High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability.

Abstract

Cancer stem cells (CSCs) have recently been identified in leukaemia and solid tumours; however, the role of CSCs in metastasis remains poorly understood. This dearth of knowledge about CSCs and metastasis is due largely to technical challenges associated with the use of primary human cancer cells in pre-clinical models of metastasis. Therefore, the objective of this study was to develop suitable pre-clinical model systems for studying stem-like cells in breast cancer metastasis, and to test the hypothesis that stem-like cells play a key role in metastatic behaviour. We assessed four different human breast cancer cell lines (MDA-MB-435, MDA-MB-231, MDA-MB-468, MCF-7) for expression of prospective CSC markers CD44/CD24 and CD133, and for functional activity of aldehyde dehydrogenase (ALDH), an enzyme involved in stem cell self-protection. We then used fluorescence-activated cell sorting and functional assays to characterize differences in malignant/metastatic behaviour in vitro (proliferation, colony-forming ability, adhesion, migration, invasion) and in vivo (tumorigenicity and metastasis). Sub-populations of cells demonstrating stem-cell-like characteristics (high expression of CSC markers and/or high ALDH) were identified in all cell lines except MCF-7. When isolated and compared to ALDH(low)CD44(low/-) cells, ALDH(hi)CD44(+)CD24(-) (MDA-MB-231) and ALDH(hi)CD44(+)CD133(+) (MDA-MB-468) cells demonstrated increased growth (P < 0.05), colony formation (P < 0.05), adhesion (P < 0.001), migration (P < 0.001) and invasion (P < 0.001). Furthermore, following tail vein or mammary fat pad injection of NOD/SCID/IL2gamma receptor null mice, ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells showed enhanced tumorigenicity and metastasis relative to ALDH(low)CD44(low/-) cells (P < 0.05). These novel results suggest that stem-like ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells may be important mediators of breast cancer metastasis.

Full text

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High aldehyde dehydrogenase and expression of cancer stem cell markers
selects for breast cancer cells with enhanced malignant and metastatic
ability

Alysha K. Croker1,2,3, David Goodale1, Jenny Chu1, Carl Postenka1, Benjamin D. Hedley1,
David A. Hess4,5, and Alison L. Allan1,2,3*

London Regional Cancer Program1; Departments of Oncology2, Anatomy & Cell Biology3,
and Physiology & Pharmacology4, University of Western Ontario; Robarts Research
Institute5, London, Ontario CANADA


Author Affiliations:

Alysha K. Croker: London Regional Cancer Program1; and Departments of Oncology2, and
Anatomy & Cell Biology3, University of Western Ontario; London Ontario CANADA

David Goodale: London Regional Cancer Program1; London Ontario CANADA

Jenny Chu: London Regional Cancer Program1; London Ontario CANADA

Carl Postenka: London Regional Cancer Program1; London Ontario CANADA

Benjamin D. Hedley: London Regional Cancer Program1; London Ontario CANADA

David A. Hess: Department of Physiology & Pharmacology4, University of Western Ontario;
and Robarts Research Institute5, London, Ontario CANADA

Alison L. Allan*: London Regional Cancer Program1; and Departments of Oncology2, and
Anatomy & Cell Biology3, University of Western Ontario; London Ontario CANADA



* Correspondence/reprint requests should be addressed to:
Alison L. Allan, Ph.D.
London Regional Cancer Program
790 Commissioners Road East
London, Ontario CANADA N6A 4L6
Tel: (519) 685-8600 x55134
Fax: (519) 685-8616
Email: alison.allan@lhsc.on.ca















Please cite this article as a “Postprint”;10.1111/j.1582-4934.2008.00455.x



approved for publication in the Journal of Cellular and Molecular Medicine, but
has yet to undergo copy-editing and proof correction. See
http://www.blackwell-synergy.com/loi/jcmm for details.





This is an Accepted Work that has been peer-reviewed and
Original Article
Received: 20-Mar-08
Accepted: 28-Jul-08

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ABSTRACT
Cancer stem cells (CSCs) have recently been identified in leukemia and solid tumors;
however, the role of CSCs in metastasis remains poorly understood. This dearth of
knowledge about CSCs and metastasis is due largely to technical challenges associated with
the use of primary human cancer cells in preclinical models of metastasis. Therefore, the
objective of this study was to develop suitable preclinical model systems for studying stem-
like cells in breast cancer metastasis, and to test the hypothesis that stem-like cells play a key
role in metastatic behavior. We assessed four different human breast cancer cell lines (MDA-
MB-435, MDA-MB-231, MDA-MB-468, MCF-7) for expression of prospective CSC
markers CD44/CD24 and CD133, and for functional activity of aldehyde dehydrogenase
(ALDH), an enzyme involved in stem cell self-protection. We then used fluorescence-
activated cell sorting (FACS) and functional assays to characterize differences in
malignant/metastatic behavior in vitro (proliferation, colony-forming ability, adhesion,
migration, invasion) and in vivo (tumorigenicity and metastasis). Sub-populations of cells
demonstrating stem-cell like characteristics (high expression of CSC markers and/or high
ALDH) were identified in all cell lines except MCF-7. When isolated and compared to
ALDHlowCD44low/- cells, ALDHhiCD44+CD24- (MDA-MB-231) and ALDHhiCD44+CD133+
(MDA-MB-468) cells demonstrated increased growth (p<0.05), colony formation (p<0.05),
adhesion (p<0.001), migration (p<0.001), and invasion (p<0.001). Furthermore, following
tail vein or mammary fat pad injection of NOD/SCID/IL2γ receptor null mice,
ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ cells showed enhanced tumorigenicity and
metastasis relative to ALDHlowCD44low/- cells (p<0.05). These novel results suggest that
stem-like ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ cells may be important mediators
of breast cancer metastasis.
Key Words: breast cancer, metastasis, cancer stem cells, ALDH, CD44

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INTRODUCTION
Breast cancer is a leading cause of death in women, due primarily to ineffective
treatment of metastasis. Metastasis is a multi-step process that involves tumor cell escape
from the primary tumor, migration through the body, adhesion and extravasation at the
secondary site, initiation of micrometastatic growth, and maintenance of growth into
clinically detectable macrometastases [1]. Given the onerous nature of this process, it is not
surprising that metastasis is highly inefficient, with the main rate-limiting steps being
initiation and maintenance of growth at secondary sites [1-3]. Taken together with the
heterogeneous nature of solid tumors, this metastatic inefficiency suggests that only a small
subset of cells can successfully navigate the metastatic cascade and eventually re-initiate
tumor growth to form metastases. However, the ability to specifically identify and target this
deadly subset of cells has remained elusive.
In light of several pivotal studies demonstrating the existence of “stem-like” cells or
cancer stem cells (CSCs) in leukemia [4, 5] and several types of solid cancer [6-11], we and
others have hypothesized that CSCs may represent the subset of tumor cells responsible for
metastatic disease [12, 13]. However, while several studies have demonstrated the ability of
CSCs to initiate and maintain a primary tumor [6-11], the functional contribution of CSCs to
metastatic behavior remains poorly understood.
In breast cancer, stem-like cells have been prospectively isolated from primary
tumors and pleural effusions based on a CD44+CD24- phenotype [6]. Subsequent
experimental studies have also isolated CD44+CD24- breast cancer cells and demonstrated
increased in vitro expression of stem cell markers and enhanced capacity for mammosphere
formation, invasion, and resistance to radiation [14-16]. Furthermore, clinical studies indicate
that CD44+CD24- tumor-initiating cells express an invasive gene signature and may be
associated with distant metastases [17-19]. Additional putative CSC phenotypes have also

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been identified in other solid cancers, including CD133+ (brain, colon, pancreatic cancer) [8,
9, 11] and CD44+CD133+ (prostate cancer) [7]. However, because of the heterogeneous
nature of solid cancers, the reliability of using cell surface markers as the sole way to isolate
CSCs remains controversial [20-23]. Furthermore, human stem cell sources (including
tumors) may contain alternate stem/progenitor cell lineages not efficiently isolated using
variably expressed cell surface markers.
A complementary strategy for identifying stem-like tumor cells involves
measurement of aldehyde dehydrogenase (ALDH) activity, an enzyme involved in
intracellular retinoic acid production [24]. Retinoic acid signaling is linked to cellular
differentiation during development and plays a role in stem cell self-protection throughout an
organism’s lifespan [25]. We and others have previously used this isolation strategy alone or
in combination with cell surface markers (i.e. CD133) to successfully isolate normal human
hematopoietic progenitor cells [26-28], and ALDH activity has also been used to identify
stem-like subsets in human hematopoietic cancers [29-31]. A recent study by Ginestier et al.
(2007) elegantly demonstrated that expression of ALDH1 in breast tumors was a predictor of
poor clinical outcome, and that high ALDH activity selects for both normal and tumorigenic
human mammary epithelial cells with stem/progenitor properties [32]. Therefore, the use of
ALDH activity as a purification strategy allows non-toxic and efficient isolation of human
stem-like cells based on a developmentally conserved stem/progenitor cell function.
While there is growing evidence supporting the existence of CSCs in solid tumors,
what remains less clear is the impact of CSCs on metastatic behavior. We believe that the
dearth of knowledge about CSCs and metastasis is due largely to technical challenges
associated with the use of primary human cancer cells in preclinical models of metastasis:
even in NOD/SCID mice, it is very difficult to grow primary cells as xenograft tumors, much
less as metastases. Therefore, workable alternative model systems must be developed in order

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to address this need. It has long been observed by tumor biologists that even with cancer cell
lines, large numbers of cells need to be injected to form a tumor and/or metastasize in
experimental animals [1, 2, 33]. This suggests that not all cells within a cell line population
are equal, and only a small subset of cells is capable of tumor initiation and progression to
metastasis. Indeed, studies have shown that commonly used cell lines contain subpopulations
of cells with stem-like properties [14-16]. Thus, the purification of stem-like cells from cell
lines may provide a valuable model system for starting to investigate the role of such cells in
breast cancer metastasis.
In the current study, we assessed four commonly used human breast cancer cell lines
(including highly metastatic MDA-MB-435 [see note in Materials and Methods], moderately
metastatic MDA-MB-231, weakly metastatic MDA-MB-468, and non-metastatic MCF-7) for
expression of the prospective CSC markers CD44/CD24 and CD133, and for functional
activity of ALDH. We then used fluorescence-activated cell sorting (FACS) and standard
functional assays to characterize differences in malignant/metastatic behavior between cell
populations, including cell growth, adhesion, migration, and invasion in vitro, and
tumorigenicity and metastasis in vivo. The novel findings presented here suggest that high
ALDH activity and CSC marker expression select for stem-like breast cancer cells with
enhanced malignant and metastatic properties, and that ALDHhiCD44+CD24- and
ALDHhiCD44+CD133+ stem-like cells may be important contributors to breast cancer
metastasis.

MATERIAL AND METHODS
Cell Culture
MDA-MB-231 and MCF-7 cells were obtained from ATCC (Manassas, VA). MDA-
MB-435 and MDA-MB-468 cells were obtained from Dr. Janet Price, M.D. Anderson Cancer

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Center, Houston, TX [34]. MDA-MB-435 and MDA-MB-468 cells were grown in αMEM +
10% fetal bovine serum (FBS), MCF-7 cells were grown in DMEM + 10% FBS, and MDA-
MB-231 cells were grown DMEM:F12 + 10% FBS. Media was obtained from Invitrogen
(Carlsbad, CA). FBS was obtained from Sigma (St. Louis, MO). It should be noted that the
MDA-MB-435 cell line was originally isolated from the pleural effusion of a woman with
metastatic breast adenocarcinoma [34]. Recently, a debate has arisen over the origins of this
cell line, whether it was derived from the M14 melanoma cell or is in fact a true breast cancer
cell line [35, 36]. Whether melanoma or breast in origin, the MDA-MB-435 cell line does
represent a highly metastatic cell line, and thus provides an opportunity to relate functional
metastatic ability to expression of CSC-related markers.

Cell Surface Marker Analysis
MDA-MB-435, MDA-MB-231, MDA-MB-468, and MCF-7 cells (1 x 105) were
incubated with fluorescently-conjugated antibodies, including anti-CD24 (clone ML5; BD
Biosciences Canada, Mississauga, ON) or anti-CD133 (clone AC133; Miltenyi Biotec,
Auburn, CA) conjugated to phycoerytherin (PE); and anti-CD44 (clone IM7; BD
Biosciences) conjugated to fluorescein isothiocyanate (FITC). For supplemental studies,
MDA-MB-231 and MDA-MB-468 cells were also incubated with anti-CXCR4 (clone 12G5;
R&D Systems, Minneapolis, MN) conjugated to PE. Fluorescently-conjugated IgG isotype
controls (BD Biosciences) were used as negative controls. Cells were analyzed using a
Beckman Coulter EPICS XL-MCL flow cytometer.

Analysis of ALDH Activity
To assess ALDH activity of the different cell lines, the ALDEFLUOR® assay kit
(StemCell Technologies, Vancouver, BC) was used. The basis for this assay is that uncharged
ALDH substrate (BODIPY-aminoacetaldehyde [BAAA]) is taken up by living cells via

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passive diffusion. Once inside the cell, BAAA is converted into negatively-charged
BODIPY-aminoacetate (BAA) by intracellular ALDH. BAA- is then retained inside the cell,
causing the cell to become highly fluorescent. Only cells with an intact cell membrane can
retain BAA-, so only viable cells can be identified [24]. The ALDEFLUOR® assay was
performed essentially as described previously [26-28]. Briefly, MDA-MB-435, MDA-MB-
231, MDA-MB-468, and MCF-7 cells were harvested, placed in ALDEFLUOR® assay buffer
(2 x 106/ml), and incubated with the ALDEFLUOR® substrate for 45 minutes @37oC to
allow substrate conversion. As a negative control for all experiments, an aliquot of
ALDEFLUOR®-stained cells was immediately quenched with 1.5-mM
diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor. Cells were analyzed using
the green fluorescence channel (FL1) on a Beckman Coulter EPICS XL-MCL flow
cytometer. Human umbilical cord blood (discard product) was labeled and assayed for ALDH
activity as described previously [27] in order to optimize and validate the flow cytometry
protocol.

FACS Isolation of Cells
Based on cell surface marker phenotype and ALDH activity, MDA-MB-468 and
MDA-MB-231 cell lines were chosen for further cell isolation by FACS and functional
analysis. Cells were concurrently labeled with 7-Amino-actinomycin (7-AAD), fluorescent
antibodies (CD44-APC + CD24-PE [MDA-MB-231] or CD44-APC + CD133-PE [MDA-
MB-468]) and the ALDEFLUORTM assay kit as described above. Cell subsets were isolated
using a four-color analysis protocol on a FACS Vantage/Diva cell sorter (BD Biosciences),
including ALDHhiCD44+CD24- and ALDHlowCD44low/-CD24+ subsets (from MDA-MB-231
cells) and ALDHhiCD44+CD133+ and ALDHlowCD44low/-CD133- subsets (from MDA-MB-
468 cells). For both cell lines, ALDH activity was used as the primary sort criteria (top
~20%=ALDHhi; bottom ~20%=ALDHlow) and CD44+CD24- (MDA-MB-231 cells) or

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CD44+CD133+ (MDA-MB-468 cells) phenotype as the secondary sort criteria (top ~10%
gated on ALDHhi; bottom ~10% gated on ALDHlow). Cell viability was assessed by 7-AAD
staining during cell sorting, and confirmed by trypan blue exclusion post-sorting. Following
FACS isolation, cells were used immediately for functional in vitro and in vivo assays.

Cell Proliferation Assays
Sorted cell populations (ALDHhiCD44+CD24- and ALDHlowCD44low/-CD24+ from
MDA-MB-231 cells; ALDHhiCD44+CD133+ and ALDHlowCD44low/-CD133- from MDA-MB-
468 cells) were plated at a density of 5.0 x 104 cells/60 mm plate (n=3 for each time point)
and maintained in regular growth media. Every 48 hours for 14 days, triplicate cultures were
trypsinized and counted by hemocytometer. Doubling time of each cell population was
estimated during the exponential growth phase according to Td = 0.693t/ln (Nt/N0), where t is
time (in hr), Nt is the cell number at time t, and N0 is the cell number at initial time. Lag time
was taken as the time for each population to reach the exponential growth phase.

Colony Forming Assays
In preparation for the assay, 60 mm dishes were coated with 1% agarose (Bioshop;
Burlington, ON) in normal growth media and allowed to set for 1 hr. Cell suspensions (1.0 x
104
cells/60 mm plate) for each sorted population (ALDHhiCD44+CD24-
and
ALDHlowCD44low/-CD24+ from MDA-MB-231 cell line; ALDHhiCD44+CD133+ and
ALDHlowCD44low/-CD133- from MDA-MB-468 cell line) were prepared using 0.6% agarose
in normal growth media and plated on top of the 1% agarose base layer (n=4 for each time
point). Normal growth media was added on top of the cell layer and changed every 3-4 days
for 4 weeks, at which time media was removed and plates were fixed in 10% neutral-buffered
formalin (EM Sciences, Gladstone, NJ). Five fields of view (100x) were counted for each
dish, and mean number of colonies/field and mean colony diameter was calculated.

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Cell Adhesion Assays
Cells were plated onto sterile 96-well non-tissue culture plates (Titertek, Flow
Laboratories Inc.; McLean, VA) treated with either 10 µg/ml of human fibronectin (Sigma)
(MDA-MB-231 cells), 5 µg/ml of human vitronectin (Sigma) (MDA-MB-468 cells), or PBS
(negative control), using 1x104 cells/well (n=3) for each sorted cell population
(ALDHhiCD44+CD24-
and ALDHlowCD44low/-CD24+
from MDA-MB-231 cell line;
ALDHhiCD44+CD133+ and ALDHlowCD44low/-CD133- from MDA-MB-468 cell line).
Vitronectin and fibronectin were chosen based on previous experiments in our laboratory that
have demonstrated that MDA-MB-231 and MDA-MB-468 cells differentially express
integrin receptors for fibronectin and vitronectin respectively ([37] and our unpublished data).
Cells were allowed to adhere for 5 hours, after which the media was removed and non-
adhered cells were rinsed away. Adhered cells were fixed with 2% gluteraldehyde and stained
using Harris’ hematoxylin. Five high powered fields (HPF) (400x) were counted for each
well, and mean numbers of adhered cells/field were calculated.

Cell Migration and Invasion Assays
Transwell plates (6.5mm, 8µm pore size; Becton Dickinson; Franklin Lakes, NJ) were
coated with 6 µg/well of gelatin (Sigma) (migration assay) or 10 µg/well of Matrigel (Becton
Dickinson) (invasion assay) as described previously [38, 39]. Control (0.01% BSA) or
chemoattractant (5% FBS) media was placed in the bottom portion of each well. 3.5 x 104
cells of each sorted population (ALDHhiCD44+CD24- and ALDHlowCD44low/-CD24+ from
MDA-MB-231 cell line; ALDHhiCD44+CD133+ and ALDHlowCD44low/-CD133- from MDA-
MB-468 cell line) were plated on top of the transwells. Cells were allowed to migrate or
invade for 24 hours, after which the upper transwell was removed, inverted, fixed with 1%
gluteraldehyde, and stained with Harris’s hematoxylin. Non-migrated or non-invaded cells on

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the inner surface of the transwell were carefully removed using a cotton swab. Five HPF were
counted for each well, and mean numbers of migrated or invaded cells/field were calculated.

In Vivo Animal Experiments
All in vivo work was carried out using NOD/SCID-IL2γ receptor null mice, which
permit increased tissue engraftment of human cells without rejection due to reduced innate
immunity (NOD mutation), complete T and B cell deficiency (SCID mutation), and reduced
NK-cell function (IL-2Rγ mutation) [40]. Animal procedures were conducted in accordance
with the recommendations of the Canadian Council on Animal Care, under a protocol
approved by the University of Western Ontario Council on Animal Care.
Following cell sorting, sorted populations (ALDHhiCD44+CD24-
and
ALDHlowCD44low/-CD24+ from MDA-MB-231 cell line; ALDHhiCD44+CD133+ and
ALDHlowCD44low/-CD133- from MDA-MB-468 cell line) were resuspended in sterile Hank’s
buffered salt solution (HBSS) (MDA-MB-231) or PBS (MDA-MB-468) at 5 x 106 cells/ml.
Using established models of experimental metastasis (tail vein injection) or spontaneous
metastasis (mammary fat pad injection) [33], 100 µl (5 x 105 cells) of each sorted cell
population were injected into the lateral tail vein or right thoracic mammary fat pad of 7-10
week old female NOD/SCID-IL2Rγ null mice (n=4 mice/group). Primary tumor growth was
evaluated weekly by caliper measurement in two perpendicular dimensions, and tumor
volume was estimated using the following formula: [volume = 0.52 × (width)2 × (length)] for
approximating the volume (mm3) of an ellipsoid. Metastatic growth in the lung and
extrapulmonary organs was allowed to develop for 5-7 weeks (MDA-MB-231) or 12 weeks
(MDA-MB-468), at which point the mice were euthanized and assessed for metastatic
burden. Endpoints and cell numbers for injection were chosen based on preliminary
experiments using unsorted cell populations and NOD/SCID-IL2Rγ null mice (data not

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shown).
Tissues and organs were examined superficially for evidence of gross macroscopic
metastases at necropsy. Tissues collected at necropsy were then fixed in 10% neutral-
buffered formalin, embedded in paraffin, sectioned randomly (4 µm sections; at least 100 µm
apart; 5 sections/tissue), and subjected to standard hematoxylin and eosin (H&E) staining.
Stained slides were evaluated by light microscopy in a blinded fashion in order to identify
and characterize incidence and regions of micrometastatic involvement. Metastatic tumor
burden in the lung (tumor area/total organ area) was determined quantitatively by manual
outlining and analysis by ImageJ software (NIH) as described previously [38].

Data Analysis
Experiments analyzing CSC marker phenotype and ALDH activity were performed a
minimum of three times. In vitro experiments with FACS-isolated cells were performed twice
(after four separate cell sorts) with at least three biological replicates included within each
experiment. In vivo studies were carried out using multiple animals (n=4 per cell population)
and using cells obtained from 5 separate cell sorts. In all cases, quantitative data was
compiled from all experiments. Statistical analysis was performed using GraphPad Prism 4.0
software© (San Diego, CA) using either t-test (for comparison between two groups) or
ANOVA with Tukey post test (for comparison between more than two groups). Differences
between means were determined using the Student's t-test when groups passed both a
normality test and an equal variance test. When this was not the case, the Mann-Whitney
Rank-Sum test was used. Linear Regression was used to assess the relationship between
primary tumor size and metastatic burden in the lung. Unless otherwise noted, data is
presented as the mean ± SEM. P values of ≤0.05 were regarded as being statistically
significant.

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RESULTS
Human breast cancer cell lines contain sub-populations of cells expressing prospective
CSC markers
Flow cytometry was used to assess the expression of prospective CSC markers,
including CD44/CD24 and CD133. Interestingly, the most aggressive cell line (MDA-MB-
435) had the highest proportion of CD44+CD24- cells (96.6 ± 1.2%) (Fig 1A), while the least
aggressive cell line (MCF-7) had the lowest proportion of CD44+CD24- cells (0.05 ± 0.01%)
(Fig. 1D). The moderately metastatic MDA-MB-231 cell line contained 79.5 ± 2.7%
CD44+CD24- cells (Fig. 1B). In contrast, the weakly metastatic MDA-MB-468 cell line
contained very few cells (0.09 ± 0.05%) with a CD44+CD24- phenotype, however the
majority of cells (92.9 ± 4.4%) did express both CD44 and CD24 (CD44+CD24+) (Fig. 1C).
Quantitative analysis demonstrated that the MDA-MB-435 cell line contained significantly
more cells with a CD44+CD24- phenotype than any of the other cell lines (p<0.01), and that
the MDA-MB-231 cell line had significantly more cells with a CD44+CD24- phenotype than
either the MDA-MB-468 or MCF-7 cell lines (p<0.01) (Supplemental Figure 1).
MDA-MB-435, MDA-MB-231, and MCF-7 cell lines did not demonstrate expression
of CD133 (Fig. 1A,B,D). However, MDA-MB-468 cells did consistently express CD133 on
their surface, at a level significantly higher than the other cell lines (Fig. 1C).

Human breast cancer cell lines contain sub-populations of cells with enhanced ALDH
activity
A complementary strategy for identifying cells with a stem/progenitor phenotype
involves measurement of ALDH activity [24]. Human umbilical cord blood was assayed for
ALDH activity in order to provide a control for setting up the flow cytometry protocol and
for confirming that the assay was working appropriately (Supplemental Fig. 2). The MDA-
MB-435 cell line did not demonstrate any significant increase in ALDH activity (Fig. 2A).
Interestingly, both the MDA-MB-231 and MDA-MB-468 cell lines showed two definitive

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subpopulations of cells: one that had ALDH activity at the level of the DEAB control, and
one that had increased ALDH activity (Fig. 2B,C). The MCF-7 cell line did not demonstrate
any increased ALDH activity (Fig. 2D).

Strategy for isolation of stem-like human breast cancer cells
MDA-MB-231 and MDA-MB-468 cell lines were chosen for further characterization
and functional analysis because they had the most distinct subpopulations of cells with stem-
like characteristics (CSC marker expression, ALDH activity). Subsets of cells were isolated
from MDA-MB-231 and MDA-MB-468 cell lines by FACS, using ALDH activity as the
primary sort criteria and CD44+CD24- (MDA-MB-231 cells) or CD44+CD133+ (MDA-MB-
468 cells) phenotype as the secondary sort criteria. The resulting cell subsets were designated
as ALDHhiCD44+CD24- (MDA-MB-231) or ALDHhiCD44+CD133+ (MDA-MB-468) (“stem-
like”) and ALDHlowCD44low/-CD24+ (MDA-MB-231) or ALDHlowCD44low/-CD133- (MDA-
MB-468) (non “stem-like”). Examples of the cell sorting strategy are represented in Fig. 3
(MDA-MB-231) and Fig. 4 (MDA-MB-468). From this point forward in the study, the non-
“stem-like” cells from both cell lines will be collectively referred to as respective
ALDHlowCD44low/- subsets.
ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ breast cancer cells demonstrate
enhanced cell growth and colony formation in vitro
Differences in cell growth characteristics in vitro between sorted subpopulations for
both cell lines were assessed (Fig. 5). ALDHhiCD44+CD24- (MDA-MB-231) and
ALDHhiCD44+CD133+ (MDA-MB-468) cells demonstrated increased growth in normal
culture relative to respective ALDHlowCD44low/- cell subsets. Lag times (time to reach
exponential growth phase) were also observed to be shorter for ALDHhiCD44+CD24- (MDA-
MB-231=1 day) and ALDHhiCD44+CD133+ (MDA-MB-468=2 days) cells versus respective
ALDHlowCD44low/- cell subsets (7 days for both cell lines) (Fig. 5A). Differences in colony-

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forming ability and anchorage-independent growth between sorted subpopulations was
assessed using a soft agar assay (Fig. 5B,C). Both ALDHhiCD44+CD24- (MDA-MB-231) and
ALDHhiCD44+CD133+ (MDA-MB-468) cell subsets formed significantly more colonies
(p<0.001) (Fig. 5B) and larger colonies (p<0.05) (Fig. 5C) than respective ALDHlowCD44low/-
cell subsets.

ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ breast cancer cells demonstrate
enhanced adhesion, migration, and invasion in vitro
In vitro assays were used to compare sorted subpopulations from the perspective of
differences in cell adhesion, migration, and invasion (Fig. 6). ALDHhiCD44+CD24- (MDA-
MB-231) and ALDHhiCD44+CD133+ (MDA-MB-468) cells were observed to be significantly
more adherent to fibronectin (MDA-MB-231) or vitronectin (MDA-MB-468) (Fig. 6A),
significantly more migratory towards serum (Fig. 6B), and significantly more invasive
through Matrigel (Fig. 6C) than respective ALDHlowCD44low/- cell subsets (p<0.001).

ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ breast cancer cells demonstrate
enhanced tumorigenicity and metastasis in vivo
Standard experimental or spontaneous metastasis assays were used to compare the
ability of sorted subpopulations to establish themselves and grow in vivo following cell
injection into the tail vein (Fig. 7) or mammary fat pad (Fig. 8) of NOD/SCID-IL2Rγ null
mice. Following tail vein injection, metastatic tumor burden in the lung (% of lung occupied
by tumor) was significantly higher in mice injected with ALDHhiCD44+CD24- (MDA-MB-
231) or ALDHhiCD44+CD133+ (MDA-MB-468) cells versus respective ALDHlowCD44low/-
cell subsets (Fig. 7AB) (p<0.05). Analysis of incidence of metastases (% of mice
demonstrating metastatic growth) in the lung and extrapulmonary organs revealed that,
although all subpopulations of cells were able to establish themselves in the lung (Fig. 7C),
only ALDHhiCD44+CD24- (MDA-MB-231) and ALDHhiCD44+CD133+ (MDA-MB-468)

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cells were able to maintain that growth into larger metastases (Fig. 7A,B). Furthermore,
metastases in extrapulmonary organs such as the pancreas, liver, spleen, and/or kidney were
only observed in mice injected with ALDHhiCD44+CD24-
(MDA-MB-231) or
ALDHhiCD44+CD133+
(MDA-MB-468) cells, and not in mice injected with
ALDHlowCD44low/- cell subsets (Fig. 7C).
In order to compare tumorigenicity and metastatic ability in a more clinically relevant
model system, we next used a spontaneous model of metastasis involving orthotopic injection
of breast cancer cells into the mammary fat pad. We observed that ALDHhiCD44+CD24-
(MDA-MB-231) and ALDHhiCD44+CD133+ (MDA-MB-468) cells demonstrated enhanced
primary tumor growth relative to respective ALDHlowCD44low/- cell subsets (Fig. 8A).
Spontaneous metastatic tumor burden in the lung was significantly higher in mice injected
with ALDHhiCD44+CD24- (MDA-MB-231) and ALDHhiCD44+CD133+ (MDA-MB-468)
cells versus respective ALDHlowCD44low/- cell subsets (Fig. 8B) (p<0.05). Interestingly, we
did not observe a significant correlation between primary tumor size and metastatic burden in
the lungs of individual mice, either in the “stem-like” populations (ALDHhiCD44+CD24- and
ALDHhiCD44+CD133+) (R2=0.365) or in the respective ALDHlowCD44low/- populations
(R2=0.530). Similar to the observations following tail vein injection, although all
subpopulations of cells were able to establish themselves in the lung (Fig. 8C), only
ALDHhiCD44+CD24- (MDA-MB-231) and ALDHhiCD44+CD133+ (MDA-MB-468) had an
enhanced capacity to maintain that growth into larger metastases (Fig. 8B), and to
spontaneously metastasize to extrapulmonary organs such as the pancreas and spleen (Fig.
8C).