Summary. Increasing data support cancer as a stem cell-
based disease. Cancer stem cells (CSCs) have been
found in different human cancers, and recent evidence
indicates that breast cancer originates from and is
maintained by its own CSCs, as well as the normal
mammary gland. Mammary stem cells and breast CSCs
have been identified and purified in in vitro culture
systems, transplantation assays and/or by cell surface
antigen identification. Cell surface markers enable the
functional isolation of stem cells that can initiate and
propagate tumorigenesis in mammary gland. These
observations have dramatic biological and clinical
significance due to increasing evidence suggesting that
the recurrence of human cancer and treatment failure
may reflect the intrinsic quiescence and drug resistance
of CSCs. Thus, the CSC hypothesis provides
fundamental implications for understanding breast
carcinogenesis and for developing new strategies for
breast cancer prevention and therapy for advanced
disease. Further strategies to isolate breast CSCs, to find
additional trustworthy surface markers, and to compare
gene expression pathways profiles with their normal
stem cells counterparts are necessary to more accurately
define putative breast cell-lineage markers for the
different cell types present in the mature mammary
gland and to identify potential therapeutical targets in
breast cancer. This review discusses the current
knowledge about stem cells and CSCs, focusing on
mammary stem cells and breast CSCs, and their
consequences for breast tumorigenesis and implications
for breast cancer susceptibility, prognosis, and treatment.
Key words: Breast, Cancer, Stem cell, CD44, CD24
All tissues in the human adult organism are derived
from organ-specific stem cells with specific properties
that maintain tissue integrity and are defined mainly by
their capacity to undergo self-renewal, as well as
differentiation into the cell types that comprise each
organ (Charafe-Jauffret et al., 2008; Shipitsin and
The malignant neoplasias are believed to result from
sequential mutations that can occur as a consequence of
progressive genetic instability and/or environmental
factors. Experimental and clinical data has recently been
accumulating which supports the hypothesis that cancer
may arise from mutations in stem cell populations (Reya
et al., 2001).
The cancer stem cell (CSC) hypothesis states that
normal stem cells may be the cells of cancer origin, and
that a specific subset of cancer cells with stem cell
characteristics can lead to tumor initiation, progression,
and recurrence (Campbell and Polyak, 2007).
One property that all cancers have in common is a
striking variability among the cancer cells within a
single tumor. These cells differ in characteristics such as
morphology, size, membrane composition, and antigen
expression, as well as behaviors such as proliferation
rate, metastatic potential, and sensitivity to therapeutic
agents (Campbell and Polyak, 2007). In diverse solid
tumor types, like breast cancer, the existence of a
hierarchical distribution of cancer cells is widely
accepted. It is theorized that only a small subpopulation
of replenishing stem-like cells can give rise to the
diversity of differentiated cells that comprise the bulk of
the tumor, and some reports have identified a small sub-
population of highly tumorigenic cells within primary
and metastatic breast tumors, as well as in some breast
cancer cell lines (Al-Hajj et al., 2003; Abraham et al.,
2005; Collins et al., 2005; Li et al., 2007; O’Brien et al.,
The recent research interest in CSCs arises from
Stem cells in human breast cancer
Lucinei Roberto Oliveira, Stefanie S. Jeffrey and Alfredo Ribeiro-Silva
1Department of Pathology, Ribeirão Preto Medical School, University of São Paulo, Campus Universitário Monte Alegre, Ribeirão
Preto, SP, Brazil and 2Department of Surgery, Stanford University School of Medicine, Stanford, USA
Histol Histopathol (2010) 25: 371-385
Offprint requests to: Alfredo Ribeiro-Silva. Department of Pathology,
Ribeirão Preto Medical School, University of São Paulo, Avenida
Bandeirantes 3900, Campus Universitário Monte Alegre, 14049-900,
Ribeirão Preto, SP, Brazil. e-mail: firstname.lastname@example.org
Cellular and Molecular Biology
experiments suggesting that cells with stem-like
properties can be sorted from solid tumors based on the
expression of specific surface markers. The
consolidation of CSCs knowledge into our current view
of multistep cancer development has important
implications for defining the target population for
transformation and the specific events required for
realization of malignant potential.
The human mammary gland
The human mammary gland is a specialized organ
that undergoes most of its development after birth and
follows hormonal events during its entire life (Charafe-
Jauffret et al., 2008). Mammary gland evolution is
regulated at three critical periods during life:
embryogenesis, puberty, and pregnancy (Kakarala and
After birth, the mammary gland develops from a
small number of invading cells derived from the
ectoderm, which form a lobulo-alveolar structure and are
composed of three cell lineages when adult:
myoepithelial cells, which are contractile cells that form
the basal layer of ducts and alveoli; ductal epithelial
cells, which line the lumen of ducts; and alveolar
epithelial cells, which synthesize milk proteins
(Hennighausen and Robinson, 2001; Kakarala and
Wicha, 2008). The functional structures produced in the
mature mammary gland are composed of a continuous
epithelium, consisting of an outer basal layer of
contractile myoepithelial cells and an inner layer of
luminal cells (Stingl et al., 2006).
The breast is a structurally dynamic and specific
organ. Hormonal changes during life induce episodes of
extensive proliferation, remodeling and differentiation in
the mammary epithelium according to age, menstrual
cycle and reproductive status (Navarrete et al., 2005). To
achieve this great plasticity and the large cell number
expansion associated with it, there is substantial
evidence of a differentiating cell hierarchy in the adult
mammary gland that includes developmentally distinct
stem and progenitor cell types, driving the proliferative
process within the normal mammary epithelium in a
hierarchical and organized way (Stingl et al., 2006;
Cariati and Purushotham, 2008).
Breast cancer is a major cause of death in women.
The majority of breast cancer deaths occur as a result of
recurrent or metastatic disease rather than from the
effects of the primary tumor (Croker et al., 2009). In the
past decade, earlier detection and improved treatments
have reduced breast cancer mortality, but the recurrence
and metastases rates for the disease remain high.
Moreover, current breast cancer therapy is not optimally
individualized, and is often associated with undesirable
side effects (Thornton et al., 2005).
Breast tumors may be classified by dissemination
patterns such as in situ, invasive, or metastatic lesions
(Simpson et al., 2005). However, breast cancer is not a
disease determined by a single tumorigenesis pathway,
but rather a heterogeneous group of diseases at both the
molecular and clinical level, and each subtype has its
own stable phenotype maintained during tumor
evolution (Sorlie et al., 2001; Korsching et al., 2002;
Dontu et al., 2004; Polyak, 2006). Distinct gene
expression profiling of a large set of breast tumors
performed by independent groups have demonstrated
five main molecular subtypes of breast cancer: basal
subtype, luminal A and B (also known as highly
proliferating luminal) (Langerød et al., 2007), HER-
2+/ER-, and normal breast-like. This classification
correlates with clinical outcome, with distinct responses
to treatment and prognostics, and there is increasing
evidence that risk factors can be different for each tumor
subtype (Hu et al., 2006; Polyak, 2006; Sorlie et al.,
Stem cells represent only a minuscule fraction of the
cells that constitute each tissue but they are the only cells
with self-renewal capacity. Stem cell divisions occur in
an asymmetric way, in which a stem cell is able to
produce an exact copy of itself, as well as a daughter cell
that leaves the stem cell niche to differentiate and
generate multipotent progenitors, which in turn can give
rise to committed progenitors and differentiated cells
(Dontu et al., 2003; Ginestier et al., 2007). The
deregulation of this self-renewal process leading to stem
cell expansion may be a key event in carcinogenesis, and
while self-renewal can drive tumorigenesis, the
differentiation process can contribute to tumor
phenotypic heterogeneity (Kakarala and Wicha, 2008)
(Fig. 1). According to Charafe-Jauffret et al. (2008),
genetic and epigenetic mechanisms in the progenitor cell
type and environmental influences in the niche where
these cells grow may contribute to the cellular
heterogeneity found in the malignant neoplasms.
The epithelial tissues are subject to continuous
remodeling and renewal. According to Smalley and
Ashworth (2003), this tissue renewal involves a
hierarchy of cells, including slowly proliferating stem
cells, rapidly proliferating transit-amplifying cells and
various terminally differentiated cells with specialized
functions. The slowly proliferating stem cells are long-
lived, and for this reason are more exposed to damaging
agents than the more differentiated cells, and can thus
accumulate mutations that are then transmitted to the
rapidly proliferating progeny (Dontu et al., 2003).
Mammary epithelial stem cells
The existence of mammary epithelial stem cells was
first suggested by DeOme et al. (1959), who originally
devised the technique of tissue fragment transplantation
into mammary fat pads cleared of mammary epithelium.
Stem cells and breast cancer
It was shown through mouse transplantation studies that
epithelium isolated from different regions of the
mammary gland at various stages of postnatal
development could recapitulate the entire glandular
structure upon transplantation into a cleared mammary
fat pad, generating complete differentiated and
functional mammary epithelial branching ducts, lobules
and myoepithelial cells. Later, Hoshino (1967) also
demonstrated and confirmed that a fragment could be
serially transplanted from this regenerating gland to
another cleared mammary fat pad.
More recently, considerable progress has been made
towards identification of human mammary stem cells,
but the exact phenotype of these cells still remains
indefinite. Using immunofluorescent monoclonal
antibodies for basal cytokeratin 5, glandular cytokeratins
8/18 or myoepithelial smooth muscle actin, Nagle et al.
(1986) identified progenitor cells of luminal and
myoepithelial cell lineages and described heterogeneous
populations in the normal human adult mammary gland.
Dairkee et al. (1988) also showed that normal, benign,
and malignant breast epithelium expressed
complementary patterns of reactivity against basal and
luminal cell-specific antibodies. They went so far as to
suggest that some basal tumors may have originated in
“undifferentiated basally located precursor cells often
referred to as ‘stem cells’”. Further support for the
pluripotent epithelial stem cell existence in the
mammary gland comes from transplantation studies, and
this in vivo assay remains the gold standard for testing
functional stem cell properties (Chepko and Smith,
In 1998, the existence of stem cells in rodent
mammary glands was corroborated by Kordon and
Smith (1998). They established the existence of
mammary stem cells through experiments with mouse
mammary stem cells marked with the mouse mammary
tumor virus (MMTV), which were serially transplanted
in random fragments of mammary epithelium into
cleared mammary fat pads, and they observed then its
capacity to regenerate a new gland tissue in vivo and
display self-renewal activity upon transplantation.
However, Kordon and Smith (1998) highlighted that any
mutation in the mammary epithelial stem cell population
could be highly relevant for the entire mammary
epithelial population because it will be conserved and
repeatedly inherited in all the subsequent progeny of the
stem cell, as well as during self-renewal.
Several other studies of clonality and implantation
performed in vitro and in vivo have shown that a subset
of pluripotent human adult mammary epithelial cells,
called mammary epithelial stem cells, are capable of
forming colonies in vitro and give rise to both luminal
epithelial and myoepithelial cells, appearing to represent
bipotential mammary epithelial progenitors (Böcker et
al., 2002; Asselin-Labat et al., 2006; Shackleton et al.,
Stingl et al. (2006) reported the use of
multiparameter cell sorting to purify a subset of adult
mouse mammary stem cells that are capable individually
of regenerating an entire mammary gland within six
weeks in vivo. These cells were designated as mammary
repopulating unit and expressed markers like cytokeratin
5 and 14, smooth muscle actin, vimentin, and smooth
muscle myosin, which are associated with
Stem cells and breast cancer
Fig. 1. Hypothetical ways of normal stem cells.
A. Stem cells can originate committed
progenitor cells that later arise differentiated
tissular cells. B. Oncogenic mutations can
transform normal stem cells in cancer stem
cells. The cancer stem cells can accumulate
genetic alterations that can drive tumor
initiation, progression and heterogeneity. C.
Stem cells can generate an idential cell to itself
through its self-renewal capacity.
Stem cells and cancer
The stem cell origin of cancer hypothesis considers
that stem cells or cells that acquired the self-renewal
ability tend to accumulate genetic alterations over long
periods of time, evading the strict control of their
microenvironment, and giving rise to tumoral evolution
(Shipitsin and Polyak, 2008).
The resemblance between stem cells and cancer was
observed a long time ago. The first register concerning
the hypothesis of cancer origin from a rare population of
normal cells with stem cell properties was proposed
almost 150 years ago (Durante, 1874; Wicha et al.,
2006). At that time, Cohnheim (1875) also proposed the
hypothesis that stem cells could be misplaced during
embryonic development, being the source of tumors that
would be formed later in life.
The studies about this subject returned over 40 years
ago, when some investigations confirmed the CSC
hypothesis showing that a single tumor cell could
generate a heterogeneous progeny and give rise to a new
tumor, through investigations performed in tumors
derived from ascites fluid in rats, and teratocarcinomas
and leukemias in mice (Makino, 1956; Bruce and Van
Der Gaag, 1963, Kleinsmith and Pierce, 1964).
In this way, Park et al. (1971) observed through a
primary cell culture assay some myeloma tumor stem
cells in mouse, and Hamburger and Salmon (1977)
corroborated the hypothesis that some cancers could
contain a small subpopulation of cells similar to normal
stem cells, because they observed in primary bioassays
that the expansive growth of malignant lesions could
suggest the presence of a CSC population with stem cell
properties, including indefinite proliferation.
In animal models, the ability of a small population of
cells to originate a new malignant neoplasia was
demonstrated in a classic experiment through
transplantation of cells from human acute myeloid
leukemia that expressed some cell surface markers
associated with normal hematopoietic stem cells
(Lapidot et al., 1994). Lapidot et al. (1994) showed that
these transplanted cells could initiate leukemia in
nonobese diabetic/severe combined immunodeficient
(NOD/SCID) mice while other isolated cells could not.
Since then, this assay has become the standard method
for determining whether cell populations isolated from
solid tumors are CSCs.
Based on the ability of diverse purified populations
to form leukemia in NOD/SCID mice, investigations
started to search for stem-like cells in leukemias. Bonnet
and Dick (1997) showed that the injection of leukemic
cells with a primitive hematopoietic progenitor
phenotype resulted in leukemias that could be
transplanted into secondary recipients, and also observed
its ability to perpetually self-renew. Since then, putative
CSCs have been isolated from many other tumors
including brain, breast, colon, pancreas, prostate, lung,
and head and neck cancer (Collins et al., 2005; Kim et
al., 2005; Dalerba et al., 2007; Li et al., 2007; Prince et
Stem cells and breast cancer
Fig. 2. Neoplastic cells from
an invasive ductal carcinoma
of the breast positive for
membrane staining). The
CD44 cell-surface marker has
been used to identify putative
cancer stem cells in breast
The main hallmarks of CSCs are their properties of
self-renewal, their ability to generate tumors from very
few cells, their slow cell division, their ability to give
rise to a phenotypically diverse progeny, and their
selective resistence to radio- and chemotherapy (Reya et
al., 2001). The self-renewal and differentiation
characteristics lead to the production of all cell types in a
tumor, thereby generating tumor heterogeneity
(Campbell and Polyak, 2007). The differentiated cells
constitute the bulk of the tumor, but are not usually
tumorigenic, due to their lack of self-renewal capacity
and limited proliferation potential (Ginestier et al.,
2007). However, it has been shown that the switch to
carcinogenesis can occur in either the stem cells or one
of their progeny, which acquires the ability to self-renew
(Dontu et al., 2003). Some studies involving both
transforming viruses and the direct introduction of
oncogenes derived from human tumors suggested that
the majority of cultured cells are susceptible to
malignant transformation and that the clonality of
tumors could be the end result of continuous selection
for cells harboring favorable combinations of
transforming gene mutations (Weinberg, 1995;
Vogelstein and Kinzler, 2004). Furthermore, in several
tissue systems, it has been proposed that some
committed progenitor cells might become a CSC
through a dedifferentiation process, which would occur
by the acquisition of stem cell properties (Ponti et al.,
2005; Krivtsov et al., 2006; Cobaleda et al., 2007).
Additional confirmation that stem cells can play a
role in carcinogenesis are the homologies found between
normal stem cells and cancer cells. In addition to self-
renewal capacity, these characteristics include the
production of differentiated cells, activation of
antiapoptotic pathways, induction of angiogenesis,
resistance to apoptosis and drugs (due to active
telomerase expression and elevated membrane
transporter activity), and the ability to migrate and
spread in metastasis (Wicha et al., 2006).
Most breast cancers arise within a relatively short
segment of terminal ductules that might be the location
of normal stem cells (Villadsen, 2005). The
demonstrations that normal mammary stem cells do exist
indicate that stem cells could play an influence in breast
cancer. The first proposal of breast CSC existence was
made by Pierce et al. (1977) through an ultrastructural
comparison of breast CSCs and their normal mammary
stem cell counterparts. This proposal was reinforced by
the observation that early first full-term pregnancy is
associated with lower lifetime breast cancer risk. This
might be explained if transformation preferentially
occurs in normal mammary stem cells, and these may be
present in fewer numbers once the mammary gland
differentiation associated with pregnancy has occurred
(Russo et al., 2005).
MMTV-induced breast carcinogenesis models have
provided further evidence that stem/progenitor cells may
be targets for malignant transformation. The MMTV
infects mammary epithelial cells and randomly inserts its
proviral DNA into somatic cell DNA during its
replicative cycle (Ringold et al., 1979). According to
Stem cells and breast cancer
Figure 3. Cancer stem cell hypothesis
implications for breast cancer treatment.
Although cancer stem cells comprise a small
amount of the cells within a tumor, they can be
resistant to radiotherapy and chemo-
therapeutical agents, probably because of their
quiescency. This therapeutical resistance
combined with the stem cells property of self-
renewal might be the cause of tumor relapse
years after the clinical remission.
Callahan (1996), some of these random DNA insertions
have been demonstrated to cause deregulation of specific
stem cell genes leading to premalignant transformation
and later to tumor progression. Nowadays, similar
mammary fat pad transplantation models have been used
to prospectively identify mouse mammary cells with
breast CSC properties (Shackleton et al., 2006).
Mammary epithelial stem cells and breast cancer
The long lifetime of stem cells makes them more
susceptible than other cells to acquire multiple mutations
required for carcinogenesis (Wicha et al., 2006). In the
mammary gland, the lifetime accumulated hormonal
exposure is one of the most important risk factors for
breast cancer. Mammary epithelial stem cells can be the
potential targets of these malignant transforming events
because hormonal changes during a specific
developmental period may determine the size of the
breast stem cell pool and thereby influence
carcinogenesis (Dontu et al., 2003; Kakarala and Wicha,
2008). Epidemiological investigations in populations
submitted to radiation, like those from Hiroshima and
Nagasaki, indicate that breast cancer develops years or
decades after the oncogenic initiation (Tokunaga et al.,
1979) and the normal mammary stem cells are the only
specific population of cells that could survive enough to
accumulate all the transforming events necessary for the
attainment of the cancer phenotype (Cobaleda et al.,
Furthermore, the terminal differentiation of
mammary stem cells following pregnancy decreases the
number of target cells susceptible to oncogenic events.
Russo et al. (2005) demonstrated in animal studies that
pregnancy or short-term treatment of virgin rats with
pregnancy-associated hormones protects against
chemically induced mammary carcinogenesis.
According to Ahlgren et al. (2004), the rate of
increase in height during adolescence, largely regulated
by growth hormone, is also strongly associated with
subsequent risk of breast cancer, and the production of
growth hormone was already previously associated as a
paracrine regulator of mammary stem cells (van
Garderen and Schalken, 2002).
The hormone susceptibility of putative mammary
epithelial stem cells has been extensively investigated.
Notwithstanding, there have been conflicting reports
regarding the hormone receptor status of these cells in
both mouse and human experiments. In mouse, based on
pulse-chase methods, Booth and Smith (2006) predicted
that the putative mouse mammary epithelial stem cells
were ER+, while Asselin-Labat et al. (2006) found that
stem cells identified by specific cell surface markers
were negative for both HER-2 and hormone receptors
(triple negative). Similarly, other divergent results have
been described for human mammary epithelial stem
cells, shown by Clarke et al. (2005) as enriched for
steroid receptors, and cited as hormone receptor negative
cells by Clayton et al. (2004).
Isolation and Purification of CSCs
Although the concept that cancers arise from stem
cells was first proposed more than 150 years ago, it is
only recently that advances in stem cell biology have
allowed for more direct testing and validation of the
It is well settled that CSCs share some properties
expressed by normal stem cells. Current methods for
determining whether cell populations isolated from solid
tumors are CSCs, consist of purification of these cells
from tumor samples based on the properties of normal
stem cells, such as their ability to form spheres in culture
(Dontu et al., 2003), membrane efflux activity (Goodell,
2002), specific cell surface molecule expression (Al-Hajj
et al., 2003), and enzymatic activity detection of
aldehyde dehydrogenase 1 (ALDH1) (Nagano et al.,
2007). Purified cells are then tested for the capacity to
originate tumors when injected into immunodeficient
In vitro culture of spheres cells
Characterization of stem cells in both human and
rodent systems has been facilitated by the development
of in vitro culture systems that allow for propagation of
stem cells in an undifferentiated state. In the in vitro
culture system, cells grow in unattached conditions into
round balls called “spheres”. This system can be used
for enrichment and propagation of stem cells (Jensen and
Contrary to the paradigm that epithelial cell survival
is anchorage-dependent, Soule and McGrath (1986)
showed for the first time the ability of undifferentiated
human mammary epithelial cells to survive in
suspension. After that, Dontu et al. (2003) showed that
the in vitro cultivation and propagation of these
undifferentiated human mammary epithelial cells
generates floating spherical colonies with anchorage-
independent growth, which were named mammospheres.
They also observed that these mammospheres could
reconstitute an entire mammary ductal tree, because they
were enriched with cells with functional properties of
stem/progenitor cells. These cells were suitable for self-
renewal testing and were capable of differentiating into
all three lineages present in the mammary gland when
cultured under differentiating conditions. Moreover,
Dontu et al. (2003) confirmed that the in vitro culture
systems and gene expression profiles of human breast
stem cells correlated with the genetic programs of other
tissue types of stem cells.
The pattern of immunostaining found within
mammospheres was consistent with previously reported
data of mammary epithelial progenitor cells in normal
adult human breast tissue (Stingl et al., 2001;
Gudjonsson et al., 2002), expressing CD10, α6 integrin,
and cytokeratin 5 in earlier progenitors, and epithelial-
Stem cells and breast cancer
specific antigen (ESA) and cytokeratin 14 in later but
still multipotent progenitors (Dontu et al., 2003).
Recently, Ginestier et al. (2007) showed that
mammosphere-initiating cells expressed ALDH1, a
detoxifying enzyme, and were capable of generating
human mammary structures when transplanted into the
humanized fat pad of NOD/SCID mice.
Membrane efflux activity
The Hoechst 33342 efflux property is a
discriminating feature of quiescent stem cells that is lost
when these cells are activated into cycle, and this allows
identification of a small stem-like cell population called
side population (SP), through flow cytometric analysis
(Uchida et al., 2004). It has been postulated that the SP
main characteristic is a universal stem cell phenotype
(Zhou et al., 2001). Although heterogeneous, SP cells
are observed in cardiac and primitive retinal cells
(Hierlihy et al., 2002; Bhattacharya et al., 2003), in
hematopoietic, epidermal and mammary stem cells
(Bunting, 2002; Alvi et al., 2003), in normal kidney and
renal cell carcinoma (Addla et al., 2008), and some
embryonic stem cells (Zhou et al., 2001).
Alvi et al. (2003) demonstrated that human and
mouse breast epithelial cells located within the SP retain
the potential to differentiate into typical mammary
clones in vitro and can also regenerate the organ upon
experimental transplantation in the mouse mammary
gland, because SP cells constitute an undifferentiated
subpopulation able to differentiate into myoepithelial
and luminal cell types, as well as into ductal and lobular
structures. Furthermore, the morphology of the
structures derived from these mammary SP cells
resembled those previously described by Kordon and
Smith (1998) for mammary epithelial clone types in
The SP cells are also associated with resistance to
drugs and toxins, and this property is a result of
increased expression of membrane transporter proteins
(ABC drug transporters), such as P-glycoproteins or
BCRP (breast cancer resistance proteins). In addition to
acting as functional regulators of stem cells, they
provide defense against damaging agents (Zhou et al.,
2001; Bunting, 2002). Therefore, tumors might have a
population of drug-resistant pluripotent cells that can
survive radio- and chemotherapy and then repopulate the
tumor (Charafe-Jauffret et al., 2008).
Stem cell markers
Expression of some specific cell surface markers has
been investigated to facilitate the identification and
purification of normal stem cells and CSCs, and several
stem cell markers may be shared by CSCs in multiple
human tumor types.
The standard procedures for the isolation of CSCs
have been similar in many investigations. Among the
most used in vivo models is the fractionation of tumor
cells using cell-surface markers with stem cell
characteristics, followed by their implantation into
NOD-SCID mice to assess xenograft growth and celular
composition (Shipitsin and Polyak, 2008).
The main surface marker phenotypes associated with
stem cell characteristics include CD133, CD44, and
CD24 (Al-Hajj et al., 2003; Singh et al., 2004; Lim and
Oh, 2005; Hermann et al., 2007; O'Brien et al., 2007).
The CD133 cell-surface marker, also called prominin 1
(PROM1), was discovered as a marker of normal
hematopoietic stem cells and was later used to purify
putative CSCs in several tumor types (Singh et al., 2004;
Mizrak et al., 2008). In brain tumors, Singh et al. (2004)
found that CD133+cells could successfully grow under
unattached conditions, with neurosphere-like formations,
whereas CD133-cells could not. Only the CD133+cell
fraction, isolated from human medulloblastomas and
glioblastomas and injected into the brains of NOD SCID
mice, contained cells capable of initiating tumors, with
phenotypic similarity between engrafted and original
tumor. According to other studies, CD133 has been
shown to play a role in migration and asymmetric
division of stem cells (Balic et al., 2006; Beckmann et
The CD44 glycoprotein is a cell surface receptor for
hyaluronic acid, and is involved in cell adhesion,
migration, and metastasis of cancer cells (Shipitsin et al.,
2007). The CD44 cell-surface marker has been used to
identify putative CSCs in breast tumors (Fig. 2)
(Shipitsin et al., 2007), as well as in other tumor types,
such as prostate (Collins et al., 2005), pancreatic (Li et
al., 2007), and head and neck carcinomas (Prince et al.,
2007). Shipitsin et al. (2007) found that CD44+tumoral
mammary cells were associated with more invasive,
proliferative, and angiogenic status, predicting an
aggressive tumoral cell behavior. Moreover, there was a
correlation between CD44+tumoral cells and decreased
patient survival (Shipitsin et al., 2007).
CD24 is a mucin-like adhesion molecule expressed
by neutrophils, pre B lymphocytes and a large variety of
solid tumors. Functionally, CD24 enhances the
metastatic potential of malignant-cells, because it has
been identified as a ligand of P-selectin, an adhesion
receptor on activated endothelial cells and platelets (Lim
and Oh, 2005). In the mouse mammary gland,
cytokeratin expression and PCR have revealed that
CD24-, CD24low, and CD24hipopulations correspond to
non-epithelial, basal/myoepithelial, and luminal
epithelial cells, respectively (Sleeman et al., 2006). Lim
and Oh (2005) investigated the role of CD24 in various
human epithelial neoplasias, and demonstrated that
intracytoplasmic CD24 expression was found to be
highly associated with adenocarcinoma of the colon,
stomach, gallbladder, and ovary. Positive or negative
CD24 expression has been used in set with other
markers to identify putative CSCs in tumors, and some
studies defined the phenotype of pancreatic CSCs as
CD24+/CD44+(Li et al., 2007; Zou, 2008). However, in
breast and prostate cancer, putative CSCs were found
Stem cells and breast cancer
isolated from tumors initiated by these cells were able to
transfer the tumor to secondary and subsequent hosts,
demonstrating the capacity for maintenance of self-
renewal and tumorigenic properties. The generated
tumors also reproduced the phenotypic heterogeneity of
the original tumors.
The CSC hypothesis suggests that breast cancer
initiation may take place preferentially in a normal
mammary stem or progenitor cell expressing the CD44
marker (Fig. 2) (Abraham et al., 2005). A hypothetical
model of tumor progression to metastatic disease in
breast cancer considers that metastasis can be initiated
by invasive CD44+breast cancer cells and that tumors
rich in CD44+cells have a significantly worse clinical
outcome (Shipitsin et al., 2007; Shipitsin and Polyak,
2008). In agreement with this assessment, it has been
postulated that breast cancers of basal-like phenotype,
which carry a poor outcome, are enriched with CD44+
cells (Sorlie et al., 2001; Shipitsin et al., 2007). Honeth
et al. (2008) recently demonstrated an association
between basal-like phenotype and CD44+/CD24-cells.
They found, however, that not all basal-like tumors
contain CD44+/CD24-cells, emphasizing that a putative
tumorigenic ability may not be confined to cells of this
phenotype and that other breast CSC markers remain to
In agreement with the stem cell property of
anchorage-independent growth in sphere cells, Dontu et
al. (2003) and Ponti et al. (2005) found that isolated
CD44+/CD24-human breast cancer cells can also form
tumor mammospheres and propagate in vitro. Moreover,
Dontu et al. (2003) demonstrated that these cells produce
vascular endothelial growth factor (VEGF) and are
highly angiogenic. Subsequent experimental studies
have also isolated CD44+/CD24-breast cancer cells and
demonstrated increased in vitro expression of stem cell
markers and enhanced capacity for mammosphere
formation, invasion, and resistance to radiation (Phillips
et al., 2006; Sheridan et al., 2006). Furthermore, clinical
studies indicate that CD44+/CD24-tumor-initiating cells
express an invasive gene signature and may be
associated with distant metastases (Abraham et al., 2005;
Balic et al., 2006; Liu et al., 2007).
There are some studies implicating CSCs in breast
metastasis. To investigate the association of the stem cell
phenotype to metastasis, Balic et al. (2006) examined the
expression of stem cell markers in disseminated
metastatic cancer cells detected in bone marrow sites of
breast cancer patients and found an increased number of
CD44+/CD24-expressing cells. Similarly, Sheridan et al.
(2006) demonstrated that CD44+/CD24-breast cancer
cells have enhanced invasive characteristics.
There are also other sets of markers associated with
breast stem cells and CSC phenotype. Stingl et al. (2006)
found that cells expressing CD29 and/or CD49F, as well
as CD24, displayed the stem cell properties of self-
renewal and multilineage differentiation, while
Shackleton et al. (2006) demonstrated that a single cell
from the CD29high/CD24+or CD49Fhigh/CD24+
Stem cells and breast cancer
Table 1. Cancer stem cell phenotypes according to stem cell markers in
Cancer stem cell
Al-Hajj et al., 2003
Li et al., 2007
Head and Neck
Prince et al., 2007
Hurt et al., 2008
Sinh et al., 2004
ProstateCollins et al., 2005
O’Brien et al., 2004
Lim and Oh, 2005
Li et al., 2007
Matsui et al., 2004
Ginestier et al.,
with a CD24-/CD44+phenotype (Al-Hajj et al., 2003;
Hurt et al., 2008).
These investigations suggest that diverse stem cell
markers can be expressed in different tumors by the
CSCs, and the significance of these observations in most
human cancers remains to be determined. Therefore, it is
possible that each tumor could have a prevalent and
specific CSC phenotype (Table 1).
Stem cell markers in mammary stem cells and breast
Identification of normal and malignant stem/
progenitor cells by the same marker can support the
concept that these cells are primary targets of
transformation. Research has applied knowledge
obtained in the fields of hematopoietic, neural and
epidermal stem cells, and investigated markers borrowed
from these areas to identify prospective stem/progenitor
cells in the mammary gland (Cariati and Purushotham,
In the last years, specific populations of breast
cancer cells with stem cell-like features and tumorigenic
characteristics were identified and investigated in some
in vivo models. Al-Hajj et al. (2003) isolated the first
CSCs in a solid tumor and identified human tumorigenic
breast CSCs with an enriched CD44+/CD24-/low/ESA+
antigenic phenotype. As few as 200 of these cells in a
primary invasive breast tumor, which comprised
between 1% and 10% of the total cell population, were
capable of forming new tumors when implanted in the
mammary fat pad of female NOD/SCID mice.
Conversely, 20,000 cells isolated from the same tumor
that did not display this phenotype were unable to form
tumors. Moreover, the CD44+/CD24-/low/ESA+ cells
population was capable of reconstituting a functional
mammary gland when this cell was transplanted into a
cleared mouse mammary fat pad. In a recent
investigation, Croker et al. (2009) found that
stem-like cancer cells isolated from some human breast
cancer cell lines (MDA-MB-231 and MDA-MB-468)
demonstrate enhanced malignant/metastatic behavior in
both in vitro and in vivo experiments.
Enzymatic activity detection of ALDH1
The ALDEFLUOR assay is a simple method for
identifying CSCs, and is based on enzymatic activity
detection of ALDH1, a detoxifying enzyme responsible
for the intracellular oxidation of aldehydes. According to
Sophos and Vasiliou (2003), ALDH1 may have a role in
early differentiation of stem cells through its function in
oxidizing retinol to retinoic acid. Retinoic acid signalling
is linked to cellular differentiation during development
and plays a role in stem cell self-protection throughout
an organism’s lifespan (Croker et al., 2009).
ALDH1 activity can provide a common marker for
both normal and malignant stem cells. Cells with high
ALDH1 activity have been associated with several types
of human hematopoietic and neural stem cells
(Armstrong et al., 2004; Corti et al., 2006), and the
ALDEFLUOR assay was also successfully used to
isolate CSCs from leukemia and multiple myeloma
(Matsui et al., 2004). Confirming these findings, Nagano
et al. (2007) demonstrated that the ALDH1 enzyme can
identify rapidly dividing cells that represent a progenitor
cell population in human umbilical cord blood and bone
marrow. Therefore, in agreement with Croker et al.
(2009), the use of ALDH1 activity detection as a
purification strategy allows an efficient isolation of
normal and malignant human stem cells based on a
developmentally conserved stem cell function.
In the mammary gland, Ginestier et al. (2007)
demonstrated that ALDH1 is a marker of
stem/progenitor cells of the normal human breast and
breast carcinomas. The ALDEFLUOR positive cells
isolated from both normal and tumoral human breast
have phenotypic and functional characteristics of
mammary stem cells, are capable of self-renewal and
recapitulate the heterogeneity of the parental tumor.
There was also partial overlap between the
CD44+/CD24-/lin-and ALDH1+populations, with cells
expressing the phenotype CD44+/CD24-/lin-/ALDH1+
able to form tumors from as few as 20 cells.
Furthermore, the ALDEFLUOR positive population
isolated from human breast tumors had the ability to
generate tumors in NOD/SCID mice. Its expression was
also associated with aggressiveness and poor clinical
outcome in a series of 477 breast carcinoma patients
(Ginestier et al., 2007). Detection of expanded stem cell
clusters using markers such as ALDH1 in breast biopsy
tissues may identify women with increased risk for
subsequent breast cancer development (Liu et al., 2008).
Pathways regulation in stem cell maintenance
Different mutations associated with cancer occur in
pathways that govern stem cell maintenance, suggesting
that deregulation of normal mechanisms of stem cell
functionality may be involved in carcinogenesis (Reya et
al., 2001). Similar to normal stem cells, when a CSC
divides, one daughter is an exact copy of the original and
retains the ability to divide and initiate additional
tumors, whereas the other daughter cell differentiates to
produce nontumorigenic cells. This asymmetric division
is strictly regulated by various pathways that govern the
normal stem cell self-renewal and differentiation
(Charafe-Jauffret et al., 2008). Human cancer mutations
have been identified that can cause dysregulation of
important pathways involving Hedgehog (Hh), Bmi-1,
Wnt, NOTCH, HER-2, p53 and PTEN signalling (Crowe
et al., 2004; Woodward et al., 2005).
The Hh pathway is one of the main pathways that
control stem cell fate, self-renewal, and maintenance.
Sonic hedgehog (SHH) acts as secreted morphogen in
developmental patterning. Mutations in the SHH
receptor patched (PTCH), a putative tumor suppressor,
with consequent mRNA upregulation (due to loss of
transcriptional autoregulation) were first shown in
human basal carcinomas of the skin (Unden et al., 1996).
More recently, other work has shown Hh pathway
dysfunction in other human malignant neoplasms, such
as prostate and breast carcinomas (Karhadkar et al.,
2004; Olsen et al., 2004). In human gliomas, Hh
signalling represents a new therapeutic target through its
essential control in the behavior of glioma CSCs
(Clement et al., 2007).
In the mammary gland, strict regulation of the Hh
pathway is required for normal development (Charafe-
Jauffret et al., 2008). Using both in vitro culture systems
and NOD/SCID mice, Liu et al. (2006) found that this
pathway, together with the polycomb protein Bmi-1,
play important functions in regulating self-renewal of
both normal and malignant human mammary stem cells.
More recently, Charafe-Jauffret et al. (2008) showed that
the Hh signalling pathway is activated in human breast
CSC defined as CD44+/CD24-/lowphenotype.
The capacity of angiogenesis induction can be
inherent to normal stem cells as well as their
transformed counterparts, and some investigations
suggest a role for Hh signalling in this process.
According to Byrd and Grabel (2004), Hh signalling can
target endothelial stem cells directly or stimulate blood
vessel support cells to produce vascular growth factors.
In agreement with this assessment, Fu et al. (2006)
found that Hh protein promotes bone marrow-derived
endothelial progenitor cell proliferation, migration and
Stem cells and breast cancer
VEGF production. Moreover, recent data suggest Hh
ligands produced by tumor cells activate signalling
pathways in the stromal microenvironment (Yauch et al.,
Based on these findings, the development of specific
Hh inhibitors, such as cyclopamine is currently
underway in breast cancer, and clinical trials utilizing
these chemotherapeutical agents are in the planning
stages (Liu et al., 2006; Kakarala and Wicha, 2008).
Wnt signalling is known to regulate cell fate
decisions, influencing morphology, proliferation,
apoptosis, differentiation, migration, and stem cell self-
renewal (Turashvili et al., 2006). Wnt proteins may
assist in maintaining stem cells in an undifferentiated,
self-renewing state within their niche (Nusse, 2008).
Defects in the Wnt pathway are seen early in colon
cancer carcinogenesis (Olsen et al., 2004).
In embryonic mammary development, the Wnt
pathway is implicated at several stages and has been
shown to be associated in the regulation of self-renewal
and differentiation of the mammary stem cells
(Turashvili et al., 2006). In breast cancer, Li et al. (2003)
showed that tumors induced in MMTV transgenic mice
codify components of the Wnt signalling pathway and
that Wnt-1-induced tumors displayed markers of both
epithelial and myoepithelial lineage. The authors suggest
that deregulated Wnt signalling causes excess
proliferation of mammary progenitor cells, predisposing
them to cancer. Corroborating these findings, Stingl et
al. (2006) observed that in MMTV-Wnt transgenic mice,
the number of cells displaying stem cell markers
expanded more than six-fold than in the preneoplastic
NOTCH signalling has been shown to play an
influence in cell fate in hematopoietic, neural, and
embryonic stem cells (Brennan and Brown, 2003).
Aberrant NOTCH signalling has been observed in
several human cancers, such as human T-cell acute
lymphoblastic leukemia, cervical cancer, and breast
cancer, suggesting that inhibition of NOTCH may
represent a potential therapeutic target (Dievart et al.,
1999; Nickoloff et al., 2003).
In human normal mammary stem cells, Charafe-
Jauffret et al. (2008) demonstrated that induction of
NOTCH signalling can promote self-renewal. According
to Brennan and Brown (2003), unregulated NOTCH
signalling in the mouse mammary gland leads to tumour
formation. In an investigation performed in human
breast cancer, the high expression of NOTCH
intracellular domain in ductal carcinoma in situ (DCIS)
predicted a reduced time to recurrence five years after
surgery (Farnie and Clarke, 2007).
An initial event in oncogenesis of sporadic breast
cancer can be the amplification and overexpression of
the HER-2 (human epidermal growth factor receptor 2)
gene. The HER-2 gene is amplified in about 18-25% of
human breast cancers and has been implicated in
mammary tumorigenesis as well as in mediating
aggressive tumor growth and metastasis (Korkaya et al.,
HER-2 overexpression in normal human mammary
epithelial cells, as well as in mammary carcinomas,
increases the proportion of stem cells, as indicated by
ALDH1 expression (Korkaya et al., 2008). Ginestier et
al. (2007) also found a significant correlation between
expression of the stem cell marker ALDH1 and HER-2
overexpression. The development of HER-2 inhibitors
such as trastuzumab or lapatinib, have demonstrated
clinical benefit, and the clinical efficacy of trastuzumab
may relate to its ability to directly target the CSC
population in HER-2-amplified tumors (Kakarala and
Wicha, 2008; Korkaya et al., 2008).
Tumor suppressor genes such as PTEN and p53 have
also been implicated in the regulation of CSC self-
renewal. According to Korkaya and Wicha (2007), these
genes are deregulated in CSCs, leading to uncontrolled
self-renewal, which in turn can generate tumors that are
resistant to conventional therapies.
Stem cell niche
Normal stem cells and cancer cells are both believed
to be regulated by epigenetic mechanisms and their
microenvironments. Evidence suggests that the stem cell
niche is the specialized microenvironment surrounding
stem cells that maintains their stemness and prevents
their differentiation, controlling normal stem cell
maintenance and self-renewal (Spradling et al., 2001;
Wicha et al., 2006). Moreover, stem cells that replenish
and repair adult tissues must be able to resist the stress
events associated with episodes of tissue damage.
Experimental evidence suggests that stem cells residing
in adult tissues are extremely resistant to alterations in
pH, temperature, and toxicant exposure (Woodward et
The pathophysiologic microenvironment in tumors
can be very heterogeneous. It comprises stem cells,
extracellular matrix properties, and signalling from
surrounding cells. The niche to which normal stem cells
are exposed can determine their ability to differentiate,
and can also modify their biological properties, such as
invasion and metastatic potential (Brisken and Duss,
In breast tumors, it has been stated that breast
density is an important risk factor for breast cancer
Stem cells and breast cancer
development (Filip et al., 2006). According to Savarese
et al. (2006), some growth factors produced by the breast
stromal fibroblasts appear to influence breast density and
also mammary stem cell behavior. In addition to
mammary fibroblasts, the role of endothelial cells and
adipocytes in mammary stem cell behavior is also
currently being investigated (Kakarala and Wicha,
Regarding metastatic dissemination, Karnoub et al.
(2007) showed that human mesenchymal stem cells
derived from bone marrow mixed with human breast
carcinoma cells markedly enhanced the metastatic
capacity of the tumor cells. This occurred through tumor
cell stimulation of chemokine CCL5 secretion by
mesenchymal stem cells, which then acted in a paracrine
manner on the breast cancer cells to enhance their
motility, invasion, and metastatic potential. According to
Karnoub et al. (2007), this metastatic capacity is
reversible and depends on CCL5 signalling through the
chemokine receptor CCR5.
CSC hypothesis implications for breast cancer
The heterogeneity and molecular complexity of
breast cancer are the great challenges for the
development of effective strategies to prevent and treat
this disease. Current therapeutic strategies in breast
cancer (surgery, radiotherapy, hormonal therapy, and
chemotherapy) have reduced recurrence rates; however,
disease still recurs in a significant proportion of women
after these treatments. The episodes of recurrences and
lack of curative treatment in metastatic disease raise the
question of whether current therapies target the correct
cells (Charafe-Jauffret et al., 2008; Kakarala and Wicha,
The existence of CSCs is significant to breast cancer
treatment. Beyond the limitations of current therapies in
targeting the CSC component, there is also evidence that
breast CSCs, as well as CSCs from other cancer types,
are relatively more resistant to both radio- and
chemotherapy (Sakariassen et al., 2007; Tang et al.,
2007; Kakarala and Wicha, 2008). Although CSCs
comprise only a very small percentage of the cells within
a tumor, a postulated mechanism for this resistance is
that stem cells are slowly proliferating and remain in the
G0 phase of the cell cycle for extended periods of time.
They also express increased levels of transporting
proteins that enable efflux of chemotherapeutic drugs,
making the cells tolerant to cell-cycle-based cytotoxic
therapies (Kakarala and Wicha, 2008) (Fig. 3).
Moreover, according to Smalley and Clarke (2005), the
ALDH1 enzyme that is highly expressed in stem cells is
capable of metabolizing some chemotherapeutic agents,
such as cyclophosphamide. As shown by Abraham et al.
(2005) and Glinsky et al. (2005), the breast tumors with
elevated proportion of CSCs are associated with a higher
risk of local and distant recurrences. In the breast cancer
cell line MCF7, Phillips et al. (2006) observed that cells
expressing the CD44+/CD24-/low phenotype are
associated with resistance to both radiotherapeutic and
tamoxifen treatment at clinically relevant doses, with the
size of this population increasing after short sessions of
fractionated irradiation. According to these authors,
these findings provide a possible mechanism for the
accelerated repopulation of tumor cells observed during
gaps in radiotherapy.
Different pathways may be associated with the
determination of mammary stem cell fate and can be
deregulated in cancer. The clarification of pathways that
regulate self-renewal and maturation of mammary stem
cells may provide potential targets for breast cancer
prevention and development of new therapeutic
strategies. Despite stem cell compartments constituting a
very small target population for therapy, successful
clinical treatment will require the knowledge of
particular tumor cell pathways and proteins that are
specifically expressed in these cells and that will be
susceptible to drug targeting (Wicha et al., 2006;
Campbell and Polyak, 2007). Hopes for new treatments
that selectively kill these cancer cells comes from recent
work showing the signalling pathways that control the
CSC proliferation and their local microenvironment
during cancer evolution (Korkaya et al., 2007). These
new findings have crucial implications for the aiming of
current therapeutic strategies and for the development of
the next generation of targeted therapies.
Stem cells have been documented in a variety of
normal tissues, and also identified in malignant
neoplasms. The CSC hypothesis is changing our current
understanding of breast carcinogenesis and provides a
new paradigm that should impact breast cancer risk
assessment, prevention, early detection, outcome, and
The presence of a rare CSC population among the
heterogeneous mix of tumoral cells supports the
hypothesis that CSCs can have a fundamental influence
on the local regrowth of cancers following treatment.
Despite the increased number of publications in the
CSC field over the past few years, further work is
imperative. Strategies to isolate breast CSCs, find
additional reliable cell surface markers, and perform
comparative gene expression profiling of CSCs and their
normal stem cell counterparts will be required to more
accurately define mammary stem/progenitor cells,
terminally differentiated luminal and myoepithelial cells,
and breast CSCs, and to better identify putative
therapeutic targets. It will be of enormous interest to
determine whether CD44+/CD24-/ESA+breast CSCs can
be directly targeted by specific therapies without
associated toxicity to normal stem cell niches or harmful
effects on wound healing. The use of breast cancer cell
lines and cancer stem cell cultures and xenograft models
Stem cells and breast cancer
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Accepted August 25, 2009
Stem cells and breast cancer