Group Title: Breast Cancer Research
Title: Breast cancer stem cells : implications for therapy of breast cancer
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Title: Breast cancer stem cells : implications for therapy of breast cancer
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Language: English
Creator: Morrison, Brian
Schmidt, Chris
Lakhani, Sunil
Reynolds, Brent
Lopez, J. A.
Publisher: Breast Cancer Research
Publication Date: 2008
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Abstract: The concept of cancer stem cells responsible for tumour origin, maintenance, and resistance to treatment has gained prominence in the field of breast cancer research. The therapeutic targeting of these cells has the potential to eliminate residual disease and may become an important component of a multimodality treatment. Recent improvements in immunotherapy targeting of tumour-associated antigens have advanced the prospect of targeting breast cancer stem cells, an approach that might lead to more meaningful clinical remissions. Here, we review the role of stem cells in the healthy breast, the role of breast cancer stem cells in disease, and the potential to target these cells.
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Review

Breast cancer stem cells: implications for therapy of breast cancer
Brian J Morrison1-3, Chris W Schmidt1, Sunil R Lakhani1,4,5, Brent A Reynolds3,6
and J Alejandro Lopez1'2'7


1 Queensland Institute of Medical Research, Royal Brisbane Hospital Post Office, Brisbane 4029, Australia
2Griffith University, 170 Kessels Road, Nathan 4111, Australia
3The Queensland Brain Institute, University of Queensland, QBI Building (79) St Lucia 4072, Australia
4Molecular & Cellular Pathology, School of Medicine, University of Queensland, Herston Rd, Herston 4006, QLD, Australia
5Pathology Queensland, Royal Brisbane & Women's Hospital, Brisbane 4029, Australia
6Current address: Department of Neurosurgery, McKnight Brain Institute, University of Florida, 1600 SW Archer Road, Box 100265, Gainesville,
FL 32610, USA
7School of Medicine, University of Queensland, Herston Rd, Herston 4006, Australia


Corresponding author: J Alejandro Lopez, alejl@qimr.edu.au


Published: 22 July 2008
This article is online at http://breast-cancer-research.com/content/10/4/210
C 2008 BioMed Central Ltd



Abstract
The concept of cancer stem cells responsible for tumour origin,
maintenance, and resistance to treatment has gained prominence
in the field of breast cancer research. The therapeutic targeting of
these cells has the potential to eliminate residual disease and may
become an important component of a multimodality treatment.
Recent improvements in immunotherapy targeting of tumour-
associated antigens have advanced the prospect of targeting
breast cancer stem cells, an approach that might lead to more
meaningful clinical remissions. Here, we review the role of stem
cells in the healthy breast, the role of breast cancer stem cells in
disease, and the potential to target these cells.

Introduction
The past decades have seen advances in the diagnosis and
treatment of breast cancer. Despite this progress, breast
cancer is still a leading cause of cancer-related deaths
among women, with as many as 40% relapsing with meta-
static disease [1]. Breast cancer survival rates have been
shown to plateau after 7 to 10 years, whereas most cancer
survival curves take between 2 and 5 years to plateau [2].
The length of time for the survival rate to plateau in breast
cancer might indicate the involvement of a cell type capable
of disease recurrence which is able to withstand primary
treatment and reside in the body, often undetected, for
prolonged periods. Interestingly, it has been shown that, of
the 40% of patients with lymph node involvement who did not
undergo surgical removal, only 15% had recurrence of
disease [3]. This raises the point that immune system


Breast Cancer Research 2008, 10:210 (doi:l 0.1186/bcr2l11)


surveillance of tumours or other protective mechanisms of the
body might be capable of controlling breast cancer relapses.

Prominent in the breast cancer field has been the notion of
the existence of a transformed population of cells with many
of the properties of stem cells that may be responsible for the
origin and maintenance of tumours. These stem cell-like cells,
designated as cancer stem cells, represent a minor subset of
cells in the tumour and are distinct from the more differen-
tiated tumour cells. It is thought that these cancer stem cells
may play an important role in cancer establishment,
progression, and resistance to current treatments. Traditional
cancer therapies are effective at debulking some tumours but
often fail to produce long-term clinical remissions, possibly
due to their inability to eradicate the cancer stem cell
population. Therefore, novel treatments aimed at targeting the
cancer stem cell population could find use in treating both
primary and metastatic tumours.

Therapies aimed at targeting cancer stem cells may prove
clinically relevant in inducing long-term clinical remission of
cancer. A variety of methods, including inducing differentia-
tion of the cancer stem cells or targeting of cancer stem cells
for elimination, are being studied to disrupt the cancer stem
cell pool. Cancer stem cell antigens may also provide a new
target for cancer immunotherapy. The targeting of breast
cancer stem cells (BCSCs) through immunotherapy, such as
dendritic cell (DC)-based therapies or adoptive T-cell


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ABC = ATP-binding cassette; ALDH1 = aldehyde dehydrogenase-1; BCSC = breast cancer stem cell; BMP = bone morphogenetic protein; CK =
cytokeratin; CT = cancer/testis; CTL = cytolytic T-cell response; DC = dendritic cell; EGFR = epidermal growth factor receptor; ER = oestrogen
receptor; ESA= epithelial surface antigen; Hh = Hedgehog; Lin- = lineage-negative; MDR = multidrug resistance; MHC = major histocompatibility
complex; NOD= nonobese diabetic; PgR = progesterone receptor; RA = retinoid acid; SCID = severe combined immunodeficiency disease; SP=
side population; TAA = tumour-associated antigen; TDLU = terminal duct lobular unit.








Breast Cancer Research Vol 10 No 4 Morrison et al.


transfer, has the advantage of treating the putative cells of
tumour origin and can be used in conjunction with current
treatment regimes. For these treatments to become effective,
cancer stem cells will need to be defined in terms of
antigenicity and distinctions between cancer stem cells and
stem cells must be found. This review will discuss stem cells
and their role in mammopoiesis, cancer stem cells and their
function in tumour formation, and the potential targeting of
cancer stem cells for therapy with a focus on breast cancer.

Stem cells
Somatic stem cells are responsible for tissue homeostasis in
the adult and have limited plasticity. They are responsible for
tissue renewal and repair and can become activated in
response to environmental signals such as hormones.
Somatic stem cells are part of a hierarchy of cells in the
tissue, including the slowly proliferating somatic stem cell, the
more differentiated and proliferating transit-amplifying cell
progeny, and the several lines of differentiated cells. While
stem cells are mostly quiescent, they may asymmetrically
divide to give rise to one transit-amplifying cell and another
stem cell [4]. This provides continuation of the stem cell
compartment while providing the starting material for produc-
tion of differentiated cells. Somatic stem cells may also divide
symmetrically to produce two stem cells. Understanding the
role that the maintenance of cell division and differentiation of
stem cells plays may lead to new insights into the signalling
pathways involved in cancer progression and, ultimately, yield
new approaches for cancer treatment.

Stem cells have basic defining and identifying properties. The
ability for self-renewal, in which through cycles of cell division
a stem cell gives rise to at least one daughter cell with the
same characteristics of the parent, is a critical characteristic.
The proliferative potential of cancer stem cells has been
examined in vitro, in particular for stem cell-like neural
precursors of human glioblastoma, and has been charac-
terised as capable of exponential proliferation, long-term
proliferation, self-renewal, and multipotency [5]. The plasticity
of stem cells allows them, when activated, to renew several
lineages of differentiated cells during homeostasis. In adult
tissue, the stem cell population rarely divides, but when
stimulated by hormones during development or by a loss of
transit-amplifying cells they can be rapidly activated to
undergo asymmetric cell division to renew the tissue
compartment. Transit-amplifying cells can expand rapidly,
providing progeny that differentiate into mature cells of
varying lineages. Nonproliferative differentiated cells make up
the bulk of the tissue and undergo apoptosis after a finite life
span. In this way, under most conditions, the total number of
cells in a tissue is maintained in equilibrium, with the number
of differentiated cells dying being equalled by the number of
progenitor cells dividing.

Stem cells have a relatively lineage-negative (Lin-) phenotype
with few cell surface markers identified. One property shared


by normal stem cells and cancer stem cells is the expression
of the ATP-binding cassette (ABC)-G2 transporter. The
ABCG2 is a class of drug transporters capable of pumping
out of the cell a variety of substrates, including cytotoxic
drugs, by using ATP energy [6]. High expression of these
transporters may help protect cancer stem cells from
cytotoxic agents used for cancer treatment. The ABCG2
transporter has been shown to specifically pump out the
DNA-intercalating dye Hoechst 33342 [6]. Activity of this
transporter leads to the identification of a population of cells
known as the side population (SP) by flow cytometric
analysis. This functional property has been used to study
mammary stem cells, which upon transplantation into cleared
mammary fat pads have been shown to give rise to breast
tissue [7,8]. However, other studies have called into question
the use of the SP to isolate cells with potential to reconstitute
a functional mammary gland [9].

The human breast and mammopoiesis
Mammopoiesis is the development of the cellular lineages
and functional units of the mammary gland. These units are
comprised of terminal ductules and alveoli, which together
form the terminal duct lobular units (TDLUs). Collectively,
TDLUs form the branches of a greater ductal-lobular system
composed of an inner layer of polarized luminal cells and an
outer layer of myoepithelial cells [10]. The adult human breast
is composed of 15 to 20 lobes each with multiple lobules
surrounded by adipose tissue. Additionally, the breast has a
system of lymphatic vessels responsible for draining breast
tissue leading to internal mammary lymph nodes and axillary
regional lymph nodes. The human breast is a dynamic gland
with tissue homeostasis occurring during early development,
puberty, within menstrual cycles, during pregnancy and
lactation, and eventual involution during menopause. It is
believed that certain of these processes are brought about by
the action of somatic stem cells.

The breast originates from the invagination of the epidermis
into the underlying mesenchymal tissue during the 10- to
24-week period of gestation. This process gives rise to
epithelial ducts, which in turn give rise to rudimentary
lactiferous ducts. Unlike a variety of other organs, the human
breast continuously undergoes morphological and functional
changes well into adulthood, with a secondary onset during
puberty and culminating with the greatest differentiation
occurring during pregnancy and lactation. Oestrogen
hormonal stimulation during puberty drives the ductal
elongation of the breast with stem cell activity found in the
terminal end buds [11]. Prolactin and progesterone drive
ductal branching and formation of acini, leading to the
formation of mature breast tissue [1 2]. Two major subclasses
of cells appear at this time: the outer myoepithelial (or basal)
cells and the inner luminal epithelial cells. Myoepithelial cells
are characterized by expression of common acute
lymphoblastic leukaemia antigen (CALLA) or CD10 [13],
Thy-1 [10], alpha-smooth muscle actin [14], vimentin [15],


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and cytokeratin (CK) 5 and CK14 amongst others [16].
Myoepithelial cells are contractile cells that form a sheath
around the ductal network of the breast. Luminal epithelial
cells are characterized by expression of MUC1 [17],
epithelial surface antigen (ESA) also known as EpCAM
(epithelial cell adhesion molecule) [18], and CK7, CK8,
CK18, and CK19 [16] as well as oestrogen receptor (ER)
and progesterone receptor (PgR). During periods of
pregnancy and lactation, the breast goes through further
rounds of development with an increase in cell growth and
formation from the luminal epithelial lineage of functional milk-
secreting alveoli. Following these periods and again during
menopause, there is an involution through apoptosis of the
breast tissue [19].

Mammary stem cells
In the 1950s, Deome and colleagues [20] performed one of
the earliest demonstrations of the existence of adult
mammary stem cells, using a limiting-dilution assay showing
that clonal precursor cells are capable of forming functional
mammary outgrowths in a murine transplantation model. This
cleared fat pad assay consists of clearing the endogenous
epithelium of mice followed by transplantation of a limited
dilution of cells, fewer than 2 x 104, into the gland-free
mammary fat pads of mice. Several additional lines of
evidence have indicated that mammary stem cells are active
in forming and maintaining breast tissue. X-chromosome
inactivation studies have shown that entire areas of the
breast, in particular individual TDLUs, are monoclonal in origin
[21-23]. These areas are derived from one progenitor cell
determined during early embryogenesis which has undergone
random inactivation of one chromosome around day 16.
Additional evidence indicating the clonal origin of areas of
breast tissue has come from retroviral tagging of mammary
epithelial cells. Single cells transplanted into the cleared fat
pads of mice can produce progeny capable of forming a
functional mammary gland [24]. More recently, it has been
shown that a single cell with stem cell-like features is capable
of forming a complete and functional mammary gland upon
transplantation into the cleared fat pads of female mice [9].

Role of stem cells in tumourigenesis
Several models have been used to explain the origin and
continued growth of tumours. Perhaps the most prominent
model used to describe cancer is the clonal evolution of
tumours theory. This model postulates that cancer originates
from mutations occurring in a few cells or a single cell that
eventually leads to uncontrolled and unlimited proliferation of
a population of cells [25]. Genetic alterations continue to
accumulate as the tumour progresses, leading to activation of
proto-oncogenes into oncogenes and inactivation of various
tumour-suppressor genes and ultimately giving rise to
subtypes of cells in the tumour which have acquired several
traits such as the ability to evade apoptosis, self-sufficiency in
growth signalling, tissue invasion and metastasis, and
limitless replicative potential [26]. This model further


Available online http://breast-cancer-research.com/content/1 0/4/210



postulates that mutations in various cells would allow for a
selection of cells to have a survival advantage over others,
leading them to proliferate to a greater extent and allowing
those cells to seed new tumours capable of further rounds of
clonal expansion. The clonal evolution of tumours model is
capable of explaining some of the key characteristics of
cancer growth but is perhaps too simplistic. Building upon
the clonal evolution theory, there are two different models for
how tumours develop and progress through unlimited cell
division: the stochastic and hierarchical models of tumour
development. The stochastic model postulates that all cells in
a tumour have equal potential to be tumourigenic (that is, any
cell from that tumour has an equal probability to form a new
tumour with characteristics similar to the primary tumour) [27].
The hierarchical model postulates that only a subset of the
cells in a tumour have this tumourigenic capacity and that the
rest of the tumour is populated by cells with varying degrees
of differentiation which cannot regenerate the tumour on their
own [27]. The latter model is in concordance with the cancer
stem cell hypothesis, in which the cancer stem cell is the cell
responsible for tumour self-renewal and not the differentiated
cells that make up the bulk of the tumour.

Tumours have been characterized as heterogenous, com-
posed of several types of differentiated and undifferentiated
cells. What, then, is the cell type that can be transformed and
give rise to both differentiated and undifferentiated cells?
Epithelial cells in the breast do not proliferate greatly and are
continuously being replaced and thus they do not have a
considerable opportunity to be the target of mutation events.
Though possible, it seems unlikely that well-differentiated
cells could be the source of the transformation event or that
they would then digress to form several undifferentiated cells
as well as differentiated ones. Some experimental models of
cancer have shown that tumours are initiated from cancer cell
lines only when large numbers of cells are used (that is, using
a larger amount of cells allows for the rare cancer stem cell-
like cell to be included in the transplant). In these models,
most of the cells in the original tumour or cell line lack the
ability to establish cancer. In one experiment model of colon
cancer, CD133, a stem cell antigen used for sorting cells
with stem cell-like qualities, was used to show that the rare
subpopulation in the tumour of CD133+ cells was respon-
sible for tumour maintenance. The in vitro growth charac-
teristic of CD133+ colon cancer tumour cells was compared
with CD133- cells. CD133+ cells grew exponentially as
undifferentiated tumour spheres in serum-free medium in vitro
and were more tumourigenic in vivo than CD133- cells [28].
These results indicate that there is an intrinsic difference in
the growth rate of stem cell-like cells compared with other
cell types in certain cancer models. Taken together, these
results seem to indicate the presence of cancer stem cells at
work driving tumour formation. Revising the clonal origin of
cancer theory, tumours derived from a transformation event
are seeded by a small subset of cells with cancer stem cell
characteristics. These proliferating undifferentiated cells with


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Breast Cancer Research Vol 10 No 4 Morrison et al.


stem cell-like phenotypes can then give rise to the
nondividing differentiated cells seen in tumours. Furthermore,
mutations affecting the cancer stem cell compartment are the
ones that are passed on to the descendant population and
multistep tumour progression occurs through this small
population of cells. This revision might also be too simplistic
as the hierarchical and stochastic models of tumour formation
might be working at the same time and might not be mutually
exclusive; indeed, some tumours could be the products of
transformation events in differentiated cells. Additionally, it is
important to note that for therapeutic purposes it might be
advantageous to identify cells capable of metastatic disease,
an event that can occur early in tumourigenesis, rather than
focus on populations that are responsible for initial tumour
formation. In this case, the focus is on cancer stem cells that
are able to seed new tumours.

Stem cells and cancer cells share a number of important
characteristics. They both have the capacity for self-renewal
and extensive proliferation. In the case of tumour cells, this
takes the form of self-sufficiency in growth signalling and
uncontrolled cellular proliferation, whereas for stem cells, this
is a tightly controlled process that occurs during embryo-
genesis, organogenesis, and maintenance and repair of adult
tissues. Both cell types are long-lived with active anti-
apoptotic pathways and telomerase activity [29]. This feature
makes stem cells more prone to accumulation of damaging
mutations and genomic instability despite active DNA repair
mechanisms. For tumours, there are often even higher risks of
accumulating mutations as defects in DNA repair mecha-
nisms are often present. Both cell types have a resistance to
environmental toxins, chemotherapeutic and radiation agents,
often as a result of multidrug resistance (MDR) via expression
of ABC transporter proteins and selection by chemotherapy.
Additionally, it should be noted that stem cells are relatively
resistant to radiation because they are slow-cycling. It is
thought that cells in a tumour which are able to withstand
radiation might be cancer stem cells that have this slow-
cycling quality. Stem cells and tumours also share the
characteristic of being mobile, leading to migration and
homing for stem cells and potentially to metastatic disease for
tumour and cancer stem cells [30]. Anchorage independence
is one of the most important characteristics of transformed
cells (including metastatic cells) and is a property of normal
stem cells. These stem cell characteristics that are common
to cancer cells suggest that fewer or different steps might be
involved for stem cells to transform into tumour-initiating cells
in comparison with differentiated cells.

The evidence for the existence of adult stem cells responsible
for the initiation and maintenance of cancer has been
characterized for several tumours recently. During the 1990s,
studies of acute myeloid leukaemia first confirmed that
transformed stem cell-like cells were capable of being the
origin of tumours [31,32]. These results were then confirmed
in relation to the brain [5,33] and the breast [34,35],


demonstrating that stem cells are involved in the initiation of
leukaemias and solid tumours.

Breast cancer stem cells
Some indication that stem cells play a role in breast cancer
comes from epidemiology data on breast cancer incidence
following radiation exposure. Women exposed to radiation in
their late adolescence following the Hiroshima and Nagasaki
atomic blasts had the highest susceptibility of breast cancer
20 to 30 years later compared with women exposed at other
age groups [36]. This suggests that adult mammary stem
cells accumulate genetic changes leading to transformation
over several years with the eventual development of solid
tumours. A model of tumour formation and development
which takes into account the role of stem cells as primary
targets for mutation should be incorporated into the view of
how breast cancer initiation occurs. Additionally, early or late
progenitor cells could be the targets of transforming events.
In this case, these progenitor cells would need to acquire,
through mutations or epigenetic changes, the characteristics
of stem cells such as self-renewal. Models of carcinogenesis
are contentious, but the role that cancer stem cells play in
tumour formation is beginning to be further defined. Recently,
AI-Hajj and colleagues [35] showed that human BCSCs,
identified on the basis of CD44+, CD24-l0ow, Lin- expression,
could form tumours when as few as 100 cells were injected
into nonobese diabetic/severe combined immunodeficiency
disease (NOD/SCID) mice. These cells have some of the key
characteristics of stem cells. When 20,000 cells without this
phenotype were used, they were unable to form a tumour.
These experiments indicate that tumour initiation could be
driven by rare BCSCs.

Studying mammary stem cells and breast
cancer stem cells
Advances in cell culture approaches have been important in
identifying and studying mammary stem cells. The study of
mammary stem cells in vitro has been based upon work
identifying neural stem cells through a cell culture assay
known as the neurosphere assay, which makes use of serum-
free medium supplemented with epidermal growth factor and
basic fibroblast growth factor [37,38]. Application of the
neurosphere assay culture conditions has been used to
identify undifferentiated human mammary stem cells grown in
culture [39] known as mammospheres and to identify a
candidate human BCSC [34]. These culture systems have
shown that mammospheres exhibit stem cell-like functional
properties of relative quiescence and phenotypic properties
such as ESA, CK5, and a-6-integrin expression. The use of
flow cytometry for phenotyping and isolating putative
mammary stem cells and BCSCs has also been critical in
studying these cell populations. In addition to identifying the
SP, flow cytometry has been used to show expression of
CD44 and ESA on human mammary progenitors [40] and
human BCSC populations [34,35]. ESA expression has also
been identified in both primary breast cancer tumours and


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Table 1

Phenotypic characteristics of breast cancer stem cells

Factor Characteristics Reference(s)

Cell surface markers that are expressed by putative breast cancer stem cells
ABCG2 ABCG2 (ATP-binding cassette G2) is a class of drug transporters capable of pumping [6]
cytotoxic drugs out of the cell.
CD44 CD44 is involved in cellular adhesion, motility, and metastases. [34,35]
CD10 CD10 is a common acute lymphoblastic leukaemia antigen that is overexpressed on many [39]
tumours.
EpCAM/ESA Epithelial cell adhesion molecule/Epithelial surface antigen is expressed on mammary [39]
tissue and tumours.
CD29 (pl-integrin) CD29 is a membrane receptor involved in cell adhesion and metastatic diffusion of tumour cells. [9]
CD49f (ca6-integrin) CD49f is involved in basal and endothelial cell distribution and is a candidate stem cell marker. [39]
CD133 (prominin-1) CD133 is a cell surface glyoprotein with an unknown function in cancer stem cells and its [116,117]
expression is documented for various types of cancer.
ALDH1 Aldehyde dehydrogenase-1 plays a role in the differentiation of stem cells and its activity [112]
predicts poorer clinical outcomes.
CXCR4 CXCR4 is a chemokine receptor involved in metastasis and its expression is increased [44,45]
in mammospheres.
ER Oestrogen receptor is expressed on breast cancer cells, mammary progenitors, and [54,59]
breast cancer stem cells.
Signalling pathways that play a role in cancer stem cells
Delta/Notch pathway This pathway is involved in cell fate development and is expressed in stem cells and early [60]
progenitor cells.
Notch-4 Notch-4 plays a role in mammary development and its overexpression has been shown to [45,60,61]
promote mammary tumours.
Wnt signalling pathway This pathway is involved in stem cell self-renewal and its overexpression can lead to [27,63]
epithelial and mammary tumours.
P-catenin P-catenin is a downstream target of the Wnt pathway. A pro-oncogenic role has been [63]
described.
Hedgehog/Patched pathway This pathway is involved in embryonic growth and cell fate determination. [64]
PITCH A receptor for the Hedgehog signalling family, PITCH has been connected to early [64]
embryonic tumourigenesis.
EGFR Epidermal growth factor receptor signalling has been found to be upregulated in breast [77,111]
cancer stem cells and may be required for mammosphere formation.


secondary metastasis as well as a variety of other malig-
nancies [41]. Flow cytometric analysis has been important in
demonstrating that SP cells are present in the human breast
[7,39]. The SP is further increased in mammosphere cultures
compared with freshly isolated primary tissue samples [39].
Additional candidate stem cell markers are waiting to be
identified. Table 1 lists some phenotypic characteristics of
BCSCs.

Breast cancer and metastatic disease
Results from experiments using primary human cancer tissue
have shown that not all cells in a tumour are equivalent; a
minority tumourigenic cell population exists in solid tumours


of both the breast and the brain which represents around 1%
to 2% of the total tumour burden [35,42]. It should be noted
that the cells that are capable of metastasising might have a
phenotype different from the original tumour-initiating cells.
Targeting cells that have the potential to metastasise will be
an important application of the BCSC field as these are the
cells that cause the majority of mortality from breast cancer.

Circulating tumour cells often can be detected in patients
with both primary and metastatic disease and the presence of
these cells often is associated with worse prognosis for
survival [43]. Metastasis is a nonrandom and organ-specific
process undertaken by certain cell types. In the case of


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Breast Cancer Research Vol 10 No 4 Morrison et al.


breast cancer, metastatic disease often follows a pattern
involving metastases to regional lymph nodes and then to
bone marrow, lung and liver, and brain [44]. Interestingly, it
has been shown that homing and migration pathways of
haematopoietic/leukocyte cells might be involved in BCSCs
and metastatic disease. CXCR4, a chemokine receptor
expressed by haematopoietic stem cells which binds
CXCL1 2, has been shown to be increased by a factor of four
in mammospheres and to be expressed in both metastatic
breast cancer cells and neuroblastomas [44-46]. Additionally,
the organs that form the main target of breast cancer
metastasis have the highest expression of the ligand
CXCL12 [44]. This indicates the importance of both the
BCSC metastatic 'seed' and the 'soil' of the organ of
metastasis for this process to occur. The CXCR4/CXCL1 2
pathway could provide a new target to specifically neutralise
the cells in tumours that are capable of forming new
metastases before metastatic disease occurs.

The cancer stem cell compartment is thought to be the
reason for initiation of disease, resistance to treatment, and
occurrence of metastatic disease. Hence, the identification
and targeting of any of these cancer stem cell
compartments, without toxicity to normal stem cells, will be
an important goal of immunotherapy of breast cancer. This
treatment will probably find most utility in the setting of
targeting cells with a metastatic potential. However,
disrupting the cancer stem cell compartment is made more
difficult by the realisation that disruption of supporting cells
around the stem cells, or differentiated tumour cells around
cancer stem cells, could have deleterious effects by
disrupting putative stem cell niches.

Stem cell niches
Stem cell niches are defined as locations in a tissue which
specifically can support the existence of somatic stem cells.
Niches allow the repopulation of the stem cell compartment
from migrating stem cells or even from differentiated cells if
the stem cell compartment is depleted [47-49]. It is possible
that tumour therapy that disrupts the stem cell niche through
ablation of the surrounding differentiated cells could lead to
the subsequent death of the cancer stem cells. Alternatively,
tumour therapy that depletes stem cells, but does not
eradicate the stem cell niche, could lead to repopulation of
the stem cell niche with additional cancer stem cells. Murine
mammary stem cells have been shown to be resident in the
peripheral caps of terminal end buds [50]. Identifying
candidate stem cell niches in human breast tissue has been
difficult. In humans, terminal end bud structures are not as
prominent and identifying stem cell zones has had to rely on
microdissection followed by cell sorting and functional
characterisation of putative cells to determine stem cell
niches in ducts and lobules. Villadsen and colleagues [51]
recently identified a stem cell niche in the ductal tissue with
cells with the characteristics of clonal growth, self-renewal,
and bipotency and positive staining for putative stem cell


markers K19 and K14. Identification of the properties of stem
cell niches will be important for targeting BCSCs as it will be
necessary to disrupt the inappropriate signalling that the stem
cell niche may provide to achieve lasting clinical effects.

Hormone receptor status of the putative
breast cancer stem cells
Breast cancer subtypes include at least two different cellular
phenotypes, one reminiscent of basal lineages and the other
of luminal lineages [52]. This has led to the idea that various
breast cancer subtypes might arise via mutations in different
compartments of stem cells [53,54]. In situ observations
have identified candidate cells with stem cell-like feature of
various phenotypes. Some of these observations have
identified candidate stem cells that are ER+. Indeed, ER+
stem cells have been identified as being important in adult
mammary gland homeostasis [54]. However, ER- stem cells
resident in the mammary tissue have also been identified and
might represent the more primitive mammary stem cells.
Recently, it has been shown that the mammary reconstituting
cells are ER- and PgR- [55]. Recent experiments have
demonstrated a role for BRCA1 being involved in the
differentiation of human ER- stem/progenitor cells into ER+
luminal epithelial cells [56]. The deletion of BRCA1 results in
the prevention of the transition of ER- stem cells into ER+
progenitor cells. Heterozygous mutations in the BRCA 1 gene
predispose women to breast and ovarian cancer [57], with
tumours often being of the basal-like phenotype charac-
terised by a lack of expression of ER, PgR, and HER-2. This
work suggests a model in which a block in BRCA 1-mediated
transition from stem cells to progenitor cells results in an
increase in ER- stem cells that can then be the pool of target
cells for further mutation events. However, greater than two
thirds of breast cancer tumours are ER+ and the majority of
these tumours are dependent on oestrogen for growth and
thus can be treated with hormonal therapy [58]. A model of
breast cancer origin has been proposed in which ER+
tumours are derived from ER+ stem cells or ER+ early or late
progenitor cells and ER- tumours are derived from the more
primitive ER- stem cells [59]. Other models have postulated
that ER- stem cells, which rarely divide and are also resistant
to hormonal therapy, can generate ER+ short-term transit-
amplifying cells that in turn give rise to ER+ differentiated
cells. These models suggest that the diversity seen in tumour
types among patients could be a direct result of trans-
formation events occurring in different lineages of stem cells
or progenitor cells. Further defining the hormone receptor
status of BCSCs will have important implications on the
treatment of disease as hormone receptor status can dictate
treatment options and is known to be an indicator of prognosis.

Pathways involved in self-renewal and
differentiation
Among the most important characteristics of stem cells are
the capacity for self-renewal and the regulation of the balance
between self-renewal and differentiation. Signalling pathways,


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such as Hedgehog (Hh), Wnt/p-catenin, and Notch, that play
a role in embryogenesis and organogenesis also play a role in
the maintenance of tissues in the adult through regulation of
the balance between self-renewal and differentiation of stem
cells. In the mammary gland, these three signalling pathways
play a role in stem cell self-renewal and thus represent
potential targets for therapy for BCSCs.

The Notch family of transmembrane signalling proteins are
involved in cell fate development and are expressed in stem
cells and early progenitor cells [60]. Notch-4 plays a role in
normal mammary development as a constitutively active form
of overexpressed Notch-4 has been shown to suppress
differentiation of breast epithelial cells in vitro [61] and to
suppress the development of normal mammary glands while
promoting the development of mammary tumours in vivo [62].
This suggests that alterations in Notch-4 signalling might play
a role in the transition of a healthy stem cell to a cancer stem
cell. Additionally, expression of Notch family members has
been found on mammospheres and Notch ligands are
capable of affecting the self-renewal and differentiation
capacity of healthy mammary cells, indicating a role for Notch
in breast cancer development [45].

The Wnt pathway is involved in cell fate determination in
many organs, including the developing mammary gland. A
pro-oncogenic role for P-catenin, a downstream target of Wnt
signalling, has also been described. Wnt signalling has been
shown to play a role in haematopoietic self-renewal, and
experimental evidence from transgenic mouse models has
shown that activation of the Wnt signalling pathway in stem
cells can lead to epithelial tumours [27]. Overexpression of
Wnt in mouse mammary glands can also lead to increased
mammary tumour formation [63]. Taken together, these data
indicate the involvement of Wnt signalling pathway members
and P-catenin in the deregulation of stem cells into cancer
stem cells.

The Hh/Patched pathway is important for embryonic growth
and cell fate determination during development. The PITCH
membrane protein (product of the tumour suppressor gene
Patched) is a receptor for the Hh family of signalling
molecules and has been connected to early embryonic
tumourigenesis [64]. Alterations in this pathway have been
implicated in several types of cancer, including breast,
prostate, and lung cancer.

Breast cancer stem cells as therapeutic
targets
In the past two decades, more than 30 new anticancer drugs
have been introduced, but survival rates have improved only
marginally for many forms of cancer [65]. In contrast to most
cancer cells, cancer stem cells are slow-dividing and have a
lowered ability to undergo apoptosis and a higher ability of
DNA repair, making them more resistant to traditional
methods of cancer treatment such as radiation and chemo-


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therapy. In vitro experiments comparing differentiated breast
cancer cells grown under monolayer conditions with
CD24-/lw CD44+ cancer stem cells grown under mammo-
sphere conditions showed that the stem cell-like population
was more resistant to radiation [66]. In addition, stem cells
express ABC drug transporters, which protect the cell from
cytotoxic agents and may lead to MDR [67]. Current anti-
cancer therapy is effective at debulking the tumour mass but
treatment effects are transient, with tumour relapse and
metastatic disease often occurring as a result of the failure of
targeting cancer stem cells. For therapy to be more effective,
debulking of differentiated tumours must occur followed by
targeting of the remaining surviving, often quiescent, tumour
stem cells. This could be accomplished by differentiating
BCSCs through differentiating therapy or eliminating them via
immunotherapy.

Differentiation therapy targeting cancer stem
cells
One way to target cancer stem cells is to induce the cancer
stem cells to differentiate. Targeting the cancer stem cell pool
to differentiate results in the loss of the ability for self-renewal,
a hallmark of the cancer stem cell phenotype and the reason
behind maintenance of the cancer stem cells. One
differentiation agent used in the clinic is retinoid acid (RA)
(vitamin A) [68]. RA and vitamin A analogues can promote
differentiation of epithelial cells and reverse tumour
progression through modulation of signal transduction. RA-
based therapy followed by chemotherapy has found use in
acute promyeloctyic leukaemia and could also find use in
solid tumour therapy [69]. Recently, the use of bone morpho-
genetic protein (BMP)-4 has been described as a non-
cytotoxic effector capable of blocking the tumourigenic
potential of human glioblastoma cells [70]. This therapeutic
agent is able to work by reducing proliferation and inducing
expression of neural differentiation markers in stem-like
tumour-initiating precursors. These findings are intriguing in
light of the role that BMP-4 may play in some breast tumours
[71]. Finding ways to specifically target BCSCs via
differentiation therapy is an application that needs to be
further defined.

Targeting stem cells for elimination
Much of cancer therapy research is focused on targeting
specific markers on tumour cells that are overexpressed or
mutated and that often represent essential genes/proteins or
pathways thought to be important for the development of the
tumour. For instance, traztuzamab (Herceptin) targets the
HER-2/neu (ErbB2) oncogene, a member of the epidermal
growth factor receptor (EGFR) kinase family, a protein
overexpressed on roughly 30% of breast tumours [72]. While
these approaches have seen some clinical successes, the
cancer stem cell model predicts that only by targeting the
remaining cells left over after treatment, the putative cancer
stem cells, will significant clinical remissions of the disease
occur. It is important to note not only that tumours may be


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Breast Cancer Research Vol 10 No 4 Morrison et al.


driven by mutated proteins and inappropriate signalling, but
also that epigenetic mechanisms of gene expression of genes
involved in 'stem-ness' such as Oct4, Nanog, and Sox2 could
be behind tumour formation [73]. Reversal of these
epigenetic switches of cancer stem cells could be one novel
way to target cancer stem cells. New therapeutics aimed at
eliminating cancer stem cells could also be achieved through
a variety of methods: targeting the self-renewal signalling
pathways critical for cancer stem cells, targeting the ABC
drug transporters that cancer stem cells use to evade
chemotherapy, or inducing the immune system to eliminate
the cancer stem cells through various immunotherapeutic
interventions.

Targeting of molecular signalling pathways
and drug transporters
The use of the steroid-like molecule cyclopamine to inhibit the
Hh signalling pathway has shown some promise in inhibiting
the growth of medulloblastoma and could be used in
treatments of other tumours [68]. The Wnt pathway can also
be inhibited through a variety of mechanisms. Targeting of 3-
catenin has received a lot of attention as RA has been shown
to inhibit P-catenin activity [74] and tyrosine kinase inhibitors
such as imatinib (Gleevac) have been shown to down-
regulate P-catenin signalling [75]. Finally, the Notch pathway
has also been investigated as a target. An antibody capable
of blocking Notch-4 has been used ex vivo to block the
formation of mammospheres from primary human specimens
[76]. This indicates the potential to block the self-renewal
capacity of BCSCs in the patient with this antibody and
opens up the use of other antibody therapies in the
elimination of BCSCs.

In vitro experiments have shown the resistance of BCSCs to
chemotherapy and radiation. Recent clinical evidence has
established that tumourigenic breast cancer cells with high
expression of CD44 and low expression of CD24 are
resistant to chemotherapy [77]. Breast cancer patients
receiving neoadjuvant chemotherapy had an increase in the
CD44+/CD2410w population of cells following treatment.
These cells retained the capacity to form mammospheres
(demonstrating self-renewal) and had an enhanced propensity
for forming tumours in SCID/Beige mice compared with
pretreatment samples, increasing from 4 of 14 (29%) to 7 of
14 (50%) patient samples transferred. Treatment of patients
with HER-2-positive tumours with lapatinib, an EGFR and
HER-2/neu (ErbB-2) dual-tyrosine kinase inhibitor, resulted in
nonstatistically significant decreases in the percentage of
CD44+/CD2410w population and in the ability for self-renewal
as assessed by mammosphere formation. Thus, inhibition of
regulatory pathways involved in self-renewal may confer
improved clinical outcomes by targeting BCSCs.

The high expression of ABC transporters such as breast
cancer resistance protein (BRCP-ABCG2) and MDR-
associated protein-1 (ABCB1/MDRR1) is a property of stem


cells which is also a feature of cancer stem cells [6]. These
transporters provide a protective mechanism against
xenobiotic toxins and also are partially responsible for the
resistance of cancer stem cells to traditional therapies. Pheo-
phorbide, a chlorophyll catabolite, is a specific probe for
ABCG2 which causes inhibition of ABCG2 efflux properties
[78]. The combined use of ABC transporter inhibitors and
chemotherapy could be used to increase the efficiency of
chemotherapeutic drugs to kill cancer stem cells [67]. ABC
transporter inhibitors also cause inhibition of normal stem
cells, leading to potential toxicity in the bone marrow, and
play a role in the maintenance of the blood-brain barrier [79].
However, effective targeting of this molecule could be vital
since it plays a significant role in the resistance of cancer
stem cells to treatment.

Immunotherapy targeting breast cancer stem
cells
Immunotherapy aimed at stimulating the immune system to
recognize and eliminate tumours has been explored for many
years but recently has gained renewed interest. Many
vaccines targeting solid tumours have been employed with
varying success both preclinically and clinically in the
treatment of cancer [80]. Interest in these vaccines has been
bolstered by increased understanding of the role that the
immune system plays in cancer and by the molecular
identification of tumour-associated antigens (TAAs) that can
be used as targets for therapy. In the case of breast cancer,
evidence is now coming to light that the immune system is
involved in the surveillance of cancer, is impaired by tumours
during the progression of cancer, and can recognize and
eliminate cancer. DCs are central to these processes as a
result of their role in innate immunity and in generating
humoral and cellular immune responses. DCs are profes-
sional antigen-presenting cells and initiators of adaptive
immunity through processing antigens and presenting
epitopes in the context of major histocompatibility complex
(MHC) to T cells [81]. DCs are capable of stimulating cyto-
lytic T-cell responses (CTLs) to TAAs on tumours and are
equipped with all of the necessary co-stimulatory and
cytokine signals needed to drive an effective immune
response to tumours.

Evidence for the protective role of the immune system against
cancer is seen in the increased incidence of melanoma
observed in renal transplant recipients [82]. The concept of
tumour immunosurveillance has stimulated interest in both the
interaction between tumours and the immune system and the
potential power of using immunotherapy to target tumour. For
breast cancer, it has been observed that DCs and lympho-
cytic infiltrates in tumours are associated with better
prognosis and survival, independent of tumour size [83].
However, in breast cancer, it is also apparent that DC
impairment correlates with tumour progression. For instance,
co-stimulatory molecule expression and antigen presentation
by DCs are decreased in breast cancer patients, with a


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subsequent impairment in the capacity of these DCs to
stimulate T-cell proliferation and secretion of cytokines [84].
DCs of breast cancer patients secrete less interleukin-12 in
response to maturation signals [85]. Interestingly, it has also
been shown that breast cancer can induce apoptosis in
circulating DCs but that this process is reversible when the
DCs are removed from the inhibitory cancerous environment
[86]. Additionally, precursors to DCs can be isolated from the
body before the start of treatment, cultured ex vivo with
GM-CSF (granulocyte-macrophage colony-stimulating factor)
and interleukin-4 to form DCs, and loaded with appropriate
antigens to expand functional tumour antigen-reactive T cells
[87]. These findings suggest a strong role for the immune
system in both the progression of disease and as a potential
tool to eliminate cancer. Immunotherapy in this context is
dependent on finding strategies that will effectively target
tumour, most importantly by identifying useful tumour antigens.

Breast cancer TAAs include epitopes from proteins that are
involved in tissue differentiation (such as carcinoembryonic
antigen and NYBR-1 [88]), are overexpressed in breast
cancer (such as HER-2/neu and MUC1), or are shared
among a variety of tumours (such as telomerase, survivin, and
p53) [89]. Most TAAs have been identified on the basis of
serological identification of antigens by recombinant
expression cloning (SEREX) followed by a demonstration that
patients have T cells capable of identifying the TAAs in an
MHC-restricted fashion [90]. Breast carcinomas fall into
various subtypes associated with different gene expression
patterns, phenotypes, and clinical outcomes [91]. However,
in a single patient, tumours are heterogenous, with individual
tumour cells displaying different phenotypes and TAAs. This
raises the possibility that no single antigen can be used to
effectively target and eliminate all tumour cells as there will
likely be a resistant cell not expressing the targeted antigen
that is capable of repopulating the tumour. Targeting of the
BCSC pool could potentially eliminate this population.

DC-based vaccination strategies encompass a variety of
different approaches that can be divided into two groups:
antigen-defined vaccines and polyvalent vaccines [80].
Preclinical mouse models have employed targeting of a single
tumour antigen with some success. In one model, a DC-
based vaccine prevented the outgrowth of a spontaneous
breast tumour in a mouse model when used to specifically
target a single differentiated tumour antigen, HER-2/neu [92].
This vaccine induced the production of anti-neu antibodies
and interferon-y expression by T cells. Despite preclinical
evidence that DCs induce effective antitumour T-cell
responses, clinical trials overall have been disappointing, with
a lack of objective tumour response reported in 12 of 35 trials
[80]. However, DC-based vaccines have no serious side
effects [93]. Most clinical trials using DCs have examined the
use of single antigen peptide-loaded DCs [94-97]. These
peptides are generally either wild-type sequence or altered
epitopes with better binding to the MHC and are generally


Available online http://breast-cancer-research.com/content/1 0/4/210



restricted to HLA-A*0201 [98,99]. One trial using tumour
necrosis factor-alpha-matured, monocyte-derived DCs pulsed
with MUC1 or HER-2/neu elicited peptide-specific anti-
tumour responses, but the overall clinical response was
modest [100]. Another antigen-defined approach makes use
of adoptive transfer of antitumour T cells that have been
cultured ex vivo and identified to be active against antigens of
interest on the tumour. The adoptive transfer method has
already been used with some success to target cancer [101-
103]. Recently, the examination of adoptive transfer of HER-
2-specific T-cell clones clinically suggests the potential to
use an antigen-specific therapy to eliminate specific single
tumour cells but that additional treatments are needed to
reduce the solid tumour due to the inhibiting effects of the
stroma [104]. Targeting of a single tumour antigen may allow
for regression of the tumour by gene deletion or
downregulation or by outright failure to target cells in the
tumour not expressing the targeted antigen [97,105].
Immunotherapy that targets single antigens may also fail to
target the underlying cells responsible for cancer initiation or
tumour metastasis, thus limiting the long-term success of this
treatment. To avoid these problems, it is likely that targeting
of either multiple antigens or essential antigens is going to be
required. From the available information, it appears that
several epitopes need to be targeted simultaneously for an
effective therapy through the use of a polyvalent vaccine. One
way to achieve this is to make use of whole tumours in the
vaccine. Immunotherapy for stage IV melanoma using a
DC/irradiated tumour vaccine has demonstrated a complete
remission of disease in 3 out of 46 patients and a partial
remission for an additional 3 patients [106]. Fusions of DCs
and breast tumours have been shown to elicit CTLs against
autologous tumour cells [107].

To induce long-lasting clinical responses using immuno-
therapy, targeting cancer stem cells may be required. To
achieve this, specific antigens expressed on the cancer stem
cells but ideally not by normal stem cells must be found and
targeted. While there are not yet antigens fulfilling this
description, they are likely to be present considering the
various pathways identified as different between these two
cell populations. Hence, it is important to identify as many
antigens as possible on BCSCs in order to develop a
polyvalent vaccine approach targeting several antigens.
Therefore, cancer stem cells need to be further defined in
terms of gene expression that determines stem-ness and
identifying molecules that are involved in regulating stem cell
qualities. Gene expression comparisons have been
conducted and can be used to identify what genes are
expressed by the cancer stem cell compartment compared
with normal stem cells [108,109]. Interestingly, recent work
has demonstrated that the expression profile of BCSCs more
closely resembles that of embryonic stem cells than that of
adult stem cells. An embryonic stem cell-like gene expression
pattern was found to be upregulated in the CD44+/CD2410w
tumourigenic fraction of cancer cells [110]. Additionally,


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Breast Cancer Research Vol 10 No 4 Morrison et al.


mapping the transcriptional profile of embryonic stem cell-like
genes in primary human breast cancer has revealed two
classes of tumours: those with an embryonic stem cell-like
activated program and those with an embryonic stem cell-like
repressed program. Those tumours with an embryonic stem
cell-like activated program were associated with poorer
differentiated tumours that were more likely to progress to
metastasis and death. The CD44+/CD2410w phenotype in
human breast tumours has been found to be associated with
basal-like tumours, and particularly BRCA1 hereditary breast
cancer, and has been linked to expression of CD49f, elevated
expression of CK5/14 and EGFR, and low expression of ER,
PgR, and HER-2 [111]. Basal-like tumours often have been
linked to poorer prognosis. The occurrence of the
CD44+/CD2410w phenotype was found to be lower in
tumours of luminal type, and particularly HER-2+ tumours,
irrespective of ER status.

Mammospheres have shown expression of markers ESA,
CK5, and CD49f (a6-integrin) among many others, which
potentially could be used to identify or target BCSCs [39].
The activity of aldehyde dehydrogenase-1 (ALDH1), a
detoxifying enzyme that may play a role in the differentiation of
stem cells, has been detected in both normal and malignant
human mammary stem cells and can be used as a predictor
for poor clinical outcomes [112]. High activity of ALDH1
identified the cells capable of self-renewal and high tumouri-
genicity in NOD/SCID xenografts. As previously mentioned,
another molecule used to identify or target BCSCs is CD44,
which is a membrane receptor involved in cell adhesion,
motility, and metastases and which along with P-glycoprotein
(the product of the MDR1 [ABCB1] gene of drug trans-
porters) has been linked to MDR [113]. CD44 routinely has
been used as a marker to purify and enrich BCSCs by
selecting for cells that are CD44+CD24-/lowLin- and ESA+
[35]. CD29 (131-integrin) and CD49f (a6-integrin) expression
has also been associated with murine mammary stem cells
with a Lin-CD24+ phenotype [9,114]. Additionally, neither
Sca-1 (stem cell antigen) expression nor the SP phenotype
was found to be expressed in the mammary reconstituting
Lin-CD29hlCD24+ cell population [9]. These mouse data
demonstrate that the use of Hoechst dye to identify an SP
phenotype does not accurately enrich for cells capable of
reconstituting a mammary gland when transplanted into
cleared mammary fat pads and call into question the use of
the SP phenotype to exclusively isolate mouse and human
BCSCs, which are a heterogenous population of cells.
However, one report using the human breast cancer line
MCF7 has shown that the SP phenotype can be used to
identify cells with characteristics of cancer stem cells that
express the tumour antigen MUC1, supporting a role for the
SP in further analysis of human BCSCs [115]. A recent
report has shown that BRCA l-deficient murine breast
tumours contain heterogeneous cancer stem cell populations
[116]. In that report, some tumours contained cells with a
CD44+/CD2410w phenotype, while cell lines derived from


another tumour contained CD133+ cells, a phenotype that is
associated with other cancer stem cells for brain, prostate,
and colon cancer [11 7] but that has not been described in
breast cancer. Importantly, both populations of cells expres-
sed the stem cell-associated genes Oct4, Notchi, Aldhi,
Fgfrl, and Sox1. That study shows that, although cancer
stem cell populations may be heterogenous, they in fact
share a common set of characteristics such as expression of
stem cell regulatory genes, expression of cell surface
markers, mammosphere formation, and tumourigenicity in
xenografts, which may be exploited for targeting cancer stem
cells [118].

Understanding of the biology of BCSCs will help to
determine the best way to target them. While we now have a
broader understanding of the genes and signalling pathways
involved in stem cells and putative cancer stem cells, the
application of immunotherapy targeting these molecules might
not necessarily be useful since they are often expressed by
healthy stem cells and indeed by a variety of other cells.
Determining what mutations are present in cancer stem cells,
how these mutations aid either the stem cell-like phenotype
or the tumourigenic phenotype of these cells, and how to
best target these mutations is going to be a critical
component of immunotherapy. This is made more difficult by
the role that epigenetic regulation plays in cancer stem cells.
Additionally, targeting universal TAAs such as human
telomerase reverse transcriptase and inhibitor of apoptosis
proteins might be important for effectively targeting tumours
with immunotherapy, as will combining these treatments with
ones that target unique stem-ness-related antigens [119].
Several additional markers and signalling molecules, such as
the Hh/Patched pathway, as well as the ABCG2 drug
transporters could be used as potential targets of therapy for
breast cancer [79]. Additionally, markers that are used for
migration of BCSCs, such as chemokine receptors, should
be explored as potential targets.

It is preferable that any therapy developed is able to target
metastatic disease before metastases occur. Circulating
tumour cells have been detected in the blood of patients with
metastatic and primary tumours and have been linked with a
decrease in survival times [43]. Recent evidence has
indicated that metastatic spread can be an early event in
tumourigenesis [120-122]. Gene expression studies have
shown that the profile of the primary tumour of breast cancer
patients can be used to predict disease outcome, with a
specific gene expression signature predictive of a short
interval to metastatic disease [121]. The poor prognosis
profile included genes involved in regulating the cell cycle
and angiogenesis. Studies such as this have challenged the
traditional view of metastatic cells arising late in disease and
have stressed the importance of developing assays to identify
disseminated disease early. The identification of molecular
targets on disseminated tumour cells, targets that might also
occur on BCSCs, could lead to better treatments. Preferably,


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these treatments should be applied during early stages of
tumourigenesis before overt metastasis occurs. Immuno-
therapy is certainly one approach to targeting these cells as it
has the potential to target even single cells for cell death and
has the power to target systemic disease. Another critical
component of immunotherapy targeting BCSCs is the
determination of the number of BCSCs residing in the tumour
and the ability to eradicate them with the treatment, as
tumour regression will be dictated by any escape of BCSCs.
Additionally, any immunotherapy approach will likely require
additional therapies such as cytotoxic T-lymphocyte antigen
(CTLA)-4 blocking of the T-cell regulation to overcome
tolerance of the immune system to cancer [123]. Finally, the
identification of appropriate antigens expressed on BCSCs
needs to be conducted along with identifying the best way to
stimulate an immune response using these antigens.

Cancer/testis antigens and cancer stem cells
Cancer/testis (CT) antigens are derived from proteins that
appear to be expressed only in germ cells and tumours [124].
A range of tumours are capable of making hormones such as
chorionic gonadotropin that are trophoblastic in origin [125].
A model for the trophoblastic origin of some tumours has
been postulated in which tumours arise from germ cells that
fail to reach the gonads/ovaries during development
[126,127]. CT antigens currently are being investigated for
their role in tumour formation and as potential targets of
tumour immunotherapy. CT antigens are ideal cancer
antigens because they are expressed by a proportion of cells
of the tumour and are largely absent from non-tumour tissue.
Furthermore, immunogenic CT antigens have been shown to
be present in tumours such as melanomas and breast cancer
[1 28] and have been used as the targets of tumour vaccines
such as targeting the MAGE-3A1 peptide for melanoma [97].
There are some intriguing characteristics shared between
germ cells and tumour cells, including immortalisation,
invasion, migration/metastasis, angiogenesis induction, and
immune evasion through downregulation of MHC [127].
Immunohistochemical studies have shown that CT antigens
are expressed on only a small proportion of cells in a tumour
[129]. This small proportion of cells could represent the
cancer stem cell compartment or the early progenitor cells. It
has been postulated that CT antigens could serve as markers
and potential therapeutic targets of cancer stem cells within
tumours. Melanoma cell lines enriched for stem cells express
various CT antigens and some of these antigens are present
on the majority of stem cells [130]. The relationship between
cancer stem cells and the expression of CT antigens needs
to be further defined, and the exact role of CT antigens in
both germ line and tumours remains a central question of
research. A model for cancer arising from mutations in stem
cells or early progenitor cells which gives rise to a phenotype
of expression of CT antigens has been postulated [127].
While the exact role of the CT antigens themselves might not
be fully understood, the potential to target them through
immunotherapy is exciting because they are expressed on a


Available online http://breast-cancer-research.com/content/1 0/4/210



proportion of cells in a tumour, possibly the putative cancer
stem cells, and they have been shown to be immunogenic.

Conclusions
The cancer stem cell hypothesis is a new paradigm that could
have a major impact on the treatment of disease by suggest-
ing a new target for cancer therapy. Mammary stem cell
biology needs to be understood in the context of both
mammary development and as potential sources of the
BCSCs. Transformed mammary stem cells have been
identified as a potential source of breast cancer, tumour
relapse, and tumour metastases; as such, they have gained
prominence as potential targets for immunotherapy of cancer.
Current treatments of cancer have shown efficacy in removing
the bulk of differentiated cancer cells while failing to eliminate
the cancer stem cells responsible for tumour relapse. Future
therapies will need to effectively target the cancer stem cells
to induce clinically significant remission of disease. Target
antigens for BCSCs need to be further defined so that
effective targeting of the BCSC compartment can be realized
which spares normal stem cell niches but disrupts the cancer
stem cell niche. New treatments typically will not be fully
optimal by themselves and will need to be further developed
and placed into combination therapy with existing treatments.
Therapies targeting BCSCs might be employed after
debulking of the differentiated tumour tissue. This would allow
immune surveillance to more efficiently eliminate the few
remaining cancer stem cells. Targeting BCSCs might be an
attractive approach to treat breast cancer metastasis and
relapse and could lead to significant increases in clinical
remissions and quality of life for breast cancer patients when
used in a multimodal treatment regimen.

Competing interests
The authors declare that they have no competing interests.

Acknowledgements
We acknowledge the support of the National Breast Cancer Founda-
tion. BJM is supported by a Griffith University Postgraduate Scholar-
ship and an Endeavour International Postgraduate Research
Scholarship. JAL is partially supported by a donation from Suncorp-
Metway Ride-for-Research.


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