells of which most
of them have the potential for self-renewal and over time cells
which gain beneficial mutations will exclude other tumor cells.
Adapted from Chen et al. 2012.
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2.2 GLIOMA STEM CELLS
GSCs have acquired multiple mutations over time which make them behave differently compared to
NSCs, for example that GSCs proliferate with a much higher speed than NSCs23. An overview over the
events required for transformation into GSCs is beyond the scope of this writing assignment. This
section focuses on the most important features of GSCs. The key properties of NSCs are the capacity
for self-renewal and multipotency and transformed GSCs were initially ascribed the same key
properties as NSCs. Although GSCs share the capacity for self-renew with NSCs, recent evidence
makes clear that GSCs should not be considered as multipotent cells. GSCs have the ability to
differentiate into neurons, astrocytes or oligodendrocytes in vitro24, but the differentiated progeny
has an aberrant genetic profile22. The definition now used is that GSCs can self-renew and thereby
generate a heterogeneous tumor mass16.
When cultured under serum- and matrix free conditions supplemented with epidermal growth factor
(EGF) and fibroblast growth factor (FGF), GSCs grow in clusters of cells which are called
neurospheres25. This is also characteristic of NSCs26. GSC neurospheres derived from human gliomas
express proteins which are typically found in normal NSCs, such as the cytoskeletal intermediate
filament nestin, the stem cell marker cluster of differentiation-133 (CD133), the RNA-binding protein
musashi-1 and the transcription factor Sox212,25,27. After addition of leukemia inhibitory factor (LIF) to
the medium, cultured human GSCs can differentiate into neurons, astrocytes or oligodendrocytes,
which also occurs when NSCs are cultured under these conditions24.
GSCs represent a minority of the total number of tumor cells in a glioma. Of great significance was
the finding that the CD133 expressing tumor cells, which have the capacity for proliferation, selfrenewal and multipotency and thus display the typical stem cell properties, are crucial for formation
and propagation of the tumor in vitro and in vivo, whereas CD133 negative tumor cells lack this
potential. These results support the view that CD133 expressing cells are glioma stem cells18,27. When
cells from human glioblastomas are dissociated into single-cell suspensions and the CD133 positive
fraction is purified and injected into the frontal lobe of non-obese diabetic severe combined
immunodeficient (NOD-SCID) mice, the CD133 positive cells form a tumor. Injection of 100 CD133
positive glioblastoma cells is sufficient to induce tumor formation, whereas a 500- to 1000-fold
higher number of CD133 negative cells does not induce tumor formation. Tumors formed in the
NOD-SCID mice display the same histological characteristics as the human glioblastoma from which
they derive, such as high levels of proliferation, heterogeneous staining for nestin, expression of the
astrocytic marker GFAP and the neuronal marker microtubule associated protein 2 (MAP2). CD133
positive and negative tumor cells display the same chromosomal abnormalities, which demonstrates
that these cells are presumably derived from each other. Serial transplantation of CD133 expressing
cells causes the formation of new tumors with similar characteristics as the original human tumor,
thus indicating the self-renewing potential and the potential to propagate gliomas of CD133 positive
cells in vivo. This study identifies CD133 positive cells with stem cell properties to be the causal for
glioma formation and propagation18. These data are supported by a study in which cells derived from
human glioblastomas are cultured in clonal density in a serum-free system with EGF and FGF2, so
that differentiated cells will disappear and only GSCs remain. These GSCs are capable of tumor
formation and propagation, even after serial transplantation24.
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After receiving surgery and radiotherapy, malignant gliomas often recur and this happens almost
without exception within a small margin from where the primary tumor was located. Thus, tumor
regrowth occurs in the area which receives the highest dose of radiotherapy28. This suggests that a
small group of cells, which is now thought to be the GSCs, is resistant to radiotherapy and is able to
initiate and propagate the formation of a new tumor. This is supported by a study indicating that,
following ionizing radiation exposure, the number of CD133 positive cells in cultures increases four
times compared to non-irradiated cultures. In vivo, the number of CD133 positive cells present in a
glioma xenograft increases three to five times after irradiation. The CD133 negative cells remain
CD133 negative after receiving radiotherapy. Cultures with CD133 positive cells show a lower
number of apoptotic cells compared to CD133 negative cells and nude mice transplanted with a
higher percentage of CD133 positive cells of the total number of tumor cells develop tumors earlier
and the tumors show increased growth and vascularization. Tumors also develop faster when tumor
cells from irradiated xenografts are transplanted into the brains of other mice compared to
transplantation of non-irradiated tumor cells. These data indicate that CD133 positive cells are the
cause of radioresistance. CD133 positive cells derived from human glioblastomas or from xenografts
are more resistant to therapy because of increased phosphorylation of checkpoint proteins which
respond DNA damage. This leads to activation of these proteins, which causes cell cycle arrest,
eventually leads to increased activation of DNA repair mechanisms and thereby the cell death which
would be the result of radiotherapy is evaded12.
2.3 CELL OF ORIGIN OF GLIOMA STEM CELLS
As discussed in the introduction of this chapter, GSCs potentially derive from three cell types: NSC,
restricted PCs or mature neural cells. This section gives an overview of the most important hallmarks
of these cell types and evidence that these cells can give rise to gliomas (Fig. 3).
Fig. 3. An overview of the potential cell types from which GSCs might
derive and the markers that are expressed by these cells. From Chen et
al. 2012.
NSCs are characterized by their multipotency and long-term self-renewing potential, which thereby
enables them to maintain and expand the stem cell pool and to give rise to heterogeneous progeny.
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A balance in self-renewal and differentiation is important to maintain the stem cell population and is
sustained by the niche, which provides NSCs with growth factors, blood vessels and a matrix29.
However it appears to be very difficult to distinguish NSCs and PCs, which hampers the investigation
of the cell of origin of glioma stem cells30. In the adult human and rodent brain, the main NSC niches
are the subventricular zone (SVZ) close to the lateral ventricles and the subgranular layer (SGL) of the
hippocampal dentate gyrus (DG) (Fig. 4)31–34.
Fig. 4 Overview of the main stem cell niches, the
SGL and the SVZ, in a schematic saggital section of
the adult mouse brain. The SGL and SVZ are
indicated by the red boxes. SGL; subgranular layer,
SVZ; subventricular zone. Adapted from Uchida et
al. 2000.
The murine SVZ comprises a layer of dividing cells along the lateral edge of the lateral ventricles and
contains four cell types: type A neuroblasts, type B astrocytes, type C transit amplifying progenitor
cells and ependymal cells. The ependymal cells separate the SVZ from the lateral ventricles. After
treatment with the antimitotic agent cytosine-β-D-arabinofuranoside (Ara-C), all neuroblasts and
transit amplifying cells are destroyed and start to reappear a few days after treatment. Together with
the findings that all remaining cells immediately after Ara-C treatment express GFAP and that these
cells incorporate a marker for DNA synthesis, these data demonstrate that type B astrocytes are the
only self-renewing cells in the SVZ and thus can be seen as NSCs. Type B astrocytes divide and give
rise to new stem cells and type C transit amplifying progenitors, which in turn proliferate several
times and generate type A neuroblasts. The neuroblasts migrate via the rostral migratory stream into
the olfactory bulb where they become interneurons. This process occurs under normal conditions32.
A subset of the SVZ astrocytes has the potential to differentiate into oligodendrocyte precursor cells
(OPCs) and mature oligodendrocytes in vitro and in vivo35. The human SVZ also comprises a cell
population with stem cell properties31,33, but the organization of the human SVZ is different than the
rodent SVZ. It comprises four layers of which the first layer is a layer of ependymal cells, followed by
a hypocellular layer which contains very few cell bodies and is mainly occupied by dense astrocyteand ependymal cell protrusions. The third layer is formed by a ribbon of astrocyte cell bodies of
which a subgroup is mitotically active, followed by the fourth layer of brain parenchyma which is
called the transitional zone36. The astrocytes in layer three are capable of forming neurospheres and
generating cells of the neuronal, astrocytic and oligodendrocytic lineage in vitro33,37.
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A process similar to that in the SVZ occurs in the adult hippocampus. In the SGL of the mouse
hippocampal DG, GFAP expressing astrocytes are found which have the same characteristics as type
B astrocytes in the SVZ. These SGL astrocytes divide and thereby generate GFAP-negative type D cells
and sustain the SGL stem cell pool. Type D cells in turn differentiate into new granule neurons which
will be part of the DG granule cell layer (GCL)34. This is also found in postmortem brain samples from
the hippocampus of patients who were treated with bromodeoxyuridine (BrdU), a marker which is
incorporated into the DNA during DNA synthesis and thus labels proliferating cells. In these samples,
BrdU labeled new granule cell neurons are detected in the GCL of dentate gyrus31.
Evidence that NSCs can give rise to glial tumors comes for example from a mouse model in which the
tumor suppressor genes pten, p53 and nf1 are exclusively inactivated in NSCs and PCs. In this model,
a transgene expressing cre recombinase-modified estrogen receptor ligand-binding domain fusion
protein is placed under the control of the nestin promoter. This promoter causes inactivation of the
tumor suppressor genes only in NSCs and PCs. After administration of tamoxifen, the fusion protein
translocates to the nucleus and recombines the tumor suppressor genes pten, p53 and nf1. All of
these transgenic mice develop grade III or IV astrocytomas, which suggests that deletion of pten, p53
and nf1 restricted to NSCs and PCs is sufficient to induce astrocytomas. The labeling of NSCs and PCs
with LacZ allows tracing of these cells, which reveals that NSCs and PCs from the SVZ and SGL
transform into GSCs, migrate away and infiltrate surrounding tissue where they form astrocytomas.
These GSCs express neuronal, astrocytic or oligodendroglial markers and thus show some level of
differentiation, which might explain the heterogeneity seen in human astrocytomas. Inactivation of
pten, p53 and nf1 in NSCs and their progenitors from the SVZ reveals astrocytoma formation in many
brain regions, while inactivation of these tumor suppressors in regions without NSCs do not cause
astrocytoma formation. Thus, NSCs and PCs are capable of initiating astrocytoma formation, whereas
mature cells cannot initiate the formation of astrocytomas. The mutant SVZ NSCs or PCs show, when
cultured as neurospheres, increased proliferation and diminished apoptosis38.
Evidence for gliomas arising from PCs in the adult brain is also available. In a mouse model, an
activated allele of the epidermal growth factor receptor (EGFR) is placed under the S100β promoter
to specifically target OPCs which lost the stem cell phenotype. When this is combined with p53
deficiency, oligodendrogliomas develop in white matter regions. In the corpus callosum of these
transgenic mice, three times higher numbers of OPCs are present compared to control animals,
which are identified by S100β, olig2 and NG2 expression. In this mouse model, tumors arise in white
matter regions and NG2 positive cells from these tumors show similar protein expression patterns
compared to normal OPCs and human oligodendroglioma cells. Moreover, the expression of sox10,
which is involved in OPC differentiation and the formation of gliomas, is increased in these NG2
positive tumor cells. These data indicate that the increase in NG2 positive cell number precedes
oligodendroglioma formation. Furthermore, a NG2 positive cell fraction isolated from human grade II
oligodendrogliomas can initiate oligodendroglioma formation in a mouse model, whereas NG2
negative cells are not capable of tumor formation. These data support that NG2 positive OPCs are
the cell of origin for gliomas. However, the absence of multipotency and of the potential to form
neurospheres indicates that these OPCs are not dedifferentiated into a more stem-like state39. In
addition, in another study a model is used in which platelet-derived growth factor B (PDGF-B) drives
the formation of oligodendrogliomas from OPCs. These oligodendrogliomas express OPC markers but
lack the stem cell marker nestin. One third of the mice develop an oligodendroglioma, of which all
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except one are low grade gliomas. These tumors have similar histological characteristics as human
grade II oligodendrogliomas. These results point at restricted progenitors to be a possible origin of
GSCs40.
Experimental data also demonstrate that differentiated neurons and astrocytes are capable of glioma
formation. Evidence comes for example from a mouse model in which nf1 and p53, two tumor
suppressor genes which are often mutated in human glioblastoma, are silenced exclusively in
neurons or astrocytes. This is achieved by injecting a lentiviral vector encoding short hairpin RNA
(shRNA) for both nf1 and p53 in the brains of synapsin 1-cre transgenic or GFAP-cre transgenic mice
to specifically target neurons or astrocytes respectively. Independent of whether the gene silencing
occurs in either neurons or astrocytes, all mice develop glial tumors with histological characteristics
resembling those of human glioblastomas. However, silencing of nf1 and p53 specifically in NSCs by
using sox2-cre and nestin-cre transgenic mice also causes the formation of gliomas. When the tumors
progress, the expression of stem cells and progenitor markers increases, while the expression of
differentiation markers decreases. This most likely reflects the dedifferentiation of mature cells into
stem or progenitor cells. The same results are obtained when cultured astrocytes from the mice in
which nf1 and p53 are silenced are transplanted into NOD-SCID mice. Furthermore, when astrocytes
or neurons in which nf1 and p53 are silenced are transferred from a culture with serum to a serumfree culture supplemented with FGF-2, the morphology of the cells alters and neurospheres are
formed. Cells from these neurospheres express the stem cell markers sox2 and nestin. This study
demonstrates that mature neurons and astrocytes can dedifferentiate into a cell with stem cell
properties and that NSCs, mature neurons and mature astrocytes are capable of forming gliomas41.
3.
EVIDENCE SUPPORTING THE HYPOTHESIS THAT GLIOMAS ARISE FROM GLIOMA
STEM CELLS
The identification of GSC in gliomas and the capacity of these cells to cause the formation and
propagation of tumors in serial transplantation experiments in animal models supports the idea that
the cancer stem cell hypothesis also applies to gliomas. However, the existence of GSCs and also the
more general CSCs remains controversial and opinions about these stem cells range from questioning
their existence to thinking about methods to target them in cancer therapy21. Thus, more evidence is
required to make an evidence-based consideration about the role of GSCs in glioma pathogenesis. In
this chapter, evidence for the existence of GSCs and their causal role in glioma formation are
discussed.
The niche in which stem cells reside is important for regulation of their self-renewal and
differentiation29. Thus, studying GSCs in vivo in a developing tumor can be of great significance to
elucidate the role of GSCs in the process of tumor formation and propagation. In a recent study, GSCs
in developing high-grade gliomas are traced in living mice by multiphoton intravital microscopy.
Surgically resected human high grade gliomas are divided into CD133 positive cell fractions (GSCs)
and non-stem tumor cell fractions, which are transduced with lentiviruses to make the different
fractions express different fluorescent proteins to allow tracing of these cells. The first and second
week after transplantation of GSCs into the cortices of nude mice, the GSCs reside and grow in close
proximity to blood vessels. In the third week a tumor appears and GSCs infiltrate the surrounding
tissue (Fig. 5).
14
Moreover, when GSCs and non-stem tumor cells are
transplanted into the cortices of nude mice in a ratio of 1:9,
there is a rapid and strong increase in the volume occupied
by GSCs, whereas the number of non-stem tumor cells
slightly decreases. Thus, GSCs have a higher tumor forming
potential than non-stem tumor cells. When the tumors are
allowed to grow until the mice develop neurological signs,
94.5% of all tumor cells derives from the GSCs, whereas only
0.2% derives from non-stem tumor cells. This demonstrates
that a large majority of the tumor cells are GSCs and GSCderived cells. Similarly as observed in human high-grade
gliomas, GSCs and GSC-derived cells remain close to blood
vessels. Moreover, 75% of the GSCs in culture prior to
transplantation into the mouse brain, expresses the stem
cell marker sox2, whereas only 25% of the GSCs expresses
sox2 after transplantation. Thus, the cells that expressed
sox2 in culture can generate other cell types in vivo. A
decrease is also found in the cells expressing a marker for
the M-phase of the cell cycle. These data suggest that only a
subset of the GSCS is still capable of proliferating and that
the in vivo environment regulates differentiation and
thereby creates heterogeneity within the tumor. Together,
these data demonstrate that GSCs are present in gliomas
and are causal for tumor formation and propagation in vivo42.
Fig. 5. Projection micrograph reveals
green fluorescent protein (GFP) labeled
GSCs (green) form a tumor close to
dextran labeled blood vessels (red) at
day 20 after transplantation in the
cortex of nude mice. From: Lathia et al.
2011.
Another experimental study supporting the hypothesis that gliomas arise from GSCs comes from a
study in which the Sonic hedgehog (Ssh) pathway, which is a critical stem cell signaling pathway, is
blocked by the compound cyclopamine. In glioma cell lines in which the Ssh pathway is activated,
blockade causes decreased growth of the cells and when cells from dissociated neurospheres which
received cyclopamine treatment are cultured in medium without the Ssh inhibitor, no neurosphere
formation is detected. This demonstrates that inhibition of the Ssh pathway eliminates GSC from the
tumor cells. Moreover, the number of GSCs increases following radiotherapy43, which is in line with
the previous findings demonstrating an increase in CD133 positive cells after radiotherapy12. When
human glioblastoma cells, which received cyclopamine treatment, are injected into the brains of
athymic mice, no tumor is formed. Together, these data demonstrate that following Ssh blockade,
which is a critical signaling pathway in stem cells, the GSCs are depleted43.
Moreover, the cancer stem cell hypothesis implies that if GSC exist and if they are not removed, the
tumor recurs. This is supported by the findings that following radiotherapy, the number of CD133
positive cells increases and that this subset of cells shows decreased apoptosis and increased
activation of cell cycle checkpoint proteins to repair DNA damage12. Furthermore, when samples
from primary and recurrent human glioblastomas are compared after receiving radio- and
chemotherapy, the CD133 positive cell number is increased in recurrent tumors compared to primary
tumors. In sections from primary glioblastomas, low proliferation levels are observed, whereas
15
higher proliferation is found in the recurrent samples. These data indicate that following therapy,
CD133 positive cells survive and become highly proliferative and thereby the glioma regrows44.
If GSCs are causal for the propagation of gliomas and an increase in stem cell properties means an
increase in the stem cell state, it might be expected that expression of GSC markers correlates with
disease prognosis. To assess this, markers for GSCs are required such as the transmembrane protein
leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), a recently identified marker of
brain tumor stem cells including GSCs. LGR5 overexpression is detected in all glioma tissue samples
and correlates to proliferation rates and the grade of the gliomas. Knockdown of LGR5 results in
inhibition of the cell-cycle, subsequent inhibition of proliferation and decreased neurosphere
formation in a glioma cell line. In vivo, cells in which LGR5 is inhibited show diminished tumor growth
in a xenograft model. These results indicate that depletion of the GSC marker LGR5 causes a decrease
in tumor forming potential, thus pointing at a possible role for GSCs in the formation of gliomas45.
Evidence supporting the view that GSCs are crucial in glioma pathogenesis also comes from a study in
which the role of zinc finger E-box binding homeobox 1 (ZEB1) in glioblastoma invasion and
chemoresistance is examined. ZEB1 is mainly expressed by glioblastoma cells in the most infiltrative
tumor area in xenograft mouse models. ZEB1 regulates MGMT on a transcriptional level by affecting
microRNA-200c (miR-200c) and c-MYB, thereby causing infiltration of surrounding tissue and
chemoresistance. The actions of ZEB1 lead to expression of the stem cell factors CD133, olig2 and
sox2 and increased ZEB1 expression in a neurosphere assay causes an increase in sphere forming
frequency. When ZEB1 is silenced with shRNA, tumor initiating capacity is diminished. Together,
these data implicate that through activation the ZEB1 pathway, glioma cells have a higher capacity
for tumor formation46.
4.
RESEARCH TECHNIQUES TO INVESTIGATE GLIOMA STEM CELLS
A GSC is defined by the ability to self-renew and produce a heterogeneous tumor mass. Identification
of GSCs is based on the ability to form neurospheres, on the ability to differentiate into multiple cells
types and on the ability to form a tumor in serial transplantation experiments resembling the original
tumor phenotype. The latter characteristic distinguishes GSCs from normal NSCs19. Thus, cell culture
and animal models are of great significance to assess these hallmarks of GSCs. This chapter will first
highlight the most important in vitro techniques, followed by the most promising animal models.
4.1 IN VITRO MODELS FOR HUMAN GLIOMAS
A very important tool in glioma research is culturing of glioma cells that are isolated from surgically
removed human glioma tissue. The human tissue is dissociated and suspended and the tumor cells
are cultured under serum-free conditions in the presence of FGF and EGF to promote GSC
proliferation. GSCs can be distinguished from non-stem tumor cells based on their tendency to form
neurospheres or by selecting for the stem cell marker CD133 with magnetic cell sorting18,27. GSC
cultures can be maintained for about 20 passages without observing major changes in growth rates.
However, new mutations are introduced especially at later passages, thus changing the genotype of
the GSCs compared to the genotype of the tumor from which they are derived. The GSCs can
differentiate when cultured under differentiation conditions47. The purified GSCs can be transplanted
into immunocompromised mice to study the formation and propagation of the tumor.
16
Furthermore, several glioma cell lines which derive from human gliomas are available, such as U87
and U251. These cell lines are often used for transplantation into the brains of rodents. U251, which
is one of the best studied glioma cell line, derives from a human glioblastoma48. When U251 cells are
injected intracranially in nude mice, a glioma develops that displays necrosis, neovascularization,
nuclear atypia and inflammation. However, the tumor displays no endothelial proliferation and does
not always show signs of invasion of surrounding tissue49. The U87 cell line, which also derives from
human glioblastoma, is mainly used to study neovascularization50. Tumors formed from U87 cells do
not display necrosis, endothelial proliferation and do not always display invasion of surrounding
tissue49. However, these cell lines are not suitable for studying GSCs, since they do not contain stem
cells. Glioma stem cell lines include 9L, but this cell line shows less similarities with glioblastoma cells
than the non-stem cell lines50.
4.2 IN VIVO MODELS FOR HUMAN GLIOMAS
In vitro models remove the tumor cells from their niche and thus a critical component which provides
the tumor with growth factors, oxygen and a matrix, is lacking29, whereas in vivo studies do include
the interactions with the niche. Several vertebrate animal models for different types and grades of
gliomas are available and many of these models involve mice. The two main types of mouse models
are genetically engineered mouse models and xenograft mouse models. In genetically engineered
mouse models, the mutations that are frequently found in human glioma tissue are induced in mice,
thereby allowing the investigation of the causes and pathogenesis of gliomas in vivo48. Genetic
mouse models have been developed for most genes known to be involved in human gliomas, for
example mice which are heterozygous for tumor suppressor genes nf1, pten and p53. These mice
develop high grade astrocytomas with 100% penetrance, show accelerated tumor formation and
show decreased survival compared to mice heterozygous for only nf1 and p5351. Furthermore, genes
can be controlled in a spatial manner with cre-mice, where for example the mutation is exclusively
induced in GFAP-expressing cells48.
In xenograft mouse models, glioma cells from human or murine glioma tissue or a glioma cell line are
transplanted into the brains of mice. These mice are immunocompromised to avoid rejection of the
xenograft. These models are predominantly used to study glioma pathogenesis and to test drugs 48.
The NOD-SCID mouse model is frequently used, and injection of 100 CD133 positive cells is sufficient
to induce glioma formation in this model18. Although some models are known in which the
developing tumors show similar phenotypes as the human gliomas, not all the xenograft tumors are
good representatives of the human tumors48.
Recently, a new xenograft model for human gliomas was developed in pigs. Until now, the only large
animal model for gliomas were dogs, but this model is difficult to use in experiments because the
glioma develops spontaneously. This model is developed in pigs, since testing of continuous
application of drugs over the blood-brain barrier by implanted devices is easier in large animals52 and
because the anatomy of the brain is similar in pigs and humans53. In this pig xenograft model, cells of
the human glioblastoma cell line U87 MG or the glioblastoma stem cell line G6, are implanted into
the parietal lobe of the pigs, which are immunosuppressed by administration of ciclosporine. A
glioma develops in about 95% of the animals which were transplantated with U87 MG cells, whereas
only one out of 5 pigs transplanted with G6 cells develops a tumor. The tumor which is formed shows
17
angiogenesis, necrosis and infiltration of surrounding tissue and resembles the situation seen in
human malignant gliomas. This can possibly be explained by the short follow-up period, because
mice transplanted with G6 cells develop malignant gliomas slower than mice transplanted with U87
MG cells52.
Recently an experimental setup to visualize GSCs in living mice by multiphoton intravital microscopy
was developed. This technique allows tracing of individual cell types within a developing tumor42. In
the future, this model could be expanded so that the volume occupied by GSCs before and after
treatment can be compared. This model does not only have the potential to be an important model
to study drug efficacy but also for providing the ultimate proof that GSCs are the cell of origin of
gliomas, by observing that a therapy is effective in eliminating GSCs and improves the condition of
the animals. Since animals are sacrificed after intravital microscopy, using different groups of
animals from an identical strain might help to elucidate the role of GSCs in glioma pathogenesis.
5.
REMAINING QUESTIONS AND CONCLUSION
Although there an increasing number of studies showing the presence of GSCs in gliomas and
confirming their causal role in glioma formation, there are still several remaining questions
concerning the role of glioma stem cells in glioma formation. These questions are identified and
discussed in this section.
An important point of discussion is that the terms GSC and GIC are often confused and thereby
complicates the debate. GICs are cells which undergo the initial cancerous transformation and are
then able to initiate glioma formation. GSCs are the cells capable of self-renewal and maintaining a
heterogeneous tumor. However, since GSCs can induce tumor formation in animal models, the terms
are often confused. Thus, in the future it is important to gain more knowledge on both cell types to
understand the remaining questions in the glioma stem cell field19.
Another point of confusion are contrasting findings with the stem cell marker CD133. If CD133
expressing cells, which are considered to be stem cells, are causal for glioma propagation, it might be
expected that the level of CD133 positive cells correlates with prognosis. This is investigated in
several studies, although contradictory results are reported. For example, it is found that higher
levels of CD133 are correlated with longer survival in glioblastoma patients after surgical resection
and radiotherapy54. On the contrary, in several studies in which a combination of chemotherapy and
radiotherapy are given, prognosis is worse in patients with high levels of CD13355. Related to this
subject is that if gliomas arise from GSCs, it might be expected that high grade gliomas contain more
GSCs than low grade gliomas. One study found that the number of CD133 expressing cells in a glioma
is correlated to the WHO grade assigned to the glioma in patients with primary gliomas who did not
receive radiotherapy or chemotherapy. No differences were found in the capacity for self-renewal or
multipotency between the CD133 positive cell populations from gliomas of different grades in vitro.
These data point at a role for CD133 expressing cells in glioma progression56. Moreover, it is also not
clear if benign gliomas contain a stem cell population. Based on the glioma stem cell hypothesis, it
might be assumed that benign gliomas lack GSCs because GSCs are causal for therapy resistance12
and cause the regrowth of gliomas44, which is not observed in low grade gliomas. One study failed to
identify cells capable of neurosphere formation in grade I gliomas and grade II oligodendrogliomas
after culturing these cells for 90 days in a serum-free culture system with EGF and FGF2, whereas
18
cells from high grade gliomas form neurospheres after 20 to 40 days in culture. These data indicate
that low grade gliomas might not contain GSCs24.
If the glioma hypothesis is true and GSC are crucial in glioma pathogenesis, GSC can arise from NSCs,
more restricted PCs or mature astrocytes. This means that to provide evidence for the existence of
GSCs and their role in glioma formation, is it crucial to have the appropriate markers to distinguish
these cell types. At the moment, these markers are not yet available. CD133 is frequently used as
GSC marker but the value is still controversial. One study reported that CD133 negative cells can also
initiate tumor formation57, which is not in line with the findings from another study in which CD133
positive cells are shown to initiate tumor formation, whereas 500- to 1000-fold higher numbers of
CD133 negative cells cannot18. Finding new markers for GSCs and other tumor cells and the possible
cell types of origin of GSCs might help enormously in elucidating glioma pathogenesis. Also further
investigation about the validity of current markers is needed.
Gliomas can become resistant to radiotherapy12 and following surgical resection, gliomas often
regrow28. Both approaches are focused at removing the majority of the cancer cells. According to the
cancer stem cell hypothesis, only a small subset of the tumor cells, which are the GSCs, is capable of
tumor formation and survival. This implicates that therapy which specifically targets the GSCs would
be a more specific treatment for gliomas and might decrease recurrence and improve survival22.
Thus, identification of the cell type responsible for glioma formation has major impact on therapy
development and therefore research focusing on the glioma cell of origin is of huge importance14.
To conclude, the experimental data discussed in this writing assignment provide a solid basis to
support that the cancer stem cell hypothesis also applies to gliomas. The fact that both in vitro and in
vivo studies confirm the causal role for GSCs in glioma formation strengthens the support for this
hypothesis. The main problem now seems to be the lack of a valid GSC marker. When more selective
markers are found to distinguish GSCs from NSCs and other cell types, this will help to answer the
questions mentioned in this chapter. Thereby, it opens the way to show that future therapies, which
selectively target GSCs, eliminate the tumor and improve outcome of glioma patients. This would be
the ultimate proof for the causal role of GSCs in glioma formation21. Until then, the debate about the
existence of GSCs and their role in glioma pathogenesis will be ongoing.
19
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