# The Controversial Clinicobiological Role of Breast Cancer Stem Cells

Authors: Claudia Casarsa; Saro Oriana; Danila Coradini
Journal: Journal of Oncology
 (2008)

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## Abstract

Breast cancer remains a leading cause of morbidity and mortality in women mainly because of the propensity of primary breast tumors to metastasize. Growing experimental evidence suggests that cancer stem cells (CSCs) may contribute to tumor progression and metastasis spread. However, despite the tremendous clinical potential of such cells and their possible therapeutic management, the real nature of CSCs remains to be elucidated. Starting from what is currently known about normal mammary stem/progenitor cells, to better define the cell that originates a tumor or is responsible for metastatic spread, this review will discuss experimental evidence of breast cancer stem cells and speculate about the clinical importance and implications of their evaluation.


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## Body

## 1. Introduction

Despite significant advances in diagnosis and clinical management, breast cancer
remains a leading cause of morbidity and mortality in women [1], mainly owing
to the propensity of primary breast tumors to metastasize to regional and
distant sites such as lymph nodes, lung, liver, bone, and brain [2]. When the
primary tumor is detected and removed before metastasis occurs, prognosis could
be good and the chance of disease-free survival is high. However, if cancer
cells have already begun to disseminate from the primary tumor and spread to
other organs, current therapeutic strategies largely depend on the use of
systemic cytotoxic drugs that frequently result in severe side effects on the
patient and, in many cases, do not yield long-term success.This clinical scenario is further complicated by the fact that invasive
breast cancers exhibit a wide range of morphological types, molecular profiles,
and clinical behaviors. Not only there is a large variation in the nature of cell types between cancers, but even within a single tumor a significant heterogeneity in phenotype and genotype can be observed [3]. Based on growth patterns
and cytological characteristics of the tumor cells, invasive breast cancers are
categorized by the World Health Organization into 18 different histological subtypes, each of them associated with a diverse
clinical behavior. In addition to morphology, invasive breast cancers can also be
classified according to their proliferative potential (evaluated, e.g., by Ki67
expression) or the presence of such biological factors as hormone receptors (estrogen
[ER]  and progesterone [PgR]) or HER2/neu overexpression that are currently used
in clinical practice to predict the prognosis and the response/resistance to cytotoxic
and/or hormonal therapy [4–6].Understanding the molecular causes of such a heterogeneity is therefore
of paramount importance not only for the development of new therapeutic
approaches, but also for a better knowledge of the biological bases of breast
tumorigenesis and metastatic spread. To address these questions, over the past
decade, scientists have used
innovative technical strategies and approached new intriguing directions
aimed to define the genetic and epigenetic profile of the single tumor and to
better define the cell that originates a tumor or is responsible for metastatic
spread. In particular, investigators focused their attention on the hypothesis
that breast cancer may be a stem cell disease, arising from tissue
stem/progenitor cells or driven by cells with stem-cell properties [7].


## 2. Isolation and Characterization of Mammary Stem/Progenitor Cells

In the adult,
cell loss, associated with the physiological tissue turnover, is compensated by
the activity of specific cells, termed stem cells, which are so defined by
their ability to self-renew and to generate the entire repertoire of the differentiated
cells composing a given tissue. Since 1983, the existence of such stem cells
has also been postulated in the mammary gland to explain the cellular dynamics underlying
morphological changes throughout a woman's life, particularly during and after
pregnancy [8].In general, the identification and purification of normal stem cells are  difficult tasks because of the paucity of stem cells in the
tissue of origin and to the lack of stemness-specific morphologic traits. Hence,
animal models have been used, and the murine model, in particular, has supplied
relevant data to improve our understanding of the cell biology of the mammary
gland and to clarify the presence of stem/progenitor cell and differentiated cell
compartments in the mammary gland [9, 10]. However, the information obtained
from mice cannot be directly applied to humans (as in the case of murine exclusive
cell surface markers) or may not be promptly translated to humans, as known in the
hematopoietic field, where an opposite surface-antigen profile was observed in
humans (CD34+CD38−) with respect to mice (CD34−CD38+).
Several in vitro strategies have
thus been developed to isolate and characterize human mammary stem/progenitor
cells, based on the differential expression of some cell surface markers, the
formation of mammospheres, and the use of fluorescent dyes.


### 2.1. Cell Surface Markers

The first experimental
evidence of the existence of human mammary stem/progenitor cells was obtained by
the in vitro isolation of multipotent
epithelial cells, from normal human adult breast, according to their different expression of MUC-1
glycoprotein, CALLA/CD10, and epithelial-specific antigen (ESA). Using these
markers, two epithelial-cell progenitor populations were distinguished, corresponding
to the two components of normal mammary gland (myoepithelial cells, forming the basal layer of ducts, and epithelial cells, lining the lumen of
ducts and forming the alveoli) [11–13]. Subsequently,
Gudjonsson et al. [13],
starting from the previous findings [14], provided in vivo evidence of the morphogenic potential of the MUC-1−/ESA+ subpopulation, inoculating these cells subcutaneously in nude mice after pre-embedding
them in a mixture of collagen gel and matrigel.


### 2.2. Mammosphere Formation

To identify a
human mammary stem/progenitor-cell subpopulation, Dontu et al. [15] adopted a strategy similar to that employed for
primary neural cells and based on the formation of floating spherical colonies used
to define and measure stem cell-like behavior. In fact, contrary to the dogma
that epithelial cell survival is anchorage-dependent, single cell suspensions
of human mammary epithelial cells, obtained by mechanic/enzymatic dissociation,
surprisingly survived in suspension and generated floating spherical colonies,
termed nonadherent mammospheres [15].
The mammospheres contained numerous undifferentiated cells that, once isolated
from the cluster, were able to generate new multilineage colonies, when
cultured under differentiating conditions, and, in 3D culture, to reconstitute a
functional mammary gland. However, the intrinsic dynamics of such cytospheres,
as conventionally assayed, introduces several confounders, as reported in a
recent paper by Singec et al. [16]
who underlined the need to use more accurate conditions for assessing the clonality,
number, and fate of stem cells. Although sphere formation may represent a
useful culturing tool, it is not specific to stem cell characterization; any
dividing cell from virtually any tissue will form floating cell clusters, when
cultured in a serum-free medium and on a nonadherent substrate, owing to a
predominant intercellular adhesiveness. Spontaneous sphere fusion may occur in
normal as well as in neoplastic sphere cultures. Furthermore, in agreement with
Singec et al. [16], we have observed that the mammospheres,
supposedly rich in multilineage progenitors, have a very short life span (about
3-4 weeks), making it
difficult to define the cells composing them as real mammary stem cells, which
are long-lived by definition. On account of all these criticisms, this experimental
approach, based on the ability of the supposed mammary stem cells to generate
clonal mammospheres under anchorage-independent culture conditions, has been defined
“a surrogate stem cell assay” [17].


### 2.3. Fluorescence Methods

To overcome the
challenge represented by the limited availability of stemness markers and to
take advantage of the ability of stem cells to extrude dyes, for example, Hoechst-33342
DNA-binding dye or rhodamine because the overexpression of some membrane
transporter proteins, such as P-glycoproteins or breast cancer resistance proteins
(BCRPs)  [18, 19],
fluorescent dyes have been used to identify and isolate by flow cytometry a small
fraction of cells supposed to be stem progenitors [20]. This cell fraction,
which amounts to around 0.2% of the total population, has been called side population (SP).However, several criticisms to such a sorting technique have been raised
in the recent years principally concerning the toxicity of dyes used in the
analysis [21] and the high assay variability associated with technical
modifications required for each cell population under study; these limitations
hamper the comparison of results obtained from different studies and affect
cell selection for in vitro and
in vivo growth experiments. In
addition, recent findings from two teratocarcinoma cell lines indicate that
Hoechst treatment, as performed during staining for SP analysis, can affect
cell differentiation, suggesting other potential complications in the
interpretation of data [22].Recently, another approach, based
on aldehyde dehydrogenase (ALDH) activity, has been proposed as a promising
alternative to identify and characterize the human mammary stem/progenitor
component in the mammary gland [23]. ALDH, a detoxifying enzyme responsible for the oxidation of intracellular
aldehydes, is a putative candidate marker of stemness, since it is highly expressed in
hematopoietic and neuronal stem/progenitor cells. Its presence can be evaluated
by ALDEFLUOR kit: brightly fluorescent ALDH-expressing cells are easily detected
by flow cytometry in the green fluorescence channel. Using such an approach, Ginestier
et al. [23] showed that ALDH-positive
cells formed mammospheres with high efficiency (10±3.5%) when put into
96-well plates (1 cell/well), and displayed stem-like properties in terms of bilineage
differentiation in vitro and outgrowth
potential when inoculated in the mammary fat pad of humanized mice. However,
even though the findings indicate that only ALDH-positive cells had phenotypic
and functional characteristics of mammary stem cells, immunostaining of tissue
sections using a monoclonal antibody against the first isoform of ALDH (ALDH1) did
not detect any overlapping expression of several markers (e.g., CK5/6 and
CK14), previously associated with undifferentiated mammary epithelial cells,
probably owing to the scarcity of this population. Analysis performed on mammosphere
sections have shown that ALDH1-positive cells represented approximately 5% of
the total cell population and expressed CK5/6 or CK14, supporting the
hypothesis that ALDH1-positive cells represent the stem/progenitor population [13, 15].Unfortunately, as
highlighted by these inconclusive results, the efforts to purify adult stem
cells from the human mammary gland have so far been hampered, on the one hand, by
the lack of cell surface markers specific to undifferentiated or differentiated
mammary cells and, on the other hand, by the lack of suitable in vivo assays for testing stem cell
properties, with the consequence that human breast stem cells have not yet been
extensively characterized.


## 2.1. Cell Surface Markers

The first experimental
evidence of the existence of human mammary stem/progenitor cells was obtained by
the in vitro isolation of multipotent
epithelial cells, from normal human adult breast, according to their different expression of MUC-1
glycoprotein, CALLA/CD10, and epithelial-specific antigen (ESA). Using these
markers, two epithelial-cell progenitor populations were distinguished, corresponding
to the two components of normal mammary gland (myoepithelial cells, forming the basal layer of ducts, and epithelial cells, lining the lumen of
ducts and forming the alveoli) [11–13]. Subsequently,
Gudjonsson et al. [13],
starting from the previous findings [14], provided in vivo evidence of the morphogenic potential of the MUC-1−/ESA+ subpopulation, inoculating these cells subcutaneously in nude mice after pre-embedding
them in a mixture of collagen gel and matrigel.


## 2.2. Mammosphere Formation

To identify a
human mammary stem/progenitor-cell subpopulation, Dontu et al. [15] adopted a strategy similar to that employed for
primary neural cells and based on the formation of floating spherical colonies used
to define and measure stem cell-like behavior. In fact, contrary to the dogma
that epithelial cell survival is anchorage-dependent, single cell suspensions
of human mammary epithelial cells, obtained by mechanic/enzymatic dissociation,
surprisingly survived in suspension and generated floating spherical colonies,
termed nonadherent mammospheres [15].
The mammospheres contained numerous undifferentiated cells that, once isolated
from the cluster, were able to generate new multilineage colonies, when
cultured under differentiating conditions, and, in 3D culture, to reconstitute a
functional mammary gland. However, the intrinsic dynamics of such cytospheres,
as conventionally assayed, introduces several confounders, as reported in a
recent paper by Singec et al. [16]
who underlined the need to use more accurate conditions for assessing the clonality,
number, and fate of stem cells. Although sphere formation may represent a
useful culturing tool, it is not specific to stem cell characterization; any
dividing cell from virtually any tissue will form floating cell clusters, when
cultured in a serum-free medium and on a nonadherent substrate, owing to a
predominant intercellular adhesiveness. Spontaneous sphere fusion may occur in
normal as well as in neoplastic sphere cultures. Furthermore, in agreement with
Singec et al. [16], we have observed that the mammospheres,
supposedly rich in multilineage progenitors, have a very short life span (about
3-4 weeks), making it
difficult to define the cells composing them as real mammary stem cells, which
are long-lived by definition. On account of all these criticisms, this experimental
approach, based on the ability of the supposed mammary stem cells to generate
clonal mammospheres under anchorage-independent culture conditions, has been defined
“a surrogate stem cell assay” [17].


## 2.3. Fluorescence Methods

To overcome the
challenge represented by the limited availability of stemness markers and to
take advantage of the ability of stem cells to extrude dyes, for example, Hoechst-33342
DNA-binding dye or rhodamine because the overexpression of some membrane
transporter proteins, such as P-glycoproteins or breast cancer resistance proteins
(BCRPs)  [18, 19],
fluorescent dyes have been used to identify and isolate by flow cytometry a small
fraction of cells supposed to be stem progenitors [20]. This cell fraction,
which amounts to around 0.2% of the total population, has been called side population (SP).However, several criticisms to such a sorting technique have been raised
in the recent years principally concerning the toxicity of dyes used in the
analysis [21] and the high assay variability associated with technical
modifications required for each cell population under study; these limitations
hamper the comparison of results obtained from different studies and affect
cell selection for in vitro and
in vivo growth experiments. In
addition, recent findings from two teratocarcinoma cell lines indicate that
Hoechst treatment, as performed during staining for SP analysis, can affect
cell differentiation, suggesting other potential complications in the
interpretation of data [22].Recently, another approach, based
on aldehyde dehydrogenase (ALDH) activity, has been proposed as a promising
alternative to identify and characterize the human mammary stem/progenitor
component in the mammary gland [23]. ALDH, a detoxifying enzyme responsible for the oxidation of intracellular
aldehydes, is a putative candidate marker of stemness, since it is highly expressed in
hematopoietic and neuronal stem/progenitor cells. Its presence can be evaluated
by ALDEFLUOR kit: brightly fluorescent ALDH-expressing cells are easily detected
by flow cytometry in the green fluorescence channel. Using such an approach, Ginestier
et al. [23] showed that ALDH-positive
cells formed mammospheres with high efficiency (10±3.5%) when put into
96-well plates (1 cell/well), and displayed stem-like properties in terms of bilineage
differentiation in vitro and outgrowth
potential when inoculated in the mammary fat pad of humanized mice. However,
even though the findings indicate that only ALDH-positive cells had phenotypic
and functional characteristics of mammary stem cells, immunostaining of tissue
sections using a monoclonal antibody against the first isoform of ALDH (ALDH1) did
not detect any overlapping expression of several markers (e.g., CK5/6 and
CK14), previously associated with undifferentiated mammary epithelial cells,
probably owing to the scarcity of this population. Analysis performed on mammosphere
sections have shown that ALDH1-positive cells represented approximately 5% of
the total cell population and expressed CK5/6 or CK14, supporting the
hypothesis that ALDH1-positive cells represent the stem/progenitor population [13, 15].Unfortunately, as
highlighted by these inconclusive results, the efforts to purify adult stem
cells from the human mammary gland have so far been hampered, on the one hand, by
the lack of cell surface markers specific to undifferentiated or differentiated
mammary cells and, on the other hand, by the lack of suitable in vivo assays for testing stem cell
properties, with the consequence that human breast stem cells have not yet been
extensively characterized.


## 3. Cancer Propagation Models

To explain why not every cell within a tumor is
capable of maintaining and/or reinitiating tumor growth, two models of
heterogeneity in solid cancer have been proposed: theclonal evolution model
(Figure 1(a)) and the cancer stem cell (CSC) model (Figure 1(b))  [24–26].Models of heterogeneity in solid cancer cells. (a)
The clonal evolution model assumes that every cell in a tumor is potentially
tumor-initiating. Progression is governed by rare stochastic events operating in
all cells. Cells with mutations (yellow)
that acquire growth advantage will dominate over all other cells in the tumor
and will originate a new clone containing cells characterized by a different
phenotype and having different proliferative potentials; in a clonogenicity or
tumorigenicity assay, some of these cells (blue)
would have a low probability of exhibiting this potential. (b) The cancer stem cell model states that a
particular subset of tumor cells with stem cell-like properties, called “cancer
stem cell” (CSC) (pink), drives tumor initiation, progression, and recurrence.
CSCs are able to self-renew indefinitely and to differentiate, leading to the
production of all cell types (blue) that make up the rest of the tumor. In
clonogenic assays, CSCs have the potential to proliferate extensively and can
form new tumors on transplantation.
(a)
Clonal evolution model(b)
Cancer stem cell modelThe two main aspects of theclonal evolution model, first proposed
by Nowell in 1976 [27], are (1) diversity within the tumor due to genetic
instability and (2) selection of the cells with the most advantageous phenotype.
In this respect, stem or differentiated cell characteristics (including self-renewing
capacity) are just simple phenotypes and, as such, can change. According to this
model, any cancer cell can potentially become invasive and cause metastasis or
become resistant to therapies and cause recurrence.Thecancer
stem cell (CSC) model (Figure 1(b))
states that a particular subset of tumor cells with stem cell-like properties,
called “cancer stem cells”, drives tumor initiation, progression, and
recurrence. Since CSCs are widely believed to arise from normal stem or
progenitor cells, the identification of stem cells in a tissue is of paramount
importance to understand how a tumor arises. By definition, CSCs have the
ability to self-renew indefinitely and to differentiate, which leads to the
production of all cell types composing a tumor, both tumorigenic and nontumorigenic
cells. But the latter lack the unlimited self-renewing capacity and the ability
to reproduce the phenotypically diverse cell populations that make up the tumor
bulk [28, 29]. Therefore, this model suggests that the presence of such rare
tumor-initiating cells, in the heterogeneous mix of cells composing a tumor, is
essential for neoplastic progression and metastatic spread [29, 30]. While the CSC model of carcinogenesis was
described in the context of systemic malignancies as long ago as in the 1930s,
only recently has it been extended to solid tumors, and during the last decade,
several studies have provided evidence that CSCs may also exist in solid tumors
including brain [31], lung [32], prostate [33], colon [34], liver [35], pancreas
[36], and breast [37] carcinomas,
as well as melanoma [38].In breast tumorigenesis, theCSC model also seems to be supported by
clinical observations indicating that, despite the fact that breast cancer
patients may have hundreds or thousands of single disseminated cancer cells
detectable in their bloodstream, only a very small percentage of cells progresses to form overt
macroscopic metastases [39], and that metastatic tumors tend to reproduce a
heterogeneity similar to the primary tumor [40]. Since CSCs have been supposed to
be responsible for the chemo- and radioresistance observed in several solid
tumors [41, 42], the CSC model could
explain such a finding with the ability of CSCs to escape cytotoxic drugs, via a
high expression of specific drug transporter proteins, and to resist radiotherapy
by increasing DNA repair activity.Unfortunately, the definitive proof
of the cellular origin of CSCs—they may arise either
from normal resident stem cells within the tissue bearing the malignancy or
from transformed progenitor cells that acquire the stem cell ability of self-renewal—remains elusive
and is a topic of intense debate as well as experimental investigation. In
fact, if these cells arise from normal stem cells, then cancer cells could take
advantage of the existing regulatory self-renewal pathways of stem cells. On
the other hand, if these cells arise from mature, differentiated cells,
multiple oncogenic mutations, affecting differentiation and self-renewal
pathways are required for a cell to become tumorigenic and metastatic [43, 44].
It can be argued that mature cells have a very limited life span, and thus it
is unlikely that all the necessary mutations could occur during the relatively
short life of these cells. In contrast, the unlimited self-renewing capacity of
normal stem cells could enable them to accumulate the necessary mutations,
despite the apparent paradox of the stem cell dogma, according to which a stem
cell maintains its DNA constant through symmetric division [26, 45]. Thus,
whereas the CSC model is highly
hierarchical with a unique self-renewing cell type at the apex, the clonal evolution model attributes much
of the intratumor variation to subclonal differences in mutational profile and implies
that all cells, except the terminally differentiated ones, may have
self-renewal capacity. Nevertheless, clonal
evolution and CSC models share some
aspects: in both, for instance, the tumor arises from a single cell that has
acquired multiple mutations and has gained unlimited proliferative potential.
This suggests that tumor heterogeneity could be explained by a new version of
the clonal evolution model that incorporates some features of the CSC
hypothesis [26].A more intriguing model to explain the
nature of sustained tumor growth is now emerging from theCSC model. According
to this new model (Figure 2), tumors could originally
be driven by rare CSCs (CSC1). Subsequent mutations, enhancing self-renewing
capacity, could create a dominant, more aggressive subclone (CSC2), with a phenotypical
aspect distinct from the original CSC. However, if the CSC2 subclone does not display
“stem-like” properties, it should not be able to initiate tumors with a high
frequency [46].Figure 2
Mixed model for the nature of sustained tumor growth. The tumor is originally driven by rare cells
of one phenotype (CSC1, yellow), which may have stem/progenitor cell origin and
give rise to the tumor bulk by producing terminally differentiated cells (blue).
Subsequent mutations enhancing self-renewing capacity create a dominant
subclone that is phenotypically different (CSC2, green) and more aggressive. If
the CSC2 subclone does not display “stem-like” cell properties, such a subset will
not be able to initiate tumors with a high frequency [30].Although metastasis is the predominant
cause of lethality in breast cancer patients, metastatic spread is a highly
inefficient process, since very few cells successfully colonize distant sites. A
possible explanation of this inefficiency is provided by theCSC model, according to which genetic
and epigenetic mechanisms may generate, within the primary tumor, a
self-renewing metastatic cancer stem cell (mCSC) characterized by an immunophenotype
different from the CSC that is driving tumorigenesis (Figure 3). This is
suggested by the observation that in metastatic sites, some cell subpopulations
with self-renewing features display a cell surface marker profile different
from the CSC that originated the primary tumor [47]. Through a series of
invasive processes, this new mCSC subclone could enter blood vessels and
colonize distant organs according to the “seed and soil” hypothesis.Figure 3
Metastatic evolution.
Genetic and epigenetic mechanisms may cause the generation of a self-renewing
metastatic cancer stem cell subclone (mCSC, yellow), phenotypically different
from the CSC that is driving tumorigenesis (pink). Through a series of invasive
processes, mCSC enters blood vessels and colonizes distant organs according to
the seed and soil hypothesis. Blue cells represent bulk tumor cells [47].


## 4. Isolation and Characterization of Breast Cancer Stem Cells

On account of accumulating evidence and
according to theCSC model that
assumes a tissue stem/progenitor cell as the origin of a tumor, some methods adopted
to isolate and cultivate normal mammary stem/progenitor cells have also been
applied to breast cancer tissue. However, there may be biases, inherent in the
techniques applied, that can affect experiment reproducibility. In fact, some
technical approaches, including the strong enzymatic digestion of breast tissue
necessary to disaggregate the connective tissue surrounding the mammary gland, can
cause damage to cells or loss of some particular surface markers, hampering the
identification of unique markers for the putative CSCs (authors' unpublished
data). Furthermore, since cell recovery from solid tissue is usually low (rarely
exceeding 10%), the samples obtained may not be representative of the original
lesion, owing to the rare presence of CSCs in the tumor mass, actual CSCs may be
lost, while other cells may be mistakenly identified as CSCs [26].The first experimental clues about
the existence of putative CSCs came from the observation that, even when using
immortalized cancer cell lines, large numbers of cells (in the range105–106) must be injected into
experimental animals to initiate a tumor and, in spite of that, only a very
small proportion of these cells will go on to form metastases [48]. Additional
experimental studies showed that while early steps in hematogenous metastasis (intravasation,
survival, arrest, and extravasation) can be remarkably efficient, with over 80%
of cells successfully completing the metastatic process at this point, only a
small subset of these cells (i.e., about 2%, depending on the experimental model) can
initiate growth as micrometastases and that an even smaller subset (i.e., about
0.02%, depending on the experimental model) is able to persist and grow into
macroscopic tumors. This suggests that the initial growth of micrometastases
represents the critical decision-making stage.


### 4.1. Surface Markers

After the identification of stemness specific
markers in hematopoietic tumors, considerable progress has also been made in the
elucidation of the biological properties of breast cancer stem cells. Al-Hajj et al. [37] first demonstrated the
presence of a cell subpopulation displaying stem cell properties, characterized
by the cell surface marker profile CD44+/CD24low/lin−,
in solid tissues and in pleural effusions of patients with advanced-stage
metastatic breast cancer. This phenotype displayed a 10- to 50-fold increase in
the ability to form tumors in NOD/SCID mice over unfractionated tumor cells. In
addition, the authors demonstrated that cells with such a specific cell surface
antigen profile could successfully and efficiently grow as tumor xenografts in
immunodeficient mice; the highest capacity to form tumors was observed after
injection of 200 cells with the ESA+/CD44+/CD24low/lin− phenotype.However, the heterogeneous
expression patterns of ESA, CD44, or CD24, observed by FACS analysis in
secondary lesions, support the hypothesis that the CD44+/CD24low/lin− profile could be the marker of the putative breast CSC phenotype, since
it recapitulates the heterogeneous complexity of the tumors from which it has
been isolated. Such a hypothesis could be corroborated by a study in which Ince
et al. [49] observed the presence of two different populations of
mammary epithelial cells in the tumor of a single patient. Only one population
was myoepithelial-like and was able to give rise to tumors with heterogeneous
histology and to be tumorigenic when injected into mammary fat pads of immunodeficient
mice.


### 4.2. Fluorescence Methods

Similar to normal breast stem cells, CSCs have also
been investigated according to their expression of aldehyde dehydrogenase (ALDH)
activity. Ginestier et al. [23]
analyzed the tumorigenicity of ALDEFLUOR-positive populations isolated from two
metastatic and two primary invasive ductal carcinomas (three triple negative
tumors—ER-negative,
PR-negative, and HER2-negative—and one
ER-positive, PR-positive, and HER2-negative tumor) that were transplanted into
the humanized cleared fat pad of immunodeficient mice, immediately after
surgery with no previous cultivation. To test tumorigenicity, cell sorting was
performed at early passages in animals, in order to minimize the variability
introduced by the xenotransplant model. ALDEFLUOR-positive cells represented 3%
to 10% of the total cell population, and 500 positive cells were able to
generate a tumor in as few as 40 days. Significantly, the concomitant presence
of the ALDEFLUOR-positive phenotype and of the previously described breast CSC
phenotype (CD44+/CD24−/low) was observed in a small cell
fraction of the three triple-negative tumors (range 0.08–1.16%), whereas tumor
cells generated from one metastatic tumor (pleural effusion) showed a high
percentage of overlapping cell fraction (1.16%) that gave rise to outgrowth from
as few as 20 cells. Conversely, ALDEFLUOR-negative cells, though bearing the
CD44+/CD24−/low phenotype, were not tumorigenic, even
when 50 000 cells/fat pad
were implanted. This suggests that the concomitant presence of ALDEFLUOR-positive
and CD44+/CD24−/low phenotypes may characterize
progenitor cells with proliferative potential. When assessing the potential use
of ALDH1 to detect malignant mammary stem/progenitor cells in situ on breast cancer tissue
sections, Ginestier et al. [23] found that ALDH1 expression correlated
with the histoclinical parameters, suggesting the use of this marker as a
powerful predictor of poor clinical outcome. So far, therefore, the ALDEFLUOR
assay, overcoming the limited availability of CSC-specific surface markers, seems
to represent the pivotal tool for the isolation of cell populations with high
tumor-initiating capability or cell populations with stem-like properties in
normal tissue, allowing the identification of stem/progenitor cells involved in
normal mammary development and may be in tumor transformation.As regards the use of Hoechst 33342 to detect the side population and to identify CSCs,
thus bypassing the lack of universally accepted surface-antigen markers, several
limitations are emerging in addition to the technical criticisms described for
the methods of normal mammary stem cell isolation; they include the low cell
recovery from tumor tissue that does not reflect the entire cohort of cancer
cells, and the toxicity of the dye that precludes its use for functional CSC
assays in vitro and in vivo [50].


### 4.3. Self-Renewal Pathways

Since another trait shared by normal stem cells
and CSCs is the ability to self-renew, the deregulation of key pathways
involved in such a pivotal cellular function has been presumed to be implicated
in breast carcinogenesis and more thoroughly investigated. Experimental evidence
indicates that carcinogenesis in the mammary gland, and in other solid organs,
might result in the transformation of stem and/or progenitor cells because of the
deregulation of self-renewal pathways, including Notch, Wnt, Hedgehog, and the transcription
factor Bmi1 [51] and suggests that the targeting of self-renewal pathways might
provide a specific approach to eradicate CSCs [52]. However, in developing and
testing compounds against putative CSCs, several uncertainties must be faced
and elucidated, first of all the possible instability of CSCs that could hamper
specific cell targeting.


## 4.1. Surface Markers

After the identification of stemness specific
markers in hematopoietic tumors, considerable progress has also been made in the
elucidation of the biological properties of breast cancer stem cells. Al-Hajj et al. [37] first demonstrated the
presence of a cell subpopulation displaying stem cell properties, characterized
by the cell surface marker profile CD44+/CD24low/lin−,
in solid tissues and in pleural effusions of patients with advanced-stage
metastatic breast cancer. This phenotype displayed a 10- to 50-fold increase in
the ability to form tumors in NOD/SCID mice over unfractionated tumor cells. In
addition, the authors demonstrated that cells with such a specific cell surface
antigen profile could successfully and efficiently grow as tumor xenografts in
immunodeficient mice; the highest capacity to form tumors was observed after
injection of 200 cells with the ESA+/CD44+/CD24low/lin− phenotype.However, the heterogeneous
expression patterns of ESA, CD44, or CD24, observed by FACS analysis in
secondary lesions, support the hypothesis that the CD44+/CD24low/lin− profile could be the marker of the putative breast CSC phenotype, since
it recapitulates the heterogeneous complexity of the tumors from which it has
been isolated. Such a hypothesis could be corroborated by a study in which Ince
et al. [49] observed the presence of two different populations of
mammary epithelial cells in the tumor of a single patient. Only one population
was myoepithelial-like and was able to give rise to tumors with heterogeneous
histology and to be tumorigenic when injected into mammary fat pads of immunodeficient
mice.


## 4.2. Fluorescence Methods

Similar to normal breast stem cells, CSCs have also
been investigated according to their expression of aldehyde dehydrogenase (ALDH)
activity. Ginestier et al. [23]
analyzed the tumorigenicity of ALDEFLUOR-positive populations isolated from two
metastatic and two primary invasive ductal carcinomas (three triple negative
tumors—ER-negative,
PR-negative, and HER2-negative—and one
ER-positive, PR-positive, and HER2-negative tumor) that were transplanted into
the humanized cleared fat pad of immunodeficient mice, immediately after
surgery with no previous cultivation. To test tumorigenicity, cell sorting was
performed at early passages in animals, in order to minimize the variability
introduced by the xenotransplant model. ALDEFLUOR-positive cells represented 3%
to 10% of the total cell population, and 500 positive cells were able to
generate a tumor in as few as 40 days. Significantly, the concomitant presence
of the ALDEFLUOR-positive phenotype and of the previously described breast CSC
phenotype (CD44+/CD24−/low) was observed in a small cell
fraction of the three triple-negative tumors (range 0.08–1.16%), whereas tumor
cells generated from one metastatic tumor (pleural effusion) showed a high
percentage of overlapping cell fraction (1.16%) that gave rise to outgrowth from
as few as 20 cells. Conversely, ALDEFLUOR-negative cells, though bearing the
CD44+/CD24−/low phenotype, were not tumorigenic, even
when 50 000 cells/fat pad
were implanted. This suggests that the concomitant presence of ALDEFLUOR-positive
and CD44+/CD24−/low phenotypes may characterize
progenitor cells with proliferative potential. When assessing the potential use
of ALDH1 to detect malignant mammary stem/progenitor cells in situ on breast cancer tissue
sections, Ginestier et al. [23] found that ALDH1 expression correlated
with the histoclinical parameters, suggesting the use of this marker as a
powerful predictor of poor clinical outcome. So far, therefore, the ALDEFLUOR
assay, overcoming the limited availability of CSC-specific surface markers, seems
to represent the pivotal tool for the isolation of cell populations with high
tumor-initiating capability or cell populations with stem-like properties in
normal tissue, allowing the identification of stem/progenitor cells involved in
normal mammary development and may be in tumor transformation.As regards the use of Hoechst 33342 to detect the side population and to identify CSCs,
thus bypassing the lack of universally accepted surface-antigen markers, several
limitations are emerging in addition to the technical criticisms described for
the methods of normal mammary stem cell isolation; they include the low cell
recovery from tumor tissue that does not reflect the entire cohort of cancer
cells, and the toxicity of the dye that precludes its use for functional CSC
assays in vitro and in vivo [50].


## 4.3. Self-Renewal Pathways

Since another trait shared by normal stem cells
and CSCs is the ability to self-renew, the deregulation of key pathways
involved in such a pivotal cellular function has been presumed to be implicated
in breast carcinogenesis and more thoroughly investigated. Experimental evidence
indicates that carcinogenesis in the mammary gland, and in other solid organs,
might result in the transformation of stem and/or progenitor cells because of the
deregulation of self-renewal pathways, including Notch, Wnt, Hedgehog, and the transcription
factor Bmi1 [51] and suggests that the targeting of self-renewal pathways might
provide a specific approach to eradicate CSCs [52]. However, in developing and
testing compounds against putative CSCs, several uncertainties must be faced
and elucidated, first of all the possible instability of CSCs that could hamper
specific cell targeting.


## 5. Limits to the Xenograft Approach

There are several limitations to the use of
xenograft assays as proof of “stemness”. The most relevant is that tumor growth
and stem cell phenotype are not only determined by intrinsic characteristics of
tumor cells but are also influenced by the microenvironment in which cells grow.
In this respect, the heterogeneity found in animal experimental models does not
prove that normal or malignant stem cells undergo asymmetric division but just reflects a change in cell
surface antigen expression induced by environmental conditions. Simply
injecting tumor cells into mice without measuring the time of latency required
to form a palpable tumor mass may induce to draw wrong conclusions about their
absolute tumorigenic potential, which may be influenced by environmental
conditions. Another important limitation to CSC investigation is the efficiency
of thein vivo model used. It is well known
that xenograft is less efficient than syngeneic transplant because of the
presence of animal growth factors that could interact with their equivalent
human receptors and provide confounding stimuli for the transplanted human
stem/progenitor cells. In a recent paper, Kelly et al. [53], challenging the CSC hypothesis, proposed that xenograft
may select a dominant clone capable of surviving and maintaining tumor
outgrowth in a foreign environment, and stressed the importance of performing a
tumorigenic assay using cells sorted from the patient's tumor to avoid cell variability
and the selection of a dominant cell subpopulation, after several serial
passages in animals, whereas the majority of cells die owing to the lack of
appropriate supporting factors.As regards the mammary gland, in
order to study human breast carcinogenesis, it is central to establish a model
system that more accurately recapitulates normal breast epithelial development
in rodents. For cancer cells, such a system should also correlate with the clinical
behavior of the source tumor in patients. However, the inability of human
breast epithelial cells to colonize mouse mammary fat pads represents a
constant problem.The importance of both species- and
tissue-specific influences has been highlighted in the studies by Kuperwasser et al. [54], which indicated that, although
outgrowths can be generated in the murine humanized mammary fat pads, the
repopulating frequency by normal breast stem cells remains relatively low. This
suggests that the expression of some markers of the inoculated cells could be influenced
by circulating or locally produced animal-specific factors. For example,
estrogen has been found to profoundly affect the growth of ER-negative breast
cancer cells because circulating mouse estrogens led to recruitment of bone
marrow-derived stromal cells and promoted the growth of tumors in virgin mice
[55].However, despite these limitations, xenograft
models still represent an essential tool for in vivo carcinogenesis studies. Meanwhile, the great challenge
in stem cell investigation will be the standardization
of an orthotopic model in which the whole tumor bulk could arise from a single definitively
characterized human breast CSC.


## 6. Issues Concerning Established Breast Cancer Cell Lines

Despite the intriguing results so far obtained,
the use of established breast cancer cell lines as experimental models to
collect data regarding CSCs is not without pitfalls, the main one being the
attempt to apply stem-cell concepts to breast cell lines. Established cell
lines, in fact, are cultivated in artificial conditions (depending on the
experimental model) for many generations, with the risk that the unavoidable selection
induced by the serum media blurs the distinction between tumorigenic and nontumorigenic
clones. For example, in vitro culture
conditions, such as growth with or without serum medium, could contribute to the
functional differences found between non-CSCs and CSCs, including a diverse proliferative
activity.It is unlikely that the so-called “cancer
stem cells” derived from established cell lines are the stem cells that make up
a tumor. It is more likely that the “stem cell component” indicated as
responsible for maintaining the line is in reality only a subpopulation of
cells having a high-proliferative rate and being able to form clonal aggregates
in the presence of additional techniques, for example, retroviral marking [16].
Therefore, extending the CSC concept to cell lines could be misleading as is
the case with the results reported by Sheridan et al. [56]. In a series of established breast cancer
cell lines (MDA-MB-231, TMD-436, Hs578T, SUM1315, HBL-100, and MDA-MB-468), the
authors observed a high percentage (>30%) of CD44+/CD24−/low cells which were highly efficient in initiating a tumor in experimental
settings, and concluded that those cell lines were composed mainly of cancer
stem cells. However, since the CD44+/CD24−/low phenotype
has been shown to be insufficient to confer stem-like properties [23], such an enrichment
in CD44+/CD24−/low phenotype in established cell lines could
be hypothesized to be a purification marker without functional implications. Therefore,
it is crucial to demonstrate that such a subpopulation, contained in
established breast cancer cell lines and displaying a putative stem-like
phenotype, has the real functional characteristics of CSCs: self-renewal and
differentiation capability. Awaiting these confirmations as well as more
standardized protocols for the isolation and expansion of CSCs from tissue, the
established breast cancer cell line model still remains a useful experimental tool
to test drugs, radioresistance, and antibodies against the surface markers associated
with the putative stem-like phenotype.


## 7. Microenvironment and Stem Cell Niche

Despite the extensive use of the in vivo model, in which human
tumor cells are injected into immunodeficient mice, significant
challenges are pending in experimental settings with CSCs, mostly related to
the biological and technical complexities associated with identifying,
quantifying, and longitudinally monitoring CSCs in a complex in vivo environment. In particular, as already mentioned, xenograft
may fail to reveal possible tumor growth-sustaining cells because the heterologous
microenvironment precludes essential interactions with support cells. As recently
shown in an elegant study by Naumov et
al. [57], many established human tumor cell lines, which had previously
been described as “nontumorigenic” (including the breast cancer cell line
MDA-MB-436), were potentially tumorigenic but dormant. In fact, when these
cells were injected into animals and allowed to grow for a much longer period
of time than a normal tumor growth assay (up to 12 months), spontaneous tumors
eventually began to develop after an initial dormancy period and a switch to an
angiogenic phenotype. Therefore, this study provides a conceptual framework to
approach the problem that a supportive microenvironment is required for tumor
outgrowth in CSCs-involving studies.In adults, normal stem cells reside
in a physiologically unique microenvironment called stem cellniche. This niche, mainly composed of fibroblasts and myoepithelial cells, provides
a physical anchoring site for stem cells via adhesion molecules linking the
stem cells to the extracellular matrix. Support cells act as a hub in
orientating dividing stem cells to hold one daughter cell in the niche, while
the other one exits the niche and undergoes transit amplification followed by
differentiation [58, 59]. Under normal physiological conditions, the niche
provides fine control over cell proliferation, typically balancing
proliferation and apoptosis through paracrine factors, so that the stem cell
population remains undifferentiated and maintains a constant size [60, 61]. The
effectors mediating heterotypic cell interactions within the niche include a
number of soluble factors and cell surface receptors. Interestingly, some of
these molecules, including Wnt, Notch, TGF-β, bone morphogenetic proteins
(BMPs), and others [62–65], are known to
be involved in tumor development, and emerging data support the idea that a
“cancer stem cell niche” could also exist, and that interactions with such a
tumor niche may sustain a self-renewing population of tumor cells [66].
Alterations affecting stromal cells, such as local modifications of tissue
homeostasis induced by chronic inflammation, have been shown to promote
formation of epithelial tumors, and very recent papers have described the
generation of pluripotent stem cells from fibroblasts. This evidence supports the
possibility that cancer may arise from just a few mutations in resident tissue
stem/progenitor cells or even differentiated cells leading to a stem-like
phenotype [67, 68]. Indeed, if signaling pathways are dysregulated, the niche
may be converted into a microenvironment that favors uncontrolled proliferation
and expansion of an altered stem cell population. Similarly, a cancer stem cell
could be hypothesized to remain dormant in a metastatic site until activated by
abnormal signaling from the microenvironment [66].Currently, evidence of an
anatomically and/or physiologically specialized environment that constitutes a
true CSC niche is scarce, and the identity of a stem cell niche within the
mouse mammary gland has not been defined. Conversely, a putative stem cell niche
which gives rise to at least three lineage-restricted cell types outside the
stem cell zone has been identified in the adult human breast [28]. The most
likely location of the niche in the mature gland could be the ducts, where in situ analysis identified a narrow
region of quiescent cells that were stained for putative stem cell markers including
chondroitin sulphate, K6a, CK15, and SSEA-4 [69]. However, the data published
so far do not exclude the possible existence of other niches or models of
niche. In particular, the model of the estrogen-driven stem cell niche consists
of three different cell types: the ER-positive sensor cells, the EGFR-positive
stromal cells, and the ER-negative stem cells. All of them could remain
quiescent until they are switched on by estrogens. In response to estrogens,
the ER-positive sensor cells synthesize and secrete amphiregulin that activates
EGFR-positive stromal cells which in turn activate ER-negative stem cells [70],
although the identity of the stromal factors interacting with the epithelial
components of the stem cell niche remains to be revealed. In addition, the
similarities between stem cell niches in different tissues remain poorly
understood, in particular whether tissue-specific stem cells can be regulated
by stem cell niches in other organs or whether vice versa ectopic mesenchymal
stem cells may colonize a breast niche and influence its behavior. Studies by
Hochedlinger et al. [71]
support the notion that a malignant genome can be reprogrammed to exhibit a
normal-like phenotype when transferred into a new biological context, whereas
Blanpain et al. [72] have reported that epithelial
stem cells are able to generate their own microenvironment. Tumor cells also
have the well-established ability to interact with their surrounding
environment and to influence it profoundly; examples are neoangiogenesis,
recruitment of immune cells, and modification of tissue architecture. All these
findings have important and provocative implications for understanding
metastatic growth in secondary sites.


## 8. CSCs and Metastasis

Since the majority of breast cancer deaths
occur as a result of metastatic disease rather than from the effects of the
primary tumor, one of the biggest challenges is the identification, as early as
possible, of patients harboring metastatic cells. In fact, the persistence of
disease at a low or undetectable level (the so-called “minimal residual
disease”) is a common feature of breast cancer as supported by autoptic
findings [73] as well as by the accumulating evidence that breast cancer
patients, even with no indication of
metastatic spread by current clinical parameters, have individual tumor cells
in their blood [74, 75]. Several studies have shown that detection of isolated
tumor cells in the bone marrow is an independent prognostic factor. However,
even though approximately 30% of breast cancer patients may have
micrometastatic disease in their bone marrow at the onset, only 30–50% of them will
go on to develop clinically evident metastases within 5 years [76, 77].The presence of such cells in the
bone marrow is particularly interesting since bone represents one of the most
common sites for breast cancer metastasis, together with regional lymph nodes, lung, liver, and brain, all of which may
represent putative nichesfor
disseminating tumor cells according to the hypothesis that cancer cells can arrest
and grow in favorite metastatic sites. This “seed and soil” theory, first
proposed in 1889 by Paget [78], predicts that a cancer cell (the “seed”) can survive
in and colonize only secondary sites (the “soil”) that produce growth factors capable
of significantly influencing cell behavior [79, 80], and has largely withstood
the test of time [81]. In the case of breast cancer, disseminated carcinoma
cells are detectable in the bone marrow [82], and recent findings indicate that
most cancer cells found in the bone marrow have a breast CSC phenotype [83].However, recent experimental
observations demonstrated a direct involvement of the bone marrow-derived cells
in the development of human epithelial tumors, suggesting that CSCs may
originate from bone marrow-derived cells [84]. Furthermore, a very recent paper
from Mylona et al. [85] indicated
that, in clinical breast cancer tissues, the CD44+/CD24−/low phenotype (i.e., the phenotype experimentally associated with stemness) had no significant
correlation with clinical outcome. This is in agreement with a previous paper
by Abraham et al. [86] that found
no correlation between CD44+/CD24−/low tumor cell
prevalence and tumor progression, in terms of event-free and overall survival.
Conversely and quite surprisingly, the prevalence of CD44−/CD24+ tumor cells was found to exert an unfavorable impact on patients' relapse-free
and overall survival. This suggests that, although CD44+/CD24−/low breast tumor cells may be highly efficient in initiating tumors in animal experimental
models, in patients, these cells could be associated with the development of distant
metastasis, particularly bone metastases, rather than with clinical outcome. Therefore,
CD44+/CD24−/low tumor cells could be a subclass of
tumorigenic cells characterized by a great metastatic potential, maybe due to
the role of CD44 as a homing receptor for distant tissue compartments.


## 9. Clinical Implications of CSC Paradigm and Future Directions

The hypothesis that only CSCs are capable of reinitiating
growth to form metastases in distant sites has fundamental clinical implications
in terms of both prognosis and therapy since it provides an explanation of the
limits to many current breast cancer treatments [87, 88]. In fact, the main goal
of the current therapeutic strategies is represented by the “gold standard” of
tumor shrinkage. However, if a tumor is maintained by a small subpopulation of
CSCs that is constitutively resistant to therapeutic agents, tumor shrinkage results
in the selective killing of the more differentiated, “nontumorigenic” cells
that make up the bulk of the tumor, while leaving cancer stem cells viable and
able to continue to maintain and/or reinitiate tumor growth on a metastatic
site. Thus, current therapies fail to account for potential molecular and
proliferative differences in the various subpopulations of tumor cells, and may
be ineffective on the more aggressive and dangerous subgroup that constituted
by CSCs. Since CSCs may constitute metastasis precursor cells, as suggested by the
detection of disseminated tumor cells with a breast CSC phenotype in the bone
marrow of breast cancer patients [83], it is of paramount importance to develop
reliable diagnostic tools through the identification of additional and more
specific markers for CSCs or even for niche cells, although this could be a difficult
task due to the complexity of niche composition [89]. Difficult but not
impossible, since recently Calabrese et al. [90] were able to
visualize brain CSCs surviving in a vascular niche that secretes factors which promote
their long-term growth and self-renewal. In addition, innovative technologies,
using sensitive imaging techniques, have recently permitted real-time
monitoring of CSC presence and viability as well as analysis of the angiogenic
switch [91]. However, although this imaging technique can be extremely valuable
in order to achieve a greater understanding of the biology of CSCs and their
relationship with the stromal compartment, it cannot be used, at least at
present, for intravital monitoring of such elusive cells in patients. Conversely,
more clinically relevant imaging techniques, such as high-resolution magnetic
resonance imaging (MRI) [92] and three-dimensional high-frequency ultrasound [93],
are currently being developed for the study of CSCs in preclinical models.
These techniques look promising for future clinical applications to determine
prognosis, monitor therapeutic efficacy, and possibly affect therapies.In addition to its impact on diagnosis and prognosis, the CSC hypothesis
also has significant implications for the therapy of breast cancer. Since current
therapies do not target the tumor-initiating cells effectively, as implied by
the large number of patients who relapse after adjuvant chemotherapeutic and/or
hormonal treatment [94], there is a need for therapeutic agents specifically
directed against CSCs. These specific agents could be added to conventional
cytotoxic drugs, designed to kill actively dividing cells, and be able to
eradicate the metastatic disease. Thus, they would turn cancer, if detected at
early stage, into a curable disease limited to the primary organ. Although we still
know too little about the molecular features distinguishing CSCs from the bulk
of tumor cells to develop a “smart drug”, the significant advances in the field
indicate that CSCs could soon represent a really useful target.


## 10. Conclusions

Recent findings in breast biology have provided
support for the CSC hypothesis, but researchers still face many challenges. First,
attention should be paid to the accuracy of experimental methods for the isolation
and propagation of CSCs derived from clinical samples, with a particular
emphasis on cell culture environment (substrate, atmosphere, and medium) that has
a critical role in standardizing the culture conditions for breast cancer
progression studies [49]. Secondly, more accurate techniques should be used for
the sphere formation assay, so as to determine a self-renewing capability sufficient
to classify a cancer cell as a cancer stem cell, and to avoid conflicting
results obtained by different groups. Moreover, it is necessary to pursue the
clinical demonstration that CSCs can be used as a prognostic indicator of
disease progression and to identify the mechanisms by which CSCs escape
conventional therapies in order to develop new specific therapeutic approaches.
Finally, there is the need to find the definitive evidence of the existence of
CSCs and to identify the stroma-related factors that influence the development
and spread of CSCs. Since CSCs have not yet been fully defined, their existence
in breast cancer cannot conclusively be proven, and competitive hypotheses,
which may explain some of the puzzling features of certain tumor cell
populations, should be taken into account. The most exciting of these
competitive hypotheses implies the reversible epithelial-to-mesenchymal
transition, the developmental process in which epithelial cells acquire the
migratory properties of mesenchymal cells. As shown in a very recent paper [95],
the induction of the epithelial-mesenchymal transition could stimulate breast
cells to adopt characteristics of stem cells. This suggests that CSCs are not
distinct entities but rather tumor cells that transiently acquire stem
cell-like properties as a consequence of an epithelial-mesenchymal transition.
Undoubtedly, such a link between epithelial-mesenchymal transition and stem cell
phenotype further fuels the debated question about the existence of CSCs and
holds a number of interesting implications for the biology of epithelial cells,
including the possibility that the stem cells of certain epithelial organs such
as mammary glands may acquire many of the attributes of the mesenchymal cell state
that confer them an increased tumorigenic potential.The advent of new technologies,
including gene expression profiling and proteomics, and the ability to apply
them to small numbers of cells will probably help to solve such open questions.



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