PROSPECTIVE IDENTIFICATION OF THE PRE-CANCER STEM CELL
Ninnie Marie-Louice Abrahamsson
B.S., Gothenburg University, Gothenburg, 2009
PROJECT
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF ARTS
in
BIOLOGICAL SCIENCES
(Stem Cell)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
SPRING
2011
PROSPECTIVE IDENTIFICATION OF THE PRE-CANCER STEM CELL
A Project
by
Ninnie Marie-Louice Abrahamsson
Approved by:
__________________________________, Committee Chair
Thomas Peavy, Ph.D.
_________________________________, Second Reader
Jan Nolta, Ph.D.
__________________________________, Third Reader
Thomas Landerholm, Ph.D.
____________________________
Date
ii
Student: Ninnie Marie-Louice Abrahamsson
I certify that this student has met the requirements for format contained in the University format
manual, and that this project is suitable for shelving in the Library and credit is to be awarded for
the project.
__________________________, Graduate Coordinator
Susanne Lindgren, Ph.D.
Department of Biological Sciences
iii
___________________
Date
Abstract
of
PROSPECTIVE IDENTIFICATION OF THE PRE-CANCER STEM CELL
by
Ninnie Marie-Louice Abrahamsson
Ductal Carcinoma In Situ (DCIS) is considered the earliest form of breast cancer where
cells that appear malignant develop into benign heterogneous lesions confined within the
epithelial membrane, yet progression to invasive ductal carcinoma frequently occurs.
Therefore, treatment of the disease is equal to preventing development of invasive cancer.
It has previously been suggested that the cells of DCIS may already be programmed to
become invasive, which led to the speculation that a small population of cancer stem
cells, sharing characteristics with normal stem cells, are responsible for the development
of invasive cancer. As a mammary gland stem cell population that increases in size with
tumor progression has previously been identified using FACS, along with transplantation
experiments revealing a capability to regenerate the ductal tree, the goal of this project
was to identify the pre-cancer stem cell population using a mouse model representative of
human DCIS and previously established surface markers CD24, CD29 and CD49f.
However, identification of the pre-cancer stem cells in the DCIS mouse model using the
aforementioned surface markers was proven unsuccessful, and thus novel markers CD44
iv
and CD61 were added to the selectional process to increase specificity. The results of this
study revealed a putative stem cell population with a CD24low CD29high CD49fhigh
expression profile within mammary glands of normal FVB mice as well as in the precancer mouse model. The data further revealed an increased expression of CD44 in the
same population, with respect to the DCIS mouse model as well as in normal mammary
glands. However, CD61 expression was increased in a CD24negative CD29low CD49flow
population, indicating a possible luminal epithelial origin. The results furthermore
revealed an increase in the stem cell population with carcinoma progression through
comparisons of DCIS lesions, DCIS derived tumors and a tumor cell line. In summary,
the results of this study indicates that a pre-cancer stem cell population may be
distinguished based on the expression of surface markers CD24, CD29, CD49f and
CD44, and seems to correlate with their expansion and progression to invasive cancer.
These findings should significantly improve the isolation of the pre-cancer stem cell
population in DCIS, and thus may lead to new treatments aimed at eliminating the cancer
stem cells.
, Committee Chair
Thomas Peavy, Ph.D.
______________________
Date
v
ACKNOWLEDGMENTS
I would like to thank Dr. Thomas Peavy and Dr. Alexander Borowsky for all their
dedication, support and guidance throughout this entire process, none of this would have
been possible without you. I would also like to thank Jane Chen, Neil Hubbard, Rebecca
Lobo, Andrew Fong in the Borowsky Lab for making this a great experience and for all
always being there with a helping hand or helpful advice. I would furthermore like to
thank Carol Oxford, David Boucher, Scott Ohlson and Jesse Engelberg for the time they
spent teaching me relevant techniques, protocols and maths. I would also like to thank the
committee members Dr. Jan Nolta, Dr. Thomas Landerholm and Dr. Susanne Lindgren,
as well as CIRM for providing us with the opportunity to become a part of the fascinating
field that is stem cell research.
vi
TABLE OF CONTENTS
Page
Acknowledgments…………………………………………………………………..…….vi
List of Figures…………………………………………………………………………….…….. ix
INTRODUCTION…………………………………………………………………..……...…….1
Ductal Carcinoma In Situ………………………………………………………………. 1
Tumor heterogeneity and the cancer stem cell model……………………………......3
Mammary stem cells………………………………………………………………..…... 6
Identification of mammary stem cells..…………………………………………..….... 7
Expansion of mammary stem cells with tumor progression………………….…… 12
Project Goal to Identify mammary pre-cancer stem cells using the MINO mouse
model.……………………………………………………………………………………13
MATERIALS AND METHODS………………………………………………………..….....16
Animals……………………………………………………………………...…... 16
Cell culture…………………………………………………………………….… 16
Tissue dissection……………………………………………………………….... 17
Dissociation of primary tissue………………………………………………..…..17
Flow cytometry……………………………………………………………..…… 19
RESULTS……………………………………………………………………….……….…....…20
Identification of the putative stem cell population in the normal mammary
gland……………………………………………………………………………………..20
Identification of the putative stem cell population in the MINO model…………..24
vii
The putative pre-cancer stem cell population shows a CD49fhigh profile………....29
Increased expression of CD44 in the putative stem cell population……………… 31
Restriction of CD61 expression to potential luminal epithelial populations…….. 34
Increased "stemness" with progression into carcinoma…………………………….37
DISCUSSION………………………………………………………………………...................39
Literature Cited……………...…………………………………..………...……………….........48
viii
LIST OF FIGURES
Page
Figure 1. Expression of putative cancer stem cell related cell surface markers in the
FVB mammary gland……………………………………………………........ 21
Figure 2. Full gating path of the FVB mammary gland ……………………………........ 22
Figure 3. Expression of putative cancer stem cell related cell surface markers in the
MINO………………………………………………………..……….........….25
Figure 4. Full gating path of the MINO…………………..……………………………... 28
Figure 5. Expression of the cell surface marker CD49f in the MINO...…..…………….. 30
Figure 6. Expression of putative cancer stem cell surface marker CD44 in the MINO….32
Figure 7. Expression of CD44 in the FVB mammary gland…………………………......33
Figure 8. Expression of putative cell surface marker CD61 in the MINO..…………….. 35
Figure 9. Distribution of CD61 expression in Lin- CD49f+ cells within the MINO…….. 36
Figure 10. Expression of cancer stem cell related cell surface markers with tumor
progression………………………………………………………………….... 38
ix
1
INTRODUCTION
Ductal Carcinoma In Situ
Ductal Carcinoma In Situ (DCIS) is considered the earliest form of breast cancer where
cells that appear malignant develop into benign heterogeneous lesions, confined within
the epithelium of the ductal structures and terminal lobular units of the breast. However,
even though the invasion is confined within the epithelial membrane, progression to
invasive ductal carcinoma frequently occurs. Therefore, the lesions synonymous with
DCIS are viewed as possible precursors to invasive cancer, and treatment of the disease is
equal to a treatment preventative for the development of invasive carcinoma. About 20%
of all newly detected incidences of breast cancer are diagnosed as DCIS, and there has
been a dramatic increase in the number of incidences in the last 20 years, due to a higher
frequency of mammography screenings.
DCIS follows a pattern of progression where the ultimate stage of invasive cancer
is developed from atypical hyperplasia and is graded with regards to certain
characteristics such as size of the lesion and the amount of necrosis, which in turn
determines the latency of invasion as well as the likelihood of spreading and recurrence.
Occurrences of chromosomal imbalances, where several loci may suffer gains or losses,
are examples of biological features indicating a progression of the disease. Even though
the initial pathways are not fully understood, it seems like nearly all invasive breast
cancers arise from in situ carcinomas (Ernster et al. 2002, Burstein et al. 2004, Chin et al.
2004, Leonard and Swain 2004).
2
Previous findings showing that pre-invasive lesions are sharing chromosomal
changes as well as very similar gene expression patterns with the adjacent invasive
cancers have led to speculations that the events initiating the transformation may precede
the development of invasive disease. Thus, it has been suggested that the cells of DCIS
may at the pre-malignant stage already be programmed to become invasive, undergo
metastasis, and possess therapeutic resistance. Damonte et al. (2008) provided evidence
for this model when cells from tumors and outgrowths in the pre-cancer state from
engineered mouse mammary glands were genomically analyzed revealing a shared
genomic stability as well as increased telomerase activity. This study demonstrated that
the transition of DCIS into invasive carcinoma is not dependent on genetic alterations and
subsequent genomic instability as has been previously suggested in the model of
sequential acquisition (Wellings and Jensen 1973, Burstein et al. 2004). Thus, although
genetic as well as epigenetic changes are not necessary for driving the transformation,
they may well contribute to the heterogeneity of the tumor.
The indication that the cells of DCIS may be pre-programmed to develop invasive
carcinoma led to the speculation that cancer progression in this disease may follow the
pattern of the cancer stem cell model, which states that a small population of cells sharing
characteristics with normal stem cells are responsible for the development of invasive
cancer (this model will be explained in more detail in the following section). In order to
characterize these cells for future breast cancer treatment purposes, it became relevant to
distinguish them from other cell types within the mammary gland. This should be
possible since the normal stem cell population within human breast cancers and within
3
the normal mammary gland of the mouse have previously been identified. As no findings
have been made that reveal such a population with regards to DCIS, the goal of this study
was to identify this population by elucidating the expression profile of cancer cells that
correlates with having stem cell-like properties in a mouse model representative of this
disease.
Tumor heterogeneity and the cancer stem cell model
According to the model of sequential acquisition, the progression from DCIS to
invasive cancer involves a series of genetic alterations, leading to clonal selection
favoring variant cells with specifically advantageous and aggressive behaviors. Such cells
may acquire the capability of extensive proliferation as well as the ability to invade the
surrounding tissues. This model may furthermore explain tumor heterogeneity and is
supported by the fact that there are oncogenes acting in a dominant manner, while certain
tumor-suppressor genes act as recessive (Bjerkvig et al. 2005, Feinberg et al. 2006).
However, in a number of cases there has been no success linking certain stages of tumor
to necessary mutation events. For example, in colorectal cancer it has been possible to
link changes in the expression of certain genes to incidences such as invasion and
metastasis, yet no specific mutations have been found responsible for this occurrence
(Fearon and Vogelstein 1990).
As opposed to the model of clonal evolution, a small subset of cells emerged as
being possibly responsible for the progression into invasive cancer as well as the
continuous growth of the tumor. These cells are known as cancer stem cells and share
4
characteristics with normal stem cells, such as the capability of self renewal and
asymmetric division. This population is thought to drive the expansion of the malignant
population and contribute to tumor heterogeneity in a hierarchical manner, where the
cancer stem cells are residing among their differentiated progeny. Thus, the invasion and
proliferation of the malignant cells are dependent on this subpopulation, however, it is
important to consider that the models may not act in exclusion of one another (Bonnet
and Dick 1997, Bjerkvig et al. 2005, Woodward et al. 2005, Ward and Dircks 2007, Dick
2008).
In favor of the cancer stem cell model, transplantation experiments have been
performed using the mouse as a model organism, revealing a population of putative stem
cells capable of repopulating particular tissues. This has also been done in the case of the
mammary fat pad and was shown to completely regenerate the fat pad with functional
ductal structures containing cells from the luminal, myopeithelial and alveolar lineage
(Sleeman et al. 2005, Schackleton et al. 2006, Joshi et al. 2010, Stingl et al. 2006).
Cancer stem cells have been identified within other tissues, such as the brain and the
hematopoietic system, in which the latter involves leukemia. With respect to leukemia,
cancer stem cells have readily been identified in both chronic myelogeneous leukemia
(CML) and acute myelogeneous leukemia (AML), using a cell surface marker known as
CD33. Cancer stem cells have also been identified in brain tumors, such as pediatric
meduloblastoma and glioma (Singh et al. 2003, Singh et al. 2004).
Normal stem cells within the epithelium of the mammary gland of the breast
appear to be required for generation and maintenance of the epithelium during
5
development and injury, as well as to allow for expansion of the tissue during pregnancy,
involution and lactation (Smalley and Ashworth 2003. Jordan et al. 2006). However, it is
uncertain from where the cancer stem cells originated. Tissue specific stem cells are
quiescent and long-lived, therefore serving as excellent targets for mutations and accruing
various genetic alterations. One example includes disruptions affecting the regulation of
self-renewal. Furthermore, since the similarities between cancer stem cells and normal
stem cells are striking, it is possible that the normal stem cell population is the origin of
the malignant progenitor cells. The progenitor cells are defined as having a proliferative
ability similar to that found within the cancer stem cell, yet does not have the same ability
to self-renew. However, it is not impossible that differentiated somatic cells acquire
genetic mutations that provide a stem cell like phenotype, rendering cells able to invade
surrounding tissues and act in various malignant ways. Alternatively, reports of fusion
events where stem cells have been shown to fuse with various somatic cell types, have
become more frequent and seen in both liver and pancreas (Hess et al. 2003, Wang et al.
2003). Thus, it could be possible that stem cells fuse with differentiated cells that have
acquired a set of mutations that enable it to develop into cancer (Houghton et al. 2004,
Bjerkvig et al. 2005, Li et al. 2006). Yet, it is nevertheless possible that various
mechanisms act in conjunction with each other and the cancer stem cell may arise
through a number of processes and pathways.
The primary concern with cancer stem cells involves tumor recurrence and
relapse of the invasive cancer, as suggested by the failure of current chemo- and
radiotherapy to eliminate this particularly drug resistant subpopulation. As for breast
6
cancer, an example of this has been observed in the treatment of CML, where patients
treated with the ABL kinase inhibitor imatinib mesylate (Gleevec) showed presence of
residual disease even though the treatment appeared successful. Thus, the drug only
appears to target the progeny of cancer stem cells, leaving the cancer stem cells intact.
This lingering population is thus likely to be responsible for disease recurrence upon
discontinuation of the treatment (Jordan et al. 2006).
Mammary stem cells
The mammary gland contains a network of hollow ductal structures, organized in
a tree-like shape, comprised of two primary epithelial lineages. The luminal cells, which
face the lumen, are responsible for milk secretion, while the outer layer of the epithelium
is comprised of contractile basal myoepithelial cells that secrete the basal lamina.
Residing at the end of the ductal branching are the lobular cells, which during pregnancy
and lactation develop into alveolar cells, responsible for milk secretion (Woodward et al.
2005, Stingl and Tegzes 2008). As previously mentioned, the mammary stem cells appear
to be required for regeneration of mammary tissue during processes such as development
and pregnancy, and are proposed to be residing within the basal/myoepithelial portion of
the epithelium (Shackleton et al. 2006, Stingl et al. 2006). During puberty, the stem cell
population within the murine mammary gland is thought to reside within the tips of the
terminal end buds where they make up the cap cell layer, which can develop into both a
luminal and myoepithelial lineage. However, the terminal end buds are only transient and
disappear as they have finished growing and reached the end of the fat pads. The human
7
equivalent of the terminal end buds are the terminal ductal lobular units, which are
organized as a cluster of grapes at the end of the ductal branch, as opposed to the terminal
end buds which may be globular or blunt, yet does not branch out in the same fashion as
can be seen at the end of the human ducts (Cardiff and Wellings 1999, Visvader and
Lindeman 2008).
By collecting and dissociating the epithelial tissues from normal, pre-cancer and
tumor mammary glands it becomes possible to investigate the presence of the cancer
stem cells using a variety of techniques, such as fluorescence activated cell sorting
(FACS) analysis and non-adherent mammosphere cultures. Another method commonly
used for characterization of the putative mammary cancer stem cell population is 5bromo-2-deoxy-uridine (BrdU) label-retention, which reveals increased proliferation
patterns that are known as one of the defining traits of stem cells. The initial examination
is generally performed using FACS on dissociated mammary glands, which may then be
subjected to various functional assays (Woodward et al. 2005, Joshi et al. 2010,
Molyneux et al. 2010).
Identification of mammary stem cells
Characterization of the mammary cancer stem cell population through functional
assays such as transplantation studies is dependent on their successful identification and
isolation, most commonly achieved through fluorescence activated cell sorting (FACS).
Using FACS analysis to enrich for a rare population such as mammary cancer stem cells
in a heterogeneous mix of cell types like the one in the mammary gland of the mouse, it
8
becomes important to narrow down the possibilities by depleting for undesired lineages.
Due to this fact, it has become common to deplete cells committed to the hematopoietic
lineages, which are distinguished by their expression of cell surface markers CD31,
CD45 and Ter119. CD31 is a known marker used to detect endothelial cells, while
Ter119 is specifically expressed on erythrocytes. CD45 however, is widely expressed on
cells of hematopoietic origin. Depleting the sample for these markers using FACS
renders the remaining populations as negative for the hematopoietic lineage and is
commonly referred to as Lin-. The resulting lineage negative cells are usually defined as
luminal, myoepithelial, or non-epithelial cells.
A fairly large number of studies have shown that the human mammary cancer
stem cell population may be enriched using cell surface markers CD24 and CD44, where
the marker profile contained high expression of CD44 as well as negative expression of
CD24 (Sheridan et al. 2006, Fillmore and Kuperwasser 2008, Wright et al. 2008, Wright
et al. 2008. Lewis 2010). In the mouse however, the cell surface expression profile differs
from that of the human, and the most commonly used markers currently include CD24,
CD29 and CD49f. CD24 is generally known as Heat Stable Antigen and is a marker
frequently used for the isolation of a variety of tissue specific stem cells and as
previously mentioned, found in both human and mice. It has furthermore been found
expressed within a large number of cancers and low expression levels of CD24 have
recently been associated with stem cells of human breast cancer (Schindelmann et al.
2002, Phillips et al. 2006, Sheridan et al. 2006). CD49f is regularly referred to as integrin
α6, which together with integrin β4 makes up the laminin receptor on the epithelial cell
9
surface, and is generally considered an epithelial marker which repeatedly has shown
useful as a marker for “stemness” in the mammary gland of the mouse (Schackleton et al.
2006, Stingl et al. 2006, Kouros-Mehr et al. 2008).
Stemness is here referring to stem cell like properties, such as increased
proliferation rates and the ability to repopulate the mammary gland. However, CD29,
also known as Integrin β1, is widely distributed and interacts with 12 of the 18 integrin
alpha subunits to form heterodimeric structures that are able to interact with
glycoproteins in the extracellular matrix as well as neighboring cells. Due to the vast
number of possible interactions, CD29, along with its subunits, is responsible for a
variety of biological processes such as growth, migration and adhesion. For example, it
has been reported to bind a large variety of components in the extracellular matrix,
including vitronectin, laminin and osteopeontin. CD29 has also been reported to have an
essential role in normal mammalian development during pregnancy and involution
(Brakebusch et al. 1997, Pontier and Muller 2009).
Interestingly, deletion of CD29 inhibited tumorigenesis in a MMTV-PyvMT
transgenic mouse model, while other studies have reported that depletion of the CD24low
CD29high putative stem cell population inhibits regeneration of the adult mammary gland
(Taddei et al. 2008, White et al. 2010). Thus, it is suggested that CD29 is responsible for
conserving specific characteristics that are involved with “stemness” or alternatively
contributing to the preservation of the stem cell niche (Pontier and Muller 2009).
However; even though this set of cell surface markers are commonly used for mammary
cancer stem cell isolation purposes, results from different studies show inconsistencies
10
where markers such as Sca-1, Thy-1, ALDH, CD61, and CD133 have all been reported
useful for the same purpose to varying extents. Thus, to date, the specific set of markers
needed for consistent mammary stem cell isolation is still uncertain (Kouros-Mehr et al.
2008, Molyneux et al. 2010, Wegwitz et al. 2010).
With respect to CD24, FACS analysis of murine mammary glands has shown that
it defines three distinct populations, showing a CD24high, CD24low or a CD24negative
expression which appears to correspond to luminal epithelial, myoepithelial/basal and
non-epithelial populations, respectively. The CD24high population stained almost
exclusively for the luminal marker Cytokeratin 18 and possessed limited repopulating
capacity. However, the CD24low population was able to produce outgrowths in 2 out of 5
cases and almost the entire population stained positive for the basal/myoepithelial
markers Cytokeratin 14 and smooth muscle actin (Sleeman et al. 2005, Stingl et al. 2006,
Asselin-Labat et al. 2007). Thus, the mouse mammary stem cell profile appears to
contain a low expression of CD24, an observation also common to humans. As opposed
to low expression of CD24, it appears that the mammary stem cell population is
expressing high levels of both CD29 and CD49f. In further support, CD49f has been
found to be the most highly expressed in cells of the basal epithelial layer (Schackleton et
al. 2006, Stingl et al. 2006, Vaillant et al. 2008, Joshi et al. 2010). However, expression
profiles may differ between mouse models, and depend on the environmental context and
the cellular stage of differentiation (Zhang et al. 2008, Wegwitz et al. 2010).
11
Furthermore, the surface markers used to identify the cancer stem cell population
in human breast cancers differ slightly from those used in the mouse; where in humans
the most frequently reported profile includes a high expression of CD44 and a low or
sometimes negative expression of CD24 (Sheridan et al. 2006, Fillmore and Kuperwasser
2008, Wright et al. 2008, Wright et al. 2008, Lewis 2010). CD44 is expressed in a wide
range of cell types and in a large number of splice variants depending on the tissue. This
marker is further known as a membrane receptor for the extracellular polysaccharide
hyaluronan, which supports cellular proliferation, attachment and migration. A crucial
role for this surface marker with regards to migration was discovered as correlations
between a high CD44 expression and higher rates of metastases were detected in cell
lines from human melanoma (Birch et al. 1991, Wheatley et al. 1993, Van Muijen et al.
1995). However, even though this marker has been established as a cancer stem cell
marker in human breast cancer, it has not been found to be so in the mouse.
Another surface marker that recently has gained attention as a putative luminal
epithelial surface marker is CD61, commonly known as integrin β3. This integrin can
associate with either CD41 or CD51, where it forms the gpIIb/IIIa complex or the
vitronectin receptor, of which the latter is distributed in a variety of tissues. The degree of
the distribution of the gpIIb/IIIa complex is not fully established. However, the complex
has been detected in 17 different tumor cell lines from various species and tissues, one of
which is the human breast, where screening of tumors revealed the complex on solid
tumor cells (Trikha et al. 1997, Moreno et al. 2002). Regarding the utility of CD61 as a
surface marker for identification of mammary cancer stem cells, Asselin-Labat et al.
12
(2007) found that a third of the cells with a CD24+ and CD29low expression profile also
stained positive for CD61 and were capable of growing into larger colonies compared to
cells with a CD61negative profile. The CD24+ and CD29low cell population has
previously been described as a luminal epithelial population, as indicated by expression
of CK18, which in this particular study only showed presence in cells with a CD61+
profile. Together, these findings suggest that CD61 may be a potentially important
marker that should be considered with regards to prospective identification of mammary
cancer stem cells.
In previous attempts to reproduce the expression profile described in the literature
using the MINO model, a mouse model representative of human DCIS (described in
detail on p.14), enrichment for “stemness” have had limited success as sorted populations
did not show full outgrowth potential and thus were not able to regenerate the complete
ductal tree within the mammary gland (unpublished data). It thus became apparent that
the use of a more specific sorting logic, where additional markers could aid in the
selection process, may prove to be beneficial. Based on the increased attention towards
CD61 and its value as a predictor of cells with a luminal progenitor origin, along with the
importance of CD44 as a cancer stem cell marker in human breast cancer samples, both
of these cell surface markers were added to the optimized antibody panel.
Expansion of mammary stem cells with tumor progression
The pool of cancer stem cells appears to be contained as a relatively small
subpopulation within the epithelium of the mammary gland. However, characterization of
13
the epithelial subpopulation allegedly enriched for mammary cancer stem cells in normal
FVB (Friendly Virus B) mammary glands as well as early and late PyV-mT (described
below) tumors showed a progressive increase in population size with the development of
carcinoma, with the stem cell population making up the bulk of the mass within the late
carcinomas (Kouros-Mehr et al. 2008). In addition, by performing a trans-well migration
assay, these cells were revealed to possess enhanced migratory potential compared to
control populations. Further examination showed bi-potential within the aforementioned
subpopulation, meaning that they were capable of giving rise to cells from both the
luminal and myoepithelial linage. It is possible that the increase in “stemness” with tumor
progression is due to selectional pressures, which favors their enrichment over time.
Their enhanced migratory potential may suggest that this population is responsible for the
invasion as well as the dissemination of the tumor. However, further studies are needed to
establish the specific roles of the mammary stem cells in tumor progression.
Project Goal to Identify mammary pre-cancer stem cells using the MINO mouse model
The goal of this project was to identify the pre-cancer stem cell population using
previously established markers for isolation of mammary gland stem cells, with the
addition of novel markers that recently have gained attention for their possible expression
within the murine mammary stem cell subpopulation. As identification of the cancer stem
cells using previously established surface markers CD24, CD29 and CD49f was proven
unsuccessful, the addition of novel markers to the selectional process may provide the
needed specificity needed to identify the mammary stem cell population within the
14
MINO mouse model. The panel of cell surface markers used to address this objective
include CD24, CD29, CD49f, along with novel markers CD44 and CD61.
Due to the difficulty of experimentally examining the progression towards
invasive cancer within human DCIS, the use of a representative mouse model becomes
important. Therefore, the study of DCIS has often been performed using the MINO
mouse model, where MINO is the abbreviation of mammary intraepithelial neoplasia
outgrowths. These pre-cancer outgrowths were initially generated from the FVB mouse
strain expressing the mammary polyomavirus middle T gene (PyV-mT), driven by the
mouse mammary tumor virus (MMTV) promoter. This oncogene is known to activate the
phosphatidylinositol three-kinase pathway, which is commonly upregulated in breast
cancer tissues with HER2/neu amplifications, which are detected in about half of all
DCIS lesions. PyV-mT is therefore a suitable oncogene with regards to accurate
representation of human disease progression (Burstein et al. 2004).
The MINO model consists of six lines, four, six, eleven, A, B, and D, all with
varying metastasis rates and tumor latencies, which ranges from 11 to 22 weeks. MIN
outgrowths are maintained as serial transplants within pre-cleared mammary fat pads of
mice with an FVB background and have shown consistency over more than 60
generations of transplantation (Maglione et al. 2004). The behavior of the MINO tissue is
defined by “test-by-transplantation”, meaning that the MINO tissue will further have to
meet a certain set of criteria that defines its pre-malignant behavior. The following
criteria are quoted from Damonte et al. (2008): “the tissue will grow in gland-cleared fat
pads, will not grow in the subcutis, will not senesce over many generations of
15
transplantation and will transform into a phenotype which has the ability of growing
within the subcutis”.
As previously stated, the finding that transformation from DCIS to invasive
carcinoma is not dependent on sequential genetic changes suggests that there may be a
subpopulation of progenitor cells with the information for this specific behavior
genetically or epigenetically encoded that possesses the capability of driving the
progression into invasive carcinoma (Damonte et al. 2008). These cells may be
recognized as pre-cancer stem cells. However, in order to further analyze and determine
the biological potential of this population, identification and isolation of these pre-cancer
stem cells is necessary.
16
MATERIALS AND METHODS
Animals
FVB female mice weighing 10-14 g were purchased from Charles River
Laboratories (Wilmington, MA), while the MINO serial transplant line was maintained at
University of California, Davis. MIN outgrowths originate from FVB females expressing
MMTV/PyV-mT which were initially acquired from Prof. William J. Muller (McGill
University, Montreal, Quebec, Canada) and bred at the University of California, San
Diego (La Jolla, CA). Serial transplants of the MINO tissues into pre-cleared number
four mammary fat pads of FVB females were conducted at five week intervals. Animals
were maintained in University of California, Davis, vivarium according to NIH
guidelines and all procedures were performed in accordance with approved animal
protocols.
Cell culture
Frozen Met-1 cells at passage number 17, a tumor cell line derived from
transgenic (MMTV-Pyv-mT) mice, were thawed and cultured in one T75 flask containing
15 ml of Dulbecco’s Modified Eagle Medium High Glucose 1X (Invitrogen) and
passaged after reaching a 90% confluency. Cells were cultured in 37 °C and 5% CO2,
while media was changed once a day. Cells were trypsinized for 2.5 minutes on the day
of the experiment, saturated with completed DMEM containing 10% Fetal Bovine Serum
(Invitrogen) and 5% Penicillin-Streptomycin (Invitrogen), followed by a wash with
17
Dulbecco’s phosphate buffered saline 1x (Invitrogen). Cells were resuspended in PBS
and prepared for flow cytometry by filtering through 5 ml Polystyrene Round-bottom
tubes with cell-strainer caps (BD Biosciences) in order to remove any remaining cellular
aggregates. Samples were subsequently transferred to 5 ml polypropylene round-bottom
tubes (BD Biosciences), where the cell number was counted using a hemacytometer.
Tissue dissection
All mice were anaesthetized using intraperitoneal injections of 60 mg/kg
Nembutal Sodium Solution (Ovation Pharmaceuticals, Inc.) and prepared for aseptic
surgery. One incision was made between the number four mammary fat pads and
extended upwards towards the thorax, upon which two contra-lateral incisions were
created at the base of the first incision, resembling an inverted Y. The skin was pulled
back to reveal the mammary fat pads and the MINO tissues were identified using a
stereomicroscope. All ten mammary fat pads were dissected and collected from the FVB
females, while the number four mammary fat pads were collected from the MINOs.
Tissues were placed in 100 mm x 20 mm Polystyrene cell culture dishes (Corning Inc.)
where excess blood was rinsed off with PBS. All mice were euthanized by cervical
dislocation following dissections.
Dissociation of primary tissue
Since flow cytometry requires a single cell suspension acquired through
enzymatic epithelial dissociation, cellular aggregates were treated and filtered for this
18
specific purpose. All ten mammary fat pads from a 20 week old FVB female and all
number four fat pads containing MIN outgrowths were dissected and collected from
seven week old MIN females. Tissues were lightly washed in PBS, minced using ASR
razorblades and placed in a filtered digestion buffer containing serum free Dulbecco’s
Modified Eagle Medium Nutrient Mixture F-12 (1:1) 1X (Invitrogen), 7.5% Bovine
Albumin Fraction V Solution (Invitrogen), 1 M HEPES Buffer solution (Invitrogen), 10
mg/ml Insulin in 1% GAA (Invitrogen) and 3 mg/ml Collagenase Type Four
(Worthington). After further broken up through pipetting using a serological pipette,
samples were kept on a rotator overnight.
The subsequent step in the dissociation process involved further digestion of
tissues trough pipetting and centrifugation, after which pellets were washed in serum free
DMEM/F12 1:1. Cell suspensions were washed two times in PBS and excess amount of
fat was aspirated. Pellets were trypsinized using 0.25% Trypsin 1X (Invitrogen) and
incubated in 37 °C for 2 minutes. DNase I RNase-Free (Epicentre) was added to further
reduce clumping of cells through degradation of extracellular DNA, which may have
been present due to a certain degree of cell lysis. Cells were incubated in 37 °C, for an
additional 10 minutes. Incubated samples were subsequently saturated with completed
DMEM, containing 10% Fetal Bovine Serum and 5% Penicillin-Streptomycin. Following
centrifugation, the remaining pellets were washed and resuspended in PBS. Cells were
filtered through 5 ml Polystyrene Round-bottom tubes with cell-strainer caps (BD
Biosciences), to remove remaining cellular aggregates and transferred to 5 ml
19
polypropylene round-bottom tubes (BD Biosciences), where cell numbers were counted
using a hemacytometer.
Flow cytometry
One compensation control was provided for each antibody used in the sample,
which was stained using fluorescently labeled antibodies optimized for multicolor flow
cytometry. Gating controls, where the entire panel except for one of the antibodies was
used to stain the sample, were also provided for each antibody. The antibodies used are
listed as follows: PerCP-eFluor 710 anti-mouse CD24 (eBioscience), FITC hamster antirat CD29 (BD Biosciences), APC anti-human/mouse CD49f (Biolegend), PE hamster
anti-mouse CD61 (BD Biosciences), BD Horizon V450 anti-mouse CD44 (BD
Biosciences), Pe-Cy7 rat anti-mouse CD31 (BD Biosciences), Pe-Cy7 rat anti-mouse
CD45 (BD Biosciences) and Pe-Cy7 rat anti-mouse Ter119 (BD Biosciences). The
nuclear counterstain Propidium Iodide (Invitrogen) was used as a viability dye. Cells
were covered and stained for 30 minutes, followed by a wash with PBS to remove excess
unbound antibodies. The samples were kept on ice at all times. Samples were analyzed on
the Cytomation MoFlo Cell Sorter within the Optical Core Biology Facility at University
of California, Davis, and further examined using the FlowJo 7.6 .1 software. Flow
cytometry data was analyzed statistically using the unpaired two-tailed t-test where
differences in fluorescence intensities between populations were examined with regards
to their statistical significance at a 95% confidence level.
20
RESULTS
Identification of the putative stem cell population in the normal mammary gland
As previously reported, the normal mammary gland of the mouse contains three
distinct populations when sorted based on cell surface expression of CD24 and CD29, all
of which also stain positive for the surface marker CD49f (Stingl et al. 2006). The same
analysis was performed for comparative purposes, in order to establish the accuracy of
the separation and the general methodology. The results revealed a very similar pattern
with three distinct populations being identified (Figure 1). The dissociated mammary
glands of 20 week old FVB mice were analyzed using FACS and were gated using a
number of preferences, such as exclusion of cellular debris within the forward scatter
(FSC) versus side scatter (SSC) plot, exclusion of cellular aggregates by gating for single
cells under the FSC mode versus Pulse-width plot, and removal of hematopoietic lineage
committed cells (Lin-) expressing cell surface markers Ter119, CD31 and CD45. All
cells with positive expression of CD49f were then selected and further analyzed with
respect to their expression of CD24 and CD29 (Figure 2).
21
Figure 1. Expression of putative cancer stem cell related cell surface markers in the FVB
mammary gland. Flow cytometry dot plot revealing the distribution of Lin- CD49f+
populations in 20 week old FVB mammary glands based on their expression of putative
stem cell surface markers CD24 and CD29. Distinct populations were encircled and their
relative percentages of the total cell number depicted.
22
Figure 2. Full gating path of the FVB mammary gland. Flow cytometry dot plots showing
the gating strategy used to select for Lin- CD49f+ cells in the FVB mammary gland. The
gating strategy was based on (from left to right) exclusion of cellular debris on FSC
(Forward scatter) vs. SSC (Side scatter), with subsequent exclusion of doublets on FSC
vs. pulse width. Dead cells were excluded using propidium iodide (PI), followed by
selection of Lineage- (Lin-) cells based on negative expression for CD31, CD45 and
Ter119. Further selection was based on positive staining for CD49f.
23
There were noticeable differences with respect to the percentages obtained for the various
populations in this study and previously published findings, such as 4.91% for the
CD24low CD29high population as opposed to 8.0% and 6.4% reported by Schackleton et al.
(2006). These differences most likely are due to experimental variables such as the age of
the mice, cell preparation procedures, dissociation reagents, antibody properties and their
conjugated fluorochromes, as well as the flow cytometer and software used. In addition,
other variables that needs to be considered when comparing flow cytometry results are
the gain and offset settings on the cell sorter, as well as the gating path and the overall
data analysis methodology. However, despite numerous variables, the sizes of the
populations appear to fall within an acceptable range as compared to previously reported
results.
It has previously been suggested that the luminal epithelial population has an
expression profile of CD24+ and CD29low, while the CD24low and CD29high population
within the normal mammary gland of the mouse appear to be enriched for "stemness" and
is contained within the basal/myoepithelial epithelial layer of the ductal structure
(Shackleton et al. 2006, Asselin-Labat et al. 2007). As reported from other studies, when
sorted based on CD24 expression, a CD24negative population appears to be present which
are potentially not of epithelial origin, along with populations showing a CD24low and a
CD24high profile (Sleeman et al. 2005, Schackleton et al. 2006). Current results revealed a
similar population which accounts for 35.3% of the total Lin- cell number.
Furthermore, the reported population sizes for the stem cells and the luminal
epithelial cell types in the literature are varying, however, established sizes have created
24
indicative ranges, in which the current data for these populations appear to fall. The
percentage for the putative luminal epithelial population in the normal mammary gland
was 32.6%, compared to the 30 and 31% reported by Schackleton et al. (2006). Yet, these
ranges are mainly applicable to normal mammary glands and are expected to differ
between different mouse models and will vary due to a number of variables such as age
and number of pregnancies.
Identification of the putative stem cell population in the MINO model
Analysis of the MIN- outgrowths (MINOs) from line four and MINOs from line D within
mice 6 weeks of age, revealed an absence of the CD24high CD29low population that is
possibly representing a luminal epithelial population in the FVB mammary gland.
However, an increase from 35.3% to 64.7% and 63.5% in the size of the CD24negative
CD29+ population was noted (Figure 3). However, this difference is reduced when
compared to the same population in the normal mammary gland as reported by
Schackleton et al. 2006, which reported a percentage as high as 56 and 58%. It is
uncertain which cell type this population is representing; however, previous results have
revealed the presence of several subpopulations containing this expression pattern,
slightly differing in their expression of CD24 (unpublished data). This raises the
possibility for the presence of a total of three separate Lin- CD49f+ populations, showing
either a CD24negative CD29low, a CD24low CD29low or a CD24low CD29high expression
profile.
25
Figure 3. Expression of putative cancer stem cell related cell surface markers in the
MINO. Flow cytometry dot plots depicting the distribution of cells with regards to their
expression of CD24 and CD29 within MINO line A (left) and MINO line D (right). All
cells within this plot are all gated as Lin- and gated on a positive expression of CD49f.
Distinct populations were encircled and their relative percentages of the total cell number
depicted.
26
All populations are likely to be present using the CD24 gating control. However, due to
the difficulty of distinguishing several populations within CD29low cells, it is still
unclear whether or not the CD29low population should be viewed as one population or
two distinct populations with considerably similar expression profiles. Nevertheless, the
amount of cells within this population appears to be significantly similar between the two
MINO lines. However, for clarity, this population will be referred to as having a
CD24negative CD29+ expression profile.
The putative stem cell population shows a significant variance in population size
between the two MINO lines, with the larger population found in MINO line four. 11.5%
of the CD24+ CD29+ cells fall into this population, compared to 7.5% of the same cells in
MINO line D. This furthermore correlates with an increased mean of fluorescence
intensity which was seen with regards to CD29 in the putative stem cell population within
MINO line 4, with 1067 fluorescence intensity units over 1629 events compared to 478 in
MINO line D over 7380 events (data not shown), a difference that was shown to be
statistically significant (t=125.5596, p <0.001, df=9007). The same was true with respect
to the expression of CD24, which was significantly increased in MINO line four. The
same population is further increased in both MINO lines as compared to the FVB
mammary gland. As previously mentioned, prior to analysis of CD42 and CD29
expression, cells with positive expression of the epithelial cell surface marker CD49f
have been selected. As hematopoietic lineage committed (Lin+) cells have been removed
in the previous gating path, the percentage for this population is relatively high and the
CD49f negative population is thus thought to house other cell types, such as fibroblasts or
27
adipocytes, which cannot fully be excluded by the washing procedures of the enzymatic
epithelial dissociation procedure. The full gating path involves exclusion of cell debris,
exclusion of doublets, and removal of dead and Lin+ cells prior to selection for the
CD49f+ population (Figure 4).
28
Figure 4. Full gating path of the MINO. Flow cytometry dot plots showing the gating
strategy used to select for Lin- CD49f+ cells in the MINO mammary gland. The gating
strategy was, as previously described in the FVB mammary gland, based on (from left to
right) exclusion of cellular debris on FSC (Forward scatter) vs. SSC (Side scatter), with
subsequent exclusion of doublets on FSC vs. pulse width. Dead cells were excluded using
propidium iodide (PI), followed by selection of Lineage- (Lin-) cells based on negative
expression for CD31, CD45 and Ter119. Further selection was based on positive staining
for CD49f.The data shown is a representation of MINO line D.
29
The putative pre-cancer stem cell population shows a CD49fhigh profile
According to the literature, the majority of the mouse mammary stem cell
population is located within the population expressing high levels of CD49f, while the
luminal epithelial cells are suggested to have a CD49flow profile (Schackleton et al. 2006,
Stingl et al. 2006). By comparing the CD24low CD29high and the CD24negative CD29+
population from the MINO with regards to their expression of CD49f, it becomes
apparent the majority of the CD24negative CD29+ population is showing a low expression
of CD49f (Figure 5). The CD24low CD29high population revealed a higher expression of
CD49f as shown by an almost 10 fold increase in fluorescence intensity which moves it
further right on the x-axis of the histogram plot. This was also strengthened by the fact
that CD49f showed a higher mean of fluorescence intensity in the putative stem cell
population with 88.0 units over 7380 events compared to 37.1 over 68 171 events
detected in the luminal epithelial population, which was determined to be statistically
significant (t=123.6001, p <0.001, df=75549). The CD24negative CD29+ population also
shows a small number of CD49f as revealed by the overlap with the CD24low CD29high
population, yet the majority of these cells do not show such high expression. These
results thus provide further support that the CD24low CD29high CD49fhigh population is
enriched for "stemness", as opposed to the CD24negative CD29low CD49flow population,
which is suggested to contain a luminal epithelial cell type.
30
Figure 5. Expression of the cell surface marker CD49f in the MINO. Flow cytometry
histogram comparing the expression of CD49f in the CD24- CD29low (dotted) population
and the CD24+ CD29high population (black solid line) against the CD49f expression in the
entire Lin- population (filled gray area) in mammary glands from MINO line D.
31
Increased expression of CD44 in the putative stem cell population
Although human breast cancer stem cells have been reported to express high
levels of CD44, few attempts have been made to examine the expression and the
correlation to mammary cancer stem cells in the mouse. It becomes apparent that CD44
is positively expressed in both populations found in the MINO (Figure 6), with a total
expression of 34% in all Lin- cells (data not shown). However, CD44 expression is
significantly increased in the putative stem cell population with the peak of the
population showing approximately a five fold increase in fluorescence intensity with a
mean of 37.4 fluorescence intensity units over 7380 events, compared to the putative
luminal epithelial population showing a mean of fluorescence limited to 20.3 units over
68 171 events. This difference was further shown to be statistically significant as
supported by Student’s t-test (t=107.7452, p <0.001, df=75549). The CD24negative
CD29low population shows a larger cell count with a 19.9% positive CD44 expression, yet
the CD24low CD29high population peaks at higher fluorescence intensity with a 60.9%
positive expression and thus with fewer cells within the CD44 negative portion
(percentages not shown). Furthermore, CD44 appears to be highly expressed within the
normal mammary gland, with 84.7% of Lin- CD49f+ cells staining positive for this
marker (data not shown). However, when selecting for the portion of Lin- CD49f+ cells
expressing the highest levels of CD44 (2.62%) and overlaying this subset onto the
distribution plot of cells expressing CD24 and CD29, it becomes evident that these cells
mainly appear within the putative stem cell population (Figure 7).
32
Figure 6. Expression of putative cancer stem cell surface marker CD44 in the MINO.
Flow cytometry histogram overlay comparing the level of CD44 expression within the
CD24- CD29+ population (gray solid line) and the CD24low CD29high population (black
line with filled gray area) in cells gated for Lin- and CD49f+ expression in the MINO.
Data represented by MINO line D.
33
Figure 7. Expression of CD44 in the FVB mammary gland. Flow cytometry dot plot
gating Lin- CD49f+ cells in the FVB mammary gland based on expression of CD44 (left).
The gate includes cells showing the highest levels of CD44 expression as compared to the
rest of the cells within this plot. The right dot plot is depicting an overlay of Lin- CD49f+
selected cells based on their expression of CD24 and CD29. Black dots correlate with
cells containing a CD44high expression profile included in the gate on the CD24/CD29 dot
plot on the left.
34
Restriction of CD61 expression to potential luminal epithelial populations
As previously mentioned, CD61 has been reported as a luminal epithelial
progenitor marker and in accordance with those findings (Kouros-Mehr et al. 2008),
positive expression of CD61 appears to be present in the MINO, even though the levels
appear relatively low (Figure 8). Compared to previously published findings, the
majority of the Lin- CD49f+ cells showed a negative expression of CD61, consisting of
approximately 64% of the total cell count (data not shown). However, 15% of all LinCD49f+ cells were positive for CD61, and a flow cytometry dot plot overlay of CD61,
CD24 and CD29 revealed that they could mainly be traced to the CD24negative CD29low
population (Figure 9). Thus, compared to a previous study which found that 32% of cells
with a CD24+ CD29low profile were CD61+ (Asselin-Labat et al. 2007), the expression of
CD61 appears to be significantly lower within the MINO in this study. These results
reveal 18.8% CD61+ cells in the CD24negative CD29low population, as compared to a
5.79% for the CD24low CD29high population within the MINO. The CD24negative CD29low
population shows a low mean of fluorescence intensity, measured to 4.30 fluorescence
intensity units, however; as compared to the 2.30 units found within the CD24low
CD29high population, this difference was determined as statistically significant
(t=22.9351, p<0.001, df= 75549). Thus, there appears to be a small number of cells
positive for this marker within the putative stem cell population, yet the highest levels
were detected within the population suggested to be enriched for cells of a luminal
epithelial origin.
35
Figure 8. Expression of putative cell surface marker CD61 in the MINO. Flow cytometry
dot plot showing a comparison of the total amount of CD61 in the CD24low CD29high
population (left) and the CD24negative CD29low (right) within MINO line D. All cells are
Lin- and selected for positive CD49f expression. The squared gates reveal the total
percentage of CD61+ within the respective population.
36
Figure 9. Distribution of CD61 expression in Lin- CD49f+ cells within the MINO. Flow
cytometry dot plot overlay showing the distribution of all cells expressing CD61 (black
dots) on top of the dot plot revealing the distribution of cells with regards to their
expression of CD24 and CD29 (gray dots). All cells have been gated to only include Linas well as CD49f+ cells. Data is represented by MINO Line D.
37
Increased "stemness" with progression into carcinoma
As has been previously described by Kouros-Mehr et al. (2008), the stem cell
population appears to increase with progression into carcinoma. This was investigated
using the MINO and a MINO derived tumor, compared to cells from the Met-1 cell line,
which was derived from a mammary tumor with a PyV-mT background. These results
showed a similar increase in the size of the putative stem cell population, with the largest
increase seen in the Met-1 cells (Figure 10). As previously reported, the CD24+ CD29+
population is highly increased in the carcinoma, here represented by the Met-1 tumor cell
line. Thus, with a percentage as high as 72.5%, the bulk of the carcinoma appears to be
enriched for "stemness", which may be comparable to the 80% CD24+ CD29+ cells
within the Met-1 cell line. Even though the CD24+ CD29+ population seen in the Met-1
cells appears exceptionally large in comparison to the MINO as well as the MINO tumor,
with a mean of 1272 in fluorescence intensity for CD29 in the Met-1 cell line compared
to 35.0 in the tumor (t=45.9213, p <0.001, df=16985), the tumor only shows a doubling
of the putative stem cell population, with an increase from about 7 to 20%. The
differences in fluorescence intensity for both surface markers CD29 and CD24 were
shown to be statistically significant between all three samples. It was also evident that the
number of cells with a CD24 negative profile was significantly reduced in the Met-1 cell
line, with less than 1% of cells showing no expression of this surface marker, while the
MINO as well as the MINO derived tumor revealed a larger percentage of cells with this
expression profile.
38
Figure 10. Expression of cancer stem cell related cell surface markers with tumor
progression. Flow cytometry density plots comparing the levels of CD24 and CD29
expression within the A) MINO, B) MINO derived tumor and C) the Met-1 cell line.
39
DISCUSSION
The focus of this study has been centered on the identification of the mammary
cancer stem cells within a pre-cancer mouse model named after its expression of
Mammary Intraepithelial Neoplasia Outgrowths or MINOs, which develop
heterogeneous lesions representative of human Ductal Carcinoma In Situ (DCIS). DCIS
represents about 20% of all breast cancer cases reported in the US and is considered the
earliest form of breast cancer, where the lesion is still confined within the epithelium of
the ductal structures and the terminal lobular units of the breast. As DCIS shows frequent
incidences of progression into invasive carcinoma, treatment of the condition will
significantly affect the development of invasive cancers and may thus serve as a
treatment for breast cancer in its earliest stages. Along with the popularity of the cancer
stem cell hypothesis predicting cancer progression involving drug resistant progenitor
cells driving the growth of the tumor, identification of the cancer stem cell population has
gained a great deal of attention. Identification of the cancer stem cell population
potentially by the use of specific cell surface markers would be a first step towards
characterizing and acquiring knowledge regarding the specific pathways and mechanisms
responsible for such progression. Functional studies of the cancer stem cells, such as the
measurement of proliferation rates or regenerative potential upon injection into the
mammary gland of the mouse, can thus subsequently establish the true nature of the
various populations within the mammary epithelium.
40
In previous studies using the mouse as a model, the mammary gland cancer stem cell
population has been identified and isolated using various combinations of cell surface
markers, including Sca-1, Thy1, ALDH, CD133, CD24, CD29 and CD49f, however, a
consistent method using a combination of these markers for isolation has yet to be
revealed. The cell surface expression profile of this rare population appears to be context
dependent with regards to the environmental setting and the stage of differentiation, and
may therefore reveal varying patterns when conditions change. In several previously
published studies, a putative stem cell population was isolated by FACS based on an
expression profile with low levels of cell surface marker CD24 along with high levels of
surface markers CD29 and CD49f (Schackleton et al. 2006, Stingl et al. 2006, Wegwitz et
al. 2010). It was furthermore revealed that this diminutive population exhibits expression
of the basal marker cytokeratin 14, consistent with the location of mammary stem cells
within the basal portion of the epithelium, along with findings showing that the same
cells possesses the capability to regenerate the ductal tree upon injection into pre-cleared
mammary fat pads. Additional functional assays have further detected that the putative
stem cell population can be characterized by a higher proliferative and migratory rate,
and were able to develop into mammospheres when cultured in vitro (Schackleton et al.
2006, Stingl et al. 2006, Joshi et al. 2010). Together, this collection of evidence suggests
that there is a rare subpopulation within the basal layer of the ductal epithelium of the
mammary gland showing stem-like characteristics, with the potential to be identified and
isolated with the use of specific cell surface markers.
41
Since further attempts to isolate this population within the same model have met with
limited success, an additional set of novel cell surface markers was explored to
potentially increase the specificity for mammary cancer stem cell isolation. Based on the
discovery that the adhesion molecule CD44 is an indicator of mammary stem cells in
human breast cancers, it became of interest to investigate the expression pattern and the
utility of this cell surface marker as an indicator of “stemness” in the mouse MINO
model. In addition, it became interesting to examine the expression of CD61 in specific
cell types within the mammary epithelium since it was suggested to be a putative marker
for luminal progenitor cells in mammary lesions from mice overexpressing the protooncogene Wnt-1 (Vaillant et al. 2008). However, there are conflicting reports that
instead, CD61 may be a marker for the identification of luminal epithelial cells within the
mammary gland of the mouse and therefore could be useful for the separation of the
luminal and the basal/myoepithelial populations (Kouros-Mehr et al. 2008). Thus, to
increase specificity for the identification of the pre-cancer stem cell population, the cell
surface markers CD44 and CD61 were added to an antibody panel consisting of integrins
CD24, CD29 and CD49f to fractionate the mammary gland cells using FACS.
As previously mentioned, the putative stem cell population normal mammary gland
of the mouse has been identified using CD24, CD29 and CD49f, and it therefore became
interesting to establish what role CD61 and CD44 plays with regards to the identification
this population. The results from this study revealed three distinct populations within the
normal mammary gland of the mouse, one of which is expressing low levels of CD24
along with high levels of CD29 and CD49f, previously described in the literature as a
42
putative stem cell population. This has been validated by transplantation experiments
where only the aforementioned population had the ability to repopulate the mammary fat
pad, showing enhanced migratory potential and possessed a higher rate of proliferation
(Schackleton et al. 2006, Kouros-Mehr et al. 2008).
The FACS data in this study also indicates that the FVB gland consists of a
population with CD49f+ CD24high CD29low expression profile, previously suggested to be
of luminal origin, together with a population showing CD49f+ CD24negative CD29low
expression (CD24 being the distinguishing feature), which potentially belongs to cells of
non-epithelial origin as indicated by the literature. Previous studies also described a
fourth population, with a CD49f+ CD24negative CD29 high expression profile, which does
not appear to be enriched for “stemness” since it was incapable of repopulating the
mammary fat pad (Schackleton et al. 2006, Stingl et al. 2006, Sleeman et al. 2005).
However, this population was not found in our results, possibly due to biological as well
as technical differences, such as the age of the animals and the variation introduced by
different FACS gating strategies along with different instrumentation and software. Other
factors that could have contributed to these differences are the specific antibody used
along with their respective fluorochrome conjugations. Nevertheless, compared to
previous studies, significantly similar population sizes as well as a similar distribution
were revealed when investigating cells based on their expression of CD24 and CD29.
Taken together, the population containing cells with a CD24low CD29high profile appears
to be representative of the stem cell population in the normal mammary gland of the
mouse.
43
In the mammary gland of the MINO, the expression of CD49f, CD24 and CD29
revealed two separate populations, one of which may be further subdivided into two
significantly related populations as it appears to contain cells expressing both low as well
as negative levels of cell surface marker CD24. This may possibly correspond to the
CD49f+ CD24negative CD29low population previously seen in the normal mammary gland,
but with the addition of a population containing CD49f+ CD24low CD29low positive cells.
Alternatively, this large group of cells may correspond to one single cell type with
varying CD24 expression levels; this may be caused by cells being in different stages of
differentiation that correlates with CD24 levels. However, this study also revealed that
both of these populations stain positive for the expression of CD61, which was previously
mentioned as being an identifier for luminal epithelial populations in some studies.
Together with the fact that the same cells are also expressing CD49f, but at a low level
(data not shown), one may suggest that both populations may be of a luminal origin.
However, as conflicting results have revealed CD61+ cells as luminal progenitors, it is
not yet clear at what stage of differentiation this marker is expressed. Further studies will
be needed to address this issue. Nevertheless, based on the low expression of CD24 as
well as CD49f, along with a relatively large population size, the assumption that can be
made from this study is that CD61 may be labeled as a marker for luminal epithelial cells
in contrast to the suggestion that the same marker would serve as an indicator for cancer
stem cells.
The MINO mouse model further contains a diminutive population containing a
CD24low CD29high expression profile, which showed high levels of CD49f. As this
44
population size and expression profile is comparable to the one presented for the putative
cancer stem cell population in prior studies (Schackleton et al. 2006, Stingl et al. 2006), it
suggests that this population is in fact enriched for “stemness”. This cell type was
furthermore shown to have a lower expression of surface marker CD61 as well as a
higher expression of CD44 in comparison to the CD24negative/low populations. The putative
stem cell population in the normal mammary gland also showed a similar expression
pattern, with the CD49fhigh, CD24low, CD29high population expressing the highest levels of
CD44. This is especially intriguing that human breast cancer stem cells appear to contain
high levels of the same marker, suggesting that CD44 could be a valid identifier for the
stem cell population within the mammary gland of the mouse as well. However,
expression of CD61 has yet to be examined within the normal mammary gland. Increased
levels of CD44 were also seen within the CD24low CD29high population in the MINO.
Thus, CD44 may be able to provide additional specificity to the identification of the
cancer stem cell population in combination with CD49f, CD24 and CD29 markers in the
FVB mouse as well as the mouse model representing human DCIS.
In comparing MINO lines, no differences were noted for the population size of
CD24low, CD29low cells, however considerable differences were observed for the putative
stem cell population that contain a CD24low CD29high expression profile. Furthermore, the
putative stem cell population in MINO line four appears to be increased in size as
compared to the same population within MINO line D. This may be due to characteristic
differences between the two lines, such as the fact that MINO line four is known to be
non-metastatic with a shorter tumor latency, which indicates a more rapid tumor
45
development. In turn, this correlates with the finding that the putative stem cell
population was found to be increased with tumor progression (Kouros-Mehr et al. 2008).
Thus, the increase in size of the putative stem cell population in MINO line four may be
associated with rapid tumor progression. Along this line of thinking, the equivalent
population within the MINO Line D may be smaller due to its phenotype, which includes
a longer tumor latency period. One may assume that the stem cell population within line
D has not yet reached the size of those in a system with a shorter latency to tumor
formation, yet further studies will be needed to resolve this issue.
Similarities between the MINO lines were that the CD24low CD29high population both
were higher compared to the normal mammary gland, which may be due to the transgenic
background of the MINO. The pre-disposition to development of invasive cancer caused
by the oncogene PyV-mT, along with increased likelihood for tumor formation may play
a role in mechanisms related to maintaining the stem cell niche or conservation of the
stem cell pool. An additional explanation may include genomic alterations, such as
mutation events, leading to regulatory changes causing increased proliferation, thereby
affecting the size of the stem cell population.
The size of the putative cancer stem cell population has furthermore been reported
to increase with progression to carcinoma (Kouros-Mehr et al. 2008). This was revealed
through the increased population size of the cells expressing both CD24 and CD29 within
the MINO derived tumor and the PyV-mT derived tumor cell line Met-1. However, even
though the population expressing surface markers CD49f, CD24 and CD29 appears to be
highly increased in the MINO derived tumor, this population is not expressing as high
46
levels of CD29 as can be seen in the MINO as well as in the Met-1 cell line. This
suggests that the usefulness of CD29 as a stem cell marker is still debatable, as there have
been conflicting reports in the literature. Reports of a critical role for CD29 in
tumorigenesis in a MMTV/Pyv-mT transgenic mouse model have been contrasted by the
finding that the same marker appears to be dispensable with regards to ErbB2 mammary
tumor formation (White et al. 2004, Huck et al. 2010). Thus, CD29 may be dispensable
for the identification of mammary cancer stem cells in certain contexts, but it is useful in
the models under investigation in these studies. However, further studies will be needed
to determine how consistent of an indicator of “stemness” CD29 is in the mammary gland
of the mouse.
Compared to the findings presented by Kouros-Mehr et al. (2008), the bulk of the
carcinoma appears to show a positive expression for both CD24 and CD29 and is
suggested to be enriched for “stemness”, which also becomes apparent in the Met-1 cell
line, where the majority of cells appear to be double positive with regards to expression
of the same surface markers. This population was also found to be in majority and could
represent an increased cancer stem cell population; however, it is important to consider
the fact that high expression levels may be cell culture artifacts, induced by repeated
passages and long periods of culturing. The increase in “stemness” with carcinoma
progression correlates with the observation that these cells can be continuously selected
over time, thereby promoting their expansion.
In summary, these findings have revealed a putative stem cell population with a
CD49fhigh CD24low CD29high expression profile within mammary glands of normal FVB
47
mice and in the MINO mouse model. It has yet to be revealed whether or not the rest of
the cells in the MINO, gated on the same preferences during FACS, were two different
cell types with similar expression profiles or if they were comprised of one large
population. Future studies will be needed to investigate the characteristics of these cells.
This study has further identified CD44 as a potential marker for mammary cancer
stem cells, along with the potential use of CD61 as a marker for cells of luminal epithelial
origin. Together, these findings may significantly improve the isolation of the pre-cancer
stem cell population from the MINO mouse model and therefore aid in future assays to
confirm the origin of the cancerous cells, possibly leading to increased effectiveness of
treatments aimed at eliminating cancer stem cells. Identification of the pre-cancer stem
cells and understanding their biology with respect to breast cancer progression may
provide a new basis for therapeutic treatment and diagnostics. This is especially needed
since these cells have been suggested to possess resistance to chemo- as well as radiationtherapy, consistent with the observation that current cancer treatments often are unable to
completely eliminate the entire tumor. Even though findings have demonstrated the
presence of stem cell populations within breast carcinomas as well as other cancers, their
origin and at what time point these cells could be detected is still unknown. It is
furthermore not established when these cells appear and if their behavior may be
recognized in advance by certain markers or properties (Jordan et al. 2006). Future
studies need to focus on the functional characterization of this cell type in order to reach
the goal of treating DCIS and thus treating breast cancer in its earliest stage.
48
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