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CCL2 mediates crosstalk between cancer cells and stromal
fibroblasts that regulates breast cancer stem cells
Akihiro Tsuyada1,7,8, Amy Chow1, Jun Wu2, George Somlo3, Peiguo Chu4, Sofia Loera4,
Thehang Luu3, Arthur Xuejun Li5, Xiwei Wu6, Wei Ye5, Shiuan Chen1, Weiying Zhou1, Yang
Yu1,9, Yuan-Zhong Wang1, Xiubao Ren9, Hui Li9, Peggy Scherle10, Yukio Kuroki7, and
Shizhen Emily Wang1
1Division of Tumor Cell Biology, City of Hope Beckman Research Institute and Medical Center,
Duarte, California
2Division of Comparative Medicine, City of Hope Beckman Research Institute and Medical
Center, Duarte, California
3Department of Medical Oncology, City of Hope Beckman Research Institute and Medical Center,
Duarte, California
4Department of Pathology, City of Hope Beckman Research Institute and Medical Center, Duarte,
California
5Department of Information Science, City of Hope Beckman Research Institute and Medical
Center, Duarte, California
6Department of Bioinformatics Core Facility, City of Hope Beckman Research Institute and
Medical Center, Duarte, California
7Department of Molecular Biology, Sapporo Medical University, Sapporo, Japan
8Graduate School of Medicine, Sapporo Medical University, Sapporo, Japan
9Department of Immunology & Biotherapy, Tianjin Cancer Hospital, Tianjin, China
10Incyte Corporation, Wilmington, Delaware
Abstract
Cancer stem cells (CSCs) play critical roles in cancer initiation, progression, and therapeutic
refractoriness. Although many studies have focused on the genes and pathways involved in
stemness, characterization of the factors in the tumor microenvironment that regulate CSCs is
lacking. In this study, we investigated the effects of stromal fibroblasts on breast cancer (BC) stem
cells. We found that compared to normal fibroblasts, primary cancer-associated fibroblasts (CAFs)
and fibroblasts activated by co-cultured BC cells produce higher levels of chemokine (C-C motif)
ligand 2 (CCL2), which stimulates the stem cell-specific, sphere-forming phenotype in BC cells
and CSC self-renewal. Increased CCL2 expression in activated fibroblasts required STAT3
activation by diverse BC-secreted cytokines, and in turn, induced NOTCH1 expression and the
CSC features in BC cells, constituting a “cancer-stroma-cancer” signaling circuit. In a xenograft
model of paired fibroblasts and BC tumor cells, loss of CCL2 significantly inhibited tumorigenesis
and NOTCH1 expression. In addition, upregulation of both NOTCH1 and CCL2 was associated
with poor differentiation in primary BCs, further supporting the observation that NOTCH1 is
Corresponding author: S. Emily Wang (ewang@coh.org), Division of Tumor Cell Biology, Beckman Research Institute of City of
Hope, 1500 E Duarte Road, KCRB Room 2007; Duarte, CA 91010, U.S.A., TEL: 1-626-2564673 x63118; FAX: 1-626-3018972.
Conflict of interest: The authors have declared that no conflict of interest exists.
NIH Public Access
Author Manuscript
Cancer Res
. Author manuscript; available in PMC 2013 June 01.
Published in final edited form as:
Cancer Res
. 2012 June 1; 72(11): 2768–2779. doi:10.1158/0008-5472.CAN-11-3567.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
regulated by CCL2. Our findings therefore suggest that CCL2 represents a potential therapeutic
target that can block the cancer-host communication that prompts CSC-mediated disease
progression.
Keywords
breast cancer; cancer stem cells; chemokine (C-C motif) ligand 2; tumor microenvironment;
stromal fibroblasts
Introduction
Recent studies indicate that a subset of cancer cells possessing stem cell properties, referred
to as cancer-initiating or cancer stem cells (CSCs), play crucial roles in tumor initiation,
progression and therapeutic refractoriness (1–2). Similar to embryonic and somatic stem
cells, the self-renewal and differentiation of CSCs are simultaneously regulated by intrinsic
(cancer cell-endowed) and extrinsic (microenvironmental) factors. Despite the increasing
number of studies on genes and pathways involved in cancer “stemness”, factors in the
tumor microenvironment that regulate CSCs, and how cancer cells, in turn, modify the niche
by influencing their neighboring cells remain largely uncharacterized. In this study, we
focus on the regulation of CSCs by stromal fibroblasts, an important cellular component of
the tumor-hosting niche in many human cancers, especially breast cancer (BC). Fibroblasts
release a variety of growth factors, chemokines, and components of the extracellular matrix
into the microenvironment and influence the differentiation and homeostasis of adjacent
epithelia (3). Reconstitution of human-specific mammary glands in cleared mouse mammary
fat pads using stem-cell-enriched human mammary epithelial cell organoids requires co-
injection of human mammary fibroblasts (4), suggesting a critical role for fibroblasts in
regulating stem cell functions. Fibroblasts interplay with cancer cells at all stages of cancer
progression through complex paracrine mechanisms. Cancer-associated fibroblasts (CAFs)
can promote cancer progression by modulating multiple components in the cancer niche to
build a permissive and supportive microenvironment for tumor growth and invasion.
In the current study, we observed robust induction of the stem-cell-like mammosphere-
forming phenotype in BC cells co-cultured with CAFs. The CSC-stimulating effect was
attributed to the increased secretion of CCL2 (monocyte chemotactic protein-1; MCP-1) by
CAFs when compared to normal cell-associated fibroblasts (NAFs). CCL2 signals through
the G protein-coupled receptor chemokine (C-C motif) receptors CCR2 and CCR4, and is a
potent chemoattractant for monocytes and other immune cells to areas of inflammation (5).
Both monocytes and non-monocytic cells, such as fibroblasts and endothelial cells, secrete
CCL2 in response to cytokine stimulation (6–7). Cancer cells also frequently overexpress
CCL2 in order to modify their local environment. Here, we further found that CCL2 induced
the self-renewal of CSCs by inducing NOTCH1 expression at both RNA and protein levels.
NOTCH signaling has been recognized as a key regulator in normal and malignant stem
cells from various tissues, including the breast (8–9). Upon ligand binding, NOTCH
receptors are activated by sequential cleavages involving members of the ADAM (a
disintegrin and metalloprotease domain) protease family (α-secretases) and γ-secretase.
These cleavage events result in translocation of the NOTCH intracellular domain (NICD) to
the nucleus, where it acts on downstream targets (10). Our identification of NOTCH-
mediated activation by tumor environmental factor CCL2 therefore presents a unique mode
of niche-conferred regulation of CSCs. These findings further emphasize the importance of
simultaneously targeting cancer cells and events within the tumor microenvironment, such
as production of CCL2 by stromal cells, in future anti-cancer therapies that also incorporate
the latest stem cell research.
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Materials and Methods
Tissue sources and cell purification
Human BC tissues were obtained from consented patients at City of Hope Medical Center
(Duarte, CA) under approved institutional review board protocols. Tissues were
mechanically and enzymatically dissociated, and epithelial tumor cells and fibroblasts
(CAFs) were isolated as previously described (11). Briefly, BC tissue was mechanically
minced into small pieces, placed in digestion solution containing collagenase III
(Worthington Biochemical Corporation; Lakewood, NJ) and DNase I (AppliChem; St.
Louis, MO), and incubated at 37°C for 2–3 h with occasional pipetting. The cells were
separated by differential centrifugation at 90 ×
g
for 2 min. The epithelial (tumor) cells in the
pellet were cultured in Iscove’s Modified Dulbecco’s Media (Invitrogen; Grand Island, NY)
containing 0.7 mM L-glutamine (Mediatech/Cellgro; Manassas, VA), 5 μg/ml insulin
(Lonza; Allendale, NJ), 5 μg/ml transferrin (Lonza), 5 ng/ml selenium (Lonza), and 20%
fetal bovine serum (FBS; PAA Laboratories; Dartmouth, MA). The supernatant containing
fibroblasts were centrifuged at 800 ×
g
for 10 min, resuspended and cultured in Dulbecco’s
Modified Eagle Medium (Mediatech/Cellgro) containing 10% FBS on a non-treated dish.
Purity of primary tumor cells and CAFs were confirmed by expression of Epithelial Specific
Antigen (ESA) and Vimentin, respectively, in flow cytometry and immunofluorescence
assays (Fig. S1). CAF265922 (primary CAFs) and XP265922 (primary tumor cells) were
isolated from a primary triple-negative BC that was resistant to the chemotherapy regimen
containing cisplatin, 5-fluorouracil, and docetaxel. CAF3 were isolated from a primary
HER2-positive BC for which the primary tumor cells were not available. Normal human
mammary fibroblasts (NAF2) were purchased from ScienCell (Carlsbad, CA). For
immunohistochemistry in primary breast tumors, pretreatment core biopsies or surgical
specimens were obtained from patients with HER2-positive (31 cases) or triple-negative
(ER−/PR−/HER2−; 20 cases) BC. Specimens were collected and processed for formalin
fixation and paraffin embedding in a time frame that would preserve the integrity of protein
epitopes.
Cell lines, plasmids and viruses
Human BC cell lines BT474, MDA-MB-361 (MDA361) and MCF7, and the non-cancerous
mammary epithelial cell line MCF10A were obtained from American Type Culture
Collection (Manassas, VA) and cultured in the recommended media in a humidified 5%
CO2 incubator at 37°C. Recombinant human CCL2 was purchased from R&D Systems
(Minneapolis, MN). The STAT3 inhibitor Stattic, p38 MAPK inhibitor SB202190, and γ-
secretase inhibitor DAPT were purchased from Sigma-Aldrich (St. Louis, MO). The α-
secretase inhibitor INCB3619 was provided by Incyte Corporation (Wilmington, DE). For
conditional knockdown of CCL2, the shRNA targeting the CCL2 mRNA
(TRCN0000006283) was constructed into the pTIG (pHIV7-TetR-IRES-GFP) lentiviral
vector (12) (kindly provided by Dr. Rossi) downstream of a Dox inducible U6 promoter, as
described elsewhere (13). GFP-labeled CAF265922 were generated using pBABE-GFP
retroviral vector. Production of viruses, as well as infection and selection of CAFs were
carried out as previously described (13).
Mammosphere formation assay
Please see Supplemental Materials for procedures.
RNA extraction, reverse transcription (RT) and real-time quantitative PCR (qPCR)
Please see Supplemental Materials for procedures.
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Cytokine antibody array and Western blot analyses
Please see Supplemental Materials for procedures.
Cell transfection, reporter assays, and RNAi studies
Please see Supplemental Materials for procedures.
Flow cytometry and cell sorting
Single-cell suspensions prepared from tumors or cell culture were stained with APC-
conjugated human ESA antibody (Catalog #347200; BD Biosciences, Franklin Lakes, NJ) or
analyzed by ALDEFLUOR assay kit (Catalog #01700; Stemcell Technologies, Vancouver,
BC, Canada) following the manufacturer’s protocol. Flow cytometry assays were performed
using a CyAn ADP cytometer (Dako; Carpinteria, CA) and analyzed with FlowJo software
(TreeStar; Ashland, OR). Electronic cell sorting based on intensity of GFP, APC, PKH67 or
ALDEFLUOR was done on a FACSAriaIII cell sorter (BD Biosciences).
Xenografts
All animal experiments were approved by the institutional animal care and use committee at
City of Hope. Please see Supplemental Materials for procedures.
Immunohistochemistry (IHC)
IHC staining of formaldehyde-fixed, paraffin-embedded primary or xenograft tumor tissues
was performed as previously reported (14) using the following antibodies and dilutions:
human CCL2 (Catalog #ab9669; Abcam; Cambridge, MA), 1:75 dilution; NOTCH1 (for
primary BC: Catalog #1935-1; Epitomics; Burlingame, CA; for xenograft tumors: Catalog
#3608; Cell Signaling; Danvers, MA), 1:40 dilution; and SMA (Catalog #ab5694; Abcam),
1:100 dilution. For CCL2 and SMA, cytoplasmic staining was evaluated and for NOTCH1
nuclear staining was evaluated. Stained slides were scored according to intensity of staining
(-: 0; +: 1; ++: 2; and +++: 3) and percentage of tumor cells staining positive for each
antigen (0%: 0; 1~30%: 1; 31~70%: 2; and >70%: 3). The score for the intensity of
immunostaining was multiplied by the score for the percentage of cells staining positive to
obtain a final score, which was used in the statistical correlation analysis of CCL2 and
NOTCH1.
Statistical analyses
All quantitative data are presented as mean ± standard deviation (SD). All results were
confirmed in at least three independent experiments, and data from one representative
experiment was shown. The statistical analysis was performed using a SPSS 13.0 software
package. Student
t
test or ANOVA test was used for comparison of quantitative data. Values
of
P
< 0.05 were considered significant. The differential expression of CCL2, CCR2 and
NOTCH family of receptors and ligands between Grade 3 and Grade 1 BCs were analyzed
using a logistic regression model after which an odds ratio (OR) was reported. For example,
an OR of 13.72 (CCL2) means that tumors with each unit increase in CCL2 expression are
13.72 times more likely to be Grade 3, rather than Grade 1 (95% CI = 2.66–70.89). The
linear dependence between CCL2 and NOTCH1 expression was evaluated by Pearson (for
the microarray dataset) or Spearman correlation coefficient (for the IHC dataset). The
microarray gene expression data and associated clinical data were extracted from a
published 295 BC dataset (15–16).
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Results
CAF-derived CCL2 induces the stem-cell-like mammosphere-forming phenotype in BC
cells
The mammosphere assay has been used to functionally characterize and enrich normal and
malignant stem cells from the breast, relying on the unique feature of stem cells to escape
anoikis and grow into spheres in anchorage-independent conditions (17–18). Using this
approach, we examined the sphere-forming efficiency (SFE) in BT474 BC cells in the
presence or absence of co-cultured primary human mammary NAFs or CAFs. Compared to
the BC cells that were cultured alone, co-culture with CAFs, but not NAFs, significantly
increased mammosphere formation in BC cells (Fig. 1A). When NAFs and CAFs were first
“activated”
in vitro
by co-culturing with BT474 cells before being transferred to the
mammosphere co-culture, both activated NAFs and the CAFs that were continuously
activated by BC cells
in vitro
were able to induce BT474 mammosphere formation to a
greater extent than their pre-activation counterparts (Fig. 1A). The mammosphere-inducing
effect was also observed with the conditioned medium (CM) harvested from CAFs, but not
NAFs. CM from CAFs that were further activated by BT474 or MDA361 BC cells exhibited
an enhanced capacity for inducing mammosphere formation.
In vitro
activation of NAFs
also resulted in mammosphere-inducing activity in the CM, which was more significant in
NAFs that had been co-cultured with BT474 cells for a prolonged 10-day period (Fig. 1B).
These results indicate that the continuous activation of stromal fibroblasts by BC cells leads
to secretion of fibroblast-derived soluble factors that can induce the CSC-like phenotype.
To identify these soluble factors, we performed a cytokine array assay using the CM of
untreated and BT474-treated CAFs. Significant induction of CCL2 was observed following
in vitro
activation of CAFs (Fig. 1C). At the RNA level, expression of CCL2 was induced in
CAFs 7 to 9-fold in response to activation by various BC cells, with BC-activated CAFs
producing the highest amounts of CCL2 among various cell types, including NAFs and BC
cells. Treatment of CAFs with the non-cancerous MCF10A human mammary epithelial
cells, as well as short-term (3-day) treatments of NAFs with BC cells only modestly induced
CCL2 expression (Fig. 1D). To examine the direct effect of CCL2 on CSCs, we added
increasing amounts of recombinant CCL2 to various BC cells, and observed dose-dependent
formation of mammospheres in BT474 and MDA361, but not MCF7 cells (Fig. 1E). Using a
neutralizing antibody for CCL2, we further showed that depletion of functional CCL2 from
the CM of activated CAFs abolished the CSC-inducing activity (Fig. 1F). Thus, we
concluded that BC-activated CAFs regulate CSCs through expression and secretion of
higher levels of CCL2.
CCL2 induces the self-renewing expansion of CSCs
The sphere-initiating CSCs are maintained in the primary mammospheres through self-
renewal, and are able to give rise to secondary mammospheres when cells from the primary
spheres are dissociated and allowed to grow in anchorage-independent conditions (17). We
therefore examined the effect of CCL2 on CSC self-renewal by secondary mammosphere
culture. Interestingly, compared to the control spheres that had not been treated with CCL2,
the first-passage spheres that had been treated with CCL2 contained higher numbers of
CSCs capable of initiating secondary spheres, even in the absence of continuous CCL2
treatment (Fig. 2A). Based on this result, we hypothesized that CCL2 either promoted the
self-renewal (to cause a self-renewing expansion) of existing CSCs, or, alternatively,
promoted the conversion of non-CSCs to CSCs. These possibilities were examined using a
reported approach involving the labeling of an initial cell population with PKH fluorescent
dye and tracking the serial dilution of fluorescence in single-cell-formed spheres during the
cell division events in mammosphere formation (19). Because the PKH dye binds to cell
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membranes and segregates in daughter cells after each cell division, the PKH fluorescence
intensity of each cell in a sphere reflects its proliferation history. The PKHhigh cells,
therefore, represent the stem cell population that has undergone a limited number of
divisions during sphere formation, in contrast to the non-stem, PKHlow cells that are highly
proliferative (19). Using PKH67-labeled BT474 cells, we generated primary mammospheres
containing cells with various PKH67 intensities. In the absence of CCL2 treatment, about
50% of mammospheres carried 1 PKH67high cell per sphere, and about 33% and 13%
carried 2 and 3 PKH67high cells, respectively. This distribution was altered in CCL2-treated
spheres, where the percentage of spheres carrying a single PKH67high cell decreased to 28%,
and of those carrying 3 or more PKH67high cells significantly increased (Fig. 2B). In spheres
carrying multiple PKH67high cells, the PKH67high cells also exhibited slightly dimmer
fluorescence comparing to those in spheres carrying single PKH67high cells (Fig. 2B and
S2), likely indicating one or two more rounds of division (and PKH67 dilution) of the
PKH67high cells before entering the stem-cell-like, slow-proliferating or quiescent state in
the multi-PKH67high-cell spheres. The overall population of PKH67high cells in dissociated
sphere cells was also ~2-fold higher in CCL2-treated spheres than untreated spheres, as
determined by flow cytometric analysis (Fig. 2C). When the PKH67high and PKH67low
sphere cells were purified by fluorescence-activated cell sorting, the PKH67high primary
sphere cells exhibited the highest SFE in the secondary mammosphere formation assay
when compared to the PKH67low cells (Fig. 2D), consistent with their CSC property. CCL2
treatment did not alter the SFE of PKH67low and PKH67high cells in the secondary sphere
formation (Fig. 2D), indicating that CCL2 does not cause conversion of non-CSCs
(PKH67low) to CSCs (sphere-initiating). Therefore, we conclude that CCL2 regulates CSCs
by inducing their self-renewing expansion. Interestingly, a similar effect has been previously
described for NOTCH activation on stem cells (8). The role of NOTCH pathway in
mediating CSC regulation by CCL2 was further investigated in Fig. 4.
Paracrine signaling of cancer-secreted cytokines induces CCL2 production in fibroblasts
via STAT3 activation
We then set out to identify the mechanism underlying increased CCL2 production in BC-
activated CAFs. STAT3 has been recently reported to bind to and activate the promoter of
CCL2
(20). To determine if BC cells induce CCL2 expression in fibroblasts through STAT3
activation, we first examined the effects of BC-derived CM and a STAT3 inhibitor on a
previously reported
CCL2
promoter reporter (21) and on CCL2 expression in CAFs freshly
isolated from a triple-negative (ER−/PR−/HER2−) primary BC (CAF265922). CM from BC
cells, including BT474, MDA361 and the primary BC cells isolated from the same BC
specimen (XP265922), but not from MCF10A cells, markedly induced luciferase expression
driven by the
CCL2
promoter (Fig. 3A), as well as endogenous expression of CCL2 (Fig.
3B). These effects were abrogated by addition of the STAT3 inhibitor (Fig. 3A and 3B),
indicating STAT3 involvement in mediating the induction of fibroblast-derived CCL2
expression. Similar results were also obtained using CAFs from a different primary BC (Fig.
S3). Indeed, high levels of STAT3 phosphorylation were observed in CAFs as early as 30
min following treatment with BC-derived, but not MCF10A-derived, CM (Fig. 3C). The
induction of CCL2 expression was sustained over a time course of 5 days in CAFs co-
cultured with BC cells, and was abolished by inhibition of STAT3 (Fig. S4). Although CM
from BT474, MDA361 and primary BC cells all induced STAT3 phosphorylation and CCL2
expression in CAFs, different cytokines were detected in the CM of BT474 and MDA361
(Fig. 3D). In either case, multiple cytokines with reported STAT3-activating effect were
detected, suggesting that the STAT3 activation observed in BC-treated CAFs was likely a
combined effect of various BC-secreted cytokines. Thus, BC cells derived from different
origins and secreting different cytokines activate the STAT3 core pathway in fibroblasts of
the tumor microenvironment, leading to STAT3-mediated promoter activation of
CCL2
.
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CCL2 induces CSCs by activating NOTCH signaling
To explore the molecular basis for CCL2-mediated regulation of CSCs, we surveyed the
CCL2 responsiveness of genes involved in NOTCH, Wnt/β-catenin and Hedgehog
pathways, which are known to regulate stem cells (8, 22–23). We found that expression of
NOTCH1 and a target gene of NOTCH signaling, HES1, were induced by CCL2 in various
BC cells, and that HES1 expression occurred subsequent to NOTCH1 expression (Fig. 4A
and 4B). Interestingly, the NOTCH-activating effect of CCL2 was only subtle in MCF7
cells, which also failed to respond to CCL2-induced sphere formation (Fig. 1E). This may be
related to the lower expression level of CCR2 in MCF7 compared to other cells (Fig. S5),
making MCF7 less sensitive to CCL2-triggered effects. Transfected HES1 and HEY1
reporters were significantly activated by CCL2 in primary BC cells and BT474 cells, and
these effects were abolished by inhibitors of the NOTCH-activating α- and γ-secretases
(Fig. 4C and S6). Activation of NOTCH signaling by CCL2 was thus attributed to increased
NOTCH1 expression, as CCL2 showed no effect on the activities of α- and γ-secretases as
assessed by
in vitro
substrate cleavage assays (Fig. S7). Upon CCL2 treatment, a rapid and
dramatic induction of p38 MAPK phosphorylation was observed, followed by the induction
of NOTCH1 and NICD1 (cleaved NOTCH1) proteins at a later time point (Fig. 4D). Both
p38 phosphorylation and NOTCH1/NICD1 induction were abolished by an inhibitor of p38
MAPK (Fig. 4D and 4E). Using a 6-kb
NOTCH1
promoter reporter, we observed a ~4-fold
induction of
NOTCH1
promoter activity by CCL2 in primary BC cells, and this effect was
also completely suppressed by the p38 MAPK inhibitor, but only partially suppressed by
inhibitors of PI3K and MEK1/2 MAPK (Fig. 4F). This suggests that p38 MAPK plays an
essential role in activating
NOTCH1
promoter, whereas PI3K and MEK1/2 MAPK may
play an accessory role. Induction of p38 MAPK phosphorylation and NOTCH1 proteins
(full-length and cleaved) was also observed in XP265922 BC cells treated with CM from
CAF265922 previously activated by co-culturing with XP265922. This effect was abolished
by treatment with a p38 MAPK inhibitor or CCL2 neutralizing antibody, and also by using
CM from CAF265922 previously co-cultured with XP265922 cells but in the presence of a
STAT3 inhibitor (Fig. 4G). We therefore concluded that in BC cells, CCL2 induces
NOTCH1 expression and its downstream signaling mainly through p38-dependent activation
of the
NOTCH1
promoter. In BT474 and MDA361 BC cells, CCL2-induced mammosphere
formation was efficiently blocked by inhibitors of the α- and γ-secretases and p38 MAPK,
and by RNA interference (RNAi) of NOTCH1 (Fig. 4H), indicating that activation of
NOTCH1 mediates the effect of CCL2 on CSCs.
Fibroblast-specific knockdown of CCL2 or CCL2 depletion by a neutralizing antibody
inhibits in vivo tumorigenesis
To obtain
in vivo
evidence of the CSC-regulating function of fibroblast-derived CCL2, we
established an orthotopic xenograft model of co-transplanted primary CAFs and BC cells.
CAF265922 were stably transduced to express doxycycline (Dox)-inducible short hairpin
RNA (shRNA) of CCL2, which suppressed CCL2 expression
in vitro
by ~60% in the
presence of Dox (Fig. 5A). The modified CAFs were then co-transplanted with BC cells
from the same primary tumor (XP265922) into the mammary fat pads of female NOD/
SCID/IL2Rγ-null (NSG) mice. Mammary tumor formation was monitored in transplanted
mice with or without Dox treatment. Fibroblast-specific knockdown of CCL2 in the Dox+
group resulted in significantly delayed tumor formation and reduced tumor volume,
compared to the Dox− control group (Fig. 5B and 5C). Xenograft tumors from both groups
were harvested, and the fibroblast (labeled by lentiviral-encoded GFP) and tumor cell
(positive for human ESA) components were separated for gene expression analyses. In Dox+
tumors, both fibroblast-derived CCL2 expression and tumor cell-derived NOTCH1
expression were significantly lower than their counterparts in Dox− tumors (Fig. 5D).
Immunohistochemistry (IHC) staining also indicated lower levels of CCL2 and NOTCH1
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proteins in the Dox+ tumors, compared to the Dox− tumors (Fig. 5E). We further analyzed
the CSC population within all tumor cells in each xenograft tumor using ALDEFLUOR flow
cytometry analysis. The ALDEFLUORbright CSC populations in Dox+ tumors were
significantly smaller than those in Dox− tumors (Fig. 5F; ~0.45% vs. ~1.3%), indicating a
decrease in the number of CSCs. Thus, fibroblast-derived CCL2 secretion appears to play an
important role in tumorigenesis and in regulating NOTCH1 expression and the CSC
population in BC cells. Consistent with these results, the CCL2 neutralizing antibody, but
not a control IgG or PBS, significantly suppressed tumorigenesis and decreased NOTCH1
expression in tumor cells when XP265922 and GFP-labeled CAF265922 were co-
transplanted into NSG mice (Fig. 5G, 5H and S8). Thus, based on the studies that have been
described to this point, we propose a model of CSC generation that incorporates a crosstalk
circuit involving STAT3, CCL2 and NOTCH pathways (Figure 5I).
Expression of CCL2 and NOTCH1 are correlated in primary BCs and associated with poor
differentiation of tumor cells
To extend our findings to a larger number of primary BCs, odds ratio (OR) and 95%
confidence internal (CI) were calculated using unconditional logistic regression to determine
if the microarray-determined expression levels of CCL2, CCR2 and NOTCH family of
receptors and ligands in a previously reported BC dataset (15–16) were associated with
tumor grade (Table 1). Patients with Grade 3 (poorly-differentiated) and Grade 1 (well-
differentiated) tumors were included in the analysis. We didn’t include the Grade 2 tumors
because they fell between Grade 1 and Grade 3 and exhibited highly diverse levels of
differentiation, compared to Grades 1 and 3. Patients with poor differentiation (Grade 3)
were significantly associated with higher levels of CCL2 (OR = 13.72, 95% CI = 2.66–
70.89), NOTCH1 (OR = 9.56, 95% CI = 1.54–59.27) and delta-like 3 (OR = 20.93, 95% CI
= 1.53–289.94), as well as lower level of jagged 1 (OR = 0.14, 95% CI = 0.03–0.57).
In addition, correlation coefficients were calculated between microarray-determined CCL2
and NOTCH1 expression and stratified by tumor grade and stage (Table 2, top part). A
significant linear correlation was observed between CCL2 and NOTCH1 expression among
all BCs (R = 0.18,
p
< 0.01). This association was especially pronounced in Grade 3 tumors
(R = 0.19,
p
= 0.03), and in Stage 1 (R = 0.33,
p
< 0.01) and Stage 2a disease (R = 0.19,
p
=
0.02). We further evaluated the expression of CCL2 and NOTCH1 at the protein level by
IHC in 51 HER2+ or triple-negative primary BCs. The correlation between two proteins was
approaching statistical significance (R = 0.26,
p
= 0.06) among all tumors, and was indeed
significant within HER2+ tumors (R = 0.40,
p
= 0.03), but not triple-negative BCs (Table 2,
bottom part). Although the previously reported association of CCL2 with poor clinical
outcome (24) was not detected in the current cohort, our results nevertheless support a
relationship between CCL2 and NOTCH1 with effects on CSCs in primary BCs.
Discussion
Fibroblasts are altered by cancer cells through non-genetic modifications, and in turn, effect
direct changes on cancer cells or indirect changes on the tumor microenvironment to
facilitate cancer growth and invasion. The resulting co-evolution of cancer and the hosting
niche critically influences disease progression. Our attention to the influence of fibroblasts
on CSCs, the “seeds” of cancer, showed that, upon cancer-mediated activation, human
mammary fibroblasts secreted CCL2 to induce CSC generation through activation of
NOTCH signaling. In the cancer niche, CCL2 may be produced and secreted into the
extracellular environment by almost all cell types, including cancer cells, stromal
fibroblasts, tumor-infiltrated monocytes, and endothelial cells. Nevertheless, our study
indicated that: 1) CCL2 expression was 4 to 9-fold higher in activated CAFs than in BC
cells (Fig. 1D); 2) fibroblast-specific knockdown of CCL2 significantly suppressed
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tumorigenesis and the CSC population in xenograft tumors (Fig. 5A-F); and 3) CCL2 is
detected by IHC in both tumor cells and stromal fibroblasts in primary BC (Fig. S9). These
data strongly suggest that CAFs are an important source of CCL2 in the cancer niche and a
major environmental regulator of CSCs.
The CAF populations in tumor-associated stroma are known to include both fibroblasts and
myofibroblasts. Myofibroblasts are endowed with the ability to promote tumor growth and
associated with higher-grade malignancy and poor prognosis in cancer patients (25–26).
These cells express α-smooth muscle actin (SMA) to be distinguished from fibroblasts.
Kojima
et al.
recently show that through self-sustaining autocrine signaling of transforming
growth factor β (TGF-β) and stromal cell-derived factor-1 (SDF-1), fibroblasts can
transdifferentiate into myofibroblasts during tumor progression (26). The CAFs prepared
and examined in our study indeed contained a subpopulation of SMA-expressing
myofibroblasts, as indicated by immunofluorescent assay and immunohistochemistry (Fig.
S1B and S8). However, the SMA+ myofibroblasts and SMA− fibroblasts produced CCL2 at
comparable levels in the cancer niche (Fig. S8), suggesting that the increased CCL2
production and enhanced CSC-promoting capacity of the herein examined CAFs are
unlikely related to the myofibroblast phenotype, but rather a general alteration in fibroblasts
in response to cancer-derived stimulation. Our findings also support further studies on the
regulation of CSCs and their non-cancerous counterparts by other physiological and
therapeutic conditions that locally elevate CCL2 levels. Such conditions, including wound
healing, inflammation, and chemotherapy (7, 27), are becoming increasingly appreciated for
their relevance to the biology of normal and cancerous stem cells.
Our data indicate that the CCL2-producing and CSC-promoting ability of CAFs is conferred
by BC-secreted soluble factors present in the CM, such as the cytokines listed in Fig. 3D.
Although different BC cells appeared to secrete distinct sets of cytokines to induce CCL2
production in CAFs, these cytokines ultimately functioned through STAT3, and inhibition of
STAT3 completely abrogated the induction of CCL2 by BC paracrine signaling (Fig. 3).
Therefore, compared to the diverse signals released by cancer cells, the common effector
STAT3 serves as a superior target to therapeutically block cancer-induced activation of
stromal fibroblasts. STAT3 has been identified as an important effector and target in cancer
cells and tumor-infiltrated immune cells (28). Our study now identifies STAT3-mediated
fibroblast activation as a potential therapeutic target, further supporting the idea that anti-
STAT3 therapies may exert dual effects on both cancer and host cells, halting their dynamic
and mutual activation during cancer progression.
CCL2 has been implicated in breast cancer progression and metastasis (29). In primary
breast tumors, CCL2 expression is correlated with the accumulation of tumor-associated
macrophages, and is a significant indicator of early relapse (24, 30). Overexpression of
CCL2 in BC cells promotes metastasis formation in lungs and bone through increasing
macrophage infiltration and osteoclast differentiation, respectively (31). A recent report
demonstrates that CCL2 produced by both tumor and stromal cells recruits the CCR2-
expressing inflammatory monocytes to the pulmonary metastases of mammary tumors,
where monocyte-derived factors promote endothelial permeability and extravasation of
tumor cells (32). CCL2 expression is interactively regulated in the crosstalk between tumor
and niche cells. Increased expression of CCL2 is detected in the bone marrow mesenchymal
stem cells (MSCs) following stimulation by leukemia cells, resulting in enhanced cancer-
promoting capacity of MSCs (33). Co-culture with MSCs, in turn, induces CCL2 expression
in cancer cells (34). These previous studies have therefore established an important role of
CCL2 in cancer-host crosstalk through the regulation of tumor cell homing and metastasis,
angiogenesis and the immune system.
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Here we show that CCL2 induces CSCs both
in vitro
and
in vivo
through activation of
NOTCH (Fig. 4–5). NOTCH activation has been shown to promote the self-renewal of
mammary stem cells (8). An important role for NOTCH signaling in human cancers has
been long established (35), and several γ-secretase inhibitors (GSIs) are currently in early
clinical development as potential NOTCH-targeting therapeutics. It is proposed that single-
agent GSI therapy may be effective in triple-negative BCs, which are known to harbor CSC-
like characteristics (36). Here, our data further support the use of NOTCH-targeting agents
in efficiently blocking the stimulatory effect of stromal fibroblasts on CSCs. Our data also
indicate that activation of p38 MAPK is required for CCL2-induced NOTCH1 expression
(Fig. 4). The E2A-encoded transcription factors E12 and E47 have been shown to activate
NOTCH1 expression through binding to multiple E-box sites in the 6-kb
NOTCH1
promoter
region (37). Phosphorylation of E47 by p38 MAPK and by MAPK-activated protein kinase
2 (MAPKAPK2), a kinase activated by p38, has been reported (38–39). The function of p38-
mediated E47 phosphorylation in regulating NOTCH1 promoter activity is still unclear, and
may underlie the induction of NOTCH1 by CCL2, which induces potent p38 activation in
primary BC cells (Fig. 4D). In addition, the NOTCH-activating effect of CCL2 was only
subtle in the ER+/PR+/HER2−MCF7 cells (Fig. 4A and 4B), which also failed to respond to
CCL2-induced sphere formation (Fig. 1E). Whether the lower CCR2 level in MCF7 (Fig.
S5) causes their low sensitivity to CCL2 effect, and whether levels of CCL2 receptors are
associated with BC subtypes need to be further investigated. Nevertheless, IHC staining of
primary BCs indicated a significant correlation between CCL2 and NOTCH1 in HER2+
tumors (Table 2, bottom part), suggesting that at least in these tumors, as observed in the
HER2+ BT474 and MDA361 BC cells, the regulation of NOTCH signaling by CCL2 may
indeed occur
in vivo
.
In summary, our study provides a model in which paracrine signaling initiated by BC cells
induces CCL2 production by stromal fibroblasts through STAT3 activation. The fibroblast-
derived CCL2, in turn, promotes cancer progression by regulating CSCs through NOTCH
activation (Fig. 5I). The results described herein provide novel insights into understanding
how CSCs are influenced by the tumor microenvironment during the co-evolution of cancer
and the hosting niche, and identify CCL2, STAT3 and NOTCH1 as future therapeutic
targets to efficiently block the CSC-stimulating cancer–host crosstalk to overcome CSC-
mediated disease progression and treatment resistance.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
Financial support: NCI R00 CA125892 (SEW) and P30 CA033572; National Natural Science Foundation of
China Grant Number 30872986 (XR) and 81171983 (HL)
We acknowledge Drs. Leonid S. Metelitsa and Warren S. Pear for kindly providing the plasmid constructs of
CCL2
and
NOTCH1
promoter reporters. We also thank Drs. John J. Rossi and Hua Yu for providing reagents, Drs. Susan
Kane, Mei Kong, Toshifumi Tomoda, and Takahiro Maeda and members of the Division of Tumor Cell Biology for
valuable comments, as well as the Analytical Cytometry Core, Light Microscopy Digital Imaging Core,
Bioinformatics Core, Biostatistics Core, Pathology Core and Animal Facility for professional services.
Grant Support
The project described was supported by the National Cancer Institute (NCI) Grant Number R00 CA125892 (SEW)
and P30 CA033572, and by the National Natural Science Foundation of China Grant Number 30872986 (XR) and
81171983 (HL).
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References
1. Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and
unresolved questions. Nat Rev Cancer. 2008; 8(10):755–68. [PubMed: 18784658]
2. Ailles LE, Weissman IL. Cancer stem cells in solid tumors. Curr Opin Biotechnol. 2007; 18(5):460–
6. [PubMed: 18023337]
3. Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer. 2006; 6(5):392–401. [PubMed:
16572188]
4. Kuperwasser C, Chavarria T, Wu M, et al. Reconstruction of functionally normal and malignant
human breast tissues in mice. Proc Natl Acad Sci U S A. 2004; 101(14):4966–71. [PubMed:
15051869]
5. Melgarejo E, Medina MA, Sanchez-Jimenez F, Urdiales JL. Monocyte chemoattractant protein-1: a
key mediator in inflammatory processes. Int J Biochem Cell Biol. 2009; 41(5):998–1001. [PubMed:
18761421]
6. Strieter RM, Wiggins R, Phan SH, et al. Monocyte chemotactic protein gene expression by
cytokine-treated human fibroblasts and endothelial cells. Biochem Biophys Res Commun. 1989;
162(2):694–700. [PubMed: 2787988]
7. Lazennec G, Richmond A. Chemokines and chemokine receptors: new insights into cancer-related
inflammation. Trends Mol Med. 2010; 16(3):133–44. [PubMed: 20163989]
8. Dontu G, Jackson KW, McNicholas E, Kawamura MJ, Abdallah WM, Wicha MS. Role of Notch
signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res.
2004; 6(6):R605–15. [PubMed: 15535842]
9. Lobo NA, Shimono Y, Qian D, Clarke MF. The biology of cancer stem cells. Annu Rev Cell Dev
Biol. 2007; 23:675–99. [PubMed: 17645413]
10. Kadesch T. Notch signaling: a dance of proteins changing partners. Exp Cell Res. 2000; 260(1):1–
8. [PubMed: 11010805]
11. Ghebeh H, Tulbah A, Mohammed S, et al. Expression of B7-H1 in breast cancer patients is
strongly associated with high proliferative Ki-67-expressing tumor cells. Int J Cancer. 2007;
121(4):751–8. [PubMed: 17415709]
12. Aagaard L, Amarzguioui M, Sun G, et al. A facile lentiviral vector system for expression of
doxycycline-inducible shRNAs: knockdown of the pre-miRNA processing enzyme Drosha. Mol
Ther. 2007; 15(5):938–45. [PubMed: 17311008]
13. Wang Y, Yu Y, Tsuyada A, et al. Transforming growth factor-beta regulates the sphere-initiating
stem cell-like feature in breast cancer through miRNA-181 and ATM. Oncogene. 2011; 30(12):
1470–80. [PubMed: 21102523]
14. Gu L, Lau SK, Loera S, Somlo G, Kane SE. Protein kinase A activation confers resistance to
trastuzumab in human breast cancer cell lines. Clin Cancer Res. 2009; 15(23):7196–206.
[PubMed: 19920112]
15. van de Vijver MJ, He YD, van’t Veer LJ, et al. A gene-expression signature as a predictor of
survival in breast cancer. N Engl J Med. 2002; 347(25):1999–2009. [PubMed: 12490681]
16. Chang HY, Nuyten DS, Sneddon JB, et al. Robustness, scalability, and integration of a wound-
response gene expression signature in predicting breast cancer survival. Proc Natl Acad Sci U S A.
2005; 102(10):3738–43. [PubMed: 15701700]
17. Dontu G, Abdallah WM, Foley JM, et al. In vitro propagation and transcriptional profiling of
human mammary stem/progenitor cells. Genes Dev. 2003; 17(10):1253–70. [PubMed: 12756227]
18. Scheel C, Eaton EN, Li SH, et al. Paracrine and autocrine signals induce and maintain
mesenchymal and stem cell States in the breast. Cell. 2011; 145(6):926–40. [PubMed: 21663795]
19. Cicalese A, Bonizzi G, Pasi CE, et al. The tumor suppressor p53 regulates polarity of self-
renewing divisions in mammary stem cells. Cell. 2009; 138(6):1083–95. [PubMed: 19766563]
20. Potula HS, Wang D, Quyen DV, et al. Src-dependent STAT-3-mediated expression of monocyte
chemoattractant protein-1 is required for 15(S)-hydroxyeicosatetraenoic acid-induced vascular
smooth muscle cell migration. J Biol Chem. 2009; 284(45):31142–55. [PubMed: 19736311]
21. Song L, Ara T, Wu HW, et al. Oncogene MYCN regulates localization of NKT cells to the site of
disease in neuroblastoma. J Clin Invest. 2007; 117(9):2702–12. [PubMed: 17710228]
Tsuyada et al. Page 11
Cancer Res
. Author manuscript; available in PMC 2013 June 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
22. Liu Y, Elf SE, Miyata Y, et al. p53 regulates hematopoietic stem cell quiescence. Cell Stem Cell.
2009; 4(1):37–48. [PubMed: 19128791]
23. Peacock CD, Wang Q, Gesell GS, et al. Hedgehog signaling maintains a tumor stem cell
compartment in multiple myeloma. Proc Natl Acad Sci U S A. 2007; 104(10):4048–53. [PubMed:
17360475]
24. Ueno T, Toi M, Saji H, et al. Significance of macrophage chemoattractant protein-1 in macrophage
recruitment, angiogenesis, and survival in human breast cancer. Clin Cancer Res. 2000; 6(8):
3282–9. [PubMed: 10955814]
25. Kellermann MG, Sobral LM, da Silva SD, et al. Mutual paracrine effects of oral squamous cell
carcinoma cells and normal oral fibroblasts: induction of fibroblast to myofibroblast
transdifferentiation and modulation of tumor cell proliferation. Oral Oncol. 2008; 44(5):509–17.
[PubMed: 17826300]
26. Kojima Y, Acar A, Eaton EN, et al. Autocrine TGF-beta and stromal cell-derived factor-1 (SDF-1)
signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts. Proc Natl
Acad Sci U S A. 2010; 107(46):20009–14. [PubMed: 21041659]
27. Qian DZ, Rademacher BL, Pittsenbarger J, et al. CCL2 is induced by chemotherapy and protects
prostate cancer cells from docetaxel-induced cytotoxicity. Prostate. 2010; 70(4):433–42. [PubMed:
19866475]
28. Yu H, Kortylewski M, Pardoll D. Crosstalk between cancer and immune cells: role of STAT3 in
the tumour microenvironment. Nat Rev Immunol. 2007; 7(1):41–51. [PubMed: 17186030]
29. Soria G, Ben-Baruch A. The inflammatory chemokines CCL2 and CCL5 in breast cancer. Cancer
Lett. 2008; 267(2):271–85. [PubMed: 18439751]
30. Saji H, Koike M, Yamori T, et al. Significant correlation of monocyte chemoattractant protein-1
expression with neovascularization and progression of breast carcinoma. Cancer. 2001; 92(5):
1085–91. [PubMed: 11571719]
31. Lu X, Kang Y. Chemokine (C-C motif) ligand 2 engages CCR2+ stromal cells of monocytic origin
to promote breast cancer metastasis to lung and bone. J Biol Chem. 2009; 284(42):29087–96.
[PubMed: 19720836]
32. Qian BZ, Li J, Zhang H, et al. CCL2 recruits inflammatory monocytes to facilitate breast-tumour
metastasis. Nature. 2011; 475(7355):222–5. [PubMed: 21654748]
33. de Vasconcellos JF, Laranjeira AB, Zanchin NI, et al. Increased CCL2 and IL-8 in the bone
marrow microenvironment in acute lymphoblastic leukemia. Pediatr Blood Cancer. 2011; 56(4):
568–77. [PubMed: 21298741]
34. Molloy AP, Martin FT, Dwyer RM, et al. Mesenchymal stem cell secretion of chemokines during
differentiation into osteoblasts, and their potential role in mediating interactions with breast cancer
cells. Int J Cancer. 2009; 124(2):326–32. [PubMed: 19003962]
35. Ranganathan P, Weaver KL, Capobianco AJ. Notch signalling in solid tumours: a little bit of
everything but not all the time. Nat Rev Cancer. 2011; 11(5):338–51. [PubMed: 21508972]
36. Takebe N, Harris PJ, Warren RQ, Ivy SP. Targeting cancer stem cells by inhibiting Wnt, Notch,
and Hedgehog pathways. Nat Rev Clin Oncol. 2011; 8(2):97–106. [PubMed: 21151206]
37. Yashiro-Ohtani Y, He Y, Ohtani T, et al. Pre-TCR signaling inactivates Notch1 transcription by
antagonizing E2A. Genes Dev. 2009; 23(14):1665–76. [PubMed: 19605688]
38. Meng W, Swenson LL, Fitzgibbon MJ, et al. Structure of mitogen-activated protein kinase-
activated protein (MAPKAP) kinase 2 suggests a bifunctional switch that couples kinase activation
with nuclear export. J Biol Chem. 2002; 277(40):37401–5. [PubMed: 12171911]
39. Page JL, Wang X, Sordillo LM, Johnson SE. MEKK1 signaling through p38 leads to
transcriptional inactivation of E47 and repression of skeletal myogenesis. J Biol Chem. 2004;
279(30):30966–72. [PubMed: 15159407]
40. Yu Y, Wang Y, Ren X, et al. Context-dependent bidirectional regulation of the MutS homolog 2
by transforming growth factor beta contributes to chemoresistance in breast cancer cells. Mol
Cancer Res. 2010; 8(12):1633–42. [PubMed: 21047769]
41. Wang SE, Narasanna A, Whitell CW, Wu FY, Friedman DB, Arteaga CL. Convergence of p53 and
transforming growth factor beta (TGFbeta) signaling on activating expression of the tumor
Tsuyada et al. Page 12
Cancer Res
. Author manuscript; available in PMC 2013 June 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
suppressor gene maspin in mammary epithelial cells. J Biol Chem. 2007; 282(8):5661–9.
[PubMed: 17204482]
42. Wang SE, Narasanna A, Perez-Torres M, et al. HER2 kinase domain mutation results in
constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine
kinase inhibitors. Cancer Cell. 2006; 10(1):25–38. [PubMed: 16843263]
43. Farmery MR, Tjernberg LO, Pursglove SE, Bergman A, Winblad B, Naslund J. Partial purification
and characterization of gamma-secretase from post-mortem human brain. J Biol Chem. 2003;
278(27):24277–84. [PubMed: 12697771]
Tsuyada et al. Page 13
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. Author manuscript; available in PMC 2013 June 01.
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Fig. 1. CCL2 secreted by cancer-activated fibroblasts induces mammosphere formation in BC
cells
(A) Mammosphere formation assay of BT474 cells co-cultured with various fibroblasts.
NAFs and CAFs were first co-cultured with BT474 cells growing in transwell inserts for 3
days (NAF2BT474 and CAF3BT474), or cultured alone (NAF2ctrl and CAF3ctrl), before being
transferred to transwell inserts and co-cultured with freshly plated BT474 cells for
mammosphere assays. Spheres were counted on day 10, and sphere forming efficiency
(SFE) was calculated. *
p
<0.01. (B) Mammosphere formation assay of BT474 cells exposed
to the conditioned media (CM) of differentially treated fibroblasts. Fibroblasts were first
treated with BT474- or MDA361-derived CM for 3 or 10 days as indicated by “3d” or “10d”
in the superscript, or with regular medium (NAF2ctrl and CAF3ctrl). Treated fibroblasts were
then cultured in sphere-forming media for 24 h to prepare CM subsequently used in BT474
sphere formation assays. *
p
<0.01 compared to the control (the first column). (C) CM was
collected from CAF3 that had been previously treated for 3 days with BT474 CM
(CAF3BT474), and from untreated CAF3 as a control. Concentrated CM was subjected to a
cytokine antibody array. Induction of CCL2 protein was observed in the CM of CAF3BT474.
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(D) Relative expression of CCL2 mRNA in various cell types was determined by RT-qPCR.
CAF265922 (primary CAFs) and XP265922 (primary tumor cells) were isolated from the
same BC specimen. The superscripts denote CM treatments as in (B). The time length of
CM treatment was 3 days in CAF3 and 10 days in NAF2. *
p
<0.01 compared to the
untreated control CAF3 or NAF2. (E) BT474, MDA361, and MCF7 BC cells were assayed
for sphere formation in the presence of CCL2 at the indicated concentrations. *
p
<0.01
compared to the control (no CCL2 treatment) in each cell line. (F) Effect of CAF CM (as
indicated in B) on BT474 sphere formation in the presence or absence of a CCL2
neutralizing antibody. *
p
<0.01 compared to the control (the first column). Each bar
represents the mean ± S.D. of 3 wells.
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Fig. 2. CCL2 induces the self-renewing expansion of CSCs
(A) Primary and secondary sphere formation in the presence or absence of CCL2 (10 ng/ml).
*
p
<0.01 compared to the control (the first column in each cell line). (B) Left: Confocal
images of representative spheres formed by PKH67-labeled BT474 cells in the presence or
absence of CCL2. PKH67high cells are indicated by arrows. Scale bar equals 20 μm. Right:
Summary of the numbers of PKH67high cells per sphere by counting 150 spheres formed in
the presence or absence of CCL2. (C) A representative histogram indicating the flow
cytometric profile of dissociated primary sphere cells and gating of PKH67low and
PKH67high cells. Percentage of PKH67high cells was summarized in the bar graph. Each bar
represents the mean ± S.D. of 3 independently treated sphere cultures. *
p
<0.01. (D)
Secondary mammosphere formation of sorted PKH67high and PKH67low primary sphere
cells. Each bar represents the mean ± S.D. of 3 wells.
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Fig. 3. Paracrine signaling of cancer-secreted cytokines induces CCL2 production in fibroblasts
via STAT3 activation
(A) A luciferase reporter containing a 2.8-kb CCL2 promoter was transfected into
CAF265922 cells. Luciferase activity was analyzed at 4 h post CM exposure in the presence
of DMSO or Stattic (a STAT3 inhibitor; 5 μM). Each bar represents the mean ± S.D. of 3
independently transfected wells. *
p
<0.01 compared to the control (the first column). (B)
Total RNA isolated from CAF265922 that had been treated with CM from indicated BC
cells for 4 or 24 h was analyzed for CCL2 mRNA level by RT-qPCR. Data were normalized
to 18S in each sample. Each bar represents the mean ± S.D. of 3 wells. *
p
<0.01 compared
to the control (the first column). (C) CAF265922 cells were treated with CM from indicated
cells and analyzed by Western blot. (D) Summary of the cytokine array data identifying
cytokines constitutively secreted by BT474 and MDA361 BC cells. Cytokines that are
known to activate STAT3 are in bold.
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Fig. 4. CCL2 regulates CSC phenotype in BC cells by activating NOTCH signaling
(A) Total RNA isolated from various BC cell lines treated with CCL2 or vehicle for 24 h
were analyzed for the expression of HES1, a target gene activated by NOTCH signaling.
Data of RT-qPCR were normalized to 18S in each sample. Each bar represents the mean ±
S.D. of 3 wells. *
p
<0.001 compared to the control (the first column). (B) Expression of
NOTCH1 and HES1 mRNAs upon CCL2 treatment in the indicated time course. The
mRNA level at each time point was compared to that in untreated cells, which was set as 1.
Each bar represents the mean ± S.D. of 3 wells. *
p
<0.01 compared to untreated cells. (C)
Luciferase reporters of HES1 and HEY1 were transfected into XP265922 primary BC cells.
Luciferase activity was analyzed at 24 h post CCL2 treatment +/− inhibitors of γ-secretase
(DAPT; 10 μM) or α-secretase (INCB3619; 5 μM). Each bar represents the mean ± S.D. of
3 independently transfected wells. *
p
<0.001 compared to the control (the first column). (D)
Western blot analysis of p38 and NOTCH1 at indicated time points following treatment
conditions in XP265922 cells. (E) Expression of NOTCH1 mRNA in XP265922 cells
treated with CCL2 +/− SB202190 (5 μM) for 24 h. *
p
<0.001 compared to the control (the
first column). (F) A luciferase reporter containing a 6-kb NOTCH1 promoter was
transfected into XP265922 cells to analyze NOTCH1 promoter regulation by CCL2 and
various inhibitors (Wortmannin: 1 μM; U0126: 10 μM; SB202190: 5 μM). Each bar
represents the mean ± S.D. of 3 independently transfected wells. *
p
<0.001 compared to the
control (the first column). (G) XP265922 cells were treated with CM from indicated sources
and analyzed for NOTCH1 and p38 expression by Western blot. (H) Mammosphere
formation assay of BC cells treated with various inhibitors in the presence or absence of
CCL2. Each bar represents the mean ± S.D. of 3 wells. *
p
<0.01 compared to the control
(the first column in each cell line).
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Fig. 5. Fibroblast-specific CCL2 knockdown or CCL2 depletion by neutralizing antibody
inhibits in vivo tumorigenesis
(A) CAF265922 cells stably expressing Dox-inducible CCL2 shRNA were examined for
CCL2 mRNA expression by RT-qPCR upon 48 h treatment of Dox (1 μg/ml) or vehicle. *
p
<0.001. (B)
In vivo
tumor formation was examined by co-transplanting unmodified
XP265922 primary BC cells and the CAF265922 cells tested in (A) into the mammary fat
pads of NOD/SCID/IL2Rγ-null mice, as described in Materials and Methods. The time-
course of xenograft tumor formation in Dox-treated mice (Dox+) and control mice (Dox−)
was compared.
p
<0.001 between the two groups. (C) Tumor volume determined in Dox+
and Dox− mice. *
p
<0.001 at each available time point starting from day 22. (D) Total RNA
was isolated from fibroblasts and epithelial tumor cells purified from the Dox+ and Dox−
xenograft tumors, and subjected to RT-qPCR for CCL2 and NOTCH1 expression,
respectively. *
p
<0.001. (E) Representative immunohistochemistry images of Dox+ and
Dox− xenograft tumor sections stained with antibodies against CCL2 and NOTCH1 (40×;
bar=20 μm). (F) Representative flow cytometry dot plots indicating the ALDEFLUOR-
bright tumor cells from Dox+ and Dox−xenograft tumors. Diethylaminobenzaldehyde
(DEAB), an inhibitor of ALDH, was added in the left two panels. Bar graph: Averaged
percentages of the ALDEFLUOR-bright population from bulk tumor cells in 4 Dox+ and 4
Dox− xenograft tumors. *
p
<0.001. (G) Tumor volume determined in mice treated with
PBS, IgG, or CCL2 neutralizing antibody. *
p
<0.001. (H) Total RNA was isolated from
epithelial tumor cells purified from the three groups of xenograft tumors, and subjected to
RT-qPCR for NOTCH1 expression. *
p
<0.001. (I) A model of the CSC-stimulating
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crosstalk circuit that involves STAT3, CCL2 and NOTCH pathways. In the tumor
microenvironment, paracrine signaling initiated by BC cells induces CCL2 production by
stromal fibroblasts through STAT3 activation, and the fibroblast-derived CCL2, in turn,
promotes cancer progression by regulating CSCs through activation of the NOTCH
pathway.
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Table 1
Relative odds of having BC Grade 3 (
N
= 119) vs. Grade 1 (
N
= 75) associated with CCL2 and NOTCH
related genes.
Gene Symbol Gene Name OR*CI** p
CCL2 chemokine (C-C motif) ligand 13.72 2.66 – 70.89 < 0.01
CCR2 chemokine (C-C motif) receptor 26.32 0.87 – 45.82 0.07
NOTCH1 Notch homolog 1 9.56 1.54 – 59.27 0.02
NOTCH2 Notch homolog 2 0.76 0.15 – 3.90 0.74
NOTCH3 Notch homolog 3 2.51 0.36 – 17.29 0.35
NOTCH4 Notch homolog 4 0.39 0.03 – 4.33 0.44
DLL1 delta-like 1 0.48 0.10 – 2.30 0.36
DLL3 delta-like 3 20.93 1.53 – 286.94 0.02
DLL4 delta-like 4 1.16 0.03 – 49.36 0.94
JAG1 jagged 1 0.14 0.03 – 0.57 < 0.01
JAG2 jagged 2 0.31 0.08 – 1.19 0.09
*
Odds ratio of Grade 3 vs. Grade 1;
**
95% Confidence interval.
Cancer Res
. Author manuscript; available in PMC 2013 June 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Tsuyada et al. Page 22
Table 2
Correlation between CCL2 and NOTCH1 in primary human BC.
Microarray-based expression
Group N (%) R* p
All 295 (100) 0.18 < 0.01
Grade
1 75 (25.4) 0.21 0.07
2 101 (34.2) 0.02 0.81
3 119 (40.3) 0.19 0.03
Stage
1 82 (27.8) 0.33 < 0.01
2a 142 (48.1) 0.19 0.02
2b 41 (13.9) 0.05 0.76
3–4 30 (10.2) 0.13 0.49
IHC-based expression
Group N (%) R** p
All 51 (100) 0.26 0.06
Subtype
triple-negative 20 (39.2) 0.04 0.86
HER2+ 31 (60.8) 0.40 0.03
*
Pearson correlation coefficient.
**
Spearman correlation coefficient.
Cancer Res
. Author manuscript; available in PMC 2013 June 01.
