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Carcinogenesis vol.32 no.10 pp.1441–1449, 2011
doi:10.1093/carcin/bgr134
Advance Access publication July 18, 2011
Deletion of cyclooxygenase 2 in mouse mammary epithelial cells delays breast cancer
onset through augmentation of type 1 immune responses in tumors
Nune Markosyan, Edward P.Chen, Victoire N.Ndong,
Yubing Yao, Christopher J.Sterner1, Lewis A.Chodosh1,
John A.Lawson, Garret A.FitzGerald and Emer M.Smyth
Department of Pharmacology, University of Pennsylvania, Institute for
Translational Medicine and Therapeutics, Philadelphia, PA 19104, USA and
1
Department of Cancer Biology, University of Pennsylvania, Abramson
Family Cancer Research Institute, Philadelphia, PA 19104, USA
To whom correspondence should be addressed. University of Pennsylvania,
421 Curie Boulevard, 808 Biomedical research Building II/III, Philadelphia,
PA 19104, USA. Tel: þ215 573 2323; Fax: þ215 573 9004;
Email: [email protected]
Inhibition of cyclooxygenase (COX) 2, which is associated with
>40% of breast cancers, decreases the risk of tumorigenesis and
breast cancer recurrence. To study the role of COX-2 in breast
cancer, we engineered mice that lack selectively mammary epithelial cell (MEC) COX-2 (COX-2 KOMEC). Compared with wild type
(WT), MEC from COX-2 KOMEC mice expressed >90% less COX2 messenger RNA (mRNA) and protein and produced 90% less of
the dominant pro-oncogenic COX-2 product, prostaglandin (PG)
E2. We confirmed COX-2 as the principle source of PGE2 in MEC
treated with selective COX-2 and COX-1 inhibitors. Tumors were
induced in mice using medroxyprogesterone acetate and 7,12-dimethylbenz[a]anthracene. Breast cancer onset was significantly delayed in COX-2 KOMEC compared with WT (P 5 0.03), equivalent
to the delay following systemic COX-2 inhibition with rofecoxib.
Compared with WT, COX-2 KOMEC tumors showed increased
mRNA for Caspase-3, Ki-67 and common markers for leukocytes
(CD45) and macrophages (F4/80). Analysis of multiple markers/
cytokines, namely CD86, inducible nitric oxide synthase (iNOS),
interleukin-6, tumor necrosis factor a (TNFa) and Tim-3 indicated
a shift toward antitumorigenic type 1 immune responses in COX-2
KOMEC tumors. Immunohistochemical analysis confirmed elevated
expression of CD45, F4/80 and CD86 in COX-2 KOMEC tumors.
Concordant with a role for COX-2 in restraining M1 macrophage
polarization, CD86 and TNFa expression were offset by exogenous
PGE2 in bone marrow-derived macrophages polarized in vitro to
the M1 phenotype. Our data reveal the importance of epithelial
COX-2 in tumor promotion and indicate that deletion of epithelial
COX-2 may skew tumor immunity toward type 1 responses, coincident with delayed tumor development.
Introduction
Cyclooxygenase (COX)-2, the inducible COX isoform, has been convincingly linked with colon, prostate, lung and breast cancer (1,2) across
basic, animal and human studies. In breast tumors, increased COX-2
expression correlates with parameters of aggressiveness and poor outcome (3). Inhibition or deletion of COX-2 reduced tumor incidence in
animal models of mammary tumorigenesis (4–6), whereas targeted overexpression of COX-2 to the mammary epithelium was sufficient to
induce tumor formation (7). Concordantly, preventive effects of nonsteroidal anti-inflammatory drugs, which inhibit both the COX-1 and
COX-2 isoforms (8), or COX-2 selective inhibitors (1,9) have been
consistently reported. These and other studies have raised interest in
Abbreviations: BMDM, bone marrow-derived macrophages; COX, cyclooxygenase; LPS, lipopolysaccharide; MEC, mammary epithelial cell; mmtv,
mouse mammary tumor virus; mRNA, messenger RNA; PCR, polymerase
chain reaction; PG, prostaglandin; Q-PCR, quantitative polymerase chain reaction; TAM, tumor-associated macrophages; TNFa, tumor necrosis factor a;
WT, wild type.
targeting COX metabolic pathways, COX-2 in particular, for chemoprevention and/or therapy. Indeed, celecoxib, a selective COX-2 inhibitor, is approved for the prevention and treatment of familial adenomatous
polyposis, a condition that frequently proceeds to colon cancer (10).
However, clinical use of the COX-2 inhibitors in chemoprevention
and/or therapy is limited because of an increased cardiovascular risk
associated with this class of drugs (11,12).
COX-2 uses arachidonic acid as a substrate to catalyze the production of the prostaglandins (PGs) and thromboxane A2. PGE2, a ubiquitous and well-studied product of COX-2, has proven pro-oncogenic
roles in many tissues (1). Thus, PGE2 enhanced intestinal adenoma
growth via activation of the Ras–MAPK cascade (13), whereas cell
migration was stimulated through activation of the epidermal growth
factor receptor (14). PGE2 also promoted survival and growth of human colon cancer cells via induction of the anti-apoptotic protein Bcl-2
(15) and the Gs–axin–ß-catenin signaling axis (16). Recent studies in
mice with enhanced mammary COX-2 expression established the role
of PGE2 in the ’angiogenic switch’ that is necessary for cancer
development, growth and invasion (17).
Primary tumor cells are surrounded and strongly influenced by stromal cells, including fibroblasts, adipocytes and immune cells, and a supportive microenvironment appears critical for the tumor to progress
toward malignancy (18,19). Environmental cues, tumor-derived stimuli
and contact with tumor cells direct stromal cell differentiation and function (20,21). COX-2 contributes to this epithelial-stromal interplay. For
example, the pro-tumorigenic effects of fibroblasts and macrophages
have been linked to COX-2 upregulation in tumor cells (22,23), whereas
COX-2-derived PGE2, in turn, contributed to matrix remodeling and
suppressed immune surveillance (24). Tumor-associated immune responses can be generalized to type 1, in which Th1 lymphocytes and
M1-polarized macrophages limit tumor progression, and type 2, in
which Th 2 lymphocytes and M2 macrophages favor immune escape
and disease progression (25). PGE2 has been reported to augment protumorigenic type 2 lymphocyte and myeloid cell functions (26,27).
However, the precise contribution of COX-2 to tumor-immune responses
within the microenvironment remains ill-defined.
Strategies that interrupt global COX-2 function, either via inhibitors
or systemic deletion of the COX-2 gene, make it difficult to decipher its
role in orchestration of tumor cell’s interaction with its microenvironment. In the present study, we report generation of mice that lack COX-2
only in mammary epithelium. Selective deletion of mammary epithelial
COX-2 was sufficient to delay the onset of breast cancer in mice and
equivalent in benefit to systemic COX-2 inhibition. This was associated
with elevated expression of type 1 inflammatory and immune cell
markers/cytokines, indicating a role for epithelial COX-2 in limiting
antitumorigenic immune responses in mammary tumors.
Materials and methods
Mice
All procedures involving mice were conducted in accordance with National
Institutes of Health regulations and were approved by the Institutional Animal
Care and Use Committee of the University of Pennsylvania.
Floxed COX-2 mice, generated by flanking the region of the COX-2 gene
between introns 5 and 8 with loxP sites (COX-2flox/flox) (28), were backcrossed
fully (.9 generations) onto an FVB background (29). COX-2flox/flox mice were
crossed with FVB mice expressing Cre-recombinase under control of the
mouse mammary tumor virus (mmtv) promoter (Cremmtv), which is used
widely to target transgene expression to the mammary epithelial cells
(MEC) (29). The resulting mice were termed COX-2 KOMEC.
Genotyping
Genomic DNA was extracted from mouse tails and used for polymerase chain
reaction (PCR). Primers are listed in supplementary Materials and Methods
(available at Carcinogenesis online).
Ó The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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MEC, peritoneal macrophage and bone marrow-derived macrophage isolation
and culture
Mouse MEC were isolated and cultured according to the protocol from StemCell
Technologies (Vancouver, BC Canada). Reagents used were from StemCell Technologies, unless otherwise stated. Cells were plated on 6- or 12-well plates (50 000
or 100 000 cells/well, respectively) in EpiCult-B medium, supplemented with
recombinant human epidermal growth factor, recombinant human basic fibroblast
growth factor, Heparin and 5% fetal bovine serum; 24 h later, cells were transferred to serum-free EpiCult-B medium, to prevent overgrowth by stromal cells.
Cells, at 80% confluency, were or were not treated with 5 lg/ml lipopolysaccharide (LPS; Sigma, St Louis, MO) for 24 h. For inhibitor studies, rofecoxib (COX-2
selective inhibitor, 107 M; Sequoia Research, Berkshire, UK) or FR122047
(COX-1 selective inhibitor, 107 M; Cayman Chemicals, Ann Arbor, MI) were
added at the beginning of the LPS treatment. RNA was extracted from cells in 12well plates (see below); 1 ml conditioned medium from each well of the six-well
plates was stored at 80°C for prostanoid measurements, and cells were lysed
with radio-immunoprecipitation assay buffer (Sigma) for western blotting.
Ice-cold sterile phosphate-buffered saline was injected into the peritoneal
cavity for peritoneal macrophage collection. Peritoneal fluid was collected
through a small midline incision. After centrifugation, cells were resuspended
and plated on a 60-mm dish in 3 ml RPMI1640 medium (Invitrogen) with 10%
fetal bovine serum, 1% Pen/Strep and 1% L-glutamate. After an overnight incubation at 37°C, the cells were placed in serum-free medium for 24 h and then
treated with or without 5 lg/ml LPS for an additional 24 h. Media were harvested
for prostanoid measurements. Cells were lysed for RNA and protein extraction.
Bone marrow-derived macrophages (BMDM) were isolated as described
(30). Femurs and tibias were removed from female mice and flushed with
DMEM. Cells were pelleted (300g) and incubated at 37°C for 24 h, in DMEM
medium containing 10% fetal bovine serum, 1% Pen/Strep and 1% L-glutamate.
L929 cells (American Type Culture Collection, Manassas, VA) were plated in
DMEM; 24 h later, L929 cell conditioned medium was collected and mixed 1:5
with DMEM (L-cell conditioned medium). Non-adherent cells from BMDM
cultures were collected and plated in L-cell conditioned medium. Purity
(99%) was verified by flow cytometry for F4/80 and CD11b (data not shown).
Once confluent, conditioned media were collected and BMDM were detached
using 10 mM lidocaine in DMEM and lysed for RNA extraction. Conditions for
polarization of BMDM are described below.
Measurements of eicosanoids
PGs and their metabolites were measured by ultra high-pressure liquid chromatography/tandem mass spectrometry, using negative electrospray ionization
and multiple reaction monitoring (MRM) techniques, as described previously
(31). Additional details are given in the supplementary Materials and Methods
(available at Carcinogenesis online).
Real-time RT-PCR
Total RNA from MEC, macrophages and tumors was isolated (RNeasy; Qiagen,
Germantown, MD) and reverse transcribed (TaqMan Reverse Transcriptase;
Applied Biosystems, Carlsbad, CA), according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction (Q-PCR) of all genes,
including 18S ribosomal RNA, was performed using inventoried gene expression assays and TaqMan Universal PCR Master Mix from Applied Biosystems.
PCR products were detected in ABI-PRISM 7900 sequence detection systems
(Applied Biosystems). Results were analyzed using the comparative Ct method
(32) and normalized to 18S RNA.
Western blotting
MEC and macrophages were lysed and homogenized in radio-immunoprecipitation
assay buffer. Proteins were quantified (Pierce BCA Assay; Thermo Scientific,
Rockford, IL), resolved by SDS-PAGE (4–12% Bis-Tris gel; Invitrogen, Carlsbad,
CA) and electrophoretically transferred to nitrocellulose membranes. After blocking, the membranes were incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Immune complexes were
detected by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ). Details of the antibodies used are given in the supplementary Methods (available at
Carcinogenesis online).
Tumor induction
Subcutaneous inter-scapular implants of medroxyprogesterone pellets (75 mg/
pellet, 21 day release; Innovative Research of America, Sarasota, FL) were
administered to 6-week-old virgin female mice. Starting at 9 weeks, 1 mg/100
ll of 7,12-dimethylbenz[a]anthracene (Sigma) in sesame oil was given, via
gavage, weekly for 4 weeks. This regime causes predominantly mammary
tumors (33). Mice were fed either with regular chow, or starting from the
implant date, diets (Harlan Laboratories, Inc., Madison, WI) containing 10
mg/kg/day rofecoxib or 3 mg/kg/day SC560 (COX-1 selective inhibitor; Tocris
Biosciences, Ellisville, MO). Mice were checked weekly and considered tumor
bearing if a palpable mammary mass persisted for .1 week.
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Immunocytochemistry and immunohistochemistry
MEC cultured in Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) were fixed with 10% buffered formalin, blocked with 3% donkey
serum (Jackson Immunoresearch, West Grove, PA) and incubated overnight
with primary antibodies followed by a fluorophore-conjugated secondary antibody (as detailed in supplementary Materials and Methods, available at Carcinogenesis online). Cells were visualized by fluorescent microscopy.
For immunohistochemistry, after deparaffinization and rehydration, endogenous peroxidase was blocked with 3% hydrogen peroxide, on 5-lm thick
tumor sections. Heat-induced epitope retrieval was performed with 1 mM
EDTA. Primary antibody treatments were for 1 h at 37°C, followed by immune
complex visualization using the Polink-2 HRP Plus AEC System (Golden
Bridge International, Inc., Mukillteo, WA). Additional details are given in
supplementary Materials (available at Carcinogenesis online).
BMDM polarization
BMDM were plated (0.5 106 cells/well) in L-cell conditioned medium, which
was replaced, 24 h later, with BMDM medium. After a further 24 h, cells received
either vehicle or M1 polarizing mix of 100 ng/ml LPS (Sigma) and 20 ng/ml
interferon c (Peprotech, Rocky Hill, NJ) with or without 10, 250 and 1000 nM
PGE2 (Cayman Chemicals). Supernatants were removed, 18 h later, and cells
were lysed for RNA isolation (see above). Tumor necrosis factor a (TNFa) in
supernatants was measured by ELISA (R&D Systems, Minneapolis, MN).
Statistical analysis
Statistical analyses were performed using Prism (GraphPad Software, Inc., La
Jolla, CA). As appropriate, comparisons were made using Mann–Whitney test,
log-rank analysis, Student’s t-test, or, for multiple group comparisons, analysis
of variance followed by Dunnet’s multiple comparison test.
Results
Characterization of COX-2 KOMEC mice
Mouse genotypes—loxP null (COX-2þ/þ), heterozygous (COX-2flox/þ)
or homozygous (COX-2flox/flox), and Cremmtv positive or negative—were
determined by PCR (Figure 1A). COX-2flox/flox mice lacking Cre expression (Figure1A, lane 4) were considered wild type (WT), whereas
COX-2flox/flox mice positive for Cremmtv (Figure 1A, lane 3) were considered COX-2 KOMEC. We estimated that .95% of cells cultured from
the single-cell suspensions obtained from the mammary glands of WT
and COX-2 KOMEC mice expressed either a luminal epithelial cell
marker CK18 or a basal/myoepithelial cell marker CK14, or both, (Figure 1B) and were negative for a fibroblast marker vimentin (supplementary Figure 1 is available at Carcinogenesis online), confirming their
predominantly epithelial identity. COX-2 expression in cultured MEC
appeared to be constitutive and marginally responsive to LPS induction
(Figure 1C). This was in contrast to peritoneal macrophages in which
COX-2 expression was strongly induced by LPS treatment (Figure 1E).
In MEC from COX-2 KOMEC mice, COX-2 messenger RNA (mRNA)
and protein expression were dramatically decreased under vehicle or
LPS-treated conditions (Figure 1C and D, left panels). COX-1 mRNA
and protein were unaltered (Figure 1C and D, right panels). Importantly,
COX-2 and COX-1 expression was not different between peritoneal
macrophages obtained from WT and COX-2 KOMEC mice, with or
without LPS (Figure 1E). COX-2 protein in peritoneal macrophages
similarly was not affected by COX-2 deletion in MEC (Figure 1F).
These data confirmed selective loss of COX-2 in MEC.
Genomic or pharmacological inhibition of PGE2 production delays
mammary tumor onset
PGE2 in conditioned media from vehicle or LPS-treated WT MEC
exceeded other COX-2 products by 100-fold (Figure 2A). Genomic
deletion of COX-2 in MEC led to a 90% reduction in PGE2 production
(Figure 2B), despite the presence of COX-1 (Figure 1D). Similarly,
treatment with COX-2 selective inhibitor rofecoxib markedly decreased
PGE2 production by untreated or LPS-treated MEC (Figure 2B). In
contrast, the COX-1 selective inhibitor FR122047 did not affect
PGE2 production, whether alone or in combination with rofecoxib,
in MEC from WT or COX-2KOMEC mice. Together these data indicate
that COX-1 does not contribute to PGE2 generation in MEC. Systemic
COX function, as indicated by urinary prostanoid metabolite levels,
was unaltered in COX-2 KOMEC mice compared with WT (Figure 2C).
Deletion of mammary epithelial COX-2 delays tumors
Fig. 1. MEC-specific COX-2 gene deletion. (A) PCR detection of COX-2þ/þ (lane 1), COX-2flox/þ (lane 2), COX-2flox/flox (lanes 3 and 4), Cremmtv/ (lower panel,
lanes 1 and 4) and Cre mmtvþ/ (lower panel, lanes 2 and 3) in tail DNA. (B) Immunocytochemistry for CK14 (myoepithelial marker, red) and CK18 (luminal
epithelial marker, green) in MEC isolated from WT and COX-2 KOMEC (KO) mice. Background staining in the absence of primary antibody is shown in KO cells
(inset) and was similarly negative in WT cells. Nuclei are stained blue with DAPI. Data are representative of similar results from n 5 3. (C) Real-time Q-PCR
quantification of COX-2 (left panel) and COX-1 (right panel) mRNA expression in untreated and LPS-treated MEC isolated from WT or COX-2 KOMEC (KO) mice
(n 5 6). (D) COX-2 protein in untreated or LPS-treated MEC (left panel) or COX-1 protein in LPS-treated MEC (right panel) from WT and COX-2 KOMEC (KO)
mice. (E) COX-2 (left panel) and COX-1 (right panel) mRNA in untreated and LPS-treated peritoneal macrophages from WT and COX-2 KOMEC (KO) mice
(n 5 5–6). (F) COX-2 protein in LPS-treated peritoneal macrophages from WT and COX-2 KOMEC (KO) mice. In (C) and (E) data are mean ± SE. P , 0.005.
Despite unchanged systemic PGE2 levels, mammary tumor onset was
delayed by 8 weeks in COX-2 KOMEC mice (Figure 2D-1). In the WT
group, treatment with the COX-2 selective inhibitor rofecoxib, but not
the COX-1 selective inhibitor SC560, similarly delayed tumorigenesis
(Figure 2D-2). Pharmacological inhibition of either COX isoform did
not modify the delayed tumor onset in COX-2 KOMEC mice (Figure
2D-3). These data indicate a dominant role for epithelial COX-2 in
mammary tumorigenesis in this model.
Proliferative, apoptotic, angiogenic and extracellular matrix
remodeling pathways in mammary tumors from WT and COX-2
KOMEC mice
To identify the potential mechanism through which COX-2 in MEC
promotes mammary tumorigenesis, we next examined expression of
a range of genes in WT and COX-2 KOMEC tumors. Messenger RNA
levels for both the apoptotic enzyme Caspase 3 and a proliferation
marker, Ki67, were increased in tumors from COX-2 KOMEC mice
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(Figure 3A and B), suggesting an increase in cell turnover in KO
tumors but not indicating a mechanism for the delay in tumorigenesis in these animals. No changes were detected in the expression of
the endothelial marker CD31, either by Q-PCR (Figure 3C) or immunohistochemistry (Figure 5K and L), two angiogenic factors vascular endothelial growth factor (VEGF) A and VEGFC (Figure 3D
and E) or their receptors VEGFR 1, 2 and 3 (Figure 3F–H), suggesting that modified angiogenesis did not underlie reduced disease in
COX-2 KOMEC animals. Similarly, matrix metalloproteinase enzymes 2 and 9 were unaltered in COX-2 KOMEC compared with
WT (Figure 3I and J), indicating that deletion of COX-2 only in
MEC did not alter extracellular matrix remodeling.
Fig. 2. Inhibition of PGE2 production in MEC delays tumor onset in mice. (A) Prostanoids in conditioned media from cultured MEC treated with or without LPS
(n 5 6). (B) PGE2 in conditioned media from MEC obtained from WT and COX-2 KOMEC (KO) mice and incubated with either rofecoxib, or FR12207, or both,
with or without LPS (n 5 4–6). (C) Urinary prostanoid metabolites measured in WT and COX-2 KOMEC (KO) disease-free 6-week-old female mice (n 5 10–12).
For (A–C), data are mean ± SE, P , 0.05, P , 0.0002. (D) Percent of tumor-free WT and COX-2 KOMEC (KO) mice on regular chow (1, n 5 24–30) or chow
containing rofecoxib (2, n 5 17) or SC560 (3, n 5 13–14).
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Deletion of mammary epithelial COX-2 delays tumors
COX-2 deletion in MEC modifies the tumor-immune
microenvironment
We observed, by Q-PCR and immunohistochemistry, significant increases in expression of a common leukocyte marker, CD45 (Figures
4A-1 and 5E and F), and a common macrophage marker, F4/80 (Figures 4A-2 and Fig. 5G and H) in COX-2 KOMEC tumors compared with
WT. Macrophages can be anti- (M1 phenotype) or pro- (M2 phenotype)
tumorigenic, with tumor-associated macrophages (TAM) displaying
predominantly M2 characteristics; thus, we examined a range of phenotypic macrophage markers in WT and COX-2 KOMEC tumors. Expression of M2-type macrophage markers Scaf1 (Figure 4B-1) and
Arginase-1 (Figure 4B-2), as well as the M2-type cytokine transforming growth factor b (Figure 4B-3), were not different between WT and
COX-2 KOMEC tumors. Another M2-type macrophage cytokine, interleukin-10, was low/undetectable in tumors from both animal groups. In
contrast, expression of the M1-type marker CD86 was increased in
COX-2 KOMEC tumors as assessed by Q-PCR (Figure 4C-1) and immunohistochemistry (Figure 5I and J). Similarly, the M1 macrophage
cytokine TNFa was significantly higher in COX-2 KOMEC tumors
(Figure 4C-2). Two additional characteristic M1 macrophage-associated genes, inducible nitric oxide synthase and interleukin-6, were not
significantly altered (Figure 4C-3 and 4C-4) although interleukin-6
correlated strongly with CD86 or F4/80 in COX-2 KOMEC, but not
WT, animals (supplementary Figure 2 is available at Carcinogenesis
online). Taken together, these data are consistent with polarization
of macrophages associated with COX-2 KOMEC tumors toward the
antitumorigenic M1 phenotype.
We also examined markers for other tumor-associated immune
cells. Significantly higher levels of CD2, a shared marker for T-lymphocytes and natural killer (NK) cells, were evident in COX-2 KOMEC
tumors (Figure 4D-1). No significant changes were seen in expression
of the NK-specific marker CD49b (Figure 4D-2), the regulatory T cell
marker CD25 (Figure 4D-3) or the cytotoxic T cell marker CD8
(Figure 4D-4). In contrast, CD4 was increased in COX-2 KOMEC
tumors (Figure 4E-1), indicating increased presence of CD4expressing cells. Among CD4þ cells, Th1 cells are known to direct
macrophage polarization toward M1 (34) leading us to speculate that
the apparent increase in M1-type responses in COX-2 KOMEC tumors
could be secondary to the influence of CD4þ Th1-derived mediators.
Indeed, although expression of Th1 markers, TIM3 and TRANCE
(35,36) was not significantly different in tumors from COX-2 KOMEC
mice compared with WT (Figure 4E-2 and E-3), TIM-3 levels showed
almost perfect correlation with both F4/80 and CD86 in COX2MECKO, but not WT, tumors (Figure 4F). Together, these data are
consistent with a shift toward type 1 innate and adaptive immune
responses in COX-2MECKO tumors.
PGE2 limits in vitro M1 Polarization of BMDM
Our study suggests that delayed tumorigenesis in COX-2MECKO mice
is at least partially due to interruption of the limit placed by COX-2derived PGE2 on type 1 macrophage polarization. We examined
whether PGE2 modifies M1 polarization of isolated macrophages.
As expected, compared with non-polarized cells, significant increases
in mRNA for CD86 and TNFa were evident in M1-polarized BMDM
(Figure 6A). PGE2 did not significantly alter M1 markers in nonpolarized cells (supplementary Figure 3 is available at Carcinogenesis
online). The M2 marker Arginase-1 expression was at or below the
detection level in control and PGE2-treated non-polarized BMDM.
However, concordant with our in vivo data, when PGE2 was included
at the beginning of the polarization procedure, the M1-associated
CD86 and TNFa mRNA expression, as well as TNFa protein secretion, were substantially reduced in dose-dependent manner consistent
with a PGE2-mediated restraint of M1 polarization (Figure 6B and C).
Discussion
Tumorigenesis involves a complex and intricate interplay between
tumor and stromal cells (21). Although multiple pro-tumorigenic
events are associated with COX-2 (2,37), the contribution of COX2-derived prostanoids to autocrine and paracrine signaling in tumor
cells and the surrounding microenvironment are poorly understood.
We used a novel model of COX-2 deletion in mammary epithelium to
examine the role of COX-2 in epithelial-stromal interplay and its
consequence for mammary cancer. We found that deletion of MEC
COX-2 induced a substantial delay in tumorigenesis that was equivalent to systemic inhibition of the COX-2 enzyme. We detected no
change in proliferative, apoptotic, angiogenic or matrix remodeling
pathways to explain the reduced disease in COX-2 KOMEC mice.
Fig. 3 Expression of genes associated with proliferation, apoptosis, angiogenesis and matrix remodeling. Real-time Q-PCR quantification of mRNA for all genes
are relative to 18S in tumors harvested from WT and COX-2 KOMEC (KO) mice. Data are mean ± SE; n 5 15–16, P , 0.01.
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N.Markosyan et al.
Fig. 4. Increased association of cells expressing M1-type macrophage markers and cytokines with tumors from COX-2 KOMEC mice. (A–E) Real-time Q-PCR
quantification of mRNA for all genes is relative to 18S mRNA. Data presented are mean ± SE; n 5 15–16, P , 0.05. (F) Correlations of TIM3 mRNA expression
with F4/80 and CD86 genes in COX-2 KOMEC (KO) and WT tumors (n 5 14).
Rather, we observed an increase in markers/cytokines for antitumorigenic M1 macrophages, and possibly Th1 lymphocytes, in COX-2
KOMEC tumors suggesting augmentation of type 1 polarized inflammatory/immune cells in the absence of epithelial COX-2. Consistent
with this hypothesis, PGE2, the dominant MEC COX-2-derived prostanoid, restrained M1 polarization of BMDM in vitro. Thus, we conclude that deletion of COX-2 in mouse MECs delayed tumor onset, at
least in part, by shifting the tumor inflammatory/immune microenvi-
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ronment toward antitumorigenic type 1 responses. We propose a role
for epithelial COX-2-derived prostanoids in modulating the tumorimmune microenvironment to favor tumor growth and progression.
Multiple prostanoids can be generated via COX-2, and each has been
implicated in tumorigenesis. PGE2 is the dominant pro-tumorigenic
prostanoid (2,14) although pro-tumorigenic effects of thromboxane
A2 have been reported (38). PGI2 and PGD2 are generally considered
antitumorigenic (38,39), whereas the role of PGF2a in cancer remains
Deletion of mammary epithelial COX-2 delays tumors
Fig. 5. COX-2 KOMEC tumors show more M1 macrophage infiltration by
immunohistochemistry. Tumors, originated from luminal epithelial cells and
harvested from WT and COX-2 KOMEC (KO) mice, were selected based on
hematoxylin and eosin staining and immune staining for myoepithelial (red
staining for CK14, A and B) and luminal epithelial (red staining for CK18, C
and D) cells. Red-brown color represents specific staining for CD45 (E and
F), F4/80 (G and H), CD86 (I and J) and CD31 (K and L), in tumors from
WT (left column) and COX-2 KOMEC (KO, right column) animals. In panels
(E–J), areas within dashed squares are shown as insets with arrowheads,
indicating positively stained cells. The scale bar in all images indicates 40
magnification. Images are representative of at least n 5 3.
poorly examined (40). In cultured MEC, PGE2 is the most abundant
COX-2-derived mediator (Figure 2A), with little or no contribution by
COX-1 (Figure 2B). It is highly likely, therefore, that loss of COX2-derived PGE2 in MEC underlies the delayed disease we observed.
Deletion of COX-2 in MEC was equivalent to systemic COX-2
inhibition in delaying carcinogen-induced mammary tumorigenesis
(Figure 2D). This is perhaps surprising given the theoretical advantage afforded by depression of PGE2 generation not only in MEC but
also in surrounding stromal cells in rofecoxib-treated animals. Further, reported COX-independent anticarcinogenic effects of rofecoxib
(37) would be expected in drug-treated, but not COX-2 KOMEC, mice.
The degree to which COX-2 pathways in stromal cells, or COX-independent events, contribute to mammary tumor progression has yet
to be elucidated although the pro-tumorigenic effects of fibroblasts
(22) and macrophages (23) may be realized through induction of
epithelial cell COX-2. The equivalence of mammary epithelial
COX-2 deletion with systemic COX-2 inhibition indicates a dominant
role of COX-2 in the epithelial cell itself in tumor promotion.
The pro-tumorigenic effects of PGE2, which include increased cell
proliferation, cell survival, angiogenesis and decreased apoptosis, are
well described (2,37). In our COX-2 KOMEC mice, mRNA for the
proliferation marker Ki67 and apoptotic enzyme Caspase-3 were both
upregulated, indicating increased cell turnover in tumors lacking epithelial COX-2. We sought to determine, by immunohistochemistry,
whether epithelial or specific stromal cells were undergoing proliferation or apoptosis. However, Ki67 and Caspase-3 protein localization
and expression intensity were very variable across WT and KO tumors,
preventing definitive conclusions. Given the established pro-angiogenic
role of COX-2, we were surprised to observe similar expression of
genes for angiogenic factors and their receptors in WT and COX-2
KOMEC tumors. It may be that mRNA quantitation in heterogeneous
tumor samples did not reveal localized changes in angiogenesis. We do
not believe this to be the case, however, because no difference was
observed between WT and COX-2 KOMEC tumor levels of CD31, an
endothelial marker, by PCR (Figure 3C) or immunohistochemistry
(Figure 5K and L). Similarly, expression of extracellular matrix remodeling enzyme matrix metalloproteinase 2, regulated by COX-2 in breast
cancer (41), was not different between WT and COX-2 KOMEC tumors
(Figure 3I). These data imply that non-epithelial cell COX-2 in COX-2
KOMEC tumors was sufficient to maintain angiogenesis and matrix
remodeling at levels similar to WT tumors.
Further examination revealed an apparent shift in the immune microenvironment in COX-2 KOMEC tumors. Increased expression of mRNA
for the common leukocyte marker CD45, the macrophage marker F4/80,
the T cell markers CD2 and CD4 and the M1 macrophage marker CD86
and cytokine TNFa, was evident in COX-2 KOMEC tumors (Figure 4A1, -2, C-1, -2, D-1 and E-1). Elevated CD45, F4/80 and CD86 in the KO
tumors were also evident by immunohistochemistry (Figure 5E-J). Confirmation of the M1 versus M2 status of TAM, as well as phenotypic
analysis of other tumor associated immune cells by flow cytometry, was
not possible in this carcinogen-induced model of mammary tumorigenesis because of limitations in the size and number of tumors obtained
and the cellular yield per tumor. However, our data from Q-PCR and
immunohistochemistry were consistent with the augmented presence of
M1-type macrophages in COX-2 KOMEC tumors. We verified a PGE2dependent restraint on in vitro M1 polarization of BMDM, consistent
with the lower levels of M1 markers/cytokines in WT versus COX2MECKO tumors. One putative source of a principle M1 polarizing
cytokine, interferon c, is the pool of CD4þ Th1 cells (34,42). In the
present study, COX-2 KOMEC tumors also showed increased expression
of T cell markers CD2 and CD4, whereas TIM3, a Th1 marker, trended
toward elevation (P 5 0.062). It may be that a slight increase in the Th1
cell population in COX-2 KOMEC tumors was sufficient to shift TAM
polarization to the M1 phenotype, although this remains to be verified
experimentally. Alternatively, M1 polarization of macrophages in COX2 KOMEC may be Th1 independent, relying instead on mediators
elaborated from other cells.
A permissive microenvironment is necessary for the tumors to progress fully to the malignancy (43). The tumor microenvironment consists
of many cells and the composition varies depending on the tumor type.
Macrophages populate the microenvironment of most if not all tumors.
Independent studies have demonstrated a correlation of TAM with poor
prognosis, particularly when associated with high microvascular density (44). Further, the local tumor microenvironment may educate macrophages to perform non-immune, trophic functions similar to the
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N.Markosyan et al.
Fig. 6. PGE2 limits M1 polarization of BMDM. CD86 and TNFa mRNA expression change, measured by real-time Q-PCR in (A) M1-polarized BMDM (M1)
versus non-polarized (control—ctrl) or (B) M1-polarized BMDM treated with 10, 250 and 1000 nM PGE2 versus untreated (0 nM), M1-polarized BMDM. (C)
TNFa protein measured by ELISA in conditioned media of non-polarized and untreated (control—ctrl), M1-polarized and untreated (0 nM PGE2) and M1polarized and PGE2-treated (10, 250 and 1000 nM PGE2) BMDM. For all panels, data are mean ± SE, n 5 5, P , 0.05, P , 0.005, P , 0.0005.
promotion of epithelial outgrowth and invasion during development
(21,45). It appears that once they have infiltrated the tumor, the macrophage phenotype is switched from an activated, immunological M1
type to an alternatively activated M2 type that promotes tumor progression (46). However, clinical studies have shown that, in some cases,
elevated macrophage infiltration is associated with a beneficial outcome. Thus, resident and newly recruited macrophages displaying
M1 characteristics limited the development of peritoneal metastases
from colon carcinoma (47). Similarly, in patients with resected nonsmall cell lung carcinoma, tumor islet macrophage infiltration was
identified as a strong favorable independent prognostic marker for survival (48). Further, it was shown that antigen-presenting and cytotoxic
functions of M1-polarized macrophages can destroy tumor cells (49).
The tumor milieu can direct macrophage polarization to hinder tumor
growth and progression. Indeed, in the PyMT mouse model of breast
cancer, augmented type 1 polarization of both Th cells and TAM was
associated with reduced tumor invasiveness and limited metastasis of
the primary disease (42). Our data suggest a putative novel role for
COX-2 in contributing to macrophage phenotype in mammary tumors.
We believe that MEC-derived COX-2 products, most probably PGE2,
restrain M1 polarization of macrophages in tumors, limiting their antitumorigenic functions and promoting tumor progression. It is worth
noting that macrophages, whose COX-2 expression was strongly inducible by LPS (Figure 1E), may contribute significantly to tumor
microenvironmental PGE2 levels with unexplored paracrine and autocrine effects. The contribution of macrophage COX-2 to mammary
tumorigenesis is currently under investigation in our laboratory.
The mechanism through which mammary epithelial COX-2derived products impact immune cell polarization in mammary tumors is not clear. There is evidence that prostanoids can modulate
T cell and macrophage differentiation and polarization (27,50,51).
Our data in isolated BMDM argue for a direct effect of PGE2 on
macrophages although this remains to be verified in TAM. Our current
findings support the concept that the COX-2/PGE2 biosynthetic pathway may provide novel targets for therapeutic modulation of the
tumor-immune microenvironment. Further, in our head-to-head comparison of mammary epithelial versus systemic interruption of COX-2
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activity, similar benefit was seen using the targeted approach. Refined
approaches to inhibit specific functions of COX-2 in the tumor microenvironment may yield beneficial antitumorigenic effects while
avoiding deleterious systemic events that limit clinical use of systemic
COX-2 inhibition in cancer prevention or treatment.
Supplementary material
Supplementary Figures 1–3 and other Materials and Methods can be
found at http://carcin.oxfordjournals.org/
Funding
American Cancer Society [RSG-08-024-01-CNE to E.M.S.].
Acknowledgements
The assistance of Dr Robert Cardiff, UC Davis, with immunohistochemistry is
gratefully acknowledged. We also thank Ms Jenny Bruce for her technical
assistance.
Conflict of Interest Statement: None declared.
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Received April 28, 2011; revised June 20, 2011; accepted July 5, 2011
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