COX-2 inhibition improves immunotherapy and is associated with decreased numbers of myeloid-derived suppressor cells in mesothelioma. Celecoxib influences MDSC function.
ABSTRACT Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature cells that accumulates in tumour-bearing hosts. These cells are induced by tumour-derived factors (e.g. prostaglandins) and have a critical role in immune suppression. MDSC suppress T and NK cell function via increased expression of arginase I and production of reactive oxygen species (ROS) and nitric oxide (NO). Immune suppression by MDSC was found to be one of the main factors for immunotherapy insufficiency. Here we investigate if the in vivo immunoregulatory function of MDSC can be reversed by inhibiting prostaglandin synthesis by specific COX-2 inhibition focussing on ROS production by MDSC subtypes. In addition, we determined if dietary celecoxib treatment leads to refinement of immunotherapeutic strategies.
MDSC numbers and function were analysed during tumour progression in a murine model for mesothelioma. Mice were inoculated with mesothelioma tumour cells and treated with cyclooxygenase-2 (COX-2) inhibitor celecoxib, either as single agent or in combination with dendritic cell-based immunotherapy.
We found that large numbers of infiltrating MDSC co-localise with COX-2 expression in those areas where tumour growth takes place. Celecoxib reduced prostaglandin E2 levels in vitro and in vivo. Treatment of tumour-bearing mice with dietary celecoxib prevented the local and systemic expansion of all MDSC subtypes. The function of MDSC was impaired as was noticed by reduced levels of ROS and NO and reversal of T cell tolerance; resulting in refinement of immunotherapy.
We conclude that celecoxib is a powerful tool to improve dendritic cell-based immunotherapy and is associated with a reduction in the numbers and suppressive function of MDSC. These data suggest that immunotherapy approaches benefit from simultaneously blocking cyclooxygenase-2 activity.
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ABSTRACT: Malignant gliomas are heavily infiltrated by immature myeloid cells that mediate immunosuppression. Agonistic CD40 monoclonal antibody (mAb) has been shown to activate myeloid cells and promote antitumor immunity. Our previous study has also demonstrated blockade of cyclooxygenase-2 (COX-2) reduces immunosuppressive myeloid cells, thereby suppressing glioma development in mice. We therefore hypothesized that a combinatory strategy to modulate myeloid cells via two distinct pathways, i.e., CD40/CD40L stimulation and COX-2 blockade, would enhance anti-glioma immunity. We used three different mouse glioma models to evaluate therapeutic effects and underlying mechanisms of a combination regimen with an agonist CD40 mAb and the COX-2 inhibitor celecoxib. Treatment of glioma-bearing mice with the combination therapy significantly prolonged survival compared with either anti-CD40 mAb or celecoxib alone. The combination regimen promoted maturation of CD11b(+) cells in both spleen and brain, and enhanced Cxcl10 while suppressing Arg1 in CD11b(+)Gr-1(+) cells in the brain. Anti-glioma activity of the combination regimen was T-cell dependent because depletion of CD4(+) and CD8(+) cells in vivo abrogated the anti-glioma effects. Furthermore, the combination therapy significantly increased the frequency of CD8(+) T-cells, enhanced IFN-γ-production and reduced CD4(+)CD25(+)Foxp3(+) T regulatory cells in the brain, and induced tumor-antigen-specific T-cell responses in lymph nodes. Our findings suggest that the combination therapy of anti-CD40 mAb with celecoxib enhances anti-glioma activities via promotion of type-1 immunity both in myeloid cells and T-cells.Cancer Immunology and Immunotherapy 05/2014; · 3.94 Impact Factor
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ABSTRACT: An elevated number of myeloid-derived suppressor cells (MDSCs) in tumor-bearing hosts has been recognized as a crucial mediator of tumor progression due to the cells potent ability to suppress antitumor immunity. Cluster of differentiation (CD) 40, as a suppressive phenotype expressed in MDSCs, is essential for MDSC-mediated immune suppression and the expansion of T regulatory cells. However, whether CD40 exerts a direct effect on the accumulation of MDSCs remains unclear. In the present study, CD40 was observed to be highly expressed on the MDSCs obtained from mice bearing gastric tumors. Notably, a significant decrease in the level of CD40 expression was observed in addition to an increased number of MDSCs during tumor progression. Further analysis revealed that the MDSC levels were found to positively correlate with tumor progression and that CD40 expression levels inversely correlate with the accumulation of MDSCs. To confirm the potent correlation between CD40 expression and the accumulation of MDSCs, the apoptosis of the MDSCs was detected using agonistic anti-CD40 treatment. The results indicated that CD40 activation induces apoptosis in MDSCs and that the downregulation of CD40 expression may contribute to MDSC accumulation by facilitating MDSC resistance to apoptosis. Thus, these observations provide a novel mechanism to improve our understanding of the involvement of CD40 in MDSC accumulation during cancer development.Oncology letters 08/2014; 8(2):775-780. · 0.99 Impact Factor
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ABSTRACT: We tested the hypothesis that a novel vaccine developed from autologous dendritic cells (DC) loaded with cells from a unique allogeneic brain tumor cell line (GBM6-AD) would be well-tolerated and would generate an immune response. Patients with recurrent primary brain tumors underwent vaccination with GBM6-AD/DC vaccine. Subjects were treated at escalating DC cell doses: 5 × 10(6) (one patient), 10 × 10(6) (one patient) and 15 × 10(6) (6 patients). Subcutaneous injections were planned for days 0, 14, 28, 42, 56, and monthly thereafter. The primary endpoint was the safety of the GBM6-AD/DC vaccination. The secondary endpoints were immune response, measured by flow cytometry, and the clinical outcome of tumor response defined by time to progression and overall survival. Eight patients were treated. The first three patients were treated in the dose escalation phase of the trial; the remaining five patients received the maximum dose of 15 × 10(6) DC. No dose limiting toxicity was observed. The best response per modified McDonald criteria was partial response in one patient. Flow cytometric immune profiling revealed significant differences in CD4(+)IL17(+) lymphocytes and myeloid derived suppressor cell populations between patients characterized as having stable vs. non-stable disease. This first-in-human study shows that the GBM6-AD/DC vaccine was well tolerated and was associated with an immune response in a subset of patients. No MTD was achieved in this trial. This small-scale pilot provides information for larger scale investigations into the use of this allogeneic vaccine source.Journal for immunotherapy of cancer. 01/2014; 2:4.
RESEARCH ARTICLEOpen Access
COX-2 inhibition improves immunotherapy and is
associated with decreased numbers of myeloid-
derived suppressor cells in mesothelioma.
Celecoxib influences MDSC function
Joris D Veltman, Margaretha EH Lambers, Menno van Nimwegen, Rudi W Hendriks, Henk C Hoogsteden,
Joachim GJV Aerts, Joost PJJ Hegmans*
Background: Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature cells that
accumulates in tumour-bearing hosts. These cells are induced by tumour-derived factors (e.g. prostaglandins) and
have a critical role in immune suppression. MDSC suppress T and NK cell function via increased expression of
arginase I and production of reactive oxygen species (ROS) and nitric oxide (NO). Immune suppression by MDSC
was found to be one of the main factors for immunotherapy insufficiency. Here we investigate if the in vivo
immunoregulatory function of MDSC can be reversed by inhibiting prostaglandin synthesis by specific COX-2
inhibition focussing on ROS production by MDSC subtypes. In addition, we determined if dietary celecoxib
treatment leads to refinement of immunotherapeutic strategies.
Methods: MDSC numbers and function were analysed during tumour progression in a murine model for
mesothelioma. Mice were inoculated with mesothelioma tumour cells and treated with cyclooxygenase-2 (COX-2)
inhibitor celecoxib, either as single agent or in combination with dendritic cell-based immunotherapy.
Results: We found that large numbers of infiltrating MDSC co-localise with COX-2 expression in those areas where
tumour growth takes place. Celecoxib reduced prostaglandin E2 levels in vitro and in vivo. Treatment of tumour-
bearing mice with dietary celecoxib prevented the local and systemic expansion of all MDSC subtypes. The
function of MDSC was impaired as was noticed by reduced levels of ROS and NO and reversal of T cell tolerance;
resulting in refinement of immunotherapy.
Conclusions: We conclude that celecoxib is a powerful tool to improve dendritic cell-based immunotherapy and is
associated with a reduction in the numbers and suppressive function of MDSC. These data suggest that
immunotherapy approaches benefit from simultaneously blocking cyclooxygenase-2 activity.
MDSC are a heterogeneous population of immature
myeloid cells. These cells can inhibit anti-tumour
responses in an antigen-specific and in a non-specific
way [1,2]. Antigen-specific suppression takes place in
lymphoid organs and depends on reactive oxygen spe-
cies (ROS) production, leading to T cell tolerance. Anti-
gen-non-specific suppression takes place at the tumour
site and is mainly dependent on nitric oxide (NO) secre-
tion, causing T cell specific apoptosis [2-4]. More
recently, it has been shown that the heterogeneous
group of MDSC can be subdivided into three major
groups. Polymorph nuclei MDSC (PMN-MDSC), mono-
nuclear MDSC (MO-MDSC) and Gr-1lowMDSC [5-7].
Greifenberg et. al. showed that the MO-MDSC and a
subpopulation of Gr-1lowMDSC can inhibit T cell pro-
liferation by the production of NO, in contrast to the
PMN-MDSC that did not show this suppressive capacity
. However, the exact mechanisms that play a role in
* Correspondence: email@example.com
Department of Pulmonary Medicine, Erasmus MC Rotterdam, The
Veltman et al. BMC Cancer 2010, 10:464
© 2010 Veltman et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
the induction of T cell tolerance by MDSC subtypes
need to be further explored. As ROS production by
MDSC also contributes to the induction of tolerance we
determined the ROS producing capacity of these subpo-
pulations of MDSC. In addition, we investigated if ROS
production by these different MDSC populations can be
The production of ROS by MDSC is highly depending
upon cyclooxygenase-2 (COX-2) enzyme activity [8,9].
The inducible COX-2 enzyme is essential in the bio-
synthesis of prostaglandins. Over-expression of COX-2
has been described as an important factor in tumour
development. Therefore, high expression of COX-2 has
been correlated with poor prognosis in cancer [10-12].
In addition, several studies showed the relevance of
COX-2 inhibition in cancer progression [13-15].
Although the relation between COX-2 over-expression
and prostaglandin E2 (PGE2) synthesis in cancer has
been studied extensively, the impact on the tumour
microenvironment is still under investigation [16-18].
Increased evidence indicates that immune suppressive
cells, recruited by tumour-derived factors, are the main
cause of failure of novel anti-cancer therapies, including
immunotherapy [4,19-24]. We investigated dendritic cell
(DC)-based immunotherapy in a murine model of
mesothelioma. It was found that the efficacy of this
treatment is hampered by the highly immunosuppressive
environment in mesothelioma . Adjacent to this, it
was found that COX-2 over-expression in mesothelioma
correlates with poor prognosis . Combining the cur-
rent knowledge on MDSC, COX-2 over-expression and
immune suppression, we aimed to determine the effect
of selective COX-2 inhibition on MDSC populations in
the optimization of DC-based immunotherapy. We stu-
died the effect of celecoxib on different MDSC popula-
tions in a murine model for mesothelioma. Since ROS is
one of the principal factors leading to induction of
T cell tolerance, we focused on the effects on ROS
production by different MDSC populations during
Animals and cell lines
BALB/c mice (specific pathogen free [SPF], female, 6-
8 weeks old) were purchased from Harlan (Zeist, The
Netherlands) and were housed under pathogen-free con-
ditions at the animal care facility of Erasmus MC. All
experiments were approved by the local ethical commit-
tee for animal welfare (Erasmus University Committee
of Animal Experts, Rotterdam, the Netherlands) and
complied with the guidelines for the welfare of animals
in experimental neoplasia by the United Kingdom Coor-
dinating Committee on Cancer Research (UKCCCR),
and by the Code of Practice of the Dutch Veterinarian
Inspection. The mesothelioma AB1 cell line was kindly
provided by Professor B.W.S. Robinson (School of Medi-
cine and Pharmacology, Sir Charles Gairdner Hospital
Unit, The University of Western Australia, Perth,
Tumour growth of murine mesothelioma in BALB/c mice
BALB/c mice were divided into 4 groups. Each group
consisted of 6 mice. On day 0, all mice were injected
with a lethal dose of 0.5 × 106AB1 tumour cells. From
day 0 onwards, group 1 and 3 received a control diet
while group 2 and 4 received celecoxib diet (500 mg cel-
ecoxib/kg [Celebrex®; Pfizer, New York, NY, USA]). Both
were starch-based diets manufactured by Harlan Teklad
(Madison, WI, USA) as described by T. Hahn et al. .
Mice in group 3 and 4 received DC-based immunother-
apy consisting of 1 × 106tumour-lysate loaded dendritic
cells at day 10.
Dendritic cells were culture from bone marrow using
RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented
with 5% heat inactivated fetal bovine serum (FBS
[Hyclone, Waltham, MA, USA]), 20 ng/ml GM-CSF
(kindly provided by Kris Thielemans, VU Brussels, Bel-
gium), and 50 μM b-mercaptoethanol (Sigma-Aldrich
BV, Zwijndrecht, The Netherlands) for 9 days. At day 8,
cells were pulsed with tumour-lysate (to the equivalent
of three AB1 cells per DC) and overnight matured with
100 ng/ml LPS E. Coli 026:136 (Sigma-Aldrich BV). On
the day of vaccination, DCs were harvested and purified
by Lympholyte-Mammal (Cedarlane, Hornby, ON,
Canada) density gradient centrifugation, and washed
three times in phosphate-buffered saline (PBS [Gibco]).
Cells were resuspended at a concentration of 1 × 106
viable cells in 500 μl PBS and intraperitoneally injected.
The occurrence of tumour growth, body weight,
physical well-being and survival were measured for
2 months, as described previously .
Immunohistochemistry on tumour cells and biopsies
Tumour cells were cultured in RPMI supplemented with
5% FBS on two chambers Falcon culture slides (BD
biosciences, Erebodegem, Belgium) starting with 5 × 104
AB1 cells per well. Celecoxib was added to the culture
when cells reached a confluence of 60% for 24 hours in
a concentration of 10 μg/ml and 100 μg/ml. Resected
tumour material was obtained from the peritoneal cavity
of AB1 inoculated BALB/c mice at day 15. Tumour
biopsies were embedded in Tissue-Tek II optimum cut-
ting temperature (OCT) medium (Miles, Naperville, IL,
USA), snap-frozen in liquid nitrogen and stored at
-80°C. Tissue sections (6 μm) were cut on a HM-560
cryostat (Microm, Heidelberg, Germany).
For the COX-2 expression a rabbit affinity-purified
IgG against murine COX-2 was used (Cayman chemical,
Veltman et al. BMC Cancer 2010, 10:464
Page 2 of 13
Montigny-le-Bretonneux, France) (this antibody shows
no cross-reactivity with COX-1). For the COX-1 expres-
sion a rabbit affinity-purified IgG against murine COX-1
was used. These primary antibodies were incubated for
1 hour at room temperature. Binding of antibodies was
detected using the goat anti-rabbit alkaline phosphatase
(AP) (Sigma-Aldrich). Naphtol-AS-MX-phosphate
(0.30 mg/ml [Sigma-Aldrich]) and new fuchsine
(160 mg/ml in 2 M HCl [Chroma-Gesellschaft, Köngen,
Germany]) were used as substrate. Hematoxylene was
used as counterstaining. The specificity of the antibodies
was checked using a protein concentration-matched
non-relevant monoclonal antibody and PBS.
Mouse mesothelioma biopsies were double stained for
Gr-1-FITC (clone RB6.8C5) and COX-2. As secondary
antibodies horseradish peroxidase (HRP) conjugated
goat anti-FITC (Rockland, Gilbertsville, PA, USA) and
AP-conjugated goat anti-rabbit (Sigma-Aldrich) were
used. Naphtol-AS-MX-phosphate and 1 mM Fast Blue
(Sigma-Aldrich) were used as substrate for AP and
NovaRed was used as substrate for HRP, according to
the manufacturer’s instructions (Vector, Burlingame,
Spleens were aseptically removed, and mechanically
dispersed in cold HBSS (Invitrogen). Cell suspensions
were filtered through a 100 μm nylon cell strainer (BD
Biosciences), depleted of erythrocytes by osmotic lysis,
washed twice in RPMI medium containing 5% FBS, and
adjusted to a concentration of 1 × 106cells/ml in FACs-
Splenocytes were stained with the following optimally
diluted mAbs: Ly6c (FITC conjugated), MHCII (PE con-
jugated), CD11b (PercP-Cy5.5 conjugated) (all BD
bioscience), CD8 (FITC conjugated), F4/80 (FITC conju-
gated), CD4 (PE conjugated), CD31 (PE-Cy7 conju-
gated), B220 (Alexa fluor 700 conjugated), Ly6g (APC-
Cy7 conjugated) (all eBioscience), CD11c (PE-Texas red
conjugated [Caltag, Burlingame, CA, USA]), and a live/
dead marker (DAPI [Invitrogen]). Splenocytes were res-
timulated in the presence of GolgiStop (BD biosciences)
for 4 hours using anti-CD3 and intracellular stained for
Granzyme B (PE conjugated [Caltag]) and IFN-g (APC
conjugated [BD Biosciences]). Viability was determined
by live dead aqua (Invitrogen). Acquisition of eight to
nine colour samples was performed on a FACs LSR II
cytometer (BD Biosciences). The analysis and graphical
output were performed using FlowJo software (Tree Star
Inc., Costa Mesa, CA, USA).
PGE2levels in the peritoneal washes were determined
using a specific ELISA assay for PGE2(R&D systems,
Abingdon, UK). Manufacturer’s recommended protocols
were followed. Serum was diluted appropriately to
ensure that readings were within the limits of accurate
Nitric oxide and its reactive products (NO)
Equal volumes of peritoneal wash (150 μl) were mixed
with Greiss reagent (1% sulfanilamide in 5% phosphoric
acid and 0.1% N-1-naphthyl-ethylenediamine dihy-
drochloride in double-distilled water). After 30 min
incubation at room temperature, the absorbance was
measured at 548 nm using a microplate reader (Bio-
Rad). Nitrite concentrations were determined by com-
paring the absorbance values for the test samples to a
standard curve generated by serial dilution of a stock
solution of sodium nitrite.
Reactive oxygen species (ROS)
The oxidation-sensitive dye dichlorodihydrofluorescein
diacetate (DCFDA, Sigma-Aldrich) was used for the
measurement of ROS production by splenocytes. Cells
were incubated at 37°C in DMEM (Invitrogen) in the
presence of 1 μM DCFDA for 60 min, and washed twice
with cold PBS. Cells were stained with Abs directed
against Gr-1 (PE conjugated [BD biosciences]) and
CD11b (PercP-Cy5.5 conjugated [BD biosciences]) as
previously described. Cells were washed with cold PBS
after 20 min incubation. Acquisition of the samples was
performed on a flowcytometer.
Tumour-specific lysis assay
Spleens were aseptically removed, and mechanically dis-
persed in cold PBS. Cell suspensions were filtered
through a 100 μm nylon cell strainer (BD Biosciences),
depleted of erythrocytes by osmotic lysis, washed twice
in RPMI, and adjusted to a concentration of 4 × 106
cells/ml in RPMI medium supplemented with 5% FBS.
After 48 h of culture, spleen cells were washed exten-
sively. Mouse AB1 cells were incubated with 100 μCi of
washed three times, resuspended in culture medium at a
concentration of 5 × 104cells/ml. Splenocytes (150.000
cells per well) from either naïve mice, untreated mice,
or mice treated with DC-based immunotherapy were
mixed with 5 × 103radiolabeled AB1 target cells and
added to wells of a 96-well round-bottom microtiter
plate (0.2 ml final volume) to achieve the desired effec-
tor:target (E:T) ratios. To determine the suppressive
capacity of splenocytes from mice treated with control
diet or celecoxib diet, 150.000 splenocytes from DC
treated mice were mixed with 150.000 splenocytes from
mice treated with either control diet or celecoxib diet.
After 24 hour incubation, cells were mixed with radiola-
beled AB1 target cells and added to wells of a 96-well
51CrO4(ICN Biomedicals) for 2 hours at 37°C,
Veltman et al. BMC Cancer 2010, 10:464
Page 3 of 13
round-bottom microtiter plate (0.2 ml final volume) to
achieve the desired effector:target (E:T) ratios.
Plates were incubated for 4 hours at 37°C in a humidi-
fied atmosphere containing 5% CO2, and cell-free super-
natants were collected from each well. The amount of
51Cr released from lysed AB1 target cells was deter-
mined by g scintillation counting. Percent lysis was cal-
culated using the formula: corrected % lysis =
100×(experimental release - spontaneous release [target
cells incubated in medium alone])/(maximum release[2%
Triton X-100 as lysing agent]-spontaneous release).
Data are expressed as mean ± SD. Comparisons between
groups were made using the t-test. A two-tailed p-value
< 0.05 was considered significant. Data presented as a
percentage of tumour-free animals were analysed with
Kaplan-Meier survival-curves, using the log-rank test to
Identification of myeloid-derived suppressor cell subsets
Splenocytes from mice that were inoculated with a
lethal dose of AB1 tumour cells and placed on a control
or celecoxib diet were stained for flowcytometric
Similar to the most recent published data, three popu-
lations of Gr-1+CD11b+cells were identified [5-7]. The
Gr-1lowMDSC could be further subdivided in two
populations based on size and inner complexity of the
cells (forward [FSC]/side scatter [SSC]). The physical
characteristics combined with Ly6c expression was used
to visualize other intrinsic differences between the dif-
ferent groups. We found that Ly6c expression was lower
on the Gr-1lowMDSC with a high SSC (subset 1) in
contrast to the Gr-1lowMDSC with low SSC (subset 2).
Ly6c expression on PMN-MDSC was lower then Ly6c
expression on MO-MDSC (Figure 1A).
MDSC can induce T cell tolerance by producing ROS.
ROS production was highest in PMN-MDSC followed
by MO-MDSC. Subset 2 of the Gr-1lowMDSC had
the capability to produce ROS, whereas the majority of
the cells in subset 1 were not able to produce ROS
Tumour biopsies were obtained and stained for the
presence of Gr-1+CD11b+cells. Large areas with infil-
trating positive cells were found at the rim of the
tumour. Since COX-2 is an essential enzyme for catalyz-
ing the biosynthesis of tumour-derived prostaglandin E2
(PGE2), a molecule that induces MDSC , biopsies
were stained for COX-2 expression. COX-2 expressing
cells were found at the border of the tumour closely
located near areas with Gr-1+CD11b+infiltrating cells
Next we investigated if the enzyme activity of tumour-
derived COX-2 could be inhibited by celecoxib in vitro.
Therefore, cultured AB1 tumour cells were incubated
with different dosages of celecoxib. After 24 hours
supernatant was removed and cells were stained for the
expression of COX-2 and COX-1 as a control. COX-1
expression was not effected by the selective COX-2 inhi-
bitor celecoxib. COX-2 expression was reduced in a
doses-depended manner (Figure 1C).
In conclusion, these data show that MDSC in the
spleens of tumour-bearing mice can be subdivided into
three groups. Additionally, we demonstrate that PMN-
MDSC and MO-MDSC produce high ROS. The group
of Gr-1lowMDSC could be further subdivided into two
subsets based on FSC/SSC, Ly6c and ROS production.
Large areas with infiltrating Gr-1+CD11b+cells were
found at the rim of the tumour in close contact to
COX-2 expressing cells. COX-2 expression by AB1
tumour cells was decreased by celecoxib in vitro in a
Reduction of MDSC by dietary administration of celecoxib
We investigated the effect of celecoxib treatment on the
four MDSC subsets that were identified in the spleen of
tumour-bearing mice. Splenocytes from mice that were
inoculated with AB1 tumour cells and received celecoxib
diet or control diet were analyzed for the presence of
the MDSC subsets.
Ten days after tumour injection, the absolute number
of MDSC was significantly lower in mice receiving cele-
coxib diet compared with mice receiving control diet.
This difference was more pronounced at day 22 after
tumour injection (Figure 2A). When MDSC subsets
(MO-MDSC PMN-MDSC and the two subpopulations
of Gr-1lowMDSC as described in Figure 1A), a signifi-
cant decrease in the percentage of PMN-MDSC was
found. In addition, a shift between subset 1 and subset 2
within the Gr-1lowMDSC was observed leading to an
increase of subpopulation 1. The total numbers of Gr-
1lowMDSC did not differ between celecoxib treated and
untreated animals. More recently, it has been shown
that macrophages can be derived from MDSC [6,29].
Therefore this cell population was also analyzed. Macro-
phages were characterized by FACS based on their FSC/
SSC, in combination with the expression of CD11b, F4/
80 and MHCII on their membrane. A significant reduc-
tion in macrophages was observed in celecoxib treated
mice compared to untreated mice (Figure 2B).
To summarize, a significant decrease in the number of
MDSC in the spleen was found at day 10 after tumour
inoculation and became more diverged when tumour
progressed in celecoxib treated mice compared with
untreated animals. Furthermore, we found that the
reduction in MDSC after dietary celecoxib treatment
Veltman et al. BMC Cancer 2010, 10:464
Page 4 of 13
Figure 1 Identification of MDSC and myeloid cell types in tumour-bearing mice. A) PMN-MDSC expressed high levels of Gr-1, CD11b. MO-
MDSC have a slightly higher expression of Ly6c. A third population was identified showing a low expression of Gr-1. Based on FSC/SSC this
population was further subdivided into two subsets; SSClowexpressing high Ly6c and SSChighexpressing low Ly6c. ROS production was
measured for each subtype. B) Histology on tumour sections showed Gr-1+(red) at the rim of the tumour in close contact with COX-2
expressing cells (blue). [Magnification: 200× (left) and 400× (right)]. C) Inhibition of COX-2 activity was tested in vitro by adding celecoxib to AB1
cell cultures. AB1 cells were cultured for 24 hours with medium, 10 μg/ml, or 100 μg/ml celecoxib and stained with anti-COX-2 and anti-COX-1
(as control) antibodies.
Veltman et al. BMC Cancer 2010, 10:464
Page 5 of 13
Figure 2 Reduction of MDSC in tumour-bearing mice after celecoxib-treatment. A) Accumulation of MDSC (characterized by the expression
of Gr-1 and CD11b) in tumour-bearing mice receiving control diet or celecoxib diet: day 0 n = 4/4, day 10 n = 5/5, day 15 n = 3/2, day 22 n =
3/3). A significant difference was found at day 10 and day 22. * p = 0.034, ** p = 0.031. B) Splenocytes from mice receiving control diet or
celecoxib diet were analyzed 10 days after tumour injection (n = 5/5). Subtypes were identified as described in Figure 1. A significant reduction
was found in PMN-MDSC (p = 0.021) and in subset 2 from the Gr-1lowMDSC population (p = 0.041) in mice receiving celecoxib diet. A
significant increase was found in subset 1 from the Gr-1lowMDSC population (p = 0.007), leading to a shift in ratio (p = 0.020) in celecoxib
treated mice. The percentage of F4/80+MHCII+cells was significantly reduced in mice receiving celecoxib diet (p = 0.022).
Veltman et al. BMC Cancer 2010, 10:464
Page 6 of 13
was mainly caused by a reduction of PMN-MDSC and a
shift in subsets of the Gr-1lowMDSC.
Reduced ROS production by MDSC subsets
We next investigated if dietary celecoxib treatment not
only influenced the number of MDSC in the spleen but
also affects the function of the MDSC subsets. It has
been shown that MDSC can down regulated the ζ-chain
on T cells by ROS production in the lymphoid organs
and thereby induce T cell tolerance [19,30]. We ana-
lyzed splenocytes of dietary celecoxib treated and
untreated mice for their capability to produce ROS.
Celecoxib treatment reduced ROS production in all
MDSC subsets, especially in the Gr-1lowMDSC subset 2
and MO-MDSC. There was a trend toward a reduction
of ROS production by macrophages, though this reduc-
tion was not significant (p = 0.14). Analyzing ROS pro-
duction by all myeloid cells revealed that ROS
production was decreased in celecoxib treated mice
compared to untreated animals (Figure 3).
In summary, ROS production was most pronounced
in PMN-MDSC. Dietary celecoxib treatment reduced
ROS production in all MDSC subtypes but is most
effective in the MO-MDSC and Gr-1lowMDSC subset 2
both in percentage as well as the median fluorescence
Reduction of COX-2 expression in tumour tissue after
dietary celecoxib treatment
After we observed that celecoxib treatment reduced the
number and function of MDSC in the spleen of
tumour-bearing mice we investigated if dietary treat-
ment of mice with celecoxib also affects MDSC in the
tumour environment. Therefore tumour biopsies were
analyzed for the expression of COX-2 enzyme and the
presence of infiltrating MDSC. In addition, the perito-
neal cavity of tumour-bearing mice was washed with
PBS and analyzed for tumour-derived PGE2and NO
Tumour was infiltrated with massive amounts of Gr-
1+CD11b+cells, mainly at the border of the tumour.
These cells were in close contact to COX-2 expressing
tumour cells. Treatment with celecoxib showed that
the Gr-1+CD11b+cells were more restricted and that
COX-2 expression at the rim of the tumour was lost
Additionally, tumour-derived PGE2was decreased in
the peritoneal wash of mice receiving celecoxib diet
compared to animals on control diet. Also a reduction
in NO concentrations was found (Figure 4B).
These data show that treating mice with celecoxib
decreased the expression of COX-2 and thereby reduced
MDSC both systemically as well as in the local
Figure 3 Decreased ROS production by MDSC and myeloid cell types after celecoxib-treatment. Production of reactive oxygen species
(ROS) by (immature) myeloid subtypes from the spleen of tumour-bearing mice was measured. Splenocytes were obtained from tumour-bearing
mice receiving either control diet or celecoxib diet for 10 days after tumour injection. Subtypes were identified based on characteristics as
described in Figure 1. ROS production was reduced by MDSC in tumour-bearing mice treated with celecoxib diet. Mean fluorescent intensity
(MFI) of ROS was significantly lower in MO-MDSC (p = 0.015) and Gr-1lowSubset 2 MDSC (p = 0.017) after celecoxib-treatment. The MFI of ROS
on macrophages did not significantly change (p = 0.14).
Veltman et al. BMC Cancer 2010, 10:464
Page 7 of 13
Figure 4 Reduction of COX-2 expression after celecoxib-treatment. A) Tumour sections of untreated (n = 7) and celecoxib treated mice (n
= 8) were immunohistochemically double-stained for the presence of Gr-1 (red) and COX-2 expression (blue). No expression of COX-2 was
found in tumour sections from mice with dietary celecoxib. Gr-1+cells were reduced in tumour sections of mice treated with celecoxib
[magnification 200× left and 400× right]. B) PGE2levels were measured in peritoneal washings of untreated (n = 7) versus celecoxib treated mice
(n = 8) (p = 0.0061). NO was detected using Greiss reagent.
Veltman et al. BMC Cancer 2010, 10:464
Page 8 of 13
microenvironment of the tumour. This reduced COX-2
expression is accompanied by with a reduction in
PGE2and NO levels. Moreover, the infiltration of Gr-1
sup>+CD11b+cells in tumour areas was reduced by
Reduction of immune suppression after celecoxib
In our previous study we have shown that DC-based
immunotherapy in this murine model leads to the
induction of a strong anti-tumour response . How-
ever, the effectiveness of the anti-tumour response was
negatively influenced when tumours sizes increased .
It has been addressed that one of the main reasons for
immunotherapy failure is the induction of T cell toler-
ance by immune suppressive cells. To determine if treat-
ment with celecoxib also intercepts the induction of T
cell tolerance the following experiments were per-
formed. A chromium release assay was performed to
investigate the effects on tumour specific lysis in differ-
There was no specific lysis of tumour cells when sple-
nocytes of naïve mice or tumour-bearing mice were co-
cultured with radioactive labelled tumour cells. When
splenocytes from mice treated with DC-based immu-
notherapy were co-cultured with tumour cells a massive
induction of tumour lysis was observed, demonstrating
that DC-based immunotherapy induces tumour specific
recognition by immune cells. To examine the suppres-
sive effect of MDSC in the spleen, splenocytes from
DC-treated mice were co-cultured with splenocytes of
tumour-bearing mice treated with either control diet or
celecoxib diet. After 4 hour incubation at 37°C, the sple-
nocyte mixtures were co-cultured with the radioactive
labelled tumour cells. Although the total amounts of
splenocytes were equal in all conditions, a significant
reduction in anti-tumour activity was found when sple-
nocytes of control diet animals were added. In contrast,
the addition of splenocytes from tumour-bearing mice
treated with dietary celecoxib did not reduce the lytic
capacity of splenocytes from mice treated with DC-
immunotherapy (Figure 5A).
IFN-g and granzyme B production by CD8+T cells
showed similar results. CD8+T cells from the spleen of
naïve and tumour-bearing mice were not capable of
IFN-g and granzyme B production. Production of IFN-g
and granzyme B was significantly increased after DC-
treatment. Co-culture of splenocytes from DC-treated
mice with splenocytes from tumour-bearing mice trea-
ted with control diet or celecoxib diet, showed that
CD8+cells were affected by splenocytes of mice that
had received the control diet while splenocytes of cele-
coxib treated mice did not affect the capability of CD8+
cells to produce IFN-g and granzyme B (Figure 5B).
In conclusion, these data show that anti-tumour
responses induced by DC-treatment, are affected by sup-
pressive cells in the spleen of tumour-bearing mice.
However, the anti-tumour activity as indicated by AB1
lysis and IFN-g/granzyme B production by CD8+T cells
was no longer influenced when co-cultured with Spleno-
cytes of mice receiving celecoxib diet, indicating that
COX-2 inhibition leads to a reduction in suppressive
Dietary celecoxib improves DC-based immunotherapy
Next, we wanted to know if combining dietary celecoxib
and DC-based immunotherapy would lead to an
increased survival benefit. Previous studies showed that
mice treated with DC-immunotherapy one day after
tumour injection leads to 100% survival in DC-treated
mice by inducing anti-tumour T cell activity. However,
the efficacy of DC-treatment decreases with increasing
tumour burden. To study the possible synergistic effect
of celecoxib and immunotherapy a suboptimal DC-treat-
ment protocol was used. Therefore in this protocol mice
received tumour lysate-loaded DC 10 days after tumour
injection. Mice receiving AB1 tumour cells developed
signs of terminal illness after 12 days.
Treatment with celecoxib alone prolonged survival to
some extent; however this prolongation was not signifi-
cant. No side effects were observed in mice receiving
the celecoxib diet. The combination of dietary celecoxib
and DC-immunotherapy led to a significant improve-
ment of the immunotherapy (p = 0.038). In addition,
combined treatment compared to no treatment
significantly improved survival (p = 0.027) were single
treatment with celecoxib or suboptimal DC-treatment
did not improve survival (p = 0.305 and p = 0.455)
This experiment showed that the combination of diet-
ary celecoxib and immunotherapy is superior to single
treatment in mice with high tumour burden.
We show that dietary administration of celecoxib leads
to a significant decrease in the number and suppressive
function of immature myeloid cells in the spleens and
tumours of tumour-bearing mice. Subdivision of the
heterogeneous group of immature myeloid cells revealed
that three types could be identified in tumour-bearing
mice, reflecting findings done by others [5,7]. The
PMN-MDSC population was most frequently present in
the spleen of tumour-bearing mice. This population was
also most capable of ROS production. Previous studies
showed that ROS production by MDSC is the main fac-
tor for inducing T cell tolerance by down regulation of
the ζ-chain on activated T cells [19,30]. Celecoxib-treat-
ment especially influenced this PMN-MDSC population
Veltman et al. BMC Cancer 2010, 10:464
Page 9 of 13
compared to other MDSC subtypes in the spleen of
tumour-bearing mice. Greifenberg et. al. recently
showed that this population was not capable of NO pro-
duction and therefore showed no suppressive effect on
T cell proliferation in a T cell mixed leukocyte reaction
(MLR) . However, our data provides evidence that
PMN-MDSC do play a significant role in tumour-
induced immune suppression, since PMN-MDSC are
most capable of producing ROS and thereby contribute
to the induction of tolerance. Induction of tolerance can
influence novel treatment strategies like DC-based
immunotherapy negatively. Others have shown that
immune suppression in the spleen in mainly dependent
on ROS production whereas immune suppression in the
tumour area is predominately depending on NO pro-
duction by myeloid cells indicating that the increased
PMN-MDSC population suppresses the anti-tumour
T cell responses by producing ROS.
The Gr-1lowMDSC population can be divided into
two subsets. We observed a shift in the Gr-1lowMDSC
population of celecoxib treated mice leading to less ROS
producing cells in this fraction. Greifenberg et al.
showed that FSC/SSC low subset had suppressive capa-
city and were able to produce NO after LPS/IFN-g resti-
mulation. Next to this, we were able to show that this
subset is also capable of ROS production whereas the
FSChighfraction within the Gr-1lowMDSC could not.
Furthermore, we showed that celecoxib-treatment not
only decreased the amount of immature myeloid cells in
tumour-bearing mice, but that treatment also impaired
ROS production by the different subpopulations of
MDSC. Given the fact that the number of MDSC is
Figure 5 Improved T cell function after celecoxib-treatment. A) The amount of51Cr released from lysed radiolabeled AB1 target cells was
determined to compare cytolytic activity of splenocytes isolated from naïve mice (n = 4) tumour-bearing mice (n = 5) and DC-treated mice (n =
4). To determine the inhibitory effect of MDSC in the spleen, splenocytes from mice receiving control diet or celecoxib diet were co-cultured
with splenocytes of DC-treated mice (the number of DC-treated splenocytes was equal in all conditions 150.000 cells/well). Percentage of lysis
was calculated using the formula: corrected % lysis = 100 × (experimental release - spontaneous release [target cell incubated in medium
alone])/(maximum release - spontaneous release). Splenocytes from DC-treated mice were significantly better capable of lysing tumour cells
compared to naïve (p < 0.0001) and tumour-bearing mice (p < 0.0001). The addition of splenocytes from a tumour-bearing mice treated with
control diet significantly reduced the lytic capacity of splenocytes from DC-treated mice (p = 0.0002) whereas the addition of splenocytes from
tumour-bearing mice treated with celecoxib diet did not affect the lytic capacity of splenocytes from DC-treated mice (p = 0.887). B)
Percentages of IFN-g+CD8+cells within the spleen were determined by intracellular FACs staining. DC-treatment significantly improved IFN-g
production by CD8+cells compared to naïve mice (p < 0.0001) and tumour-bearing mice (p = 0.001). Conditions as described above. C) IFN-g/
Granzyme B production by CD8+cells was measured by intracellular FACs staining revealing a significant difference between naïve (p < 0.0001)
and tumour-bearing mice(p < 0.0001) compared to CD8+cells in the spleen of DC-treated mice.
Figure 6 Dietary celecoxib improves DC-based immunotherapy.
Mice were inoculated i.p. with 0.5 × 106AB1 tumour cells at day 0.
A suboptimal DC-treatment protocol was used. Mice were treated
with DC-based immunotherapy at day 10 after tumour injection.
Mice received control diet or celecoxib diet from day 1 onwards. All
groups consisted of 6 mice. Survival was measured using Kaplan-
Meier survival analysis. Combining DC-based immunotherapy with
dietary celecoxib improved survival (p = 0.027), compared to a
single treatment with celecoxib (p = 0.305) or DC-based
immunotherapy (p = 0.456). All compared to no treatment.
Veltman et al. BMC Cancer 2010, 10:464
Page 10 of 13
reduced and the production of ROS by these cells is
diminished makes them less capable of inducing T cell
tolerance. The fact that T cell tolerance was no longer
impaired after celecoxib-treatment was confirmed in a
cytotoxicity assay showing that AB1 lysis was hampered
when cells were co-cultured with splenocytes from mice
who had received control diet while tumour specific
lysis occurred when co-culture was performed with sple-
nocytes from tumour-bearing mice treated with cele-
Histological analyses revealed that COX-2 expression
was mainly present in those areas where Gr-1+CD11b+
cells were found. Almost all AB1 tumour cells express
the COX-2 enzyme in culture. An explanation for this
finding could be that tumour cells secrete high levels of
PGE2in order to attract MDSC. The other option is
that COX-2 expression in culture is necessary for prolif-
eration. However, by adding celecoxib to tumour cells
the expression of COX-2 was reduced without affecting
the metabolic activity of the cells (data not shown).
In vivo COX-2 expressing tumour cells were present at
the rim of the tumour tissue co-localizing with the
Gr-1+CD11b+areas. COX-2 expression is diminished in
tumour when mice were treated with celecoxib. The
decrease in COX-2 enzyme expression has been
observed in other studies [31-33]. The mechanism by
which celecoxib perturbs COX-2 protein expression is
not known. It has been suggested that PGE2functions
as a feedback on COX-2 protein expression and that
celecoxib inhibits this loop . It is also possible that
the inhibitory effect of celecoxib on the NF?B pathway
results in a reduced production of COX-2 proteins .
The reduction in COX-2 expression was accompanied
by a reduction of PGE2levels in peritoneal wash of cele-
coxib treated mice. Unfortunately no clear division can
be made between the MDSC types in tumour tissue.
Since Gr-1 (Ly6g) and Lyc6 are both expressed on all
subtypes, no histological subdivision can be made using
these markers. New histological markers are necessary
for better classification of the different MDSC subtypes
in tumour tissue.
Currently, we are investigating DC-immunotherapy in
mesothelioma patients. Although we were able to induce
an immune response in 4 out of 6 patients, the induced
anti-tumour responses are opposed by immune suppres-
sive cells like MDSC. Ochoa and colleagues have sug-
gested that prostaglandin E2 produced by tumour cells
induced the arginase I expression in MDSC . For
this reason, COX-2 inhibition to reduce MDSC in num-
ber and function has been proposed as promising strat-
egy to improve immunotherapy. We have previously
shown that upon injection of antigen-loaded DCs an
effective immune response can be induced in a mouse
model for mesothelioma depending on the timing of the
immunotherapy . In this study we were able to show
that the reduction of MDSC in vivo was associated with
an improved anti-tumour response and resulted in pro-
longation of survival in combination with DC-based
immunotherapy. Same results were reported by others
. We were not able to verify that the improvement
of the anti-tumour response in tumour-bearing mice
treated with celecoxib was directly caused by the reduc-
tion in MDSC. Because MDSC are depending on cyto-
kines, chemokines, prostaglandins and growth factors
produced by surrounding cells for their function, we
choose to mix total splenocytes instead of sorted
Gr-1+CD11b+cells. For example MDSC are critically
depending on IFN-g production by T cells . To deter-
mine that the abolishment of suppression in celecoxib
treatment mice was most likely caused by the reduction
of MDSC number and function, we screened spleno-
cytes of all mice for other cell-types. No significant dif-
ferences in the number of B cell, T cell (including Tregs
and gδT cells) or macrophages between untreated and
celecoxib treated tumour-bearing mice were observed.
The number and function of MDSC did differ between
the two groups (as described on in the result section).
Therefore we conclude that the improved anti-tumour
response is most likely caused be the reduction in
MDSC number and function, though no firm conclu-
sions on direct relations can be made.
We determined the effect of celecoxib treatment on
MDSC; however, since COX-2 (and PGE2) is known to
contribute to variety of cellular processes it is difficult
to determine the solitary effect of a COX-2 inhibitor on
MDSC in vivo. The role of COX-2 expression and
tumour-derived PGE2in cancer has been studied inten-
sively. Significant correlations between the levels of
COX-2 expression and survival were observed in many
human cancers [38-40], including malignant pleural
mesothelioma. COX-2 over-expression was found in the
majority of mesothelioma (73% epithelial mesothelioma,
50% of mix-variants and 37% sarcomatoid mesothe-
lioma). Survival analysis revealed that over-expression of
COX-2 was related to worse prognosis. For this reason
COX-2 inhibition has been proposed as potential thera-
peutic target for mesothelioma [10,11]. Also PGE2con-
migration and invasion of tumour cells .
A rapid induction of MDSC was found during
tumourigenisis in our mouse mesothelioma model.
Although it is generally accepted that MDSC are part of
the tumour microenvironment , their numbers or
function may differ in various tumour cell-lines or other
tumour model. This is caused by differences in the cyto-
kine, chemokine and growth factor production of
tumour cells that determines the specific microenviron-
ments [42,43]. To determine which patients may benefit
Veltman et al. BMC Cancer 2010, 10:464
Page 11 of 13
from celecoxib treatment alone or in combination with
immunotherapy, more research is needed.
Large numbers of infiltrating MDSC co-localise with
COX-2 expression in tumour biopsies. Selective COX-2
inhibition by celecoxib reduced prostaglandin E2 levels
in vitro and in vivo. Treatment of tumour-bearing mice
with dietary celecoxib prevented the local and systemic
expansion of all MDSC subtypes and also their suppres-
sive function was impaired. Combining celecoxib with
DC-based immunotherapy demonstrated highly acti-
vated cytotoxic T lymphocytes with superior immuno-
stimulatory potency and anti-tumour activity because of
the reduced MDSC expansion. This leads to a significant
benefit in overall survival.
We conclude that celecoxib is a powerful tool to
reduce the numbers and suppressive function of MDSC,
which was associated with a beneficial effect of dendritic
cell-based immunotherapy. Future studies will demon-
strate the effectiveness of celecoxib treatment combined
with dendritic cell-based immunotherapy in a clinical
setting for cancer patients.
This study was funded by the “Stichting Asbestkanker Rotterdam” and
JV: acquisition and interpretation of data; writing the manuscript. ML: carried
out animal studies. MvN: carried out flowcytometric analysis. RH: performed
the statistical analysis and data analysis. HH: have given final approval to the
manuscript submission, revised the manuscript. JA: have made substantial
contributions to conception and design, data interpretation. JH: have made
substantial contributions to conception and design, data interpretation,
revised the manuscript. All authors read and approved the final manuscript.
Authors have recently published a clinical trial in the American Journal of
Respiratory and Critical Care Medicine on the use of dendritic cell-based
immunotherapy in mesothelioma patients. We showed for the first time the
safety and feasibility of tumor lysate-pulsed dendritic cells as therapeutic
adjuvants in mesothelioma patients and found distinct immune responses
and antitumor responses in these patients. Now we are focusing on
refinement of this approach.
The authors declare that they have no competing interests.
Received: 24 March 2010 Accepted: 30 August 2010
Published: 30 August 2010
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The pre-publication history for this paper can be accessed here:
Cite this article as: Veltman et al.: COX-2 inhibition improves
immunotherapy and is associated with decreased numbers of myeloid-
derived suppressor cells in mesothelioma. Celecoxib influences MDSC
function. BMC Cancer 2010 10:464.
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