Published OnlineFirst August 14, 2012; DOI: 10.1158/1541-7786.

MCR-12-0275

Macrophages Promote Fibroblast Growth Factor Receptor-Driven

Tumor Cell Migration and Invasion in a Cxcr2-Dependent Manner
Laura R. Bohrer and Kathryn L. Schwertfeger
Mol Cancer Res 2012;10:1294-1305. Published OnlineFirst August 14, 2012.

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Published OnlineFirst August 14, 2012; DOI: 10.1158/1541-7786.MCR-12-0275

Angiogenesis, Metastasis, and the Cellular Microenvironment

Molecular
Cancer
Research

Macrophages Promote Fibroblast Growth Factor ReceptorDriven Tumor Cell Migration and Invasion in a
Cxcr2-Dependent Manner
Laura R. Bohrer and Kathryn L. Schwertfeger

Abstract
Inltration of immune cells, specically macrophages, into the tumor microenvironment has been linked to
increased mammary tumor formation and progression. Activation of growth factor receptor signaling pathways
within mammary epithelial cells, such as the broblast growth factor receptor 1 (FGFR1) pathway, induces
recruitment of macrophages to the mammary epithelium. These macrophages promote increased epithelial cell
proliferation and angiogenesis. However, the specic mechanisms by which these macrophages are regulated by the
preneoplastic epithelial cells and the mechanisms of action of the macrophages within the developing FGFR1driven tumor microenvironment remain unknown. In this study, we show that activation of inducible FGFR1 in
mammary glands leads to decreased activity of the TGFb/Smad3 pathway in macrophages associated with early
stage lesions. Further studies show that macrophages have increased expression of inammatory chemokines that
bind Cxcr2 following exposure to conditioned media from mammary epithelial and tumor cells in which the FGF
pathway had been activated. The increase in these ligands is inhibited following activation of the TGFb pathway,
suggesting that decreased TGFb signaling contributes to the upregulation of these chemokines. Using coculture
studies, we further show that macrophages are capable of promoting epithelial and tumor cell migration and
invasion through activation of Cxcr2. These results indicate that macrophage-derived Cxcr2 ligands may be
important for promoting mammary tumor formation regulated by FGFR signaling. Furthermore, these results
suggest that targeting Cxcr2 may represent a novel therapeutic strategy for breast cancers that are associated with
high levels of inltrating macrophages. Mol Cancer Res; 10(10); 1294305. 2012 AACR.

Introduction
Breast tumor formation and progression involve complex
interactions between tumor cells and their surrounding
environment. Inltration of immune cells into the tumor
microenvironment has been linked to tumor formation and
progression (1). Specically, increased numbers of tumorassociated macrophages are linked to poor prognosis in
breast cancer patients (2). Although macrophages were
initially expected to inhibit tumor growth via their cytotoxic
functions, it is now clear that exposure to the tumor
microenvironment polarizes macrophages towards a
tumor-promoting phenotype (3). Therefore, obtaining a
better understanding of the mechanisms through which
Authors' Afliation: Department of Laboratory Medicine and Pathology
and Masonic Cancer Center, University of Minnesota, Minneapolis,
Minnesota
Note: Supplementary data for this article are available at Molecular Cancer
Research Online (http://mcr.aacrjournals.org/).
Corresponding Author: Kathryn L. Schwertfeger, Department of Laboratory Medicine and Pathology and Masonic Cancer Center, University of
Minnesota, 420 Delaware St. SE, Minneapolis, MN 55455, USA. Phone:
612-626-9419; Fax: 612-626-2600; E-mail: schwe251@umn.edu
doi: 10.1158/1541-7786.MCR-12-0275
2012 American Association for Cancer Research.

1294

macrophages regulate tumor growth and progression may

result in the development of strategies that either inhibit the
activities of tumor-promoting macrophages or reprogram
the macrophages to increase their tumor cytotoxic activities.
Numerous functions have been ascribed to macrophages
during tumor progression, including promotion of tumor
cell invasion, angiogenesis and immune suppression (4).
Although published studies have identied specic mechanisms through which macrophages contribute to mammary
tumor metastasis in mouse models (5, 6), less is known
regarding the mechanisms of macrophage function during
mammary tumor initiation.
Macrophages associated with late stage tumors have been
shown to express high levels of TGFb (7). TGFb, which is a
key regulator of development, immune function and wound
healing, acts primarily through a core-signaling pathway
involving the Smad family of transcription factors (8).
During mammary gland development, TGFb is a potent
inhibitor of epithelial cell proliferation and branching morphogenesis (9). In premalignant lesions, TGFb acts as a
tumor suppressor by inhibiting proliferation and promoting
apoptosis. Paradoxically, malignant tumors are associated
with high levels of TGFb, which promote later stages of
tumor progression and metastasis (8, 10). Pleiotropic effects
of TGFb have also been observed in macrophages,

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Cxcr2 Contributes to Macrophage-Induced Tumor Cell Migration

depending on their stage of differentiation. Although TGFb

acts as a chemoattractant for monocytes, it inhibits phagocytosis and production of proinammatory mediators in
differentiated macrophages (11). TGFb expression levels are
high in macrophages associated with late stage tumors (7),
which is thought to inuence many aspects of malignant
progression including invasion, angiogenesis and immune
suppression (10). However, the functional consequences of
regulating the TGFb/Smad signaling pathway within
macrophages during different stages of tumorigenesis have
not been investigated.
Inappropriate activation of growth factor signaling pathways has been strongly linked to breast cancer formation and
progression. Recent studies have implicated the broblast
growth factor (FGF) pathway in tumor growth, progression
and resistance to standard therapies (12, 13). Amplication
of the chromosomal region of 8p12 that includes the FGF
receptor 1 (FGFR1) gene is associated with poor prognosis,
and approximately 10% of breast cancers exhibit amplied
FGFR1 (13, 14). Transgenic mice expressing an inducible
FGFR1 (iFGFR1) transgene in mammary epithelial cells
develop early epithelial lesions that progress to alveolar
hyperplasia, ultimately resulting in mammary tumor formation (15). Activation of iFGFR1 leads to alterations in the
microenvironment, including increased angiogenesis and a
rapid inammatory response characterized by inltrating
macrophages (15, 16). Macrophage depletion in this model
leads to reduced epithelial cell proliferation and angiogenesis
associated with early stage lesions showing that in an
FGFR1-dependent model of mammary tumor formation,
macrophages are capable of promoting the development of
early stage epithelial lesions (16).
In these studies, we have further used the iFGFR1 model
to identify mechanisms that regulate the protumor functions
of macrophages during early stage tumor formation. We
show here that macrophages associated with iFGFR1-driven
early stage epithelial lesions exhibit decreased activation of
the TGFb/Smad3 pathway. The decrease in TGFb-associated genes within macrophages correlates with increased
expression of macrophage-derived chemokines that bind to
the chemokine receptor Cxcr2. Restoration of TGFb signaling leads to inhibition of expression of these chemokines
in macrophages. These studies suggest that repressed TGFb/
Smad3 signaling may be functionally important for regulating the protumorigenic function of macrophages in early
stages of tumor formation. Furthermore, these studies show
that macrophage-derived chemokines, specically Cxcr2
binding chemokines, promote migration and invasion of
preneoplastic mammary epithelial cells, suggesting a potential therapeutic target for early stage breast tumors.
Materials and Methods
Animals
Generation of mouse mammary tumor virus (MMTV)iFGFR1 transgenic mice has been described previously (15)
and the mice were obtained from Dr. Jeffrey Rosen (Baylor
College of Medicine, Houston, TX). Animal care and

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procedures were approved by the Institutional Animal Care

and Use Committee of the University of Minnesota and
were in accordance with the procedures detailed in the Guide
for Care and Use of Laboratory Animals.
Cell sorting and RT-PCR analysis
Six-week-old female MMTV-iFGFR1 transgenic mice
and nontransgenic littermates were injected intraperitoneally (i.p.) with 1 mg/kg B/B dimerizer (Clontech). Mice were
sacriced 48 hours later and mammary glands were collected
for analysis. The tissue was dissociated using 2 mg/mL
collagenase A (Roche Applied Science) for 45 minutes at
37
C with rocking at 200 rpm. The solutions were vigorously shaken every 15 minutes and the dissociated cells were
collected by centrifuging for 5 minutes at 1,500 rpm. The
cells were washed 3 times with DMEM/F12 containing 5%
FBS at 1,500 rpm and 2 times at 800 rpm for 5 minutes each.
The cells were stained with either Cd11b-APC (Life Technologies) at a dilution of 1:200 or isotype control antibody
at the same concentration for 1 hour at RT. The cells
were then washed, ltered through a 40 mm lter and sorted
using a triple laser MoFlo (Cytomation). RNA was isolated
from Cd11b-positive cells sorted from 6 mice per timepoint
as described above and pooled into duplicate samples. RNA
was extracted using the Arcturus PicoPure RNA Isolation
Kit (Life Technologies) and RT-PCR analysis was carried
out using primers specic for ArgI and iNOS as described
below. qRT-PCR analysis was carried out for TGFb1 as
described below. Primer sequences are listed in Supplementary Table 1. Cyclophilin was used to normalize gene
expression levels.
Immunouorescence
Mammary glands from MMTV-iFGFR1 transgenic mice,
or nontransgenic littermate controls, that were treated with
B/B for 48 hours as described above were xed for 2 hours in
4% paraformaldehyde and embedded in parafn. Five
micrometers sections were used for immunouorescent
staining using the following antibodies and dilutions: rat
monoclonal F4/80 (Invitrogen), 1:50; phospho-Smad3
(Cell Signaling), 1:200. Immunostaining was carried out
as described previously (17) in the absence of antigen
retrieval. F4/80 and phospho-Smad3 positive cells were
counted and double positive cells were calculated relative
to the total number of F4/80 positive cells. A minimum of
600 cells from a total of 3 mice per treatment group was
counted for each dataset. All statistical analyses were carried
out using the unpaired Student t test to compare 2 means.
Cell culture
HC-11 cells were maintained in RPMI containing 10%
FBS (Invitrogen), 1% penicillin-streptomycin, 5 mg/mL
insulin (Sigma-Aldrich) and 10 ng/mL EGF (Invitrogen).
HC-11 cells stably expressing the iFGFR construct (HC-11/
R1) were generated as previously described (18) and were
acquired from Dr. Jeffrey Rosen. HC-11/R1 cells were
maintained in HC-11 medium with the addition of 0.7
mg/mL puromycin (Sigma-Aldrich). We received SUM225

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Bohrer and Schwertfeger

and MCF10DCIS.com from Dr. Fariba Behbod (University

of Kansas City Medical Center, Kansas City, KS) and
maintained as described (19). All other cell lines were
obtained from the American Type Culture Collection
(ATCC) and maintained in the suggested media. All cell
lines were used for fewer than 6 months after resuscitation.
HC-11 cells and HC-11/R1 cells, which are not commercially available, are not maintained for longer than 20
passages and are tested for mycoplasma, b-casein expression
and iFGFR1 expression regularly.
Two-dimensional coculture assays
HC-11/R1 cells were grown to conuence, washed with
PBS and incubated overnight in serum-free RPMI. The cells
were treated with 30 nmol/L B/B or an equal amount of
ethanol as solvent control for 24 hours. Conditioned media
was collected (R1-CM) and added to RAW 264.7 cells that
had been incubated overnight in serum-free DMEM. To
determine gene expression, cells were collected in Trizol after
2 hours of treatment with R1-CM supplemented with or
without 10 ng/mL recombinant TGFb (R&D Systems,
Minneapolis, MN, USA) and analyzed as described below.
For signaling studies, R1-CM was added to RAW264.7 cells
for the indicated time and lysates were collected for immunoblotting. Then, RAW 264.7 cells were pretreated for 30
minutes with 2.5 or 10 mmol/L of the MEK1 inhibitor
U0126 (Cell Signaling) or solvent control (DMSO) before
incubation with R1-CM and the inhibitor or control for an
additional 2 hours. For migration assays, RAW 264.7 cells
were incubated for 24 hours with R1-CM and CM was
collected (R1/RAW-CM). Migration assays were carried out
using cell culture inserts with 8 mm pore size (BD Biosciences, San Jose, CA, USA). HC-11 cells were washed with
PBS and incubated overnight in serum-free RPMI. Cells
were plated on the top of the insert and either R1-CM or R1/
RAW-CM was placed on the bottom of the insert as a
chemoattractant. The CXCR2 inhibitor, SB225002 (20 or
200 nmol/L, Cayman Chemical), or the solvent control,
ethanol, was added with the HC-11 cells to the top of the
well. After 18 hours, cells on the bottom of the insert were
xed with 4% paraformaldehyde and stained with hematoxylin. The number of cells migrated in 4 elds of the insert
were counted. Similar experiments were done with human
cell lines in which MCF-7 cells were treated with 50 ng/mL
basic broblast growth factor (bFGF, Invitrogen). After 24
hours, the conditioned media (MCF-CM) was then incubated with the human monocyte cell line THP-1 that had
been treated with 5 ng/mL PMA 24 hours and then starved
overnight. Gene expression of CXCR2 ligands in macrophages was determined by collecting THP-1 cells treated
with MCF-CM for 2 or 4 hours and analyzed as described
below. For migration assays, MCF-CM was added to THP-1
cells for 24 hours and the conditioned media (MCF/THPCM) was used as a chemoattractant for MCF-7 cells in the
cell culture inserts as described above. The CXCR2 inhibitor, SB225002 (100, 200, or 400 nmol/L) or solvent
control, ethanol, was added with MCF-7 cells in the top
of the well.

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Quantitative reverse transcription-PCR

RNA was extracted from primary macrophages or macrophage cell lines (RAW 264.7 and THP-1, ATCC) using
Trizol (Invitrogen) as described in the manufacturer protocol. cDNA was prepared using the Quantitect reverse
transcription kit (Qiagen). Quantitative RT-PCR (qRTPCR) was carried out using SYBR green (Bio-Rad) and the
Bio-Rad iQ5 system. The 2DDCt method (20) was used to
determine the relative quantication of gene expression
normalized to cyclophilin. The primers used for these studies
are listed in Supplementary Table 1.
qRT-PCR array
RNA was isolated from RAW 264.7 cells treated with R1CM for 2 hours in the presence or absence of 10 ng/mL
TGFb, and cDNA was made, as described above. The RT2
Proler PCR Array: Mouse chemokines and receptors from
SA Biosciences was carried out as described for other qRTPCR experiments.
ELISA analysis and activity assays
R1-CM was added to starved RAW 264.7 cells in the
presence or absence of 10 ng/mL TGFb. After 2 hours
the media was removed and serum free RPMI was added.
The R1/RAW-CM was collected after an additional 24
hours and ELISAs were carried out to quantify the level of
Cxcl1, Cxcl5, IL-10, and IL-12 following the manufacturer's
protocol (R&D Systems). In addition, the RAW 264.7 were
collected in lysis buffer (10 mmol/L Tris-HCl [pH 7.4]
containing 1 mmol/L pepstatin A, 1 mmol/L leupeptin, and
0.4% Triton X-100) and the supernatant was used for an
arginase activity assay (BioAssay Systems). For the Smad3
luciferase assay, RAW 264.7 cells were transduced with a
lentivirus expressing a Smad3 reporter construct (Qiagen)
and were then treated with conditioned media from B/Btreated HC-11/R1 cells for 6 hours. Luciferase activity was
measured using standard procedures (Promega). For analysis
of human chemokine expression, THP-1 cells were incubated with MCF-CM for 2 hours and then serum free
DMEM for 24 hours. The levels of secreted CXCL1 and
CXCL8 in the MCF/THP-CM were measured by ELISA
following the manufacturer's protocol (R&D Systems).
Immunoblot analysis
Cells were lysed in RIPA buffer and equal amounts of
protein were analyzed by SDS-PAGE. Immunoblotting was
done using the following antibodies: CXCR2 (AHR1532Z;
Biosource), b-tubulin (2146; Cell Signaling), pERK1/2
(9101; Cell Signaling), and ERK1/2 (sc-94; Santa Cruz
Biotechnology).
Three-dimensional coculture assay
Mammary glands were isolated from 8- to 12-week-old
MMTV-iFGFR1 transgenic mice as described above. Bone
marrow was collected from femurs of 6- to 10-week-old
wild-type (WT) mice. Cells were differentiated into macrophages with the addition of 20% conditioned media from
L929 cells that have a high concentration of granulocyte

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Cxcr2 Contributes to Macrophage-Induced Tumor Cell Migration

colony-stimulating factor (G-CSF). A total of 10,000 mammary epithelial cells (MECs) were plated on growth-factor
reduced matrigel (BD Biosciences) in 8 well chamber slides
as described previously (21). After 2 days, 3000 bone
marrow derived macrophages (BMDM) were added to the
3D culture. Also, the following treatments began at this
time: 30 nmol/L B/B and 200 nmol/L SB225002,
CXCR2 inhibitor (with () being ethanol, solvent control).
After 10 days, cells were xed with 2% paraformaldehyde
and costained for macrophages (F4/80, Invitrogen) and
epithelial cells (cytokeratin 8 (ab59400- Abcam) and
mounted with ProLong Gold antifade reagent with DAPI
(Invitrogen). Images were taken at the confocal microscopy
facility at the Masonic Cancer Center (University of Minnesota, Minneapolis, MN).
Results
Regulation of pro- and anti-inammatory genes in
macrophages following exposure to conditioned media
from iFGFR1-activated HC-11/R1 cells
As described previously, activation of iFGFR1 in mammary epithelial cells promotes rapid recruitment of macrophages to epithelial structures in vivo and in vitro (16).
Furthermore, depletion of macrophages leads to delayed

formation of early hyperplastic lesions in vivo (16). Therefore, we further used this model to delineate the mechanisms
that mediate the interactions between epithelial cells and
macrophages. We have previously shown that exposure of
macrophages to conditioned media from HC-11/R1 cells
following activation of iFGFR1 leads to increased production of the proinammatory cytokine IL-1b by macrophages
in vitro and that IL-1b contributes to the formation of early
stage lesions in vivo (17). To extend our analysis of macrophage response to iFGFR1 activation in epithelial cells, we
further analyzed the expression of various genes known to be
preferentially regulated in tumor associated macrophages,
including IL-10, IL-12, Arginase I (ArgI), and TGFb. For
these studies, RAW 264.7 cells were exposed to conditioned
media from HC-11/R1 cells treated with either the B/B
homodimerizer, which activates the iFGFR1, or ethanol as a
solvent control. After exposure to conditioned media,
RAW 264.7 cells were analyzed for expression and activity
of Arg1, IL-10, IL-12, and TGFb. As shown in Fig. 1,
expression levels of IL-10 and gene expression and activity of
ArgI, which are typically increased in tumor associated
macrophages, were induced whereas expression levels of
IL-12, which are normally reduced in tumor associated
macrophages, were decreased. Interestingly, expression of

Figure 1. Exposure to conditioned media from mammary epithelial cells with activated iFGFR1 leads to regulation of inammation-associated genes in vitro.
RAW 264.7 cells were exposed to conditioned media from B/B-treated HC-11/R1 cells for 2 hours and either cells were collected for gene expression or
serum free media was added for 24 hours to examine secreted protein expression or activity. A, qRT-PCR analysis of ArgI gene expression levels
(left panel) and arginase activity (right panel). B, qRT-PCR analysis of IL-10 gene expression levels (left panel) and secreted protein level by ELISA (right panel).
C, qRT-PCR analysis of IL-12 gene expression levels (left panel) and secreted protein level by ELISA (right panel). D, qRT-PCR analysis of TGFb1
gene expression levels (left panel) and Smad3 transcriptional activity by luciferase assay (right panel). Expression levels were normalized to cyclophilin for
qRT-PCR. Error bars represent standard error of the mean (SEM).  , P < 0.05;   , P < 0.005;    , P < 0.001.

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Bohrer and Schwertfeger

the TGFb1 gene, which is usually induced in tumor associated macrophages, was reduced in these studies. In addition, the transcriptional activity of Smad3 also decreased
(Fig. 1D) suggesting an overall decrease in the TGFb/Smad3
pathway. These results, together with our previously published studies (17), show that exposure of macrophages to
conditioned media from epithelial cells with activated
iFGFR1 leads to regulation of a number of genes associated
with macrophage polarization.
Decreased TGFb gene expression and Smad3 activity in
macrophages from MMTV-iFGFR1 transgenic mice
To examine gene expression of macrophages in vivo,
macrophages were isolated from the mammary glands of
WT and MMTV-iFGFR1 transgenic mice following 48
hours of B/B treatment, at which time macrophage recruitment to epithelial structures is consistently observed (16).
Following a brief enzymatic digestion, macrophages were
isolated by sorting with anti-Cd11b, which stains both
monocytes and macrophages. Approximately 8.5% of the

cells isolated from the mammary glands were positive for the
Cd11b antigen compared with the isotype control antibody
(Supplementary Fig. 1A). These cells were collected for both
immunostaining and RNA extraction. To determine the
percentage of the population of sorted cells that represents
mature macrophages, staining was carried out using the F4/
80 antibody (Supplementary Fig. 1B). Approximately 85%
of the sorted cells were positive for F4/80, suggesting that the
preparation was signicantly enriched for macrophages. To
examine gene expression in the macrophages, RT-PCR
analysis was carried out and expression levels of ArgI, a
marker of protumor macrophages, and iNOS, a marker of
tumor-inhibitory macrophages, were examined. As shown
in Fig. 2A, ArgI expression was increased and iNOS expression was decreased in macrophages isolated from MMTViFGFR1 transgenic mice treated with B/B, consistent with a
polarization of the macrophages towards the tumor-promoting phenotype. Further studies using qRT-PCR revealed a
decrease in expression of TGFb1 in these macrophages (Fig.
2B), consistent with the results observed in vitro (Fig. 1).

Figure 2. Alterations in the TGFb gene expression and Smad3 activity in macrophages isolated from mammary glands following FGFR1 activation. A, Cd11bpositive sorted cells were analyzed for expression of the indicated genes using RT-PCR. The lanes represent macrophages sorted from 3 MMTV-iFGFR1
transgenic (Tg) or 3 nontransgenic WT littermates and pooled. B, expression of TGFb1 in macrophages isolated from MMTV-iFGFR1 transgenic
mice or nontransgenic WT littermates treated with B/B for 48 hours. Expression levels were normalized to cyclophilin. C, nontransgenic (i, ii) and
transgenic (iii, iv) mice were treated with B/B for 48 hours and mammary glands were collected. Sections were costained with the F4/80 and phospho-Smad3
antibodies. F4/80 positive cells (red) are found in the stroma surrounding the epithelial structures (blue DAPI staining of nuclei). Phospho-Smad3
(green) is detected in nuclei of F4/80 positive cells in sections from the nontransgenic (arrow, ii) but not transgenic (arrow, iv), mice. Scale bars 50 mm.

D, quantication of the percentage of F4/80 /phospho-Smad3 cells. Error bars represent SEM.  , P < 0.05;    , P < 0.0001.

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Cxcr2 Contributes to Macrophage-Induced Tumor Cell Migration

To determine whether decreased TGFb production

correlated with altered Smad3 activity in the macrophages,
mammary gland sections from WT and MMTV-iFGFR1
transgenic mice following 48 hours of B/B treatment were
immunostained with an antibody to phosphorylated
Smad3 (pSmad3; Fig. 2C). The sections were costained
with an F4/80 antibody to visualize the macrophages and
the percentage of F4/80 cells positive for pSmad3 expression was determined (Fig. 2D). In WT mice, approximately 25% of the F4/80 cells located within the stroma
surrounding the ductal structures were positive for
pSmad3. Interestingly, there was a signicant decrease in
the number of cells positive for pSmad3 present in the
stroma following 48 hours of B/B treatment of iFGFR1
mice. This observation suggests that along with decreased
expression of TGFb in the macrophages, there is also
decreased TGFb signaling within macrophages following
iFGFR1 activation in mammary epithelial cells, consistent
with the in vitro studies.
Effects of TGFb on chemokine expression in
macrophages
On the basis of our results, we hypothesized that decreased
TGFb signaling in macrophages may be linked to protumorigenic macrophage function in early stages of tumorigenesis. Activation of the TGFb/Smad3 pathway has been
shown to inhibit the expression of inammatory cytokines
and chemokines (22, 23), suggesting that repression of this
pathway leads to induction of these factors. Furthermore,
recent studies showed that loss of signaling through Tgfbr2
led to increased expression of chemokines in a model of
pancreatic ductal adenocarcinoma (24). Therefore, we used a
qRT-PCR-based chemokine array to analyze chemokine
gene expression in macrophages exposed to conditioned
media from B/B treated HC-11/R1 cells in the absence or
presence of exogenous TGFb. Several chemokines were
found to be either up- or downregulated in the macrophages
following exposure to these conditions (Table 1). The
chemokines were divided into 5 clusters based on the change
in their expression patterns. Although a number of genes
were either induced or repressed with B/B alone and with B/
B and TGFb combined, we focused on genes in cluster 1,
which were upregulated with B/B treatment and downregulated with the addition of TGFb. Interestingly, 4 of
these chemokines (Cxcl1, Cxcl2, Cxcl5, and Cxcl7) are
ligands for the Cxcr2 receptor. Before validating expression
levels of these chemokines, we conrmed that Cxcr2 is
expressed in the HC-11 mammary epithelial cell line by
immunoblot analysis (Fig. 3A). We validated the results
from the array using distinct primer sets to determine gene
expression and ELISAs to measure secreted proteins. We
observed similar gene and protein expression patterns for
both Cxcl1 and Cxcl5 (Fig. 3B). Induction of Cxcl2 and
Cxcl7 was not detected by ELISA (data not shown). These
data suggest that macrophages respond to FGFR1-induced
soluble factors by increasing the production of chemokines
that bind Cxcr2, primarily Cxcl1, and Cxcl5, and that this
induction can be inhibited by restoring TGFb signaling.

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Table 1. Macrophage chemokines regulated by

conditioned media from FGFR1-induced
mammary epithelial cells in the presence or
absence of TGFb

Gene ID
Cluster 1
CCL8
CCR1
CCR1/1
CXCL1
CXCL2
CXCL5
CXCL7
Cluster 2
Bmp6
CCL2
CCL20
Rgs3
Cluster 3
CX3CL1
Trem1
Cluster 4
Ccbp2
CCR8
Ecgf1
Cluster 5
CCR6
TNfsf14
Xcl1

Fold change

Fold change

B/B CM

B/B CMTGFb

3.9
1.86
2.04
1.44
1.79
3.25
1.47

0.93
0.863
0.833
0.81
0.716
1.85
0.94

0.65
0.172
0.429
0.425

1.07
0.776
1.18
2.02

1.76
2.32

3.15
8.05

0.39
0.54
0.42

0.3
0.53
0.55

3.14
1.97
1.82

3.75
1.73
2.02

Next, we wanted to determine the signaling pathway by

which the Cxcr2 ligands are regulated in the macrophages.
Expression of Cxcr2 ligands can be regulated by various
signaling pathways depending on cell type and stimulus,
including the NFkB and ERK pathways (24, 25). Interestingly, we were unable to detect an increase in NFkB activity
in macrophages exposed to conditioned media from B/Btreated HC-11/R1 cells (data not shown). Further analysis
focused on examining the ERK signaling pathway. For these
studies, RAW 264.7 cells were exposed to conditioned
media from HC-11/R1 cells treated with or without B/B.
Protein lysates were collected at different time points and the
expression of phosphorylated and total ERK1/2 was examined. There was a rapid induction of ERK1/2 activation in
macrophages treated with B/B conditioned media in comparison to solvent controls (Fig. 3C). Further studies were
done to determine the contribution of the ERK1/2 pathway
to expression of the Cxcr2 ligands. As shown in Fig. 3D,
blocking ERK activation with the MEK1 inhibitor U0126
led to a decrease in gene expression of the Cxcr2 ligands.
Inhibition of Cxcl1 was observed at the lowest concentration
of U0126, whereas there was a dose-dependent decrease in
Cxcl5 expression (Fig. 3D). These results suggest that
although both ligands are regulated by the ERK pathway,

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Figure 3. Activation of iFGFR1 in mammary epithelial cells leads to increased expression of Cxcr2-binding chemokines in macrophages. A, HC-11 cells were
treated with R1/RAW-CM with or without B/B. Whole cell lysates were collected and the expression of Cxcr2 and b-tubulin (loading control) was analyzed by
immunoblotting. B, RAW 264.7 cells were treated with R1-CM in the presence or absence of 10 ng/ml TGFb for 2 hours. Expression of the given
genes was determined by qRT-PCR and normalized to cyclophilin (left panel). For protein expression, RAW 264.7 cells were incubated with R1-CM for 2 hours
followed by serum free media for 24 hours. The conditioned media samples were analyzed using ELISAs (middle and right panel). C, RAW 264.7
cells were treated with R1-CM for the given time. Whole cell lysates were collected and the expression of pERK1/2 and ERK1/2 (loading control) was
analyzed by immunoblotting. D, RAW 264.7 cells were pretreated with DMSO (solvent control) or U0126 for 30 minutes and then treated for an
additional 2 hours with the same conditions in the presence of B/B R1-CM. The expression of the given genes was analyzed by qRT-PCR and normalized
to cyclophilin. Error bars represent SEM.  , P < 0.05;   , P < 0.01;    , P < 0.001.

they may be regulated via slightly different mechanisms.

Overall, these results indicate that the ERK pathway contributes to the regulation of Cxcr2 ligand expression.
Macrophages induce migration of epithelial cells, which
is blocked by Cxcr2 inhibition
To determine the effects of Cxcr2 ligand secretion from
macrophages, we developed a coculture migration assay (Fig.
4A). In this assay, conditioned medium from the HC-11/R1
cells containing FGFR1-induced soluble factors was added
to RAW 264.7 cells and allowed to incubate overnight. The
ability of the soluble factors collected from the treated RAW
264.7 cells to promote migration of parental HC-11 cells,
which do not respond to B/B treatment, was determined
using a transwell assay. The ability of macrophages to
promote epithelial cell migration following exposure to
conditioned media from cells with an activated FGFR1
reects the ability of the stimulated macrophages to act in
a tumor-promoting manner. Conditioned media from B/B
treated HC-11/R1 cells alone was able to induce migration
of HC-11 cells, suggesting that activation of FGFR1 in these
cells leads to the production of migratory factors (Fig. 4B).

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However, the conditioned media isolated from the treated

macrophages led to a signicant increase in migration of
HC-11 cells compared with conditioned media from the
HC-11/R1 cells alone (Fig. 4B). Therefore, treatment of
macrophages with conditioned media following FGFR1
activation leads to an increase in the ability of macrophages
to produce migratory factors. To determine whether the
increase in migration required Cxcr2, we further assessed the
effects of blocking Cxcr2 activity on HC-11 cell migration
using the Cxcr2-specic inhibitor SB225002. As shown
in Fig. 4B, the increase in migration was blocked when
SB225002 was incubated with the HC-11 cells in the
presence of the macrophage conditioned media. These
results show an important role for macrophage-derived
Cxcr2 ligands in the promotion of mammary epithelial cell
migration.
Macrophages promote invasion of primary mammary
epithelial cells in a 3D coculture assay
Primary mammary epithelial cells (MEC) form acinar-like
structures when grown in 3D culture and more closely
represent characteristics of mammary epithelium in vivo

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Cxcr2 Contributes to Macrophage-Induced Tumor Cell Migration

Figure 4. Cxcr2 inhibition leads to decreased macrophage-induced

migration of HC-11 cells. A, model of the migration coculture assay. B,
R1-CM, / B/B, was exposed to RAW 264.7 cells for 24 hours and the
conditioned media, R1/RAW-CM was used as the chemoattractant for a
transwell migration assay. HC-11 cells were plated on top of the insert
with ethanol (solvent control) or CXCR2 inhibitor (, 20 nmol/L and ,
200 nmol/L). After 18 hours, the migrated cells on the bottom of the insert
were stained with hematoxylin and counted. Error bars represent SEM.
 
, P < 0.001.

(21). We took advantage of the ability of primary MECs

isolated from MMTV-iFGFR1 transgenic mice to grow in
3D culture (21) and developed a coculture model to study
the interactions between MECs and macrophages. For these

studies, we isolated MECs from iFGFR1 transgenic mice

and plated them in growth factor reduced Matrigel. After 2
days, bone marrow derived macrophages (BMDM) that
were isolated from WT mice, were added to MECs. The
structures were grown in the presence or absence of B/B and
the CXCR2 inhibitor SB225002. After 10 days of treatment,
cells were xed and stained for epithelial (cytokeratin 8) and
macrophage (F4/80) markers. As shown in Fig. 5A, addition
of B/B led to larger MEC structures, consistent with previously published studies (21). Although coculture of
MECs with BMDM did not signicantly affect size of
the structures (data not shown), a signicant increase in
the number of invasive structures was observed (Fig. 5A
and B). With the addition of the CXCR2 inhibitor, macrophages were still recruited to the MECs (Fig. 5A), but the
percentage of invasive structures was signicantly inhibited
(Fig. 5A and B). These data suggest that Cxcr2 ligands
secreted from primary macrophages induce invasion of
primary MECs.
Human macrophages secrete CXCR2 ligands that
promote migration of human breast cancer cells
To verify the results of the mouse iFGFR1 system, we
used human breast cancer cell lines. We rst examined
the expression of CXCR2 in different breast cell lines:
normal (MCF10A), pre-invasive (MCF10DCIS.com and
SUM225), estrogen receptor positive (MCF-7 and T47D)
and triple negative (MDA-MB-231, 435A, and 468)

Figure 5. Macrophages induce

invasion of primary mammary
epithelial cells grown in 3D culture. A,
primary MECs from iFGFR1 mice
were grown in Matrigel in the
absence or presence of BMDM.
Every 2 to 3 days fresh media was
added with the treatments: 30
nmol/L B/B and 200 nmol/L CXCR2
inhibitor. After 10 days of treatment,
cells were xed and immunostained
with cytokeratin 8 (green) and F4/80
(red). DAPI (blue) labeled nuclei.
Arrows indicate invasive structures.
Left images light microscopy. Scale
bars 50 mm. B, for each treatment,
approximately 80 structures were
examined for invasion from 3
independent experiments. Error bars
represent SEM.  , P < 0.05;

, P < 0.01.

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Bohrer and Schwertfeger

the MCF-7 conditioned media was added to the human

monocyte cell line THP-1 that had been differentiated to
macrophages with PMA as described (27). After 2 and 4
hours, THP-1 cells were collected for gene expression
analysis. In addition to the Cxcr2 ligands in mice, human
cells also express CXCL8. Gene expression of all CXCR2
ligands, with the exception of CXCL7 (data not shown),
increased following exposure to conditioned media from
MCF-7 cells treated with bFGF (Fig. 6B). Further analysis of
protein expression showed signicant increases in secretion
of both CXCL1 and CXCL8 (Fig. 6B), whereas the other
ligands were not expressed at detectable levels by ELISA
(data not shown). bFGF treatment of THP-1 cells alone did
not induce expression of these chemokines, suggesting that
these results are not due to the direct action of bFGF on
macrophages (data not shown). The MCF-7/THP-1 conditioned media was used as the chemoattractant in a transwell migration assay to examine the migration of MCF-7
cells. Media from MCF-7 cells exposed to bFGF increased
migration compared to no treatment and there was an
additional increase when the media was also exposed to
THP-1 cells (Fig. 6C). Finally, migration was inhibited with
a CXCR2 inhibitor (Fig. 6C), showing that the CXCR2
ligands are important for macrophage-induced breast cancer
cell migration.

Figure 6. The migratory ability of MCF-7 cells increases with macrophage

secreted CXCR2 ligands. A, whole cell lysates were collected from the
different cell lines and equal amounts were used for immunoblot analysis.
The expression of CXCR2 was determined and the membrane was
stained with Ponceau S for a loading control. B, THP-1 cells were
exposed to conditioned media from MCF-7 cells treated with 50 ng/mL
bFGF or no treatment (NT). Cells were collected after 2 or 4 hours, and
RNA was isolated for qRT-PCR for the given genes that were normalized
to cyclophilin (upper panel). For protein expression, THP-1 cells were
incubated with MCF-CM for 2 hours and then serum free media for 24
hours. The conditioned media was analyzed using ELISAs (lower panel).
C, MCF-CM, / bFGF, was exposed to THP-1 cells for 24 hours, and
the conditioned media, MCF/THP-CM was used as the chemoattractant
for a transwell migration assay. MCF-7 cells were plated on top of the
insert with ethanol (solvent control) or CXCR2 inhibitor (, 100 nmol/L;
, 200 nmol/L; and , 400 nmol/L). After 18 hours, the migrated cells
on the bottom of the insert were stained with hematoxylin and counted.
Error bars represent SEM.  , P < 0.05;   , P < 0.01;    , P < 0.001.

(Fig. 6A). As MCF-7 cells express CXCR2 and they have

previously been shown to respond to FGF treatment (26),
we chose to use these cells to determine the ability of bFGF
stimulation to promote CXCR2 ligand induction in macrophages. MCF-7 cells were treated with or without bFGF and

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Discussion
Recent studies have showed that FGFR signaling contributes to breast cancer growth and resistance to conventional therapies (12, 13). Our studies focus on understanding how activation of FGFR1 in tumor cells leads to
protumorigenic alterations in the tumor microenvironment.
We describe here a novel mechanism of paracrine interaction
between tumor cells and macrophages that is driven by
FGFR1 activation in the tumor cells and chemokine expression in the macrophages. We have previously showed that
activation of iFGFR1 in the HC-11/R1 cells results in the
induction of a number of secreted factors that can inuence
macrophage recruitment (16) and cytokine production (17).
The studies described here further analyze the mechanisms
through which macrophages contribute to early stage tumorigenesis. Although carrying out coculture studies, we found a
number of genes to be regulated in an expected manner
based on published studies of gene expression in tumor
associated macrophages, including increased ArgI and IL-10
and decreased IL-12 (28, 29). Interestingly, however,
decreased expression of TGFb gene expression was consistently observed in the macrophages both in vivo and in vitro,
which is not consistent with the phenotype of macrophages
associated with late stage tumors (30). Although levels of
TGFb protein were not detectable by ELISA in the in vitro
coculture model (data not shown), a decrease in Smad3
activity was observed in the macrophages, consistent with
the decrease in pSmad3 observed in vivo. Together these
studies show an overall decrease in the TGFb/Smad3 signaling pathway in macrophages in response to factors from
FGFR1-driven mammary tumor cells. The specic factors
and mechanisms responsible for this decrease are likely

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Cxcr2 Contributes to Macrophage-Induced Tumor Cell Migration

complex and remain to be determined. Early in mammary

tumor formation, TGFb is known to be growth suppressive
(8), so it is possible that decreased TGFb in the microenvironment, either from epithelial cells, inltrating macrophages or other stromal cells, is critical for the development
of early stage lesions. Consistent with this, we found that
adding exogenous TGFb to the media of primary MECs in
3D culture completely inhibited iFGFR1-induced acinar
growth (Supplementary Fig. 2). These results suggest that in
the early stages of tumor formation, the presence of TGFb
producing macrophages, such as those associated with the
promotion of late-stage tumors, might actually result in
tumor inhibition. Therefore, the association of macrophages
with decreased levels of TGFb expression and pathway
activity, as observed in our studies, may be important for
successful early-stage tumor formation.
Our results show that decreased expression of genes
associated with the TGFb pathway correlates with increased
expression of inammatory chemokines. Furthermore, restoration of TGFb signaling with exogenous TGFb inhibits
the expression of these chemokines. An inverse correlation
between the TGFb pathway and expression of inammatory
chemokines has been observed in other studies. For example,
decreased TGFb responsiveness in prostatic broblasts
caused an upregulation of chemokines including CXCL1,
leading to the adhesion of prostate cells to the bone matrix
(31). Also, loss of TGFb signaling in mammary broblasts
resulted in increased production of the proinammatory
chemokine Ccl2, which contributed to growth and metastasis of 4T1 mammary tumors (32). In addition, recent
studies using a mouse model of pancreatic ductal adenocarcinoma also showed an increase in Cxcr2 ligands associated
with loss of Tgfbr2 in the pancreas (24). Interestingly, these
studies showed that induction of the Cxcr2 ligands was
dependent on the NFkB pathway, whereas induction in
macrophages in our model is dependent upon the ERK
signaling pathway. These results possibly represent cell-type
specic differences in CXCR2 ligand regulation. Taken
together, our results, along with published studies, show
that decreased TGFb signaling is associated with increased
expression of inammatory chemokines, and that these
chemokines are capable of contributing to tumor formation
and progression.
Interactions between chemokines and chemokine receptors are known to contribute to breast cancer progression
(33). Recent studies have implicated CXCR2 and its
various ligands in breast cancer. A number of studies have
focused on CXCL8, also known as IL-8, which binds to
both CXCR1 and CXCR2 and appears to be the primary
CXCR2 binding chemokine induced in response to
bFGF-treated breast cancer cells (Fig. 6B). Expression of
CXCL8 has been linked to increased tumor grade and
experimental studies have showed its ability to regulate
angiogenesis and tumor progression (3335). CXCR2
expression itself has been detected on breast cancer cells
(34) and inhibition of CXCR2 decreased mammary tumor
cell invasion in vitro (36). Furthermore, knockdown of
CXCR2 in metastatic mammary tumor cells led to

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decreased metastasis in orthotopic transplantation models

(36). The CXCR2 ligand CXCL3 was identied in a
screen for genes associated with basal-like breast cancers
and a CXCR2 inhibitor was shown to decrease viability of
basal-like breast cancer cell lines in vitro (37). Furthermore, polymorphisms in the CXCR2 gene have been
identied in breast cancer patients and these polymorphisms correlated with larger tumor size, higher tumor grade,
and increased lymph node metastasis (38). However, the
functional consequences of this polymorphism on
CXCR2 expression and/or activity remain to be determined. Our studies show that activation of epithelial cellspecic CXCR2 by chemokine-producing cells within the
tumor stroma may play a role in early stages of tumorigenesis. Taken together, these studies suggest that the
CXCR2 axis may represent a viable pathway to target
during both early and late stage tumor development.
Our studies have focused on the use of mammary epithelial cells expressing an iFGFR1 construct. To show that
the results were not specic to the iFGFR1 model, we
validated our results using the MCF-7 cell line, which has
been previously used to study endogenous FGF signaling
(26, 39, 40). Similar to the results obtained with the iFGFR1
model, activation of the FGF signaling pathway in MCF-7
cells also led to an increase in CXCR2 ligand gene expression
in macrophages. Interestingly, FGFR1 activity has been
shown to promote resistance of estrogen receptor positive
breast cancer cells to endocrine-based therapies (13).
Although it has been reported that estrogen receptor positive
cells, including MCF-7, express lower levels of CXCR2 than
more invasive breast cancer cell lines (34), our studies as well
as another report (41) suggest that estrogen receptor positive
cells express the same or even more CXCR2 than more
invasive breast cancer cell lines. Furthermore, our studies
show that MCF-7 cells are capable of responding to CXCR2
ligands in chemotaxis assays and that inhibition of CXCR2 is
sufcient to inhibit migration towards the stimulated THP1 cells. These results suggest that activation of the FGF
pathway may contribute to enhancement of tumor progression by regulating the expression of tumor-promoting
chemokines in inltrating immune and other stromal cells.
In addition, it is possible that FGF signaling in tumor cells
may contribute to breast cancer growth and therapeutic
resistance by regulating both the tumor cells and the
microenvironment.
Although some studies have showed that breast cancer
cells produce CXCR2 ligands, which feed back to regulate
tumor cell activity in an autocrine manner, our studies show
that cells within the stroma might also represent an important source of CXCR2 ligands. Similarly, studies by Halpern
and colleagues showed that mesenchymal stem cells are also a
source of CXCR2 ligands, which are capable of promoting
migration of cells in vitro and suggest that CXCR2 ligands
may be important for homing of tumor cells to bone (42).
Our results show that the production of CXCR2 ligands is
enhanced in macrophages in response to soluble factors
released from transformed mammary epithelial cells and
that these factors are then capable of promoting migration

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Bohrer and Schwertfeger

and invasion of noninvasive cells. These results offer a novel

mechanism by which macrophages associated with the
invasive edges of developing tumors might promote invasion
through the basement membrane. Together, these results
suggest that targeting CXCR2 in breast cancer patients may
be effective at both early and late stages of tumor formation
and progression.
In summary, our studies have focused on the effects of
tumor cell-specic FGFR1 activation on alterations within
the tumor microenvironment. We have previously showed
a rapid inammatory response characterized by recruitment of macrophages to the epithelial structures (16). We
have also showed that anti-inammatory drugs are capable
of inhibiting the initiation of FGFR1-driven epithelial
lesions, showing that inammation is an important promoter of FGFR1-driven tumorigenesis (17). Using the
iFGFR1 model, we have developed novel coculture models to identify specically how FGFR1-driven tumor cells
communicate with the microenvironment. We have identied a paracrine mechanism in which soluble factors from
the FGFR1-activated cells lead to increased production of
CXCR2 ligands, which then feed back to promote migration and invasion of the tumor cells. Importantly, these
ndings were validated using an FGF-responsive breast
cancer cell line, showing that activation of endogenous
FGF signaling leads to similar results as the iFGFR1
model. These results suggest that in FGF-driven breast
cancers, targeting CXCR2 activity, possibly in conjunction with specically targeting FGFR activity, may lead to
an effective therapeutic strategy. Further studies are

required to determine which patients might benet from

this type of therapy.
Disclosure of Potential Conicts of Interest
No potential conicts of interest were disclosed.

Authors' Contributions
Conception and design: L.R. Bohrer, K.L. Schwertfeger
Development of methodology: L.R. Bohrer, K.L. Schwertfeger
Acquisition of data (provided animals, acquired and managed patients, provided
facilities, etc.): L.R. Bohrer, K.L. Schwertfeger
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L.R. Bohrer, K.L. Schwertfeger
Writing, review, and/or revision of the manuscript: L.R. Bohrer, K.L. Schwertfeger
Administrative, technical, or material support (i.e., reporting or organizing data,
constructing databases):
Study supervision: K.L. Schwertfeger
Performed the cell culture 2D and 3D coculture assays: L.R. Bohrer
Performed the in vivo macrophage isolation and expression studied: K.L.
Schwertfeger

Acknowledgments
We would like to thank Dr. Jeff Rosen for providing reagents and advice for these
studies and Dr. Fariba Behbod for providing reagents used in this study. Also, we
would like to thank Johanna Reed and Lindsey Bade for critical reading of the
manuscript. In addition, we would like to acknowledge the use of the confocal
microscope at the Masonic Cancer Center made available through an NCRR Shared
Instrumentation Grant (#1 S10 RR16851).

Grant Support
Funding for these studies was provided by grants from the American Cancer Society
(RSG-09-192-01-LIB) and NCI (1R01CA132827) to KLS.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Received May 3, 2012; revised July 5, 2012; accepted July 18, 2012;
published OnlineFirst August 14, 2012.

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