DNA Repair of Cancer Stem Cells
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Contents
1 Introduction to Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Chengzhuo Gao, Robert E. Hollingsworth and Elaine M. Hurt
2 DNA Repair Pathways and Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Thomas S. Dexheimer
3 Resistance and DNA Repair Mechanisms of Cancer Stem Cells:
Potential Molecular Targets for Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Aamir Ahmad, Yiwei Li, Bin Bao and Fazlul H. Sarkar
4 DNA Repair in Normal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Olga Momčilović and Gerald Schatten
5 DNA Repair Mechanisms in Glioblastoma Cancer Stem Cells . . . . . . . 89
Monica Venere, Jeremy N. Rich and Shideng Bao
6 DNA Repair Mechanisms in Breast Cancer Stem Cells . . . . . . . . . . . . . 105
Hong Yin and Jonathan Glass
7 DNA Repair Mechanisms in Other Cancer Stem Cell Models . . . . . . . 125
Mihoko Kai
8 Pancreatic Cancer Stem Cells in Tumor Progression, Metastasis,
Epithelial-Mesenchymal Transition and DNA Repair . . . . . . . . . . . . . . . 141
Nagaraj S. Nagathihalli and Erika T. Brown
9 Targeting Cancer Stem Cell Efficient DNA Repair Pathways:
Screening for New Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Lesley A. Mathews, Francesco Crea and Marc Ferrer
10 The Future of DNA Repair and Cancer Stem Cells . . . . . . . . . . . . . . . . . 173
Stephanie M. Cabarcas
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
vii
Chapter 1
Introduction to Cancer Stem Cells
Chengzhuo Gao, Robert E. Hollingsworth and Elaine M. Hurt
Abstract A wealth of data points to the existence of a subset of tumor-initiating cells
that have properties similar to stem cells, termed cancer stem cells (CSCs). CSCs
are thought to be at the apex of a cellular hierarchy, where they are capable of dif-
ferentiating into the other cells found within a tumor. They may also be responsible
for both patient relapse due to their relative resistance to chemotherapy as well as
metastasis. In recent years, much research has focused on these cells, their proper-
ties and potential targets within these cells for cancer treatment. This chapter will
introduce the CSC theory, discuss important properties of these cells, and highlight
the need to target them for improved patient outcome.
1.1 The Etiology of Cancer
Many hypotheses have been put forth through the years that attempt to explain
the etiology of cancer. They have come from divergent fields of study; pathology,
molecular biology and genetics but they all attempt to explain how a normal tissue
can go from homeostatic equilibrium to something that grows without the checks
and balances that govern normal biology. These theories include, but are not limited
to, a viral basis of disease, clonal expansion, and the cancer stem cell hypothesis.
While each of these theories attempts to explain the etiology of cancer, it is most
likely that some of these independent theories work together to give rise to not only
tumors, but tumors that are able to evade treatment strategies.
E. M. Hurt ()
MedImmune, LLC., Oncology Research MedImmune, Gaithersburg, MD 20878, USA
e-mail: [email protected]
C. Gao · R. E. Hollingsworth
Oncology Research, MedImmune, LLC., Gaithersburg, MD 20878, USA
L. A. Mathews et al. (eds.), DNA Repair of Cancer Stem Cells, 1
DOI 10.1007/978-94-007-4590-2_1, © Springer Science+Business Media Dordrecht 2013
2 C. Gao et al.
1.2 The Cancer Stem Cell Hypothesis
Since the early pathologists could look at tissues microscopically the origins and
progression of cancer has been pondered. It was noted that advanced tumors often
presented with diverse areas of differentiation, proliferation, invasion and vascularity.
It was during this time of early microscopic inspection that led Cohnheim, a student
of Virchow, to propose his embryonal rest hypothesis ([1] and references therein).
Cohnheim had noted that cancer tissues, teratomas in particular, had many of the
same properties as embryonic tissue. This lead him to speculate that carcinogenesis
occurs from dormant remnants of the embryo that are later reactivated. However,
this theory was largely ignored. The spirit of this hypothesis reappeard in the 1970s
when Barry Pierce et al. examined differentiation in teratomas (reviewed in [1]) and
determined that a stem cell was responsible for initiation of the teratomas. They then
furthered these observations into a theory that all epithelial cancers arise as a result
of differentiation-paused adult tissue stem cells [2].
On the heels of this hypothesis, came some of the first evidence that leukemias
may have a CSC origin. John Dick and colleagues showed that a sub-population of
acute myelogenous leukemia (AML) cells, which shared a phenotype with normal
hematopoietic stem cells, could confer cancer when transplanted into immunocom-
promised mice. Furthermore, the cells that did not have the stem cell-phenotype
could not transfer AML to recipient mice [3, 4]. Several years later, CSCs were iden-
tified in breast cancer [5], followed by a myriad of solid malignancies (discussed in
more detail below in “1.4 Identification of CSCs”).
1.3 Properties and Cell of Origin of CSCs
Cancer stem cells are so named because they share many of the same properties as nor-
mal stem cells. They are capable of tumorigenesis, self-renewal and can differentiate
to form the heterogeneous cell types present in tumors (Fig. 1.1a). These functional
properties led to the CSC moniker; however, many argue that “tumor-initiating cells”
would be a better description of these cells. For the purposes of this book, we will
refer to these cells as CSCs, where a CSC has been defined as “a cell within a tumor
that possess the capacity to self-renew and to cause the heterogeneous lineages of
cancer cells that comprise the tumor” [6]. It has also been observed that CSCs are rel-
atively resistant to chemotherapy and therefore may be responsible for patient relapse
following treatment (Fig. 1.1b, discussed in detail in Chap. 3). Thus, the CSC hy-
pothesis attempts to explain several observations of tumors, including the frequency
at which tumor cells can give rise to new tumors, the generation of cells with multiple
genetic alterations, and the heterogeneity of cell types present within tumors.
In theory, cancer arises from a single cell that has somehow subverted normal
growth restrictions. However, experimental evidence has shown a requirement of
many cells in order to seed a tumor. For example the growth of tumor cells in im-
munocompromised mice, typically requires that 1–10 million cells are implanted in
1 Introduction to Cancer Stem Cells 3
Fig. 1.1 Schematic representation of CSC-driven tumor formation. (a) CSCs can either divide
symmetrically (self-renewal) to give rise to two CSCs or they can divide asymmetrically (differen-
tiation) giving rise to one CSC and one differentiated progeny. The ability to give rise to a tumor,
to self-renewal and differentiate into the heterogeneous cell populations found in the tumor are the
defining characteristic of a CSC. The differentiated progeny of a CSC are often more proliferative
than the CSC itself, but they have a finite replicative capacity. (b) CSCs are resistant to conventional
therapies, including chemotherapy and radiation. These treatments can eliminate the more rapidly
dividing differentiated cells but leave behind the CSC (discussed in more detail in Chap. 3). Once
treatment is stopped, the remaining CSCs can begin to divide again and form a new tumor resulting
in patient relapse
order to see tumor formation. This observation has many possible explanations, in-
cluding injury to cells upon injection, the requirement for the right microenvironment
(and the difference of this between human and mouse), as well as the requirement
to have a variety of cell types present in order to efficiently induce tumor formation.
However, even in experiments where human cancer patients were reinjected with
their own tumor cells at different sites within their bodies, it took large numbers of
cells in order for a new tumor to establish. Again, this could be consistent with the
role of the tumor microenvironment, this time from one site to another; it is also
consistent with the notion that only a small proportion of cells are capable of giving
rise to a tumor. The idea that only a small proportion of the cells found within a
tumor are capable of giving rise to a tumor is consistent with the CSC hypothesis.
The frequency of CSCs in most cases has been reported to be low (typically less than
5 %) except in the case of melanoma where a variety of cells were shown to have
equally high tumorigenicity [7, 8].
The origin of the cancer stem cell is still under debate. The requirement for
multiple genetic insults in order to drive tumor formation has been recognized for a
long time [9]. It is suggested that in order for a cell to live long enough to sustain
4 C. Gao et al.
the genetic insults required to drive tumor formation, it is likely that a normal adult
stem cell is the originator of a CSC phenotype. Mathematical models support this
theory [10]. Furthermore, the leukemic stem cells (LSCs) of CML patients express
BCR-ABL, the common translocation that drives cellular transformation [11]. This
provides experimental evidence that CSCs sustain the genetic insults that are seen
to drive carcinogenesis. The identification in CSCs of oncogenic driver mutations is
also beginning to emerge for solid tumors. For example, the TMPRSS:ERG fusion
has been found in prostate CSCs [12].
However, it is still possible that a more differentiated cell undergoes these genomic
rearrangements and additionally picks up further mutations that impart the ability
to self-renew as well as differentiate. Indeed fibroblasts can acquire the properties
of pluripotent stem cells with the activation of just a few genes [13–15]. Moreover,
it was recently shown that a catastrophic event in the cell can lead to many genetic
alterations at a single time [16]. It may also be that environmental cues can trigger
CSC properties even independent of DNA rearrangements. For example, it has been
noted that cells undergoing epithelial-to-mesenchymal transition (EMT), a normal
developmental process that promotes cancer invasion and metastasis, can acquire
characteristics of CSCs [17]. These ilines of evidence suggest that a cell does not
need to be long-lived in order to sustain many genetic alterations and that CSC
properties can be bestowed by biological processes, and would therefore argue that
any cell may be the fodder for CSCs.
1.4 Isolation of CSCs
Currently, there are several commonly used approaches for the isolation of cancer
stem cells, including: (1) sorting of a side population (SP) by flow cytometry based
on Hoechst dye efflux, (2) sorting of CSCs by flow cytometry based on cell surface
marker expression, (3) enriching of CSCs by non-adherent sphere culture and (4)
sorting of CSCs by flow cytometry based on aldehyde dehydrogenase (ALDH) ac-
tivity (Fig. 1.2). All of these approaches enrich for CSCs to varying degrees, and
each of them has its own advantages and limitations, which will be discussed below.
1.4.1 Side Populations
Goodell and colleagues, while analyzing murine bone marrow cells, discovered a
small and distinct subset of whole bone marrow cells that were unstained by Hoechst
33342, a vital dye [18]. This Hoechst 33342 low population is termed SP. They
found that the SP had the phenotypic markers of multipotential hematopoietic stem
cells and were able to repopulate the bone marrow. Following their work, the SP
has been extended to a variety of cancer types, including leukemia [19, 20], ovar-
ian cancer [21], hepatocellular carcinoma [22], brain tumors [23–25], lung cancer
[26, 27], thyroid cancer [28], nasopharyngeal carcinoma [29], mesenchymal tumors
1 Introduction to Cancer Stem Cells 5
Fig. 1.2 Methods of CSC isolation. CSCs have been isolated using one of four methods. This is
a pictorial representation of these isolation techniques and how they are used to identify the CSC
versus the non-CSCs
[30], colon cancer [31], prostate cancer [32], breast cancer [33–35], head and neck
cancer [36] and other cancers [37–40]. The SP cells purified from these tumor types
harbor cancer stem cell-like cells with properties such as a “stemness” gene signature
[23, 26, 28, 29, 35], self-renewal capacity [25, 26, 28, 29, 33, 35] and tumorigenicity
[19, 21, 24–26, 28, 29, 35].
The ATP dependent transporter, ATP-binding cassette sub-family G member 2
(ABCG2, BRCP1), is generally believed to be responsible for Hoechst 33342 efflux
by the SP. Several lines of evidence support this hypothesis. First, ABCG2 knockout
mice show significantly decreased numbers of SP cells in both the bone marrow
as well as in skeletal muscle [41]. Second, overexpression of ABCG2 dramatically
increases the SP percentage of bone-marrow cells and reduces maturing progeny both
in vitro and in vivo [42]. Third, ABCG2 is highly expressed in a wide variety of stem
cells including the SP cells of neuroblastoma patients [23, 42]. Fourth, ABCG2 may
be responsible for conferring drug resistance to the SP and CSCs. ABCG2 is a
multidrug resistant pump expressed at variable levels in cancer cells, which can
bind and expel cytotoxic drugs [43]. Thus, ABCG2 may lower intracellular levels
of anticancer agents below the threshold for cell death in tumors, leaving resistant
cells to repopulate the tumor. Indeed, inhibition of the ABC transporters sensitized
SP cells of various cancer types to chemotherapeutic agents [44, 45].
However, there is not always a correlation between ABCG2 expression and the SP
phenotype. For example, erythroblasts highly express the ABC transporter ABCG2
but do not have an SP phenotype [46]. Furthermore, the expression of ABCG2, and
6 C. Gao et al.
the presence of the SP in general, do not always define the CSC population. For
instance, in prostate cancer both purified ABCG2+ and ABCG2− cancer cells have
similar tumorigenicity to the SP cells in vivo, although the SP cells express higher
level of ABCG2 mRNA than the non-SP cells [32]. Additionally, ABCG2 is not the
only multi-drug resistance gene identified in SP stem-like cells. The expression of
P-glycoprotein (ABCB1, MDR1) is also significantly up-regulated in SP cells of an
oral squamous cell carcinoma cell line [47], although Feuring-Buske et al. found
that there is no correlation between the expression of ABCB1 and the SP in acute
myeloid leukemia [19]. Instead, Zhou et al. demonstrated that Hoechst 33342 efflux
activity is compensated by ABCG2 in Abcb1 null mice [42], indicating that SP cells
may utilize different drug transportation machineries in different environments. In
addition, the presence of the SP may instead be the result of inefficient dye uptake as
a reflection of the presence of largely quiescent cells, another characteristic of stem
cells [26, 48].
Compared to other methods, isolation of the side population has two advan-
tages. First, it carries additional information about the functional status of the cells,
since this assay is based on an active metabolic process. Second, it is highly sensi-
tive, with even rare SP events (<0.5 % of the total cell population) detected within
heterogeneous samples [49].
But this method also has many disadvantages. Some of the limitations of this
method have to do with the Hoechst 33342 dye itself. Owing to the fact that Hoechst
33342 is a DNA binding dye, it is toxic to cells. Shen and colleagues found that
Hoechst 33342 staining for a prolonged periods of time increases apoptosis in C6
cells [25]. Furthermore, it is highly sensitive to slight variations in staining conditions.
Hoechst concentration, the staining time, and the staining temperatures all are critical
for the success of this approach.
Other disadvantages of SP isolation are due to the ability of this method to ac-
curately define and purify CSCs. Importantly, the SP is not always necessary or
sufficient for a CSC phenotype. In glioblastoma multiforme (GBM), the SP from
the GBM lines did not enrich for stem-like activity in vitro, and tumorigenicity was
lower in sorted SP compared with non-SP and parental cells [50]. Equally important,
is that SP cells represent a heterogeneous cell population. Wan et al. demonstrated
that SP cells purified from a laryngeal cancer cell line does harbor cancer stem cell-
like properties, but they are heterogeneous indicating that SP cells are not identical
to stem cells [51]. Combining SP detection with cell surface marker selection, may
lead to a more efficient and reliable isolation of CSCs.
Despite the limitations existing in SP isolation, the presence of the SP population
has some clinical relevance in certain disease indications. For example, the SP pop-
ulation can be identified in gastric cancer tissue and correlates with patient survival
[37]. A limited clinical study in ovarian cancer also revealed a higher SP frequency
in recurrent or metastatic tumors compared with primary tumors, suggesting a good
correlation between the presence of SP and recurrence in ovarian cancer [52].
Besides the use in prognosis of cancer, the SP may also serve as a potential thera-
peutic target for cancer. Several studies have pioneered the possibility of specifically
1 Introduction to Cancer Stem Cells 7
targeting SP cells by exploiting pathways involved in drug resistance and differenti-
ation [53]. For instance, Praveen et al. have isolated SP cells from multiple cancer
cell lines and found that these SP cells are resistant to cytochrome C release and
apoptosis. Based on this finding, they developed a high-throughput imaging assay,
in which the cytochrome C-EGFP translocation is monitored in the sorted SP cells.
Through this assay, the heat shock protein 90 inhibitors have been identified to sen-
sitize the SP cells to some antitumor agents, such as cisplatin [54]. Moreover, an
autologous vaccine for B-cell chronic lymphocytic leukemia (B-CLL) was made
using a patient’s SP cells. Following vaccination, the study showed an increase in
(B-CLL)-reactive T-cells followed by a corresponding decline in circulating B-CLL
SP cells [55]. This indicates that the SP may be a valid ground for cancer therapy.
1.4.2 Surface Markers
Initially used to identify and isolate normal stem cells, surface markers are now
extensively used for the identification and isolation of CSCs in many malignances.
Lapidot and colleagues were the first to isolate leukemia-initiating cells based on
cell-surface marker expression and found CD34+ CD38− cells, but not the CD34+
CD38+ and CD34− cells, harbored serial leukemic transplantation potential [3].
Following this initial prospective isolation of leukemia stem cells, breast CSCs were
identified as CD44+ CD24−/low by Al-Hajj and colleagues [5]. Later, based on their
surface marker expression, CSCs have been isolated from various tumors (Table 1.1).
Among all the surface markers used for CSCs isolation, CD133 and CD44 are the
most commonly used in a variety of tumor types.
CD133 (Prominin 1), a five transmembrane glycoprotein, was originally identified
both in the neuroepithelium and in various other epithelia of the mouse embryo [77].
Later, a novel monoclonal antibody recognizing the AC133 antigen, a glycosylation-
dependent epitope of CD133, detected that CD133 is restricted in CD34+ progenitor
populations from adult blood, bone marrow and fetal liver cells [78]. In addition,
CD133 expression is rapidly down regulated upon cell differentiation [79]. These
characteristics of CD133 make it a unique cell surface marker for the identification
and isolation of various CSCs (Table 1.1). The biological function of CD133 is
still largely unknown. A single nucleotide deletion, which caused the truncation
of CD133, is linked to an inherited form of human retinal degeneration [80]. A
recent report has linked CD133 with endocytosis. In this study, CD133 knockdown
improved Alexa488-transferrin (Tf) uptake in Caco-2 cells, while cell treatment
with the AC133 antibody resulted in down regulated Tf uptake, [81]. Despite its
utility as a marker of CSCs, CD133 does not appear to play a significant role in
the maintenance of at least some CSCs. In colon cancer cells isolated from patients,
CD133 knockdown did not affect their tumorigenicity in vitro and in vivo [82].
Instead, CD44 knockdown prevented tumor formation of the same cells.
CD44, a single transmembrane glycoprotein, is a major component of the extra-
cellular matrix [83]. Besides acting as an adhesion molecule, it also functions as
8 C. Gao et al.
Table 1.1 Cell surface Tumor type Phenotype Reference
markers used in the isolation
of various CSCs AML CD34+ CD38− [3, 4]
Breast CD44+ CD24− [5]
Brain CD133+ [56, 57]
SSEA-1 [58]
Prostate CD44+ CD133+ α2 ßhi
1 [59]
CD44+ CD24− [60]
Head and neck CD44+ [61]
Liver CD133+ [62]
CD90+ [63]
Colon CD133+ [64, 65]
EpCAMhi CD44+ [66]
CD44+ /CD166+ [67]
Pancreatic CD44+ CD24− EpCAM+ [68]
CD133+ [69]
Squamous cell carcinoma Podoplanin [70]
Lung CD133+ [71]
Melanoma ABCB5+ [72]
Gastric CD44+ [73]
Ovarian CD133+ [74]
CD44+ /CD177+ [75]
CD44+ [76]
a principle receptor for hyaluronan (HA) [84]. HA is enriched in the pericellular
matrices of many malignant human tumors and plays an important role in tumor
progression via regulation of receptor tyrosine kinases (RTKs), such as ERBB2 and
EGFR [85]. Thus, as a critical receptor for HA, CD44 plays an important role in
cell proliferation and survival via activation of the MAPK and PI3K/AKT pathways,
respectively [85]. Furthermore, CD44 also plays an important role in the invasion
of a variety of cancer cells, including breast [86], prostate [87], hepatoma [88],
and mesotheliomas [89], and has been significantly correlated with the circulating
prostate tumor cells [90]. Therefore, CD44 stands out as a surface marker for CSCs,
as first shown by Al-hajj and colleagues in breast cancer [5]. Following their work,
CD44 has been utilized as a surface marker to isolate CSCs from a variety of different
tumors (Table 1.1).
CD44 was also explored as a potential diagnostic target for cancer detection as
well as a drug target for cancer therapy [91–93]. For instance, in 2003, the humanized
anti-CD44 antibody (bivatuzumab) labeled with rhenium-186 was used in phase I
studies in patients with head and neck squamous cell carcinoma (HNSCC) [94, 95].
In this trial, these radiolabeled CD44 antibodies showed promising anti-tumor effects
with low toxicity. Further, a different trial with the non-radiolabeled CD44 antibody
(bivatuzumab mertansine) also had good patient response rates, although the devel-
opment of this drug was terminated due to the death of a patient [96]. Recently, CD44
was also shown to target CSCs. In 2006, Jin et al. found that interruption of CSCs
interaction with their microenvironment by monoclonal antibody directed against
CD44 markedly decreased the number of the AML LSCs in vivo, indicating a key
regulatory role of CD44 in AML LSCs [97].
1 Introduction to Cancer Stem Cells 9
While CD44 and CD133 appear to have broad tumor applicability, the choice of
the cell surface markers tends to be tissue specific and is often based on previous
knowledge of the development of that tissue. For instance, CD34 and CD38, the
markers used for isolation of AML CSCs, are also the markers used to identify
normal early hematopoietic progenitor cells [3, 4]. Another good example is CD138.
CD138 is a marker for terminally-differentiated B cells (plasma cells). It has been
shown that CSCs from multiple myeloma, a plasma cell malignancy, are CD138− .
Compared to other methods of isolation, cell surface marker isolation has a major
advantage of obtaining a precise population. However, the selection of which surface
markers to use is one of the greatest pitfalls of this approach. As discussed earlier,
many times markers selected are based on previous knowledge of the development of
the tissue. For this reason, it may not be easy to find the right markers. For example,
until recently there were no markers identified for CSCs of human primary gastric
tumors [98]. Furthermore, the choice of markers made by researchers has not been
unified, even within the same tumor type. As shown in Table 1.1, different markers
have been used for the same tumor type. To further complicate the picture, many
times there have not been careful comparisons done within the same study of all the
proposed CSC markers in order to definitively test which marker combination is the
best at identifying CSCs. A further complication is that the surface markers may be
heterogeneous between patients even within the same tumor type. A recent study
from 16 AML patients shows that the majority of LSCs are in the minor CD34+
CD38− fraction in 50 % of the subjects, and in the CD34+ CD38+ fraction in the
other 50 % [99]. Similar findings were also obtained from breast cancer patients.
When Park et al. [92] evaluated the expression of stem cell-related markers at the
cellular level in human breast tumors of different subtypes and histologic stages,
they found that the cancer stem cell markers vary according to tumor subtype and
histologic stage [100]. Ali et al. also demonstrated that breast CSC markers, such as
CD44/CD24, ALDH and ITGA6, do not identify identical subpopulations in primary
tumors [101].
Other limitations of choosing surface markers for the identification of CSCs are
methodological. First, a large number of cells is required to sort and the number of
CSCs identified by this approach is usually low (<1 − 10 %). Furthermore, when
using tumor samples, the cells must first be dissociated typically with collagenase
and/or other proteolytic enzymes. This dissociation step may damage the presentation
of the cell surface antigens [102, 103].
1.4.3 Nonadherent Sphere Culture
Initially, nonadherent sphere culture was used to culture neural stem cells [104]. The
cells isolated from the stratum of adult mouse by this method could generate both
neurons and astrocytes. Later, it was shown that purified stem cells were capable of
growing as spheres. For instance, CD133+ cells isolated from normal human fetal
10 C. Gao et al.
brain formed spheres in vitro [105]. These observations were then extended to can-
cerous tissues where further studies demonstrated that CD133+ cells from the human
brain tumors are also capable of forming neurospheres [57]. Since then, the ability
of CSCs to form spheres in culture has been shown for most solid malignancies,
including breast [106], melanoma [107], pancreatic [108], prostate [109], ovarian
[75] and colon CSCs [110].
Most importantly, researchers found that the nonadherent sphere culture condition
can enrich cells with CSC phenotypes [106]. They demonstrated that, in breast
cancer, the CSC population with CD44+ CD24− phenotype increased more than
two-fold after culturing under nonadherent sphere culture conditions. In addition,
the spheres were more tumorigenic. Since then, researchers have used sphere to
formation to enrich CSCs from brain [56, 57, 111, 112], colon [110], pancreas [108],
bone sarcomas [113] melanomas [107] and prostate [109]. The enrichment of CSCs
by growing cells under sphere culture conditions has been confirmed by surface
marker expression in all mentioned cases, except bone sarcoma, in which the surface
marker remain to be determined.
Compared to the other methods of isolating CSCs, this method is easy to perform
and allows researchers to obtain a larger number of CSCs. However, like all methods
of isolation, this method also suffers from several disadvantages. The spheres are a
heterogeneous population of cells containing both CSCs and non-CSCs. For example
it has been shown that only a portion of the spheres are capable of self-renewal
[106, 114]. Immunostaining of spheres from prostate cancer cell lines indicated that
the spheres are heterogeneous for CSC markers [115]. Also, the conditions of sphere
formation are critical to the overall success of CSC enrichment. In neurosphere
cultures, it has been shown that the composition of spheres can be different due to
the differences in sphere size, passage, culture medium, and technique [116].
1.4.4 ALDH Activity
In addition to the above three isolation methods, isolation of CSCs based on their alde-
hyde dehydrogenase (ALDH) activity is also commonly used. ALDH is a detoxifying
enzyme responsible for the oxidation of intracellular aldehydes [117]. It functions
in drug resistance, cell differentiation and oxidative stress response [118]. ALDH
activity may be easily assessed in living cells using the ALDEFLUOR kit (Stem Cell
Technologies). This kit utilizes an ALDH substrate BODIPY aminoacetaldehyde
(BAAA), which is converted in the cytoplasm into a florescent molecule by ALDH
enzymatic activity [119]. Recent studies have demonstrated a positive correlation
between ALDH expression and overall survival of patients with different cancers
[120]. To date, ALDH activity has been used to identify and isolate CSCs from AML
[121], breast [122], melanoma [123], prostate [124], liver [71], ovarian [125], lung
[126] and osteosarcoma [127].
Like other isolation methods, ALDH is also not a universal marker for cancer
stem cells in any tumor type. In a CSC marker profiling study, the breast cancer
1 Introduction to Cancer Stem Cells 11
cells selected by ALDH activity did not always identify the most tumorigenic cells
[128]. Furthermore, in prostate cancer, Yu et al. [14] found that ALDHlo CD44−
cells were also able to develop tumors with similar frequency as the ALDHhi CD44+
cells, although with longer latency periods [129].
To date, there is not a universal isolation method in the CSC field. Researchers
have to select their isolation method depending on the purpose of their studies, and
sample source. Recently, Zhou et al. demonstrated that SP rather than CD133(+)
cells indicate enriched tumorigenicity in hTERT-immortalized primary prostate can-
cer cells [130]. Sometimes, two different methods may need to be combined to isolate
CSCs. For instance, combining SP determination with cell-surface marker pheno-
typing leads to efficient, reliable characterization of the HSC subset [131]. Further,
Eppert and colleagues found that AML LSCs from different patient samples have
diverse surface marker profiles and frequency [99]. However, these LSCs shared a
core gene signature, indicating that the stemness gene signature might be a more
accurate way to identify CSCs.
1.5 Implications of the CSC Hypothesis for Cancer Treatment
The discovery of cancer stem cells has important implications for oncology, and
many groups are now working to understand and exploit CSC biology to improve
cancer treatment. Despite tremendous progress in the identification of new cancer
targets and drug development, most cancers relapse after treatment and the goal of
increasing overall survival of cancer patients has been largely unmet by most new
drugs [132]. One explanation for this lack of sustained effect is that most current
therapies do not effectively inhibit CSCs.
Indeed, CSCs are resistant to chemo- and radiation therapies that effectively kill
other cells that comprise the tumor (discussed in detail in Chap. 3). It is for this
reason that CSCs are thought to regenerate cancer when therapy is discontinued.
This hypothesis holds that, because CSCs are a small population within the tumor
mass, drugs developed to kill the cells comprising the bulk of the tumor are initially
effective in shrinking tumor size but do not eradicate the cellular source and tumors
with higher CSC content and perhaps harboring additional drug-resistance mutations
regrow. This has been termed the “dandelion hypothesis,” analogous to the regrowth
of a weed if the root is not destroyed [133]. Although this hypothesis has yet to be
fully confirmed using a CSC-directed therapy in cancer patients, numerous studies
have correlated CSCs with poor prognosis in both leukemia and solid malignancies.
For example, glioblastoma multiforme patients whose tumors bear a relatively high
proportion of CD133+ cells, which have been found to possess characteristics of
CSCs, suffer from decreased response to therapy, higher malignancy, and signifi-
cantly lower survival time [134]. Additionally, gene signatures derived from CSCs
are themselves independent predictors of patient survival [99, 135] indicating the
relevance of targeting these cells.
12 C. Gao et al.
1.6 Concluding Remarks
A consensus is emerging that the CSC population represents a distinct and clinically
important population. However, there is still a further need to determine the precise
definition and cellular source of CSCs, to elucidate the appropriate cell markers to
isolate them, and to understand the pathways contributing their biological behavior.
In the end there is growing hope that this understanding will translate into clinically
useful treatments against cancer.
References
1. Pierce GB, Johnson LD (1971) Differentiation and cancer. In Vitro 7:140–145
2. Sell S, Pierce GB (1994) Maturation arrest of stem cell differentiation is a common pathway
for the cellular origin of teratocarcinomas and epithelial cancers. Lab Invest 70:6–22
3. Lapidot T, Sirard C, Vormoor J et al (1994) A cell initiating human acute myeloid leukaemia
after transplantation into SCID mice. Nature 367:645–648
4. 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
5. 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
6. Clarke MF, Dick JE, Dirks PB et al (2006) Cancer stem cells—perspectives on current status
and future directions: AACR Workshop on cancer stem cells. Cancer Res 66:9339–9344
7. Ishizawa K, Rasheed ZA, Karisch R et al (2010) Tumor-initiating cells are rare in many human
tumors. Cell Stem Cell 7:279–282
8. Shackleton MJ, Quintana E, Fullen DR, Sabel MS, Johnson TM (2009) Melanoma: do we
need a hatchet or a scalpel? Arch Dermatol 145:307–308
9. Nordling CO (1953) A new theory on cancer-inducing mechanism. Br J Cancer 7:68–72
10. Ashkenazi R, Gentry SN, Jackson TL (2008) Pathways to tumorigenesis—modeling mutation
acquisition in stem cells and their progeny. Neoplasia 10:1170–1182
11. Maguer-Satta V, Petzer AL, Eaves AC, Eaves CJ (1996) BCR-ABL expression in different
subpopulations of functionally characterized Ph+ CD34+ cells from patients with chronic
myeloid leukemia. Blood 88:1796–1804
12. Birnie R, Bryce SD, Roome C et al (2008) Gene expression profiling of human prostate cancer
stem cells reveals a pro-inflammatory phenotype and the importance of extracellular matrix
interactions. Genome Biol 9:R83
13. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic
and adult fibroblast cultures by defined factors. Cell 126:663–676
14. Yu J, Vodyanik MA, Smuga-Otto K et al (2007) Induced pluripotent stem cell lines derived
from human somatic cells. Science 318:1917–1920
15. Takahashi K, Tanabe K, Ohnuki M et al (2007) Induction of pluripotent stem cells from adult
human fibroblasts by defined factors. Cell 131:861–872
16. Stephens PJ, Greenman CD, Fu B et al (2011) Massive genomic rearrangement acquired in a
single catastrophic event during cancer development. Cell 144:27–40
17. Mani SA, Guo W, Liao MJ et al (2008) The epithelial-mesenchymal transition generates cells
with properties of stem cells. Cell 133:704–715
18. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC (1996) Isolation and func-
tional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med
183:1797–1806
1 Introduction to Cancer Stem Cells 13
19. Feuring-Buske M, Hogge DE (2001) Hoechst 33342 efflux identifies a subpopulation of
cytogenetically normal CD34(+)CD38(−) progenitor cells from patients with acute myeloid
leukemia. Blood 97:3882–3889
20. Wulf GG, Wang RY, Kuehnle I et al (2001) A leukemic stem cell with intrinsic drug efflux
capacity in acute myeloid leukemia. Blood 98:1166–1173
21. Szotek PP, Pieretti-Vanmarcke R, Masiakos PT et al (2006) Ovarian cancer side popu-
lation defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance
responsiveness. Proc Natl Acad Sci USA 103:11154–11159
22. Chiba T, Kita K, Zheng YW et al (2006) Side population purified from hepatocellular
carcinoma cells harbors cancer stem cell-like properties. Hepatology 44:240–251
23. Hirschmann-Jax C, Foster AE, Wulf GG et al (2004) A distinct “side population” of cells with
high drug efflux capacity in human tumor cells. Proc Natl Acad Sci USA 101:14228–14233
24. Setoguchi T, Taga T, Kondo T (2004) Cancer stem cells persist in many cancer cell lines. Cell
Cycle 3:414–415
25. Shen G, Shen F, Shi Z et al (2008) Identification of cancer stem-like cells in the C6 glioma
cell line and the limitation of current identification methods. In Vitro Cell Dev Biol Anim
44:280–289
26. Ho MM, Ng AV, Lam S, Hung JY (2007) Side population in human lung cancer cell lines and
tumors is enriched with stem-like cancer cells. Cancer Res 67:4827–4833
27. Salcido CD, Larochelle A, Taylor BJ, Dunbar CE, Varticovski L (2010) Molecular charac-
terisation of side population cells with cancer stem cell-like characteristics in small-cell lung
cancer. Br J Cancer 102:1636–1644
28. Mitsutake N, Iwao A, Nagai K et al (2007) Characterization of side population in thyroid
cancer cell lines: cancer stem-like cells are enriched partly but not exclusively. Endocrinology
148:1797–1803
29. Wang J, Guo LP, Chen LZ, Zeng YX, Lu SH (2007) Identification of cancer stem cell-like side
population cells in human nasopharyngeal carcinoma cell line. Cancer Res 67:3716–3724
30. Wu C, Wei Q, Utomo V et al (2007) Side population cells isolated from mesenchymal
neoplasms have tumor initiating potential. Cancer Res 67:8216–8222
31. Sussman RT, Ricci MS, Hart LS, Sun SY, El-Deiry WS (2007) Chemotherapy-resistant side-
population of colon cancer cells has a higher sensitivity to TRAIL than the non-SP, a higher
expression of c-Myc and TRAIL-receptor DR4. Cancer Biol Ther 6:1490–1495
32. Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG (2005) Side
population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2−
cancer cells are similarly tumorigenic. Cancer Res 65:6207–6219
33. Christgen M, Ballmaier M, Bruchhardt H, von Wasielewski R, Kreipe H, Lehmann U (2007)
Identification of a distinct side population of cancer cells in the Cal-51 human breast carcinoma
cell line. Mol Cell Biochem 306:201–212
34. Nakanishi T, Chumsri S, Khakpour N et al (2010) Side-population cells in luminal-type
breast cancer have tumour-initiating cell properties, and are regulated by HER2 expression
and signalling. Br J Cancer 102:815–826
35. Engelmann K, Shen H, Finn OJ (2008) MCF7 side population cells with characteristics of
cancer stem/progenitor cells express the tumor antigen MUC1. Cancer Res 68:2419–2426
36. Tabor MH, Clay MR, Owen JH et al (2011) Head and neck cancer stem cells: the side
population. Laryngoscope 121:527–533
37. Schmuck R, Warneke V, Behrens HM, Simon E, Weichert W, Rocken C (2011) Genotypic
and phenotypic characterization of side population of gastric cancer cell lines. Am J Pathol
178:1792–1804
38. Yanamoto S, Kawasaki G, Yamada S et al (2011) Isolation and characterization of cancer
stem-like side population cells in human oral cancer cells. Oral Oncol 47:855–860
39. Yang M, Yan M, Zhang R, Li J, Luo Z (2011) Side population cells isolated from human
osteosarcoma are enriched with tumor-initiating cells. Cancer Sci 102:1774–1781
40. Zhang SN, Huang FT, HuangYJ, Zhong W,Yu Z (2011) Characterization of a cancer stem cell-
like side population derived from human pancreatic adenocarcinoma cells. Tumori 96:985–
992
14 C. Gao et al.
41. Zhou S, Morris JJ, Barnes Y, Lan L, Schuetz JD, Sorrentino BP (2002) Bcrp1 gene expression
is required for normal numbers of side population stem cells in mice, and confers relative
protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci USA 99:12339–
12344
42. Zhou S, Schuetz JD, Bunting KD et al (2001) TheABC transporter Bcrp1/ABCG2 is expressed
in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.
Nat Med 7:1028–1034
43. Mao Q, Unadkat JD (2005) Role of the breast cancer resistance protein (ABCG2) in drug
transport. AAPS J 7:E118–133
44. Loebinger MR, Giangreco A, Groot KR et al (2008) Squamous cell cancers contain a side
population of stem-like cells that are made chemosensitive by ABC transporter blockade. Br
J Cancer 98:380–387
45. Katayama R, Koike S, Sato S, Sugimoto Y, Tsuruo T, Fujita N (2009) Dofequidar fumarate
sensitizes cancer stem-like side population cells to chemotherapeutic drugs by inhibiting
ABCG2/BCRP-mediated drug export. Cancer Sci 100:2060–2068
46. Yamamoto K, Suzu S, Yoshidomi Y, Hiyoshi M, Harada H, Okada S (2007) Erythroblasts
highly express the ABC transporter Bcrp1/ABCG2 but do not show the side population (SP)
phenotype. Immunol Lett 114:52–58
47. Yajima T, Ochiai H, Uchiyama T, Takano N, Shibahara T, Azuma T (2009) Resistance to
cytotoxic chemotherapy-induced apoptosis in side population cells of human oral squamous
cell carcinoma cell line Ho-1-N-1. Int J Oncol 35:273–80
48. Bhatt RI, Brown MD, Hart CA et al (2003) Novel method for the isolation and characterisation
of the putative prostatic stem cell. Cytometry A 54:89–99
49. Golebiewska A, Brons NH, Bjerkvig R, Niclou SP (2011) Critical appraisal of the side
population assay in stem cell and cancer stem cell research. Cell Stem Cell 8:136–147
50. Broadley KW, Hunn MK, Farrand KJ et al (2011) Side population is not necessary or sufficient
for a cancer stem cell phenotype in glioblastoma multiforme. Stem Cells 29:452–461
51. Wan G, Zhou L, Xie M, Chen H, Tian J (2010) Characterization of side population cells from
laryngeal cancer cell lines. Head Neck 32:1302–1309
52. Hosonuma S, Kobayashi Y, Kojo S et al (2011) Clinical significance of side population in
ovarian cancer cells. Hum Cell 24:9–12
53. Moserle L, Ghisi M, Amadori A, Indraccolo S (2009) Side population and cancer stem cells:
therapeutic implications. Cancer Lett 288:1–9
54. Sobhan PK, Seervi M, Joseph J et al (2011) Identification of heat shock protein 90 inhibitors to
sensitize drug resistant side population tumor cells using a cell based assay platform. Cancer
Lett 317:78–88
55. Foster AE, Okur FV, Biagi E et al (2010) Selective elimination of a chemoresistant side
population of B-CLL cells by cytotoxic T lymphocytes in subjects receiving an autologous
hCD40L/IL-2 tumor vaccine. Leukemia 24:563–572
56. Singh SK, Clarke ID, Terasaki M et al (2003) Identification of a cancer stem cell in human
brain tumors. Cancer Res 63:5821–5828
57. Singh SK, Hawkins C, Clarke ID et al (2004) Identification of human brain tumour initiating
cells. Nature 432:396–401
58. 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
59. Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ (2005) Prospective identification of
tumorigenic prostate cancer stem cells. Cancer Res 65:10946–10951
60. Hurt EM, Kawasaki BT, Klarmann GJ, Thomas SB, Farrar WL (2008) CD44+ CD24(−)
prostate cells are early cancer progenitor/stem cells that provide a model for patients with
poor prognosis. Br J Cancer 98:756–765
61. Prince ME, Sivanandan R, Kaczorowski A et al (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
