REVIEW Cancer stem cells: a new framework for the design of tumor therapies Boyan K. Garvalov & Till Acker Received: 14 July 2010 /Revised: 27 August 2010 /Accepted: 16 September 2010 # Springer-Verlag 2010 Abstract Modern tumor therapy has achieved considerable progress, but many tumors remain refractory to treatment or relapse following initial remission. Recent evidence points to one possible reason for this limited therapeutic efficiency: that the design of anticancer agents so far may not have been aimed at the right target. While conventional tumor therapies have targeted the main mass of tumor cells, there is now compelling evidence that tumor initiation and progression are driven by a subpopulation of tumor cells that possess stem cell properties and are resistant to traditional cancer treatments— the cancer stem cells (CSCs). CSCs have been identified in most types of cancer and can be separated from the rest of the tumor cells using appropriate markers. CSCs are regulated by molecular mechanisms and specific, perivascular, and hypox- ic microenvironments, which largely overlap with those controlling stem cells from normal tissues. Our improved understanding of CSC biology has already provided a number of novel targets and drug discovery platforms for the design of specific therapies that aim to eradicate the CSC subpopula- tion. Therapeutic approaches can be targeted either at eliminating the CSCs themselves or at disrupting the niches in which CSCs reside. Moreover, the importance of CSCs for tumor growth, resistance, and progression implies that clinical trials and preclinical studies of anticancer therapies should include as a key element an assessment of the abundance and persistence of CSCs. Thus, CSC research holds great promise for providing important new impetus to the fields of tumor biology and clinical oncology. Keywords Cancer stem cell . Hypoxia . Microenvironment . Angiogenesis . Antitumor therapy. Metastasis The hierarchy model and cancer stem cells (CSCs) The classical view of tumor formation is based on the “stochastic” or “clonal evolution” model [1, 2]. It perceives the tumor as a mass of hyperproliferative cells with similar potential for driving tumor growth. Tumor heterogeneity and progression are seen as the result of variations in the tumor microenvironment and genetic mutations in individ- ual cells, followed by selection of those that are best adapted to support the further growth of the tumor (Fig. 1a). An alternative concept that has been gaining increasing experimental support is the “hierarchy” or “cancer stem cell” model [3]. This model posits that tumors are generated and maintained in a manner similar to the physiological stem cell system operating in normal tissues, i.e., by cells with stem cell-like properties, which self-renew and differentiate into the distinct cellular subtypes of the tumor (Fig. 1b). The key novel features of this model are that only a limited population of tumor cell ...

1. REVIEW
Cancer stem cells: a new framework for the design
of tumor therapies
Boyan K. Garvalov & Till Acker
Received: 14 July 2010 /Revised: 27 August 2010 /Accepted: 16
September 2010
# Springer-Verlag 2010
Abstract Modern tumor therapy has achieved considerable
progress, but many tumors remain refractory to treatment or
relapse following initial remission. Recent evidence points
to one possible reason for this limited therapeutic efficiency:
that the design of anticancer agents so far may not have been
aimed at the right target. While conventional tumor therapies
have targeted the main mass of tumor cells, there is now
compelling evidence that tumor initiation and progression are
driven by a subpopulation of tumor cells that possess stem cell
properties and are resistant to traditional cancer treatments—
the cancer stem cells (CSCs). CSCs have been identified in
most types of cancer and can be separated from the rest of the
tumor cells using appropriate markers. CSCs are regulated by
molecular mechanisms and specific, perivascular, and hypox-
ic microenvironments, which largely overlap with those
controlling stem cells from normal tissues. Our improved
understanding of CSC biology has already provided a number
of novel targets and drug discovery platforms for the design of
specific therapies that aim to eradicate the CSC subpopula-
tion. Therapeutic approaches can be targeted either at
eliminating the CSCs themselves or at disrupting the niches

2. in which CSCs reside. Moreover, the importance of CSCs for
tumor growth, resistance, and progression implies that clinical
trials and preclinical studies of anticancer therapies should
include as a key element an assessment of the abundance and
persistence of CSCs. Thus, CSC research holds great promise
for providing important new impetus to the fields of tumor
biology and clinical oncology.
Keywords Cancer stem cell . Hypoxia .
Microenvironment . Angiogenesis . Antitumor therapy.
Metastasis
The hierarchy model and cancer stem cells (CSCs)
The classical view of tumor formation is based on the
“stochastic” or “clonal evolution” model [1, 2]. It perceives
the tumor as a mass of hyperproliferative cells with similar
potential for driving tumor growth. Tumor heterogeneity
and progression are seen as the result of variations in the
tumor microenvironment and genetic mutations in individ-
ual cells, followed by selection of those that are best
adapted to support the further growth of the tumor (Fig. 1a).
An alternative concept that has been gaining increasing
experimental support is the “hierarchy” or “cancer stem
cell” model [3]. This model posits that tumors are generated
and maintained in a manner similar to the physiological
stem cell system operating in normal tissues, i.e., by cells
with stem cell-like properties, which self-renew and
differentiate into the distinct cellular subtypes of the tumor
(Fig. 1b). The key novel features of this model are that only
a limited population of tumor cells can drive tumor
initiation and maintenance and that the heterogeneity of
tumor cells is the result of differentiation from a stem cell

3. precursor that sits on top of the tumor’s “differentiation
hierarchy” [4], which may also include some level of
plasticity (Fig. 1b). The CSC model, therefore, extends the
clonal evolution model by proposing that clonal selection
B. K. Garvalov : T. Acker
Institute of Neuropathology, Justus Liebig University,
Giessen, Germany
T. Acker (*)
Institute of Neuropathology,
University Hospital Giessen and Marburg/Justus Liebig
University,
Arndtstr. 16,
35392 Giessen, Germany
e-mail: [email protected]
J Mol Med
DOI 10.1007/s00109-010-0685-3
operates at the level of CSCs. The notion that tumors may
be initiated and maintained by aberrant cells with a stem
cell character can be traced back to the nineteenth century
and has been revived repeatedly ever since [5–7]. However,
direct experimental evidence was first provided in the mid
1990s, when it was shown that acute myeloid leukemia
could be transferred from human patients to immunodefi-
cient mice by a rare population of cells, which carry
markers of normal hematopoietic stem cells and reproduce
the hierarchy of differentiated leukemic cells [8, 9]. These
studies also set the standard by which CSCs are defined and
identified: as those tumor cells that can self-renew and are
capable of re-growing and reconstituting the heterogeneity
of the tumor from which they were isolated.

4. Based on analogous experimental paradigms, CSCs have
also been identified as a common feature of multiple solid
tumors including lung [10, 11], colon [12–15], stomach
[16], liver [17, 18], breast [19–21], prostate [22], pancreatic
[23, 24], head and neck [25], skin [26], ovarian [27–29],
bladder [30], mesenchymal cancer [31], brain tumors [32–
34], and melanoma [35–37] (Table 1). However, it has also
become evident that there could be differences between
tumor types, with some tumors containing a high propor-
tion of tumorigenic cells and little CSC-dependent hierar-
chy [38–41]. A variety of approaches are employed for the
isolation of CSCs, often based on markers characteristic of
normal stem cells, e.g., CD44, CD133, CD15, or ABC
transporter proteins. Some of these markers may not be
fully specific for CSCs, but in all cases, they provide a
means to substantially enrich CSCs, which are more
tumorigenic than the rest of the tumor cells. Importantly,
CSC markers and signature genes have proven to be
predictive of cancer progression and clinical outcome [21,
30, 37, 42–47]. Furthermore, more aggressive or refractory
cancers contain more CSCs [37, 48], underscoring the
clinical importance of this tumor cell population.
Properties and regulation of CSCs
The realization that CSCs represent a critical population in
many tumors has prompted efforts to understand their
properties in order to therapeutically target them more
efficiently. CSCs share a number of features with normal
stem cells. First of all, both CSCs and nonmalignant stem
cells are defined by their ability to self-renew and
differentiate. In vitro, CSCs are maintained in media
supplemented with growth factors, but without serum
[49]. Culturing under such conditions can also be used as
a means of enriching the CSC population [15, 49, 50]. In
such cultures, the self-renewal capacity of CSCs is typically

5. measured by their ability to form three-dimensional spheres
[51], similarly to normal neural or mammary stem cells [52,
53]. We have recently shown that the gene expression
signature of CSCs reveals substantial overlaps with genes
that are important for the maintenance of physiological
stem cells [47]. Indeed signaling mechanisms regulating
self-renewal and differentiation appear to be largely shared
between CSCs and stem cells (Fig. 2). The Notch pathway,
which plays an essential role in stem cell maintenance and
differentiation throughout development, is important for
CSC function. Activation of Notch signaling in glioma cells
increases the expression of stem cell markers and enhances
self-renewal [54, 55]. CSCs in medulloblastoma express
high levels of Notch and are sensitive to inhibitors of the
Notch pathway [56]. Similarly, Wnt signaling has a central
function in the control of multi-/pluripotency, proliferation
and differentiation in embryonic and adult stem cells and is
also necessary for CSC self-renewal and tumorigenicity.
The activation of the key Wnt downstream effector β-
catenin (Fig. 2) is enhanced in skin CSCs and, strikingly,
conditional ablation of the β-catenin gene in mouse skin
cancer models led to a loss of CSCs and complete tumor
regression [26]. Another example is the Hedgehog pathway,
Fig. 1 The stochastic and hierarchy (cancer stem cell) models of
tumor development. a According to the stochastic model, tumor
cells
have similar proliferative capacity. Genetic mutation and
selection of
mutant clones (clonal evolution) confer different tumorigenicity
to
tumor cell subpopulations. b In the cancer stem cell model, only
CSC
can self-renew and give rise to tumor progenitor cells, which in
turn

6. give rise to more differentiated tumor cells. The resulting tumor
contains a hierarchy of cells, arising through the differentiation
of
stem-like cells. The CSCs, but not the differentiated tumor
cells, can
reinitiate tumor growth. However, the hierarchy may involve a
certain
degree of plasticity with more differentiated cells converting
into less
differentiated ones (dotted arrows)
J Mol Med
which is among the key regulators of stem cells from
various tissues and also plays a critical role in CSCs.
Inhibition of Hedgehog signaling suppressed CSC prolifer-
ation and self-renewal and increased apoptosis [57].
Importantly, it was reported that glioblastoma cells remain
viable after hedgehog blockade but are no longer capable of
tumor initiation, indicating that the hedgehog pathway is
required for maintaining the CSC population [58].
CSC niches
An additional similarity between CSCs and non-transformed
stem cells is their dependence on particular niches—
specialized microenvironments in which the surrounding
cells, extracellular matrix, and soluble factors are critical for
the maintenance of cell stemness. In situ, CSCs have been
found enriched in perivascular regions [59], and similar
findings have been reported for neural stem cells [60–62].
Recent studies have started to shed light on the signaling
mechanisms regulating CSCs in the perivascular niche.

7. Medulloblastoma CSCs localized in the vicinity of blood
vessels were resistant to radiation, owing to their ability to
activate the PI3K/Akt pathway and undergo a transient, p53-
dependent cell cycle arrest [63]. Intriguingly, the tumor
vasculature in gliomas expresses high levels of endothelial
nitric oxide synthase (eNOS). NO secreted from endothelial
cells can activate a cGMP/PKG dependent pathway leading
to enhanced Notch activity. This in turn leads to an increase
of the CSC pool and its self-renewal capacity [64].
Table 1 Markers used for the identification and isolation of
cancer stem cells from different cancers
Cancer type CSC markers % of CSC cells in tumor Efficiency of
tumor formationa
(transplanted cells)
Reference
Lung Sca-1+CD45–PECAM–CD34+ 0.008–0.064 ND [11]
CD133+ 0.3–22.0 ND [10]
Colon CD133 1.8–24.5 83% (500) [14, 15]
EpCAMhighCD44+ 0.03–38.0 (mean 5.4) 75% (200) [12]
ALDH1 3.5±1.0 ND (25) [13]
Breast CD44+EpCAM+CD24–Lineage– 0.6–5.0 100% (1,000)
[19]
CD44+CD24-ALDH1+ 0.1–1.2 100% (20) [21]
Thy-1+CD24+CD45- 1.0–4.0 80% (50) [20]

8. Prostate CD44+α2β1
highCD133+ 0.1–0.3 ND [22]
Brain CD133 19–29 100% (100) [33]
CD15 5.6–70.5 100% (1,000) [34]
SP 0.15–1.2 ND [32]
Melanoma ABCB5 1.6–20.4 50% (100,000) [37]
Pancreatic CD44+CD24+EpCAM+ 0.2–0.8 50% (100) [24]
CD133 0.7–3.2 80% (500) [23]
Liver CD90+CD45– 0.7–6.2 50% (5000) [18]
Head and neck CD44 0.1–41.7 50% (5,000) [25]
Skin CD34 13.0–20.0 50% (1,000) [26]
Ovarian CD44+CD117+ 0.1–0.2 100% (100) [29]
CD133+ 0.3–35.0 (median 8.9) 83% (500) [28]
Bladder CD44 3.1–36.3 50% (1,000–3,000) [30]
Mesenchymal SP 0.07–10.5b 55% (100) [31]
Acute myeloid leukemia (AML) CD34++CD38– 0,02–2,00c
100%d (100,000–500,000) [8, 9]
Acute lymphoblastic
leukemia (ALL)
CD34+CD10–/CD34+CD19– 8±4/3±1c 100%d (50,000–200,000)

9. [116]
a The efficiency represents the percentage of immunodeficient
mice that form tumors following injection of the number of
cells indicated in brackets
b Data presented only for a subset of tumors
c Percentage among mononuclear cells
d Percentage of engraftment
EpCAM epithelial cell adhesion molecule (also called
epithelial-specific antigen), Lineage lineage markers: CD2,
CD3, CD10, CD16, CD18,
CD31, CD64, and CD140b, SP side population
J Mol Med
Moreover, genetic ablation of eNOS interfered with Notch
activity and repressed tumor formation [64].
Importantly, the perinecrotic hypoxic micronenviron-
ment was identified as a second niche where CSCs are
concentrated, as we have recently demonstrated [47] again
in analogy to normal stem cells [65]. Low oxygen levels
(hypoxia) drive tumor progression by triggering a set of
adaptive transcriptional responses that regulate tumor
angiogenesis, metabolism, motility, and survival [66].
These cellular responses are primarily controlled by the
transcription factor system of the hypoxia-inducible factors
(HIFs). Interestingly, several reports have identified critical
stem cell regulators such as Oct4 [67], c-myc [68], and
Notch [69] as direct or indirect HIF targets. Importantly,
several studies by us and others have directly demonstrated
that the CSC phenotype is also promoted by hypoxia and

10. that the HIFs provide an intriguing link between the well-
established function of hypoxia in tumor growth and stem
cell biology. Hypoxia enhances the self-renewal of CSCs
[47, 70, 71], while HIF knockdown blocks this effect and
reduces CSC-mediated tumor growth [47, 72–74]. Interest-
ingly, HIF-2α, but not HIF-1α, is highly expressed and
strongly upregulated by hypoxia in glioma CSCs [72],
enhances the expression of a set of CSC marker genes [47],
and promotes tumor growth [70]. Taken together these
findings underscore a critical role for the perivascular and
the hypoxic niche in the maintenance and regulation of
CSCs. The identity of the regulatory cellular components
and molecular signals within these niches is one of the
crucial open questions of CSC research.
Fig. 2 Principal pathways regulating CSCs. The scheme depicts
the
main components and clinically relevant targets involved in
pathways
that have been shown to control CSCs. The Notch pathway
(orange)
is activated by binding of transmembrane ligands such as
Delta/Delta-
like proteins or Jagged to the membrane receptor Notch. This
induces
cleavage of Notch by γ-secretase, releasing the Notch
intracellular
domain (NICD), which translocates to the nucleus and activates
transcription in complex with cofactors of the CSL family.
Different
growth factors (e.g., EGF, FGF, PDGF) can activate receptor
tyrosine
kinases (RTKs, green) that subsequently promote the activity of
phosphatidylinositol-3-kinase (PI3K), Akt and mammalian
target of
rapamycin (mTOR), among others, leading to enhanced protein

11. translation, cell growth, and proliferation. Binding of Hedgehog
(Hh,
cyan) ligands to the receptor Patched (PTCH) relieves an
inhibition of
Smoothened (SMO), triggering a cascade that leads to the
transloca-
tion of glioma-associated oncogene homologue (Gli) into the
nucleus
and transcription of target genes. The Wnt pathway (yellow) is
initiated by binding of Wnt ligands to a complex of the Frizzled
(Fz)
and lipoprotein receptor-related protein (LRP) receptors. This
sets off
a series of signaling steps leading to stabilization of β-catenin,
which
can proceed to activate gene expression together with the
TCF/LEF
transcription factors. Endogenous Wnt inhibitors like Dickkopf
proteins (DKK) and secreted Frizzled-related proteins (SFRPs)
regulate the pathway and could be explored as tools for its
pharmacological blockade. Specific factors in the CSC niches
also
play critical roles in regulating CSC self-renewal and
differentiation.
For example, NO produced by endothelial nitric oxide synthase
(eNOS, red) can stimulate the production of cyclic guanosine
monophosphate (cGMP) and activate protein kinase G (PKG),
resulting in enhanced Notch signaling. Low oxygen tension in
the
hypoxic CSC niche suppresses the activity of prolyl hydroxylase
domain-containing proteins (PHDs, blue), leading to
stabilization of
hypoxia-inducible factors (HIFs) and the transcription of HIF
target
genes. Notably, the HIF pathway interacts with other pathways,

12. such
as Notch [69], allowing for an intricate oxygen dependent
crosstalk
between CSC pathways
J Mol Med
CSCs have enhanced chemo-/radioresistance
and metastatic potential
A common property of CSCs with major therapeutic
significance is their enhanced resistance to standard anti-
tumor treatments such as chemo- and radiotherapy [23,
63, 75–79]. Irradiation or chemotherapy in vivo enriches
the fraction of cells expressing CSC markers, which also
have an enhanced self-renewal capacity and tumorigenic-
ity compared to the tumor bulk [23, 75, 77]. In addition,
sorted CSC cells from different types of tumors survive
such treatments in culture much better than unsorted or
negative cells [75, 76, 78]. This is due to a combination of
factors, including the high expression of ABC drug
pumps, relative quiescence, resistance to oxidative DNA
damage, and enhanced DNA repair capacity [37, 75]. The
increased resistance of CSCs, combined with the ability of
only a very small number of CSCs to reinitiate tumor
growth [21, 33] is thought to be a major reason for cancer
persistence and relapse after treatment. If CSCs are
required for the initiation and growth of primary tumors,
as posited in the hierarchy model, it is natural to assume
that metastasis formation may also depend on CSCs. This
is an issue of major clinical relevance since metastases are
responsible for 90% of cancer related deaths, and indeed
the involvement of CSCs in tumor dissemination has
received experimental support in recent years. CSCs from

13. mammary carcinoma have a phenotype similar to that of
cells which have undergone epithelial–mesenchymal tran-
sition, a process that plays a key role in tumor cell
invasion and metastasis [80]. In addition, the early
disseminating cells in the bone marrow of breast cancer
patients have a CSC phenotype [81]. In pancreatic cancer
CSCs are found at the invasive tumor front and determine
the metastatic potential of the tumor [23]. Furthermore, a
subpopulation of CD26+ colorectal CSCs possesses
exclusive metastatic ability and is predictive of distant
metastasis in colon cancer patients, in addition to exhibit-
ing enhanced chemoresistance [82]. Taken together, these
findings indicate that CSCs may be at the heart of the two
key problems in tumor treatment—resistance to therapy
and metastasis.
Consequences for cancer research and development
of anticancer drug design platforms
The CSC paradigm has brought a fundamental shift in
our understanding of cancer biology. It prompts a re-
assessment of cancer as not simply a proliferative but
also a differentiation disorder, in which tumor cells with
aberrant stem cell characteristics that differentiate into
abnormal progeny play a central role. From a technical
point of view, the discovery of CSCs has stimulated the
development of better defined and more physiologically
relevant experimental models. Our knowledge about
CSCs questions the utility of long established cancer
cell lines and culture systems, as well as standard
subcutaneous tumor transplantations, as reliable tools
for the study of tumor behavior. Established tumor cell
lines have provided an easily tractable system to
characterize many basic properties of CSCs, in particular
when cultured under stem cell conditions [18, 47, 83,

14. 84]. However, primary, low passage tumor cells isolated
from tumor biopsies, continuously grown in non-
differentiating conditions are both closer to the original
tumors and maintain a subpopulation with characteristic
CSC properties [49, 50, 85]. In addition, orthotopic, rather
than subcutaneous tumor transplantation is the method of
choice for assaying the defining property of CSCs, tumor
propagation [19, 33].
The CSC model has a major impact on the focus of
antitumor therapies, since it highlights the importance of
eliminating the small but crucial and resilient subpopulation
of CSCs (Fig. 3). In general, traditional strategies for drug
design, which focus on the tumor bulk, have not proven to
be suitable for the discovery of agents that eliminate CSCs.
As discussed above, a hallmark of CSCs is the elevated
resistance to classical chemo- and radiotherapy. Novel bulk
tumor-targeted agents such as imatinib are also often
ineffective against the CSC pool [86–88]. Conventional
approaches for anticancer drug discovery target prolifer-
ation, rather than self-renewal or differentiation. Standard
end points include overall survival and proliferation in
two-dimensional cell culture, as well as tumor size,
metastasis and survival in vivo, predominantly in
subcutaneous xenograft models. These parameters mostly
reflect the behavior of the tumor bulk and are poor
indicators of the drug’s efficacy against the CSC
component. Therefore, to improve the clinical relevance
of traditional drug screening analyses, it is important to
complement them with an evaluation of treatment impact
on CSCs. The influence of different agents on CSCs can
be experimentally assessed using a number of established
assays, such as analysis of CSC markers, signature
genes, self-renewal assays (e.g. sphere formation [51]),
and tumorigenicity assays in orthotopic xenotransplanta-
tion models. Monitoring of the effect of anti-tumor

15. treatments on the CSC pool should additionally be
included as an important element in preclinical studies
and clinical trials. CSCs can be assessed using biomarkers,
e.g. surface markers (Table 1) or specific genetic signa-
tures [47, 89]. The clinical relevance of CSCs for tumor
growth and therapy responses underlines the necessity to
develop new criteria and platforms for the design and
assessment of CSC-targeted therapies.
J Mol Med
Novel, CSC-based drugs and drug targets
Our growing knowledge of the biology of CSCs and the
application of improved drug discovery approaches, as
discussed above, have provided a number of targets for the
design of CSC depleting agents. In principle, two types of
approaches targeting CSCs can be envisaged: eliminating
the CSCs themselves by either killing or differentiating
them, and disrupting the CSC niches (Fig. 3). One group of
targets derives from pathways that are crucial in regulating
CSC self-renewal mechanisms (see Fig. 2 and Table 2). For
example, inhibition of Hedgehog signaling with cyclop-
amine, an antagonist of the Hedgehog co-receptor SMO,
killed CSCs in chronic myeloid leukemia and impaired the
growth of imatinib resistant mouse and human CML cells
[90]. A similar inhibitory effect of cyclopamine was
observed in glioma, where it synergized with the standard
chemotherapeutic temozolomide to suppress tumor growth
[57]. Likewise, blockade of Notch signaling by inhibitors of
γ-secretase, the protease required for Notch cleavage and
activation, depleted CSCs in medulloblastoma and glioma,
blocked tumor engraftment and growth and prolonged
survival [56, 91]. Furthermore, administration of neutraliz-

16. ing antibodies against the Notch ligand DLL4 reduced CSC
frequency and tumor growth [92]. Wnt signaling may
provide a further avenue for targeting CSCs, as it has been
found to be required for CSC self-renewal and tumor
growth in different cancers, including CML and squamous
cell carcinoma [26, 93]. Additional signaling molecules,
enriched or activated in CSCs, whose suppression inhibits
tumor growth and increases survival in mouse models
include the NF-κB pathway [94, 95], IL-6 receptors [96],
IL-3 receptor α (CD123) [97], integrin α6 [98], TGF-β,
and LIF [99, 100]. The PI3K/Akt/PTEN/mTOR signaling
axis (Fig. 2) is deregulated in many tumors, and in some
settings, it can be used to preferentially target CSCs. CSCs
have an increased sensitivity to Akt inhibition [101].
Additonally, inhibition of mTOR by rapamycin in a mouse
model of PTEN-deficient leukemia depleted the CSC
population and blocked the development of the disease
[102]. In many of the above cases, the effect of the inhibitors
may not depend on direct killing of CSCs but rather on
promoting their differentiation into cells that no longer support
tumor growth. For instance, skin tumor regression following
ablation of Wnt signaling is accompanied by extensive
terminal differentiation [26]. Similarly, activation of bone
morphogenetic protein (BMP)-dependent signaling by
BMP4 induced pronounced differentiation of glioblastoma
cells, depleting the CSC pool and markedly attenuating
tumorigenicity in a xenograft model [103]. Glioma CSC
differentiation induced by retinoic acid sensitized the CSCs
to therapy, impaired the secretion of angiogenic cytokines,
inhibited motility, and reduced CSC tumorigenicity [104].
The identification of characteristic CSC surface markers
(Table 1) provides further targets for the design of antibody-
based antitumor agents (Table 2) that would either activate
the body’s immune system against CSCs or would be
coupled to cytotoxic agents [105]. In addition to candidate-

17. based approaches for antitumor drug discovery, recent
developments of suitable cell culture systems and in vitro
assays have allowed high-throughput screens that produced
promising leads against CSCs [85, 106].
Fig. 3 Comparison of conven-
tional and CSC-based anticancer
therapies. a Conventional thera-
pies (brown flash) target the
tumor bulk but are inefficient
against CSCs (red), which can
subsequently regrow the tumor. b
CSC-based anticancer therapies
are aimed at eliminating CSCs.
One approach is either direct
killing of CSCs or their differen-
tiation into non-CSCs (orange
flashes) that can be combinatori-
ally targeted by standard treat-
ments. Another strategy entails
the disruption of CSC niches,
such as hypoxic regions or peri-
vascular regions, or niche-derived
signals (blue flashes), which are
required for CSC maintenance.
Both approaches are additionally
combined with conventional anti-
cancer agents (brown flash) to
destroy bulk tumor cells. Blood
vessels are depicted in pink
J Mol Med
CSC niche targeting and combinatorial therapeutic

18. approaches
Since CSCs carry the genetic mutations characteristic of the
tumors they generate [107, 108], they may also exhibit a
similar degree of genetic instability and mutation rate as the
tumor bulk. Thus, therapies that exclusively target CSCs
could run into the same problems that have overshadowed
the design of anticancer drugs for decades: emergence of
drug resistance and selection of increasingly refractory cell
clones. One approach for circumventing these problems is
presented by the dependence of CSCs on specific niches.
Since the niches are primarily generated by non-mutated
cells or cell-independent factors such as hypoxia, they may
represent a more stable target for the elimination of CSCs.
As discussed above, CSCs in solid tumors are enriched
around blood vessels and depend on signals derived from
them. Therefore, antiangiogenic therapies can be seen as a
promising strategy for depleting both the CSCs (by
compromising their niche) and the tumor bulk (by
depriving it from nutrients and oxygen). A number of
antiangiogenic agents are approved or in clinical trials [109]
(Table 2) and new targets are continuously being uncov-
ered. For example, we have recently identified the VEGFR
trafficking machinery as an additional target for inhibition
of angiogenesis in tumors [110]. However, the antiangio-
genic therapies that are currently available bring only
limited benefits [111]. One explanation for this may be
that by shrinking the tumor vasculature and blood supply,
such agents stimulate the expansion of the other type of
niche that has been described for CSCs—the hypoxic one
[47]. This suggests that an additional important approach
for targeting CSCs would involve the suppression of
hypoxia-triggered signaling mechanisms. Substantial efforts
are currently aimed at developing anticancer agents that
target the HIF pathway [112]. Moreover, several known

19. Table 2 A selection of potential cancer stem cell inhibitors in
(pre)clinical studies or approved for clinical use
Target/pathway Agent (Developer) Status Cancer indications
References
Hedgehog
SMO antagonists GDC-0449 (Genentech/Curis) Phase I/II Solid
tumors [117–119]
Cyclopamine Phase I Basal cell carcinoma [120]
Other IPI-926 (Infinity) Phase I Advanced/metastatic solid
tumors [121]
BMS-833923/XL-139
(Bristol-Myers-Squibb/Exelixis)
Phase I Skin, solid tumors [122]
Notch
γ-Secretase inhibitors MK-0752 (Merck) Phase I Breast,
pancreatic, brain cancer [123]
R4733 (Roche) Phase I/II Solid tumors [124]
PF-03084014 (Pfizer) Phase I Advanced cancer, leukemia [125]
DLL inhibitors anti-DLL4 mAbs Preclinical Solid tumors [126,
127]
Wnt

20. Inhibitors TCF/β-catenin inhibitors Preclinical Colon cancer
[128–131]
Antibodies Wnt-2 mAb Preclinical Melanoma, non-small cell
lung cancer [132, 133]
EpCAM
Antibodies Adecatumumab (Micromet) Phase II Metastatic
breast cancer [134]
Edrecolomab Phase II-III Colon cancer [135, 136]
Catumaxomab (Trion/Fresenius) Approved Malignant ascites
[137]
Angiogenesis
VEGF Bevacizumab (Genentech/Roche) Approved colon, non-
small-cell lung cancer, breast cancer [138]
VEGF receptors Sorafenib (Bayer) Approved Renal carcinoma,
hepatocellular carcinoma [139]
Sunitinib (Pfizer) Approved Renal carcinoma, gastrointestinal
stromal tumors [140]
Hypoxia/HIF
PHD activators KRH102053 Preclinical Solid tumors [141]
HIF synthesis/activity PX-478 (Oncothyreon) Phase I Advanced
solid tumors, lymphoma [142]
Topotecan (GlaxoSmithKline) Approved Ovarian, lung, cervical
cancer [143]

21. Digoxin (GlaxoSmithKline) Approved Cardiac conditions [144]
Phase I-II Breast, lung cancer
FK228 (Gloucester) Phase II Solid tumors, lymphoma, [145]
17-AAG Phase I-II Solid tumors, leukemia [146]
J Mol Med
anticancer drugs that target regulators of HIF function have
been shown to suppress HIF levels or activity [112]
(Table 2). In principle, such agents could also play a two-
fold role as both suppressors of bulk tumor growth and as
inhibitors of CSCs, by targeting the hypoxic CSC niche. It
should be noted, however, that most efforts so far have
been focused on the more widely expressed and better
studied member of the family, HIF-1α, whereas recent
studies by us and others indicate that it is rather the second
member of the family, HIF-2α, which plays a key role in
the maintenance of CSCs [47, 72]. Future studies will need
to address the activity of currently available agents
targeting hypoxia signaling on CSCs, and to attempt
designing specific HIF-2α inhibitors that block its function
in CSC maintenance.
In designing future anticancer therapeutic strategies that
include CSC eradication as a key element, it should be kept
in mind that the approaches described above are unlikely to
be successful when applied individually. In many cases, it
may be essential to combine CSC-specific agents with
traditional bulk tumor-targeting treatments (see Fig. 3).
CSC depletion alone will be expected to halt tumor growth

22. but will not necessarily reduce the size of preexisting
tumors, which could impose a substantial clinical burden on
the patient and should also be targeted, using traditional
strategies. Similarly, drugs that cause CSC differentiation
rather than death will require the elimination of the
differentiated tumor cells as an additional step. The efficient
eradication of CSCs themselves may require the combined
ablation of CSCs and their niches. Furthermore, approaches
that center on the CSC niche as a target will need to take
into account the existence of different, even opposing
(perivascular and hypoxic) niches and target them simulta-
neously. Lastly, it has to be kept in mind that CSC targeting
treatments may also deplete normal stem cells due to the
molecular and functional similarities between CSCs and
stem cells. However, recent studies have highlighted crucial
differences between CSCs and stem cells that could be
exploited for CSC specific treatments [72].
Conclusion and perspective
Based on our current knowledge, CSCs are likely to be a
key driving force of most types of cancer. It is, therefore,
justified to include the evaluation of CSC abundance and
persistence as a critical element in future preclinical studies
and clinical trials of antitumor therapies. The CSC markers
that have been identified so far will greatly facilitate such
analyses, but it is important to further improve our
knowledge of additional markers with optimal specificity
and sensitivity. The assessment of CSCs in solid tumors
will be further aided if blood CSC biomarkers for solid
tumors become available, obviating the need for invasive
tumor biopsies. Basic research into the properties of CSCs
needs to further elucidate the mechanisms regulating the
origin and maintenance of CSCs and to delineate differ-
ences between CSCs and normal stem cells that could be

23. exploited for specific CSC targeting with minimal side
effects. The inclusion of CSC analyses in clinical studies of
anticancer treatments will aid our understanding about the
extent to which conclusions obtained in murine or tissue
culture models, e.g. related to the radio- and chemo-
resistance of CSCs, can be transferred to human patients.
The functional assays used to assess CSCs should also be
better standardized for specific tumor types. Additionally,
some general issues in the CSC field remain to be clarified,
related for instance to the origin of CSCs, their proliferative
capacity, their abundance, their precise molecular charac-
terization, or the extent to which differentiation and
dedifferentiation occur within the tumor cell hierarchy
[105, 113–115]. Overall, CSCs are likely to have a more
variable and unstable character than physiological stem
cells, just like tumors are more variable and unstable than
healthy tissues. Furthermore, the differentiation hierarchy
may not necessarily be unidirectional, as suggested by a
recent in vitro study on melanoma showing that cells with
CSC properties could be generated not only by self renewal
but also by dedifferentiation from more differentiated
progeny [79]. This would have direct therapeutic con-
sequences, as CSC-based therapies would have to be
administered chronically rather than as fast acting magic
bullets to eradicate CSCs. It is also clear that the discovery
of efficient drugs against CSCs will require specially
tailored assays and screening platforms that rest upon our
improved understanding of CSC characteristics. The com-
bination of all these approaches has already started to yield
a number of promising anticancer drug candidates. This
new line of research holds great promise for significant
improvements in the treatment of cancer by focusing on a
novel and crucial type of target—the cancer stem cells.
Acknowledgments This work was supported by grants from the
DFG (AC 110/3-1,-2 (SPP1190)), LOEWE (OSF), the Deutsche

24. Krebshilfe (107231), the German Ministry of Education and
Research
(BMBF) within the National Genome Network (NGFNplus) and
Brain
Tumor Network, and VFK Krebsforschung.
Disclosure of potential conflict of interests The authors declare
no
conflict of interests related to this study.
References
1. Dalerba P, Cho RW, Clarke MF (2007) Cancer stem cells:
models and concepts. Annu Rev Med 58:267–284
2. Nowell PC (1976) The clonal evolution of tumor cell popula-
tions. Science 194:23–28
J Mol Med
3. Visvader JE, Lindeman GJ (2008) Cancer stem cells in solid
tumours: accumulating evidence and unresolved questions. Nat
Rev Cancer 8:755–768
4. Chen R, Nishimura MC, Bumbaca SM, Kharbanda S, Forrest
WF, Kasman IM, Greve JM, Soriano RH, Gilmour LL, Rivers
CS et al (2010) A hierarchy of self-renewing tumor-initiating
cell
types in glioblastoma. Cancer Cell 17:362–375
5. Bruce WR, Van Der Gaag H (1963) A quantitative assay for
the
number of murine lymphoma cells capable of proliferation in
vivo. Nature 199:79–80

25. 6. Hamburger AW, Salmon SE (1977) Primary bioassay of
human
tumor stem cells. Science 197:461–463
7. Wicha MS, Liu S, Dontu G (2006) Cancer stem cells: an old
idea–a paradigm shift. Cancer Res 66:1883–1890, discussion
1895–1886
8. Bonnet D, Dick JE (1997) Human acute myeloid leukemia is
organized as a hierarchy that originates from a primitive
hematopoietic cell. Nat Med 3:730–737
9. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T,
Caceres-
Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE (1994)
A cell initiating human acute myeloid leukaemia after
transplan-
tation into SCID mice. Nature 367:645–648
10. Eramo A, Lotti F, Sette G, Pilozzi E, Biffoni M, Di Virgilio
A,
Conticello C, Ruco L, Peschle C, De Maria R (2008)
Identification and expansion of the tumorigenic lung cancer
stem cell population. Cell Death Differ 15:504–514
11. Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I,
Vogel S, Crowley D, Bronson RT, Jacks T (2005) Identification
of bronchioalveolar stem cells in normal lung and lung cancer.
Cell 121:823–835
12. Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW,
Hoey T,
Gurney A, Huang EH, Simeone DM et al (2007) Phenotypic
characterization of human colorectal cancer stem cells. Proc
Natl

26. Acad Sci USA 104:10158–10163
13. Huang EH, Hynes MJ, Zhang T, Ginestier C, Dontu G,
Appelman H, Fields JZ, Wicha MS, Boman BM (2009)
Aldehyde dehydrogenase 1 is a marker for normal and
malignant human colonic stem cells (SC) and tracks SC
overpopulation during colon tumorigenesis. Cancer Res
69:3382–3389
14. O’Brien CA, Pollett A, Gallinger S, Dick JE (2007) A
human
colon cancer cell capable of initiating tumour growth in
immunodeficient mice. Nature 445:106–110
15. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro
M,
Peschle C, De Maria R (2007) Identification and expansion of
human colon-cancer-initiating cells. Nature 445:111–115
16. Takaishi S, Okumura T, Tu S, Wang SS, Shibata W,
Vigneshwaran
R, Gordon SA, Shimada Y, Wang TC (2009) Identification of
gastric cancer stem cells using the cell surface marker CD44.
Stem
Cells 27:1006–1020
17. Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, Zheng BJ,
Guan XY (2007) Identification and characterization of
tumorigenic liver cancer stem/progenitor cells. Gastroenterol-
ogy 132:2542–2556
18. Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P, Chu
PW,
Lam CT, Poon RT, Fan ST (2008) Significance of CD90+
cancer
stem cells in human liver cancer. Cancer Cell 13:153–166

27. 19. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ,
Clarke MF (2003) Prospective identification of tumorigenic
breast cancer cells. Proc Natl Acad Sci USA 100:3983–3988
20. Cho RW, Wang X, Diehn M, Shedden K, Chen GY, Sherlock
G,
Gurney A, Lewicki J, Clarke MF (2008) Isolation and molecular
characterization of cancer stem cells in MMTV-Wnt-1 murine
breast tumors. Stem Cells 26:364–371
21. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F,
Dutcher J,
Brown M, Jacquemier J, Viens P, Kleer CG, Liu S et al (2007)
ALDH1 is a marker of normal and malignant human mammary
stem cells and a predictor of poor clinical outcome. Cell Stem
Cell 1:555–567
22. Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ
(2005)
Prospective identification of tumorigenic prostate cancer stem
cells. Cancer Res 65:10946–10951
23. Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW,
Guba
M, Bruns CJ, Heeschen C (2007) Distinct populations of cancer
stem cells determine tumor growth and metastatic activity in
human pancreatic cancer. Cell Stem Cell 1:313–323
24. Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V,
Wicha
M, Clarke MF, Simeone DM (2007) Identification of pancreatic
cancer stem cells. Cancer Res 67:1030–1037
25. Prince ME, Sivanandan R, Kaczorowski A, Wolf GT,

28. Kaplan
MJ, Dalerba P, Weissman IL, Clarke MF, Ailles LE (2007)
Identification of a subpopulation of cells with cancer stem cell
properties in head and neck squamous cell carcinoma. Proc Natl
Acad Sci USA 104:973–978
26. Malanchi I, Peinado H, Kassen D, Hussenet T, Metzger D,
Chambon P, Huber M, Hohl D, Cano A, Birchmeier W et al
(2008) Cutaneous cancer stem cell maintenance is dependent on
beta-catenin signalling. Nature 452:650–653
27. Bapat SA, Mali AM, Koppikar CB, Kurrey NK (2005) Stem
and
progenitor-like cells contribute to the aggressive behavior of
human epithelial ovarian cancer. Cancer Res 65:3025–3029
28. Curley MD, Therrien VA, Cummings CL, Sergent PA,
Koulouris
CR, Friel AM, Roberts DJ, Seiden MV, Scadden DT, Rueda BR
et al (2009) CD133 expression defines a tumor initiating cell
population in primary human ovarian cancer. Stem Cells
27:2875–2883
29. Zhang S, Balch C, Chan MW, Lai HC, Matei D, Schilder
JM,
Yan PS, Huang TH, Nephew KP (2008) Identification and
characterization of ovarian cancer-initiating cells from primary
human tumors. Cancer Res 68:4311–4320
30. Chan KS, Espinosa I, Chao M, Wong D, Ailles L, Diehn M,
Gill
H, Presti J Jr, Chang HY, van de Rijn M et al (2009)
Identification, molecular characterization, clinical prognosis,
and therapeutic targeting of human bladder tumor-initiating
cells. Proc Natl Acad Sci USA 106:14016–14021

29. 31. Wu C, Wei Q, Utomo V, Nadesan P, Whetstone H, Kandel
R,
Wunder JS, Alman BA (2007) Side population cells isolated
from mesenchymal neoplasms have tumor initiating potential.
Cancer Res 67:8216–8222
32. Harris MA, Yang H, Low BE, Mukherje J, Guha A, Bronson
RT,
Shultz LD, Israel MA, Yun K (2008) Cancer stem cells are
enriched in the side population cells in a mouse model of
glioma.
Cancer Res 68:10051–10059
33. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide
T,
Henkelman RM, Cusimano MD, Dirks PB (2004) Identification
of human brain tumour initiating cells. Nature 432:396–401
34. Son MJ, Woolard K, Nam DH, Lee J, Fine HA (2009)
SSEA-1 is
an enrichment marker for tumor-initiating cells in human
glioblastoma. Cell Stem Cell 4:440–452
35. Fang D, Nguyen TK, Leishear K, Finko R, Kulp AN, Hotz S,
Van Belle PA, Xu X, Elder DE, Herlyn M (2005) A tumorigenic
subpopulation with stem cell properties in melanomas. Cancer
Res 65:9328–9337
36. Monzani E, Facchetti F, Galmozzi E, Corsini E, Benetti A,
Cavazzin C, Gritti A, Piccinini A, Porro D, Santinami M et al
(2007) Melanoma contains CD133 and ABCG2 positive cells
with enhanced tumourigenic potential. Eur J Cancer 43:935–
946
37. Schatton T, Murphy GF, Frank NY, Yamaura K, Waaga-
Gasser

30. AM, Gasser M, Zhan Q, Jordan S, Duncan LM, Weishaupt C et
al (2008) Identification of cells initiating human melanomas.
Nature 451:345–349
J Mol Med
38. Kelly PN, Dakic A, Adams JM, Nutt SL, Strasser A (2007)
Tumor growth need not be driven by rare cancer stem cells.
Science 317:337
39. Kennedy JA, Barabe F, Poeppl AG, Wang JC, Dick JE
(2007)
Comment on “Tumor growth need not be driven by rare cancer
stem cells”. Science 318:1722, author reply 1722
40. le Viseur C, Hotfilder M, Bomken S, Wilson K, Rottgers S,
Schrauder A, Rosemann A, Irving J, Stam RW, Shultz LD et al
(2008) In childhood acute lymphoblastic leukemia, blasts at
different stages of immunophenotypic maturation have stem cell
properties. Cancer Cell 14:47–58
41. Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson
TM,
Morrison SJ (2008) Efficient tumour formation by single human
melanoma cells. Nature 456:593–598
42. Liu R, Wang X, Chen GY, Dalerba P, Gurney A, Hoey T,
Sherlock G, Lewicki J, Shedden K, Clarke MF (2007) The
prognostic role of a gene signature from tumorigenic breast-
cancer cells. N Engl J Med 356:217–226
43. Zeppernick F, Ahmadi R, Campos B, Dictus C, Helmke BM,
Becker N, Lichter P, Unterberg A, Radlwimmer B, Herold-
Mende CC (2008) Stem cell marker CD133 affects clinical

31. outcome in glioma patients. Clin Cancer Res 14:123–129
44. Jiang F, Qiu Q, Khanna A, Todd NW, Deepak J, Xing L,
Wang
H, Liu Z, Su Y, Stass SA et al (2009) Aldehyde dehydrogenase
1
is a tumor stem cell-associated marker in lung cancer. Mol
Cancer Res 7:330–338
45. Artells R, Moreno I, Diaz T, Martinez F, Gel B, Navarro A,
Ibeas
R, Moreno J, Monzo M (2010) Tumour CD133 mRNA
expression and clinical outcome in surgically resected
colorectal
cancer patients. Eur J Cancer 46:642–649
46. Beier D, Wischhusen J, Dietmaier W, Hau P, Proescholdt M,
Brawanski A, Bogdahn U, Beier CP (2008) CD133 expression
and cancer stem cells predict prognosis in high-grade oligoden-
droglial tumors. Brain Pathol 18:370–377
47. Seidel S, Garvalov BK, Wirta V, von Stechow L, Schänzer
A,
Meletis K, Wolter M, Sommerlad D, Henze AT, Nister M et al
(2010) A hypoxic niche regulates glioblastoma stem cells
through hypoxia inducible factor 2α. Brain 133:983–995
48. van Rhenen A, Feller N, Kelder A, Westra AH, Rombouts E,
Zweegman S, van der Pol MA, Waisfisz Q, Ossenkoppele GJ,
Schuurhuis GJ (2005) High stem cell frequency in acute
myeloid
leukemia at diagnosis predicts high minimal residual disease
and
poor survival. Clin Cancer Res 11:6520–6527
49. Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM,

32. Pastorino S, Purow BW, Christopher N, Zhang W et al (2006)
Tumor stem cells derived from glioblastomas cultured in bFGF
and EGF more closely mirror the phenotype and genotype of
primary tumors than do serum-cultured cell lines. Cancer Cell
9:391–403
50. Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G,
Coradini D, Pilotti S, Pierotti MA, Daidone MG (2005)
Isolation
and in vitro propagation of tumorigenic breast cancer cells with
stem/progenitor cell properties. Cancer Res 65:5506–5511
51. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C,
Squire
J, Dirks PB (2003) Identification of a cancer stem cell in human
brain tumors. Cancer Res 63:5821–5828
52. Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF,
Kawamura MJ, Wicha MS (2003) In vitro propagation and
transcriptional profiling of human mammary stem/progenitor
cells. Genes Dev 17:1253–1270
53. Vescovi AL, Parati EA, Gritti A, Poulin P, Ferrario M,
Wanke E,
Frolichsthal-Schoeller P, Cova L, Arcellana-Panlilio M,
Colombo A et al (1999) Isolation and cloning of multipotential
stem cells from the embryonic human CNS and establishment of
transplantable human neural stem cell lines by epigenetic
stimulation. Exp Neurol 156:71–83
54. Shih AH, Holland EC (2006) Notch signaling enhances
nestin
expression in gliomas. Neoplasia 8:1072–1082
55. Zhang XP, Zheng G, Zou L, Liu HL, Hou LH, Zhou P, Yin
DD,

33. Zheng QJ, Liang L, Zhang SZ et al (2008) Notch activation
promotes cell proliferation and the formation of neural stem
cell-
like colonies in human glioma cells. Mol Cell Biochem
307:101–108
56. Fan X, Matsui W, Khaki L, Stearns D, Chun J, Li YM,
Eberhart
CG (2006) Notch pathway inhibition depletes stem-like cells
and
blocks engraftment in embryonal brain tumors. Cancer Res
66:7445–7452
57. Clement V, Sanchez P, de Tribolet N, Radovanovic I, Ruiz i
Altaba A (2007) HEDGEHOG-GLI1 signaling regulates human
glioma growth, cancer stem cell self-renewal, and
tumorigenicity.
Curr Biol 17:165–172
58. Bar EE, Chaudhry A, Lin A, Fan X, Schreck K, Matsui W,
Piccirillo S, Vescovi AL, DiMeco F, Olivi A et al (2007)
Cyclopamine-mediated hedgehog pathway inhibition depletes
stem-like cancer cells in glioblastoma. Stem Cells 25:2524–
2533
59. Calabrese C, Poppleton H, Kocak M, Hogg TL, Fuller C,
Hamner B, Oh EY, Gaber MW, Finklestein D, Allen M et al
(2007) A perivascular niche for brain tumor stem cells. Cancer
Cell 11:69–82
60. Mirzadeh Z, Merkle FT, Soriano-Navarro M, Garcia-
Verdugo
JM, Alvarez-Buylla A (2008) Neural stem cells confer unique
pinwheel architecture to the ventricular surface in neurogenic
regions of the adult brain. Cell Stem Cell 3:265–278

34. 61. Shen Q, Wang Y, Kokovay E, Lin G, Chuang SM, Goderie
SK,
Roysam B, Temple S (2008) Adult SVZ stem cells lie in a
vascular niche: a quantitative analysis of niche cell-cell inter-
actions. Cell Stem Cell 3:289–300
62. Tavazoie M, Van der Veken L, Silva-Vargas V, Louissaint
M,
Colonna L, Zaidi B, Garcia-Verdugo JM, Doetsch F (2008) A
specialized vascular niche for adult neural stem cells. Cell Stem
Cell 3:279–288
63. Hambardzumyan D, Becher OJ, Rosenblum MK, Pandolfi
PP,
Manova-Todorova K, Holland EC (2008) PI3K pathway regu-
lates survival of cancer stem cells residing in the perivascular
niche following radiation in medulloblastoma in vivo. Genes
Dev 22:436–448
64. Charles N, Ozawa T, Squatrito M, Bleau AM, Brennan CW,
Hambardzumyan D, Holland EC (2010) Perivascular nitric oxide
activates notch signaling and promotes stem-like character in
PDGF-induced glioma cells. Cell Stem Cell 6:141–152
65. Parmar K, Mauch P, Vergilio JA, Sackstein R, Down JD
(2007)
Distribution of hematopoietic stem cells in the bone marrow
according to regional hypoxia. Proc Natl Acad Sci USA
104:5431–5436
66. Bertout JA, Patel SA, Simon MC (2008) The impact of O2
availability on human cancer. Nat Rev Cancer 8:967–975
67. Covello KL, Kehler J, Yu H, Gordan JD, Arsham AM, Hu
CJ,
Labosky PA, Simon MC, Keith B (2006) HIF-2alpha regulates

35. Oct-4: effects of hypoxia on stem cell function, embryonic
development, and tumor growth. Genes Dev 20:557–570
68. Gordan JD, Bertout JA, Hu CJ, Diehl JA, Simon MC (2007)
HIF-2alpha promotes hypoxic cell proliferation by enhancing
c-myc transcriptional activity. Cancer Cell 11:335–347
69. Gustafsson MV, Zheng X, Pereira T, Gradin K, Jin S,
Lundkvist
J, Ruas JL, Poellinger L, Lendahl U, Bondesson M (2005)
Hypoxia requires notch signaling to maintain the
undifferentiated
cell state. Dev Cell 9:617–628
70. Heddleston JM, Li Z, McLendon RE, Hjelmeland AB, Rich
JN
(2009) The hypoxic microenvironment maintains glioblastoma
stem cells and promotes reprogramming towards a cancer stem
cell phenotype. Cell Cycle 8:3274–3284
J Mol Med
71. McCord AM, Jamal M, Shankavaram UT, Lang FF,
Camphausen
K, Tofilon PJ (2009) Physiologic oxygen concentration
enhances
the stem-like properties of CD133+ human glioblastoma cells in
vitro. Mol Cancer Res 7:489–497
72. Li Z, Bao S, Wu Q, Wang H, Eyler C, Sathornsumetee S,
Shi Q,
Cao Y, Lathia J, McLendon RE et al (2009) Hypoxia-inducible
factors regulate tumorigenic capacity of glioma stem cells.
Cancer Cell 15:501–513

36. 73. Mendez O, Zavadil J, Esencay M, Lukyanov Y, Santovasi D,
Wang SC, Newcomb EW, Zagzag D (2010) Knock down of HIF-
1alpha in glioma cells reduces migration in vitro and invasion
in
vivo and impairs their ability to form tumor spheres. Mol
Cancer
9:133
74. Soeda A, Park M, Lee D, Mintz A, Androutsellis-Theotokis
A,
McKay RD, Engh J, Iwama T, Kunisada T, Kassam AB et al
(2009) Hypoxia promotes expansion of the CD133-positive
glioma stem cells through activation of HIF-1alpha. Oncogene
28:3949–3959
75. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland
AB,
Dewhirst MW, Bigner DD, Rich JN (2006) Glioma stem cells
promote radioresistance by preferential activation of the DNA
damage response. Nature 444:756–760
76. Eramo A, Ricci-Vitiani L, Zeuner A, Pallini R, Lotti F,
Sette G,
Pilozzi E, Larocca LM, Peschle C, De Maria R (2006)
Chemotherapy resistance of glioblastoma stem cells. Cell Death
Differ 13:1238–1241
77. Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu
MF,
Hilsenbeck SG, Pavlick A, Zhang X, Chamness GC et al (2008)
Intrinsic resistance of tumorigenic breast cancer cells to
chemotherapy. J Natl Cancer Inst 100:672–679
78. Ma S, Lee TK, Zheng BJ, Chan KW, Guan XY (2008)
CD133+

37. HCC cancer stem cells confer chemoresistance by preferential
expression of the Akt/PKB survival pathway. Oncogene
27:1749–1758
79. Roesch A, Fukunaga-Kalabis M, Schmidt EC, Zabierowski
SE,
Brafford PA, Vultur A, Basu D, Gimotty P, Vogt T, Herlyn M
(2010) A temporarily distinct subpopulation of slow-cycling
melanoma cells is required for continuous tumor growth. Cell
141:583–594
80. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou
AY,
Brooks M, Reinhard F, Zhang CC, Shipitsin M et al (2008) The
epithelial-mesenchymal transition generates cells with
properties
of stem cells. Cell 133:704–715
81. Balic M, Lin H, Young L, Hawes D, Giuliano A, McNamara
G,
Datar RH, Cote RJ (2006) Most early disseminated cancer cells
detected in bone marrow of breast cancer patients have a
putative
breast cancer stem cell phenotype. Clin Cancer Res 12:5615–
5621
82. Pang R, Law WL, Chu ACY, Poon JT, Lam CSC, Chow
AKM, Ng L, Cheung LWH, Lan XR, Lan HY et al (2010) A
subpopulation of CD26+ cancer stem cells with metastatic
capacity in human colorectal cancer. Cell Stem Cell 6:603–
615
83. Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J,
Cervera N,
Finetti P, Hur MH, Diebel ME, Monville F, Dutcher J et al
(2009) Breast cancer cell lines contain functional cancer stem

38. cells with metastatic capacity and a distinct molecular
signature.
Cancer Res 69:1302–1313
84. Kondo T, Setoguchi T, Taga T (2004) Persistence of a small
subpopulation of cancer stem-like cells in the C6 glioma cell
line. Proc Natl Acad Sci USA 101:781–786
85. Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S,
Russell R, Bayani J, Head R, Lee M, Bernstein M et al (2009)
Glioma stem cell lines expanded in adherent culture have
tumor-
specific phenotypes and are suitable for chemical and genetic
screens. Cell Stem Cell 4:568–580
86. Bhatia R, Holtz M, Niu N, Gray R, Snyder DS, Sawyers CL,
Arber DA, Slovak ML, Forman SJ (2003) Persistence of
malignant hematopoietic progenitors in chronic myelogenous
leukemia patients in complete cytogenetic remission following
imatinib mesylate treatment. Blood 101:4701–4707
87. Copland M, Hamilton A, Elrick LJ, Baird JW, Allan EK,
Jordanides N, Barow M, Mountford JC, Holyoake TL (2006)
Dasatinib (BMS-354825) targets an earlier progenitor
population
than imatinib in primary CML but does not eliminate the
quiescent fraction. Blood 107:4532–4539
88. Graham SM, Jorgensen HG, Allan E, Pearson C, Alcorn MJ,
Richmond L, Holyoake TL (2002) Primitive, quiescent,
Philadelphia-positive stem cells from patients with chronic
myeloid leukemia are insensitive to STI571 in vitro. Blood
99:319–325
89. Günther HS, Schmidt NO, Phillips HS, Kemming D,
Kharbanda

39. S, Soriano R, Modrusan Z, Meissner H, Westphal M, Lamszus K
(2008) Glioblastoma-derived stem cell-enriched cultures form
distinct subgroups according to molecular and phenotypic
criteria. Oncogene 27:2897–2909
90. Zhao C, Chen A, Jamieson CH, Fereshteh M, Abrahamsson
A,
Blum J, Kwon HY, Kim J, Chute JP, Rizzieri D et al (2009)
Hedgehog signalling is essential for maintenance of cancer stem
cells in myeloid leukaemia. Nature 458:776–779
91. Fan X, Khaki L, Zhu TS, Soules ME, Talsma CE, Gul N,
Koh C,
Zhang J, Li YM, Maciaczyk J et al (2010) NOTCH pathway
blockade depletes CD133-positive glioblastoma cells and inhib-
its growth of tumor neurospheres and xenografts. Stem Cells
28:5–16
92. Hoey T, Yen WC, Axelrod F, Basi J, Donigian L, Dylla S,
Fitch-
Bruhns M, Lazetic S, Park IK, Sato A et al (2009) DLL4
blockade inhibits tumor growth and reduces tumor-initiating
cell
frequency. Cell Stem Cell 5:168–177
93. Zhao C, Blum J, Chen A, Kwon HY, Jung SH, Cook JM,
Lagoo
A, Reya T (2007) Loss of beta-catenin impairs the renewal of
normal and CML stem cells in vivo. Cancer Cell 12:528–541
94. Guzman ML, Swiderski CF, Howard DS, Grimes BA, Rossi
RM, Szilvassy SJ, Jordan CT (2002) Preferential induction of
apoptosis for primary human leukemic stem cells. Proc Natl
Acad Sci USA 99:16220–16225
95. Hjelmeland AB, Wu Q, Wickman S, Eyler C, Heddleston J,

40. Shi
Q, Lathia JD, Macswords J, Lee J, McLendon RE et al (2010)
Targeting A20 decreases glioma stem cell survival and tumor
growth. PLoS Biol 8:e1000319
96. Wang H, Lathia JD, Wu Q, Wang J, Li Z, Heddleston JM,
Eyler
CE, Elderbroom J, Gallagher J, Schuschu J et al (2009)
Targeting
interleukin 6 signaling suppresses glioma stem cell survival and
tumor growth. Stem Cells 27:2393–2404
97. Jin L, Lee EM, Ramshaw HS, Busfield SJ, Peoppl AG,
Wilkinson L, Guthridge MA, Thomas D, Barry EF, Boyd A et
al (2009) Monoclonal antibody-mediated targeting of CD123,
IL-3 receptor alpha chain, eliminates human acute myeloid
leukemic stem cells. Cell Stem Cell 5:31–42
98. Lathia JD, Gallagher J, Heddleston JM, Wang J, Eyler CE,
Macswords J, Wu Q, Vasanji A, McLendon RE, Hjelmeland AB
et al (2010) Integrin alpha 6 regulates glioblastoma stem cells.
Cell Stem Cell 6:421–432
99. Ikushima H, Todo T, Ino Y, Takahashi M, Miyazawa K,
Miyazono K (2009) Autocrine TGF-beta signaling maintains
tumorigenicity of glioma-initiating cells through Sry-related
HMG-box factors. Cell Stem Cell 5:504–514
100. Peñuelas S, Anido J, Prieto-Sanchez RM, Folch G, Barba I,
Cuartas I, Garcia-Dorado D, Poca MA, Sahuquillo J, Baselga J
et
al (2009) TGF-beta increases glioma-initiating cell self-renewal
through the induction of LIF in human glioblastoma. Cancer
Cell
15:315–327

41. J Mol Med
101. Eyler CE, Foo WC, LaFiura KM, McLendon RE,
Hjelmeland
AB, Rich JN (2008) Brain cancer stem cells display preferential
sensitivity to Akt inhibition. Stem Cells 26:3027–3036
102. Yilmaz OH, Valdez R, Theisen BK, Guo W, Ferguson DO,
Wu
H, Morrison SJ (2006) Pten dependence distinguishes haemato-
poietic stem cells from leukaemia-initiating cells. Nature
441:475–482
103. Piccirillo SG, Reynolds BA, Zanetti N, Lamorte G, Binda
E,
Broggi G, Brem H, Olivi A, Dimeco F, Vescovi AL (2006) Bone
morphogenetic proteins inhibit the tumorigenic potential of
human brain tumour-initiating cells. Nature 444:761–765
104. Campos B, Wan F, Farhadi M, Ernst A, Zeppernick F,
Tagscherer KE, Ahmadi R, Lohr J, Dictus C, Gdynia G et al
(2010) Differentiation therapy exerts antitumor effects on stem-
like glioma cells. Clin Cancer Res 16:2715–2728
105. Zhou BB, Zhang H, Damelin M, Geles KG, Grindley JC,
Dirks PB (2009) Tumour-initiating cells: challenges and
opportunities for anticancer drug discovery. Nat Rev Drug
Discov 8:806–823
106. Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C,
Weinberg
RA, Lander ES (2009) Identification of selective inhibitors of
cancer stem cells by high-throughput screening. Cell 138:645–
659

42. 107. Eisterer W, Jiang X, Christ O, Glimm H, Lee KH, Pang E,
Lambie K, Shaw G, Holyoake TL, Petzer AL et al (2005)
Different subsets of primary chronic myeloid leukemia stem
cells
engraft immunodeficient mice and produce a model of the
human disease. Leukemia 19:435–441
108. Magnifico A, Albano L, Campaner S, Delia D, Castiglioni
F,
Gasparini P, Sozzi G, Fontanella E, Menard S, Tagliabue E
(2009) Tumor-initiating cells of HER2-positive carcinoma cell
lines express the highest oncoprotein levels and are sensitive to
trastuzumab. Clin Cancer Res 15:2010–2021
109. Cao Y (2009) Tumor angiogenesis and molecular targets
for
therapy. Front Biosci 14:3962–3973
110. Sawamiphak S, Seidel S, Essmann CL, Wilkinson GA,
Pitulescu
ME, Acker T, Acker-Palmer A (2010) Ephrin-B2 regulates
VEGFR2 function in developmental and tumour angiogenesis.
Nature 465:487–491
111. Bergers G, Hanahan D (2008) Modes of resistance to anti-
angiogenic therapy. Nat Rev Cancer 8:592–603
112. Poon E, Harris AL, Ashcroft M (2009) Targeting the
hypoxia-
inducible factor (HIF) pathway in cancer. Expert Rev Mol Med
11:e26
113. Gupta PB, Chaffer CL, Weinberg RA (2009) Cancer stem
cells:
mirage or reality? Nat Med 15:1010–1012

43. 114. Jordan CT (2009) Cancer stem cells: controversial or just
misunderstood? Cell Stem Cell 4:203–205
115. Shackleton M, Quintana E, Fearon ER, Morrison SJ (2009)
Heterogeneity in cancer: cancer stem cells versus clonal
evolution. Cell 138:822–829
116. Cox CV, Evely RS, Oakhill A, Pamphilon DH, Goulden
NJ,
Blair A (2004) Characterization of acute lymphoblastic
leukemia
progenitor cells. Blood 104:2919–2925
117. Robarge KD, Brunton SA, Castanedo GM, Cui Y, Dina
MS,
Goldsmith R, Gould SE, Guichert O, Gunzner JL, Halladay J et
al (2009) GDC-0449-a potent inhibitor of the hedgehog
pathway.
Bioorg Med Chem Lett 19:5576–5581
118. Rudin CM, Hann CL, Laterra J, Yauch RL, Callahan CA,
Fu L,
Holcomb T, Stinson J, Gould SE, Coleman B et al (2009)
Treatment of medulloblastoma with hedgehog pathway inhibitor
GDC-0449. N Engl J Med 361:1173–1178
119. Von Hoff DD, LoRusso PM, Rudin CM, Reddy JC, Yauch
RL,
Tibes R, Weiss GJ, Borad MJ, Hann CL, Brahmer JR et al
(2009)
Inhibition of the hedgehog pathway in advanced basal-cell
carcinoma. N Engl J Med 361:1164–1172
120. Tabs S, Avci O (2004) Induction of the differentiation and
apoptosis of tumor cells in vivo with efficiency and selectivity.

44. Eur J Dermatol 14:96–102
121. Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A,
McIntyre
D, Honess D, Madhu B, Goldgraben MA, Caldwell ME, Allard
D et al (2009) Inhibition of hedgehog signaling enhances
delivery of chemotherapy in a mouse model of pancreatic
cancer.
Science 324:1457–1461
122. Tremblay MR, Nesler M, Weatherhead R, Castro AC
(2009)
Recent patents for hedgehog pathway inhibitors for the
treatment
of malignancy. Expert Opin Ther Pat 19:1039–1056
123. Olson RE, Albright CF (2008) Recent progress in the
medicinal
chemistry of gamma-secretase inhibitors. Curr Top Med Chem
8:17–33
124. Winquist RJ, Boucher DM, Wood M, Furey BF (2009)
Targeting
cancer stem cells for more effective therapies: taking out
cancer’s
locomotive engine. Biochem Pharmacol 78:326–334
125. Wei P, Walls M, Qiu M, Ding R, Denlinger RH, Wong A,
Tsaparikos K, Jani JP, Hosea N, Sands M et al (2010)
Evaluation
of selective gamma-secretase inhibitor PF-03084014 for its
antitumor efficacy and gastrointestinal safety to guide optimal
clinical trial design. Mol Cancer Ther 9:1618–1628
126. Noguera-Troise I, Daly C, Papadopoulos NJ, Coetzee S,
Boland

45. P, Gale NW, Lin HC, Yancopoulos GD, Thurston G (2006)
Blockade of Dll4 inhibits tumour growth by promoting non-
productive angiogenesis. Nature 444:1032–1037
127. Ridgway J, Zhang G, Wu Y, Stawicki S, Liang WC,
Chanthery
Y, Kowalski J, Watts RJ, Callahan C, Kasman I et al (2006)
Inhibition of Dll4 signalling inhibits tumour growth by dereg-
ulating angiogenesis. Nature 444:1083–1087
128. Chen Z, Venkatesan AM, Dehnhardt CM, Dos Santos O,
Delos
Santos E, Ayral-Kaloustian S, Chen L, Geng Y, Arndt KT,
Lucas
J et al (2009) 2, 4-Diamino-quinazolines as inhibitors of beta-
catenin/Tcf-4 pathway: Potential treatment for colorectal
cancer.
Bioorg Med Chem Lett 19:4980–4983
129. Eguchi M, Nguyen C, Lee SC, Kahn M (2005) ICG-001, a
novel
small molecule regulator of TCF/beta-catenin transcription.
Med
Chem 1:467–472
130. Lepourcelet M, Chen YN, France DS, Wang H, Crews P,
Petersen F, Bruseo C, Wood AW, Shivdasani RA (2004) Small-
molecule antagonists of the oncogenic Tcf/beta-catenin protein
complex. Cancer Cell 5:91–102
131. Takahashi-Yanaga F, Kahn M (2010) Targeting Wnt
signaling:
can we safely eradicate cancer stem cells? Clin Cancer Res
16:3153–3162
132. You L, He B, Xu Z, Uematsu K, Mazieres J, Fujii N,

46. Mikami I,
Reguart N, McIntosh JK, Kashani-Sabet M et al (2004) An anti-
Wnt-2 monoclonal antibody induces apoptosis in malignant
melanoma cells and inhibits tumor growth. Cancer Res
64:5385–
5389
133. You L, He B, Xu Z, Uematsu K, Mazieres J, Mikami I,
Reguart
N, Moody TW, Kitajewski J, McCormick F et al (2004)
Inhibition of Wnt-2-mediated signaling induces programmed
cell death in non-small-cell lung cancer cells. Oncogene
23:6170–6174
134. Naundorf S, Preithner S, Mayer P, Lippold S, Wolf A,
Hanakam
F, Fichtner I, Kufer P, Raum T, Riethmuller G et al (2002) In
vitro and in vivo activity of MT201, a fully human monoclonal
antibody for pancarcinoma treatment. Int J Cancer 100:101–110
135. Hartung G, Hofheinz RD, Dencausse Y, Sturm J, Kopp-
Schneider A, Dietrich G, Fackler-Schwalbe I, Bornbusch D,
Gonnermann M, Wojatschek C et al (2005) Adjuvant therapy
with edrecolomab versus observation in stage II colon cancer: a
multicenter randomized phase III study. Onkologie 28:347–350
136. Adams GP, Weiner LM (2005) Monoclonal antibody
therapy of
cancer. Nat Biotechnol 23:1147–1157
J Mol Med
137. Seimetz D, Lindhofer H, Bokemeyer C (2010)
Development and

47. approval of the trifunctional antibody catumaxomab (anti-
EpCAMxanti-CD3) as a targeted cancer immunotherapy. Cancer
Treat Rev 36(6):458–467
138. Willett CG, Boucher Y, di Tomaso E, Duda DG, Munn LL,
Tong
RT, Chung DC, Sahani DV, Kalva SP, Kozin SVet al (2004)
Direct
evidence that the VEGF-specific antibody bevacizumab has
antivascular effects in human rectal cancer. Nat Med 10:145–
147
139. Wilhelm S, Carter C, Lynch M, Lowinger T, Dumas J,
Smith
RA, Schwartz B, Simantov R, Kelley S (2006) Discovery and
development of sorafenib: a multikinase inhibitor for treating
cancer. Nat Rev Drug Discov 5:835–844
140. Faivre S, Demetri G, Sargent W, Raymond E (2007)
Molecular
basis for sunitinib efficacy and future clinical development. Nat
Rev Drug Discov 6:734–745
141. Choi HJ, Song BJ, Gong YD, Gwak WJ, Soh Y (2008)
Rapid
degradation of hypoxia-inducible factor-1alpha by KRH102053,
a new activator of prolyl hydroxylase 2. Br J Pharmacol
154:114–125
142. Welsh S, Williams R, Kirkpatrick L, Paine-Murrieta G,
Powis G
(2004) Antitumor activity and pharmacodynamic properties of
PX-478, an inhibitor of hypoxia-inducible factor-1alpha. Mol
Cancer Ther 3:233–244

48. 143. Rapisarda A, Zalek J, Hollingshead M, Braunschweig T,
Uranchimeg B, Bonomi CA, Borgel SD, Carter JP, Hewitt SM,
Shoemaker RH et al (2004) Schedule-dependent inhibition of
hypoxia-inducible factor-1alpha protein accumulation,
angiogen-
esis, and tumor growth by topotecan in U251-HRE glioblastoma
xenografts. Cancer Res 64:6845–6848
144. Zhang H, Qian DZ, Tan YS, Lee K, Gao P, Ren YR, Rey S,
Hammers H, Chang D, Pili R et al (2008) Digoxin and other
cardiac glycosides inhibit HIF-1alpha synthesis and block tumor
growth. Proc Natl Acad Sci USA 105:19579–19586
145. Mie Lee Y, Kim SH, Kim HS, Jin Son M, Nakajima H,
Jeong
Kwon H, Kim KW (2003) Inhibition of hypoxia-induced
angiogenesis by FK228, a specific histone deacetylase inhibitor,
via suppression of HIF-1alpha activity. Biochem Biophys Res
Commun 300:241–246
146. Kim WY, Oh SH, Woo JK, Hong WK, Lee HY (2009)
Targeting
heat shock protein 90 overrides the resistance of lung cancer
cells by blocking radiation-induced stabilization of hypoxia-
inducible factor-1alpha. Cancer Res 69:1624–1632
J Mol Med
Cancer stem cells: a new framework for the design of tumor
therapiesAbstractThe hierarchy model and cancer stem cells
(CSCs)Properties and regulation of CSCsCSC nichesCSCs have
enhanced chemo-/radioresistance and metastatic
potentialConsequences for cancer research and development of
anticancer drug design platformsNovel, CSC-based drugs and
drug targetsCSC niche targeting and combinatorial therapeutic
approachesConclusion and perspectiveReferences
<<

49. /ASCII85EncodePages false
/AllowTransparency false
/AutoPositionEPSFiles true
/AutoRotatePages /None
/Binding /Left
/CalGrayProfile (Gray Gamma 2.2)
/CalRGBProfile (sRGB IEC61966-2.1)
/CalCMYKProfile (ISO Coated v2 300% 050ECI051)
/sRGBProfile (sRGB IEC61966-2.1)
/CannotEmbedFontPolicy /Error
/CompatibilityLevel 1.3
/CompressObjects /Off
/CompressPages true
/ConvertImagesToIndexed true
/PassThroughJPEGImages true
/CreateJDFFile false
/CreateJobTicket false
/DefaultRenderingIntent /Perceptual
/DetectBlends true
/DetectCurves 0.0000
/ColorConversionStrategy /sRGB
/DoThumbnails true
/EmbedAllFonts true
/EmbedOpenType false
/ParseICCProfilesInComments true
/EmbedJobOptions true
/DSCReportingLevel 0
/EmitDSCWarnings false
/EndPage -1
/ImageMemory 1048576
/LockDistillerParams true
/MaxSubsetPct 100
/Optimize true
/OPM 1
/ParseDSCComments true
/ParseDSCCommentsForDocInfo true

50. /PreserveCopyPage true
/PreserveDICMYKValues true
/PreserveEPSInfo true
/PreserveFlatness true
/PreserveHalftoneInfo false
/PreserveOPIComments false
/PreserveOverprintSettings true
/StartPage 1
/SubsetFonts false
/TransferFunctionInfo /Apply
/UCRandBGInfo /Preserve
/UsePrologue false
/ColorSettingsFile ()
/AlwaysEmbed [ true
]
/NeverEmbed [ true
]
/AntiAliasColorImages false
/CropColorImages true
/ColorImageMinResolution 150
/ColorImageMinResolutionPolicy /Warning
/DownsampleColorImages true
/ColorImageDownsampleType /Bicubic
/ColorImageResolution 150
/ColorImageDepth -1
/ColorImageMinDownsampleDepth 1
/ColorImageDownsampleThreshold 1.50000
/EncodeColorImages true
/ColorImageFilter /DCTEncode
/AutoFilterColorImages true
/ColorImageAutoFilterStrategy /JPEG
/ColorACSImageDict <<
/QFactor 0.40
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/ColorImageDict <<

51. /QFactor 1.30
/HSamples [2 1 1 2] /VSamples [2 1 1 2]
>>
/JPEG2000ColorACSImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 10
>>
/JPEG2000ColorImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 10
>>
/AntiAliasGrayImages false
/CropGrayImages true
/GrayImageMinResolution 150
/GrayImageMinResolutionPolicy /Warning
/DownsampleGrayImages true
/GrayImageDownsampleType /Bicubic
/GrayImageResolution 150
/GrayImageDepth -1
/GrayImageMinDownsampleDepth 2
/GrayImageDownsampleThreshold 1.50000
/EncodeGrayImages true
/GrayImageFilter /DCTEncode
/AutoFilterGrayImages true
/GrayImageAutoFilterStrategy /JPEG
/GrayACSImageDict <<
/QFactor 0.40
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/GrayImageDict <<
/QFactor 1.30
/HSamples [2 1 1 2] /VSamples [2 1 1 2]
>>
/JPEG2000GrayACSImageDict <<

52. /TileWidth 256
/TileHeight 256
/Quality 10
>>
/JPEG2000GrayImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 10
>>
/AntiAliasMonoImages false
/CropMonoImages true
/MonoImageMinResolution 600
/MonoImageMinResolutionPolicy /Warning
/DownsampleMonoImages true
/MonoImageDownsampleType /Bicubic
/MonoImageResolution 600
/MonoImageDepth -1
/MonoImageDownsampleThreshold 1.50000
/EncodeMonoImages true
/MonoImageFilter /CCITTFaxEncode
/MonoImageDict <<
/K -1
>>
/AllowPSXObjects false
/CheckCompliance [
/None
]
/PDFX1aCheck false
/PDFX3Check false
/PDFXCompliantPDFOnly false
/PDFXNoTrimBoxError true
/PDFXTrimBoxToMediaBoxOffset [
0.00000
0.00000
0.00000
0.00000

53. ]
/PDFXSetBleedBoxToMediaBox true
/PDFXBleedBoxToTrimBoxOffset [
0.00000
0.00000
0.00000
0.00000
]
/PDFXOutputIntentProfile (None)
/PDFXOutputConditionIdentifier ()
/PDFXOutputCondition ()
/PDFXRegistryName ()
/PDFXTrapped /False
/Description <<
/CHS
<FEFF4f7f75288fd94e9b8bbe5b9a521b5efa76840020004100640
06f006200650020005000440046002065876863900275284e8e5c
4f5e55663e793a3001901a8fc775355b5090ae4ef653d190014ee5
53ca901a8fc756e072797f5153d15e03300260a853ef4ee54f7f752
80020004100630072006f0062006100740020548c002000410064
006f00620065002000520065006100640065007200200035002e0
03000204ee553ca66f49ad87248672c676562535f00521b5efa768
400200050004400460020658768633002>
/CHT
<FEFF4f7f752890194e9b8a2d7f6e5efa7acb76840020004100640
06f006200650020005000440046002065874ef69069752865bc87
a25e55986f793a3001901a904e96fb5b5090f54ef650b390014ee55
3ca57287db2969b7db28def4e0a767c5e03300260a853ef4ee54f7f
75280020004100630072006f0062006100740020548c002000410
064006f0062006500200052006500610064006500720020003500
2e003000204ee553ca66f49ad87248672c4f86958b555f5df25efa7
acb76840020005000440046002065874ef63002>
/DAN
<FEFF004200720075006700200069006e0064007300740069006
c006c0069006e006700650072006e0065002000740069006c0020

54. 006100740020006f0070007200650074007400650020004100640
06f006200650020005000440046002d0064006f006b0075006d00
65006e007400650072002c0020006400650072002000620065006
400730074002000650067006e0065007200200073006900670020
00740069006c00200073006b00e60072006d007600690073006e0
069006e0067002c00200065002d006d00610069006c0020006f00
6700200069006e007400650072006e00650074002e00200044006
50020006f00700072006500740074006500640065002000500044
0046002d0064006f006b0075006d0065006e00740065007200200
06b0061006e002000e50062006e00650073002000690020004100
630072006f00620061007400200065006c006c006500720020004
100630072006f0062006100740020005200650061006400650072
00200035002e00300020006f00670020006e00790065007200650
02e>
/ESP
<FEFF005500740069006c0069006300650020006500730074006
100200063006f006e0066006900670075007200610063006900f3
006e0020007000610072006100200063007200650061007200200
064006f00630075006d0065006e0074006f007300200050004400
46002000640065002000410064006f00620065002000610064006
50063007500610064006f00730020007000610072006100200076
0069007300750061006c0069007a00610063006900f3006e00200
065006e002000700061006e00740061006c006c0061002c002000
63006f007200720065006f00200065006c006500630074007200f3
006e00690063006f0020006500200049006e007400650072006e0
0650074002e002000530065002000700075006500640065006e00
200061006200720069007200200064006f00630075006d0065006
e0074006f00730020005000440046002000630072006500610064
006f007300200063006f006e0020004100630072006f0062006100
74002c002000410064006f0062006500200052006500610064006
5007200200035002e003000200079002000760065007200730069
006f006e0065007300200070006f00730074006500720069006f00
7200650073002e>
/FRA
<FEFF005500740069006c006900730065007a002000630065007
30020006f007000740069006f006e00730020006100660069006e

55. 00200064006500200063007200e90065007200200064006500730
0200064006f00630075006d0065006e0074007300200041006400
6f006200650020005000440046002000640065007300740069006
e00e90073002000e000200049006e007400650072006e00650074
002c002000e0002000ea007400720065002000610066006600690
063006800e90073002000e00020006c002700e900630072006100
6e002000650074002000e0002000ea00740072006500200065006
e0076006f007900e9007300200070006100720020006d00650073
0073006100670065007200690065002e0020004c0065007300200
064006f00630075006d0065006e00740073002000500044004600
200063007200e900e90073002000700065007500760065006e007
4002000ea0074007200650020006f007500760065007200740073
002000640061006e00730020004100630072006f0062006100740
02c002000610069006e0073006900200071007500270041006400
6f00620065002000520065006100640065007200200035002e003
0002000650074002000760065007200730069006f006e00730020
0075006c007400e90072006900650075007200650073002e>
/ITA
<FEFF005500740069006c0069007a007a0061007200650020007
10075006500730074006500200069006d0070006f007300740061
007a0069006f006e0069002000700065007200200063007200650
0610072006500200064006f00630075006d0065006e0074006900
2000410064006f00620065002000500044004600200070006900f
9002000610064006100740074006900200070006500720020006c
0061002000760069007300750061006c0069007a007a0061007a0
069006f006e006500200073007500200073006300680065007200
6d006f002c0020006c006100200070006f0073007400610020006
5006c0065007400740072006f006e006900630061002000650020
0049006e007400650072006e00650074002e00200049002000640
06f00630075006d0065006e007400690020005000440046002000
630072006500610074006900200070006f00730073006f006e006f
00200065007300730065007200650020006100700065007200740
06900200063006f006e0020004100630072006f00620061007400
200065002000410064006f0062006500200052006500610064006
5007200200035002e003000200065002000760065007200730069
006f006e006900200073007500630063006500730073006900760

56. 065002e>
/JPN
<FEFF753b97624e0a3067306e8868793a3001307e305f306f96fb
5b5030e130fc30eb308430a430f330bf30fc30cd30c330c87d4c753
1306790014fe13059308b305f3081306e002000410064006f00620
06500200050004400460020658766f8306e4f5c6210306b906930
5730663044307e305930023053306e8a2d5b9a30674f5c6210305
5308c305f0020005000440046002030d530a130a430eb306f30010
04100630072006f0062006100740020304a308830730020004100
64006f00620065002000520065006100640065007200200035002
e003000204ee5964d3067958b304f30533068304c3067304d307e
305930023053306e8a2d5b9a3067306f30d530a930f330c8306e57
cb30818fbc307f3092884c306a308f305a300130d530a130a430eb
30b530a430ba306f67005c0f9650306b306a308a307e30593002>
/KOR
<FEFFc7740020c124c815c7440020c0acc6a9d558c5ec0020d654
ba740020d45cc2dc002c0020c804c7900020ba54c77c002c0020c7
78d130b137c5d00020ac00c7a50020c801d569d55c00200041006
4006f0062006500200050004400460020bb38c11cb97c0020c791
c131d569b2c8b2e4002e0020c774b807ac8c0020c791c131b41c00
200050004400460020bb38c11cb2940020004100630072006f006
2006100740020bc0f002000410064006f00620065002000520065
006100640065007200200035002e00300020c774c0c1c5d0c11c0
020c5f40020c2180020c788c2b5b2c8b2e4002e>
/NLD (Gebruik deze instellingen om Adobe PDF-documenten
te maken die zijn geoptimaliseerd voor weergave op een
beeldscherm, e-mail en internet. De gemaakte PDF-documenten
kunnen worden geopend met Acrobat en Adobe Reader 5.0 en
hoger.)
/NOR
<FEFF004200720075006b0020006400690073007300650020006
9006e006e007300740069006c006c0069006e00670065006e0065
002000740069006c002000e50020006f007000700072006500740
0740065002000410064006f006200650020005000440046002d00
64006f006b0075006d0065006e00740065007200200073006f006
d00200065007200200062006500730074002000650067006e0065

57. 007400200066006f007200200073006b006a00650072006d00760
0690073006e0069006e0067002c00200065002d0070006f007300
740020006f006700200049006e007400650072006e00650074007
4002e0020005000440046002d0064006f006b0075006d0065006e
00740065006e00650020006b0061006e002000e50070006e00650
073002000690020004100630072006f0062006100740020006500
6c006c00650072002000410064006f00620065002000520065006
100640065007200200035002e003000200065006c006c00650072
002000730065006e006500720065002e>
/PTB
<FEFF005500740069006c0069007a00650020006500730073006
1007300200063006f006e00660069006700750072006100e700f5
0065007300200064006500200066006f0072006d0061002000610
0200063007200690061007200200064006f00630075006d006500
6e0074006f0073002000410064006f00620065002000500044004
60020006d00610069007300200061006400650071007500610064
006f0073002000700061007200610020006500780069006200690
0e700e3006f0020006e0061002000740065006c0061002c002000
7000610072006100200065002d006d00610069006c00730020006
5002000700061007200610020006100200049006e007400650072
006e00650074002e0020004f007300200064006f00630075006d0
065006e0074006f00730020005000440046002000630072006900
610064006f007300200070006f00640065006d002000730065007
2002000610062006500720074006f007300200063006f006d0020
006f0020004100630072006f006200610074002000650020006f0
02000410064006f00620065002000520065006100640065007200
200035002e0030002000650020007600650072007300f50065007
300200070006f00730074006500720069006f007200650073002e
>
/SUO
<FEFF004b00e40079007400e40020006e00e40069007400e4002
000610073006500740075006b007300690061002c0020006b0075
006e0020006c0075006f00740020006c00e400680069006e006e0
0e40020006e00e40079007400f60073007400e40020006c007500
6b0065006d0069007300650065006e002c0020007300e40068006
b00f60070006f0073007400690069006e0020006a006100200049

58. 006e007400650072006e0065007400690069006e0020007400610
072006b006f006900740065007400740075006a00610020004100
64006f0062006500200050004400460020002d0064006f006b007
5006d0065006e007400740065006a0061002e0020004c0075006f
0064007500740020005000440046002d0064006f006b0075006d0
065006e00740069007400200076006f0069006400610061006e00
20006100760061007400610020004100630072006f00620061007
40069006c006c00610020006a0061002000410064006f00620065
002000520065006100640065007200200035002e0030003a006c0
06c00610020006a006100200075007500640065006d006d006900
6c006c0061002e>
/SVE
<FEFF0041006e007600e4006e00640020006400650020006800e
4007200200069006e0073007400e4006c006c006e0069006e0067
00610072006e00610020006f006d0020006400750020007600690
06c006c00200073006b006100700061002000410064006f006200
650020005000440046002d0064006f006b0075006d0065006e007
400200073006f006d002000e400720020006c00e4006d0070006c
0069006700610020006600f600720020006100740074002000760
0690073006100730020007000e500200073006b00e40072006d00
2c0020006900200065002d0070006f007300740020006f0063006
80020007000e500200049006e007400650072006e00650074002e
002000200053006b0061007000610064006500200050004400460
02d0064006f006b0075006d0065006e00740020006b0061006e00
2000f600700070006e00610073002000690020004100630072006
f0062006100740020006f00630068002000410064006f00620065
002000520065006100640065007200200035002e00300020006f0
0630068002000730065006e006100720065002e>
/ENU (Use these settings to create Adobe PDF documents
best suited for on-screen display, e-mail, and the Internet.
Created PDF documents can be opened with Acrobat and Adobe
Reader 5.0 and later.)
/DEU
<FEFF004a006f0062006f007000740069006f006e007300200066
006f00720020004100630072006f0062006100740020004400690
07300740069006c006c0065007200200037000d00500072006f00

59. 6400750063006500730020005000440046002000660069006c006
50073002000770068006900630068002000610072006500200075
00730065006400200066006f00720020006f006e006c0069006e0
065002e000d002800630029002000320030003100300020005300
7000720069006e006700650072002d005600650072006c0061006
700200047006d006200480020>
>>
/Namespace [
(Adobe)
(Common)
(1.0)
]
/OtherNamespaces [
<<
/AsReaderSpreads false
/CropImagesToFrames true
/ErrorControl /WarnAndContinue
/FlattenerIgnoreSpreadOverrides false
/IncludeGuidesGrids false
/IncludeNonPrinting false
/IncludeSlug false
/Namespace [
(Adobe)
(InDesign)
(4.0)
]
/OmitPlacedBitmaps false
/OmitPlacedEPS false
/OmitPlacedPDF false
/SimulateOverprint /Legacy
>>
<<
/AddBleedMarks false
/AddColorBars false
/AddCropMarks false
/AddPageInfo false

60. /AddRegMarks false
/ConvertColors /ConvertToRGB
/DestinationProfileName (sRGB IEC61966-2.1)
/DestinationProfileSelector /UseName
/Downsample16BitImages true
/FlattenerPreset <<
/PresetSelector /MediumResolution
>>
/FormElements false
/GenerateStructure false
/IncludeBookmarks false
/IncludeHyperlinks false
/IncludeInteractive false
/IncludeLayers false
/IncludeProfiles true
/MultimediaHandling /UseObjectSettings
/Namespace [
(Adobe)
(CreativeSuite)
(2.0)
]
/PDFXOutputIntentProfileSelector /NA
/PreserveEditing false
/UntaggedCMYKHandling /UseDocumentProfile
/UntaggedRGBHandling /UseDocumentProfile
/UseDocumentBleed false
>>
]
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [595.276 841.890]
>> setpagedevice