Download Breast Cancer Cell–Derived GM-CSF Licenses

Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
Cancer
Research
Microenvironment and Immunology
Breast Cancer Cell–Derived GM-CSF Licenses
Regulatory Th2 Induction by Plasmacytoid
Predendritic Cells in Aggressive Disease Subtypes
€ l Zollinger1,2,
Cristina Ghirelli1,2, Fabien Reyal3,4, Marine Jeanmougin1,2, Raphae
1,2
1,2
5,6,7
Philemon Sirven , Paula Michea , Christophe Caux
, Nathalie Bendriss-Vermare5,6,7,
1,2
8
le
ne Donnadieu , Martial Caly , Virginie Fourchotte4, Anne Vincent-Salomon8,
Marie-He
Brigitte Sigal-Zafrani8, Xavier Sastre-Garau8, and Vassili Soumelis1,2,9
Abstract
Reciprocal interactions between tumor cells and their microenvironment vitally impact tumor progression. In this study,
we show that GM-CSF produced by primary breast tumor cells
induced the activation of plasmacytoid predendritic cells
(pDC), a cell type critical to anti-viral immunity. pDC that
expressed the GM-CSF receptor were increased in breast tumors
compared with noninvolved adjacent breast tissue. Tumoractivated pDC acquired na€ve CD4þ T-cell stimulatory capacity
and promoted a regulatory Th2 response. Finally, the concomitant increase of GM-CSF and pDC was significantly associated
with relatively more aggressive breast cancer subtypes. Our
results characterize the first tumor-derived factor that can
activate pDC to promote a regulatory Th2 response, with
implications for therapeutic targeting of a tumor-immune
axis of growing recognition in its significance to cancer. Cancer
Introduction
tumor cells in the form of a complex network (2, 5, 6). Deciphering this network relies in large parts on identifying links between
soluble factors and their target cells. This is important because the
soluble microenvironment, cytokines in particular, shapes the
cellular microenvironment by modulating major cellular pathways, such as survival, differentiation, and specialized effector
functions. Such cellular cross-talks may critically influence tumor
prognosis through mutual interactions between tumor cells and
their microenvironment (1, 4, 5).
Using a systematic methodologic framework based on the
study of human primary tumors, we identified breast cancerderived GM-CSF as an endogenous activating signal for plasmacytoid predendritic cells (pDC), a key cell type in anti-viral
immunity (10). Tumor-primed pDC in turn polarized the
immune response toward a regulatory Th2 profile. Importantly,
the GM-CSF/pDC axis was significantly associated to the more
aggressive breast cancer subtypes.
Cancer is associated with disruption of tissue architecture,
which, together with the oncogenic process, promotes the
elaboration of proinflammatory signals (1) that lead to a state
of chronic inflammation (2–5). There are many evidences that
inflammation plays a key role in cancer development (6).
Inflammatory cytokines, such as TNF, have been attributed
immune activating but also immune-suppressive functions in
various mouse tumor models (7, 8). IL6 produced by human
tumors inhibits DC differentiation and promotes tumor development (9). Hence, there is increasing evidence that the proinflammatory cytokine microenvironment of tumors has the
ability to shape the immune response and influence tumor
outcome.
In addition to soluble factors, the tumor is infiltrated by a
diversity of stromal cells, including immune cells such as lymphocytes, dendritic cells (DC), and macrophages, interacting with
Res; 75(14); 1–13. 2015 AACR.
Materials and Methods
1
2
INSERM U932, Institut Curie, Paris, France. Institut Curie, Department
of Immunology, Paris, France. 3UMR144 CNRS, Institut Curie, Paris,
France. 4Institut Curie, Department of Surgical Oncology, Paris,
Lyon 1, Lyon, France. 6INSERM U1052/CNRS
France. 5Universite
rologie de Lyon, Lyon,
UMR5286, Centre de Recherche en Cance
on Be
rard, Lyon, France. 8Institut Curie, Department
France. 7Centre Le
9
of Pathology, Paris, France. Center of Clinical Investigations, CurieIGR, Paris-Villejuif, France.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Author: Vassili Soumelis, Institut Curie, 26 rue d'Ulm, 75005
Paris, France. Phone: 33-44-32–27; Fax: 33-53-10-40-25; E-mail:
[email protected]
doi: 10.1158/0008-5472.CAN-14-2386
2015 American Association for Cancer Research.
Primary samples
Healthy donor human blood buffy coats were obtained from
"Etablissement Français du Sang," Paris, Saint-Antoine Crozatier blood bank through an approved convention with the
Institut Curie (Paris, France). Tumor and juxtatumor tissue
were collected during standard surgical procedures as surgical
residues. They were obtained through the Institut Curie Center
for Biological Resources, which has received regulatory approval for the creation and use of biologic sample collections. The
study was approved by the Institut Curie Institutional Review
Board, and was performed according to national regulatory
rules. All patients and healthy donors gave informed consent
for research use of their biologic material in accordance with
the declaration of Helsinki.
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
OF1
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
Ghirelli et al.
pDC purification
Peripheral blood mononuclear cells (PBMC) were isolated
using Ficoll-gradient (Amersham). pDC were isolated by flow
cytometry as LinCD11cCD4þ as previously described (11).
Purity was higher than 98%.
Cell line culture
We collected cell lines originating from different organs: 6
breast cancer cell lines (BT-20, BT-474, MDA-MB-231, MCF7,
SK-BR-3, T47D); 2 colon cancer cell lines (HT-29, LS1034); 3
ovarian cancer cell lines (IGROV-1, SHIN3, SKOV-3); 1 lung
cancer cell line (A549); 1 bladder cancer cell line (ECV-304); 1
bone osteosarcoma cell line (MG63); 1 pancreatic cancer cell line
(PANC-1) and 1 prostate cancer cell line (PC-3). MCF7, SHIN-3,
and PANC-1 were a kind gift from Dr. Claudia Cabella (Colleretto
Giacosa, Torino, Italy). ECV-304 and MG-63 were a kind gift from
Dr. Barbara Canepa (Colleretto Giacosa, Torino, Italy). All cell
lines were cultured without stimulation at the density of 0.5 106
cells/mL in complete RPMI GlutaMAX (Gibco) containing 10%
FBS (HyClone). Supernatants were collected after 48 hours of
culture, aliquoted at 80 C and then used to quantify cytokines
and stimulate pDC in culture. All cell lines were mycoplasma free.
pDC culture
pDC were cultured at the density of 106 cells/mL in complete
RPMI GlutaMAX (Gibco) containing 10% FBS (HyClone). The
culture conditions were the following: recombinant IL3, GM-CSF
(both R&D Systems), TNF (Peprotech), and IL6 (Miltenyi) were
used at 10 ng/mL to stimulate pDC for 48 hours. For GM-CSF
titration experiments, pDC were stimulated for 48 hours with
recombinant GM-CSF concentrations from 104 to 103 ng/mL.
CpG-C (a kind gift from Dr. Franck J. Barrat, Dynavax Technologies Corporation, Berkeley, California) was used to stimulate
pDC at 5 mg/mL for 48 hours. In separate experiments, pDC were
cultured for 48 hours with cell lines supernatants or primary
breast tumor and juxtatumor supernatants diluted 1/10.
pDC viability
pDC viability was assessed by measuring the percentage of 4,6diamidino-2-phenylindole (DAPI)-negative (Invitrogen) cells by
fluorescence-activated cell sorter (FACS) after 48 hours of culture
with different stimuli. The analysis was performed on an LSRII
(BD Biosciences).
DC differentiation
pDC maturation was analyzed after 48 hours of stimulation
measuring the expression of costimulatory molecules and
human leukocyte antigen (HLA)-DR levels by FACS. We used
the following antibodies: PECy7-CD80 (BioLegend), PECy5CD86 (eBioscience), PE-ICOS-ligand (eBioscience), and Alexa700-HLA-DR (BioLegend). Isotype-matched antibodies were
used as control and values of mean fluorescent intensity (MFI)
of each costimulatory molecule are expressed after subtraction
of nonspecific isotype fluorescence values (Specific MFI). The
analysis was performed on an LSRII (BD Biosciences). Pictures
of pDC in culture were taken with a Leika microscope DMI6000
B just before FACS analysis.
Blocking experiments
Recombinant GM-CSF (R&D Systems) and TNF (Miltenyi)
were used at 1 ng/mL. Tumor cell line supernatants and primary
OF2 Cancer Res; 75(14) July 15, 2015
breast tumor supernatants were diluted 1/10 before for pDC
stimulation. Exogenous cytokines, cell line supernatants, and
primary breast tumor supernatants were preincubated 300 at 37 C
with neutralizing monoclonal antibodies against GM-CSF and
TNF (both R&D Systems) or isotype-matched control antibody
(R&D Systems) at the concentration of 1 mg/mL before adding
them to pDC in culture. pDC viability and maturation were
measured after 48 hours of culture.
Primary breast tumor and juxtatumor supernatants
Breast tumor and juxtatumor tissues were cut in pieces of
40 mg. Each piece of tissue was put in one well of a 48-well
plate in 250 mL of complete RPMI GlutaMAX (Gibco) containing 10% FBS (HyClone) without any stimulation. Supernatants were harvested after 24 hours of culture and tissues
were discarded. Supernatants were diluted 1/2 with complete
RPMI GlutaMAX (Gibco) containing 10% FBS (HyClone),
filtered with a 0.22 mm filter (Millipore), aliquoted at 80 C,
and then used to quantify cytokines and to stimulate pDC in
culture.
Cytokine quantification
IL3, IL6, and IFNg were measured by Cytometric Bead Array
Flex Set (BD Biosciences). The detection limit was 40 pg/mL.
GM-CSF, TNF, IL22, TGFb (all from R&D Systems) and IFNa (PBL
Biomedical Laboratories) were measured by ELISA. The detection
limit for GM-CSF, TNF, IL22, and TGFb was 30 pg/mL. The
detection limit for IFNa was 15 pg/mL.
IHC
Consecutive paraffin-embedded breast tumor sections 5 mm
think were stained with a rabbit anti-human GM-CSF antibody
(Abcam) and an isotype control Ab (Dako), followed by
EnVision Detection Systems Peroxidase/DAB (Dako) or GMCSF antibody and BDCA2 antibody (Dendritics). Bronchoalveolar adenocarcinoma tissue was kindly provided by Dr.
Aurelie Fabre (Centre for Biological Resources, Bichat-Claude
Bernard Hospital, Paris, France) and it was used as a positive
control for GM-CSF staining. The staining was performed using
the Autostainer 480 (Labvision). Hematoxylin staining was
used to visualize cell nuclei.
Quantification and characterization of immune infiltrating
cells in primary breast cancer
Primary breast tumor and juxtatumor tissues were obtained
from the same patient. Both tissues were carefully minced
into small pieces in CO2 independent medium (Gibco) containing 5% FBS (HyClone). They were digested with collagenase
(1 mg/mL; Roche) and DNAse (25 mg/mL; Roche) for 1 hour at
37 C under agitation at 180 rpm. Cell suspension was then
filtered through a 40-mm nylon cell strainer (Falcon BD) and
washed twice in cold PBS containing 5% of human serum (Biowest) and EDTA 2 mmol/L (Gibco). Cells were stained with the
following antibodies: APCCy7-CD45 (BD Biosciences), FITC-CD3,
FITC-CD14 (both Miltenyi), FITC-CD16, FITC-CD19 (both BD
Biosciences), APC-CD4 (Miltenyi), PECy5-CD11c (BD Biosciences), PE-Cy7-CD123 (eBioscience), Alexa 700-HLA-DR
(BioLegend), PE-GM-CSF-Ra (BD Biosciences), and PE-TNFRII (R&D Systems). Isotype-matched antibodies were used as
control. DAPI (Invitrogen) was used to discriminate live and
dead cells.
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
GM-CSF Activates pDC for Th2 Priming in Aggressive Breast Cancer
CD4 T helper differentiation
CD4 T cells were enriched by magnetic negative selection (Miltenyi). CD4 na€ve T cells were then sorted as
CD4þCD45RAþCD25CD45RO by flow cytometry. Purity
was higher than 98%. pDC were cultured for 6 days with allogeneic na€ve CD4 T cells at a 1:5 ratio as previously described (12) in
Yssel's medium (a kind gift from Dr. Hans Yssel, Montpellier,
France) with 10% of FBS (HyClone). CD4 T cells were then
restimulated with anti-CD3/CD28 microbeads (Dynal) in
serum-free X-VIVO 15 medium (Lonza) at the density of 106
cells/mL. Cytokines were measured as described above in supernatants collected after 24 hours of restimulation. In parallel, CD4
T cells were lysed to extract RNA with RNAeasy micro kit (Qiagen).
mRNA transcripts of transcription factors (TBX21, GATA3, and
FOXP3) were quantified by real-time PCR on an ABI Prism 7900
sequence detector (Applied Biosystems) with Applied Biosystem
predesigned TaqMan Gene Expression Assays and Absolute QPCR
ROX mix (Thermo Fisher Scientific).
CD3/CD28 stimulation of breast cancer-infiltrating T cells
Primary breast tumor tissues were digested as described above.
After digestion the cells were resuspended at the density of 106
cells/mL and cultured in 96 round bottom well plate in complete
RPMI GlutaMAX (Gibco) containing 10% FBS (HyClone). Cells
were stimulated with anti-CD3/CD28 microbeads (Dynal) at 1:1
ratio. Cytokines were measured as described above in supernatants collected after 24 hours of culture.
Statistical analysis
Comparisons between different conditions in experiments
performed on pDC, as well as comparison between tumor and
juxtatumor samples were performed using the Wilcoxon test
(nonparametric paired test). Correlations were evaluated using
the Spearman test (nonparametric test). For statistical analysis in
Supplementary Fig. S9, the comparison between aggressive and
nonaggressive tumors with high levels of pDC and/or GM-CSF
and/or TNF was calculated using a c2 test. Statistical significance
was retained for P values lower than 0.05 for all statistical test
applied. , P < 0.05; , P < 0.005; , P < 0.001.
Results
Breast and colon cancer cell lines produce GM-CSF, IL6, and
TNF
We focused our study on cytokines that may be secreted by
tumor cells and play an important role in shaping tumor inflammation (2, 6, 13). On the basis of a previous study of cytokine
receptor expression (14), we measured cytokines with potential
effects on human pDC (Supplementary Table S1). All cancer cell
lines we tested were pathogen- and mycoplasma free, excluding
the presence of exogenous (non-self) activating signals.
GM-CSF, which was previously shown to activate pDC (14), was
detected at high levels in 1 breast cancer cell line supernatant (MDAMB-231) out of six, and in 1 colon cancer cell line supernatant
(LS1034) out of 2, with concentrations of 100 and 20 ng/mL,
respectively. We detected lower amounts of GM-CSF (0.7 ng/mL) in
the prostate cancer cell line PC3 (Supplementary Table S1). Doseresponse curves of pDC activated with recombinant GM-CSF
showed a maximum level of CD80 and CD86 expression when
pDC were treated with 1 ng/mL of recombinant cytokine. The
plateau of activation was maintained until 100 ng/mL (Supple-
www.aacrjournals.org
mentary Fig. S1), suggesting that the levels of GM-CSF measured
in MDA-MB-231 and LS1034 supernatants fall in the range that
induces pDC activation in culture.
IL6 was detected at various levels in cancer cell line supernatants: in 3 out of 6 for breast cancer (BT-20, BT-474 and MDAMB-231), in 2 out of 2 for colon cancer (HT-29 and LS1034), in 2
out of 3 for ovarian cancer (IGROV-1 and SKOV-3), and in the
bladder carcinoma cell line ECV-304 (Supplementary Table S1).
TNF was detected in the colon cancer cell line LS1034 supernatant
and in 1 ovarian cancer cell line supernatant out of 3 (IGROV-1;
Supplementary Table S1). IL3, IFNg, and IFNa, all three cytokines
having been previously shown to activate pDC (11–15), were
undetectable in all these tumor cell line supernatants (Supplementary Table S1).
Breast and colon cancer cell lines promote pDC survival and
maturation
To identify tumor-derived secreted molecules that could
function as activating signals for pDC, we systematically
assessed the potential of various tumor cell line supernatants
to activate human pDC. Culture supernatants of breast, colon,
ovary, lung, bladder, bone, pancreas, and prostate cancer cell
lines were added to primary pDC cultures for 48 hours (Fig. 1).
pDC viability was assessed by flow cytometry as percentage of
DAPI cells. Supernatants of the breast cancer cell line MDAMB-231, and the colon cancer cell line LS1034 increased the
viability of pDC by a mean factor of 4.3 and 4.1 respectively,
similar to exogenous cytokines IL3 and GM-CSF used as controls (Fig. 1A; refs. 14, 15). In addition, MDA-MB-231 and
LS1034 supernatants upregulated the costimulatory molecules
CD80 (Fig. 1B), ICOS-ligand (ICOSL), and CD86 (Supplementary Fig. S2) reflecting pDC activation. Consistently with
increased survival, MDA-MB-231 but not MCF7 supernatants,
led to cluster formation in pDC cultures (Fig. 1C). Thus, breast
and colon cancer cell line supernatants promote pDC viability
and maturation, suggesting that they contain soluble factors
able to activate pDC.
MDA-MB-231 breast tumor cell line activates pDC through
GM-CSF
The activation of pDC with MDA-MB-231–conditioned medium (Fig. 1) and the concomitant absence of detectable amounts of
IFNa in pDC supernatants (Supplementary Fig. S3) strongly
suggested a TLR-independent activation of pDC by breast cancer
cell line supernatants.
IL6 alone is not sufficient to maintain pDC viability and to
activate them in culture (14). However, it may participate in pDC
activation in combination with other tumor-derived factors present in the supernatants. We did not observe any significant
differences neither in survival (Supplementary Fig. S4A) nor in
CD80 and ICOSL expression (Supplementary Fig. S4B) when
pDC were stimulated with a combination of GM-CSF and TNF,
in the presence or absence of IL6. This led us to conclude that even
if IL6 is present in tumor cell line supernatants, pDC do not have
the ability to respond to it.
Even if MDA-MB-231 supernatants did not contain measurable
levels of TNF (Supplementary Table S1), an autocrine effect of this
cytokine could not be excluded. Autocrine TNF was shown to
induce pDC maturation when it is combined to a survival signal (11), which could contribute to pDC activation in our
system, together with GM-CSF. To evaluate the implication of
Cancer Res; 75(14) July 15, 2015
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
OF3
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
Ghirelli et al.
A
% of DAPI- pDC
100
***
***
Viability
C
GM-CSF
Medium
80
60
40
100x
20
MCF7 sup.
100x
MDA-MB-231 sup.
0
4
9 4
0 4 1 7 -3 D
9
3
-1
m 3 F
-3
-1 3 - 3
iu IL -CS T-2 -47 -23 CF BR T47 T-2 103 OV HIN OV A54 -30 G-6 NC PC
V M
B BT MB M KH LS
A
R S SK
M
C
P
M
G
S
E
IG
DA
M
ed
100x
B
CD80-specific MFI
2,000
***
*
100x
CD80
1,500
1,000
500
0
4
9
9 4
0 4 1 7 -3 D
3
F
-1
-3
-1 3 -3
m 3
iu IL -CS T-2 -47 -23 CF BR T47 T-2 103 OV HIN OV A54 -30 G-6 NC PC
V M
H LS
B BT B M KS
K
A
R
M
C
S
P
M
-M
G
S
E
IG
DA
M
ed
Figure 1.
Breast and colon cancer cell line supernatants promote pDC viability and maturation. pDC were stimulated for 48 hours with supernatants generated from cell
lines derived from different organs. A, pDC were put in culture without stimulation (Medium), with recombinant IL3 and GM-CSF as positive controls and with 16
different cell line supernatants previously diluted 1/10. pDC viability was assessed by FACS and it is expressed as percentage of DAPI cells. Data are the mean of six or
more independent experiments, each from different donors. B, pDC maturation was assessed in the same experimental settings as A. The specific MFI of CD80 is
overall viable pDC. Data are the mean of six or more independent experiments, each from different donors. Error bars represent SEM. C, pDC formed cluster in
culture when stimulated with recombinant GM-CSF and MDA-MB-231 supernatant. pDC did not aggregate after stimulation with MCF-7 supernatants as in the
untreated condition (Medium). Inset values represent the magnification. Pictures are representative of ten independent experiments. , P < 0.05; , P < 0.001.
MDA-MB-231–derived GM-CSF and potential autocrine TNF, we
blocked with specific monoclonal antibodies the effect of GMCSF (a-GM-CSF) and/or TNF (a-TNF). Blocking GM-CSF alone
decreased ICOSL and CD80 upregulation induced by MDA-MB231 supernatants (Fig. 2A and B). Blocking TNF alone also
decreased ICOSL and CD80 upregulation, showing a stronger
effect on ICOSL inhibition (Fig. 2A). This suggested that TNF has a
partial autocrine effect on pDC maturation after stimulation with
MDA-MB-231 supernatants. However, the concomitant blocking
of GM-CSF and TNF did not show additive effects. Overall, our
results show that the breast cancer cell line MDA-MB-231 activates
pDC through GM-CSF.
GM-CSF, TNF, and IL6 are produced in the human primary
breast cancer microenvironment
Our previous results suggested that tumor cells have the capacity to produce GM-CSF, which may act in turn as an endogenous
signal to activate pDC. To establish this in primary tumors, we
OF4 Cancer Res; 75(14) July 15, 2015
studied primary human breast cancer samples. Primary breast
tumor and juxtatumor samples were collected prospectively, and
validated as tumoral and normal breast tissue, respectively, after
pathologic examination. Supplementary Table S2 summarizes the
clinical characteristics of the patients included in this study.
Secreted tumor- and juxtatumor-derived cytokines were
assessed after 24 hours of culture of unstimulated tumor and
juxtatumor tissue pieces. As compared with juxtatumor
samples, tumors produced significantly higher levels of GM-CSF
(tumor median: 0.04 ng/mL; juxtatumor median: 0), TNF (tumor
median: 0; juxtatumor median: 0), and IL6 (tumor median: 13.96
ng/mL; juxtatumor median: 5.82 ng/mL; Fig. 3A and Supplementary Table S3). The majority (58.6%) of tumor samples were
positive for GM-CSF, and 27.3% were positive for TNF, as compared with 17.8% and 12.8%, respectively, for juxtatumor supernatants (Fig. 3 and Supplementary Table S3). All tumor and
juxtatumor supernatants processed were positive for IL6 (Fig. 3
and Supplementary Table S3). Among other cytokines measured,
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
GM-CSF Activates pDC for Th2 Priming in Aggressive Breast Cancer
A
B
ICOSL
CD80
115
1546
Medium
1x105
6580
100
100
80
1000
<PE-A>
10000
1x105
3853
100
60
40
1000
<PE-A>
10000
1x105
4296
60
40
100
1000
<PE-A>
10000
1x105
0
3024
40
100
1000
<PE-A>
10000
1x105
0
100
1000
<PE-A>
10000
1x105
0
**
**
100
1000
<PE-A>
10000
1x105
+
-
+
-
1000
10000
<PE-Cy7-A>
1x105
**
+
+
+
GM-CSF + TNF
+
-
0
100
1000
10000
<PE-Cy7-A>
1x105
+
+
+
MDA-MB-231 sup.
100
1000
10000
<PE-Cy7-A>
1x105
1000
10000
<PE-Cy7-A>
1x105
767
% of Max
80
60
40
20
0
0
100
1x105
1000
10000
<PE-Cy7-A>
0
100
**
*
***
***
1,500
+
-
0
100
**
**
4,000
0
100
**
***
8,000
Isotype
a-GM-CSF a-TNF
0
CD80-specific MFI
12,000
% of Max
0
0
533
60
0
0
0
60
40
20
0
1x105
1000
10000
<PE-Cy7-A>
80
20
0
1x105
875
100
100
20
20
20
0
40
0
10000
100
1x105
40
20
40
0
1000
10000
<PE-Cy7-A>
80
60
60
40
100
673
80
20
0
40
0
1000
<PE-A>
1079
898
100
80
60
0
1x105
MDA-MB-231 sup.
60
20
100
1000
10000
<PE-Cy7-A>
80
80
60
100
100
20
0
ICOSL-specific MFI
100
100
20
0
0
80
% of Max
% of Max
MDA-MB-231 sup.
100
452
60
40
20
0
0
80
40
20
0
0
100
80
60
α-GM-CSF +
α-TNF
α-TNF
% of Max
10000
α-GM-CSF
% of Max
100
1000
<PE-A>
GM-CSF + TNF
Isotype
1144
% of Max
100
60
40
20
0
0
80
40
20
0
80
% of Max
% of Max
60
40
20
100
80
60
40
100
% of Max
% of Max
% of Max
60
% of Max
100
80
80
GM-CSF + TNF
α-GM-CSF
5974
% of Max
100
% of Max
Isotype
9573
100
α-GM-CSF +
α-TNF
α-TNF
4135
1882
% of Max
Medium
***
1,000
500
0
Isotype
a-GM-CSF
a-TNF
-
+
-
+
-
+
+
+
GM-CSF + TNF
+
-
+
-
+
+
+
MDA-MB-231 sup.
Figure 2.
MDA-MB-231 supernatants activate pDC through GM-CSF. pDC were cultured with recombinant GM-CSF and TNF and with MDA-MB-231 supernatants
previously diluted 1/10. All stimuli were incubated with blocking antibodies against GM-CSF (a-GM-CSF) and TNF (a-TNF) either alone or in combination
30 minutes before adding pDC to the culture. Isotype-matched antibody was used as control. pDC maturation was assessed by FACS after 48 hours
of culture. Gray histograms represent isotype control. Black histograms represent specific staining for ICOSL (A) and CD80 (B). Data are from one
representative donor out of 8. Inset values indicate specific MFI on over all viable pDC. Unstimulated pDC (Medium) were used as negative control.
Black histograms represent the quantification of the specific MFI of ICOSL (A) and CD80 (B). Data are the mean of eight independent experiments each
from different donors. Error bars represent SEM. , P < 0.05; , P < 0.005; , P < 0.001.
IL3 and IFNg were detected only in a few samples with no
significant differences between tumor and juxtatumor supernatants, whereas IFNa was undetectable in all samples (Fig. 3
and Supplementary Table S3). This cytokine pattern paralleled
the profile observed in the two cancer cell line supernatants
(Supplementary Table S1) that activated pDC in culture (Fig. 1),
characterized mainly by high GM-CSF and IL6.
the 58.6% of tumor samples found positive for GM-CSF after ex
vivo culture.
Figure 3B shows an example of a high and moderate GM-CSF
staining, respectively (Fig. 3B, bottom right) in comparison with
the isotype control antibody (Fig. 3B, bottom left). Pathologic
examination of these sections indicated that the major source of
GM-CSF was the breast tumor epithelial cells.
Primary breast tumor cells produce GM-CSF in situ
On the basis of the study of cancer cell lines and primary tumorconditioned supernatants, we identified GM-CSF as a main candidate cytokine for pDC activation. To exclude artefacts due to the
culture of tumor pieces that may trigger the release of GM-CSF, we
decided to confirm its presence in situ by IHC (Fig. 3B). This was
also a way to further precise its cellular source. We used bronchoalveolar carcinoma as a positive control for the presence of GMCSF (Fig. 3B; refs. 16, 18).
We stained breast cancer tissues from twenty different patient
samples. GM-CSF positivity was assessed in comparison with
an isotype-matched antibody used as control (Fig. 3B, left). Our
results show that two tumors out of 20 (10%) were highly
positive for GM-CSF, 11 (55%) displayed a moderate staining,
and 7 (35%) were negative. In total, we found over 60% of
breast tumor tissue positive for GM-CSF. This result paralleled
pDC infiltrate human breast cancer
Cellularity was analyzed by flow cytometry following tissue
digestion. Gating strategy is shown in Supplementary Fig. S5.
Among viable cells, tumor tissue contained significantly higher
proportion of CD45þ leukocytes (median: 27.7% of DAPI
cells), pDC quantified as DAPICD45þLinCD123þHLA-DRþ
(median: 0.1% of DAPI cells), and DC quantified as
DAPICD45þLinCD123dimHLA-DRþCD11cþ (median: 0.2%
of DAPI cells), as compared with juxtatumor samples from the
same patient (CD45þ median: 4.7%; pDC median: 0.005%; DC
median: 0,017%; Fig. 3C). Normalization of pDC and DC to the
CD45þ fraction gave similar results (Supplementary Fig. S6).
To quantify pDC and DC using flow cytometry, we acquired at
least 100,000 viable cells and we used a cutoff of 0.1% of CD45þ
cells to define positivity, which was significantly higher than the
background isotype control. On the basis of these criteria, 93 breast
www.aacrjournals.org
Cancer Res; 75(14) July 15, 2015
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
OF5
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
Ghirelli et al.
A
TNF
IL6
(N = 111)
(N = 117)
(N = 94)
***
0.8
ng/mL
GM-CSF
**
0.8
0.5
0.4
0.5
0.4
0.2
0.2
0
0.0
0
0.0
B
Bronchoalveolar carcinoma
***
110
Isotype
GM-CSF
80
75
50
25
Tum
Juxta
Tum
IFNα
(N = 100)
(N = 95)
(N = 96)
0.2
0.1
0.06
0.1
Tum
C
0
0.00
Juxta
0.0
0
Juxta
Tum
pDC
DC
(N = 118)
(N = 118)
***
% pDC/DAPI-
15.0
50
25
***
3.5
0.7
0.6
0.3
0
0.0
Juxta
GM-CSF
Juxta
CD45+
75
Tum
Isotype
(N = 118)
***
100
Tum
Breast cancer
ns
% DC/DAPI-
ng/mL
ns
0.12
0
Juxta
IFNγ
0.2
0
0.0
Tum
IL3
ns
% CD45+/DAPI-
0
Juxta
3.0
2
1
0
Tum
Juxta
Tum
Juxta
Figure 3.
Primary breast tumor microenvironment shows increased GM-CSF production and increased pDC percentages as compared with juxtatumor tissue. A,
GM-CSF, TNF, IL6, IL3, IFNg, and IFNa were measured in primary tumor and juxtatumor supernatants. Each dot represents the measurement performed
for a tumor tissue (Tum) paired with its respective juxtatumor tissue (Juxta). N, the numbers of paired measurements performed for each cytokine.
Bars represent median. B, breast cancer tissues were stained with a rabbit anti-GM-CSF antibody (right). An isotype antibody was used as control (left).
Bronchoalveolar adenocarcinoma (top) was used as positive control tissue to validate GM-CSF staining. Middle panels represent an example of a
highly positive lobular carcinoma and bottom panels represent an example of a moderate positive ductal carcinoma. Bars represent a length of 100 mm.
Pictures were taken with a CFW-1308C color digital camera (Scion Corporation) on a Leica DM 4000 B microscope. C, immune cells were quantified
þ
þ
þ
by FACS after tumor and juxtatumor tissue digestion. pDC were quantified as DAPI CD45 Lin CD123 HLA-DR . DCs were quantified as DAPI
þ
dim
þ
þ
CD45 Lin CD123 HLA-DR CD11c . Each dot represents the measurement performed for a tumor tissue (Tum) paired with its respective juxtatumor
tissue (Juxta). N, the numbers of paired measurements performed for each cell population. Bars represent median. , P < 0.005; , P < 0.001.
tumor samples (78.8%) were positive for pDC infiltration, and 103
(87.3%) for DC infiltration, as compared with 48 (40.7%) and 84
(71.2%) positive juxtatumor tissues for pDC and DC, respectively.
Tumor-infiltrating pDC express GM-CSF-Ra (CD116) but not
TNF-RII
To evaluate the ability of tumor-infiltrating pDC to respond to
GM-CSF and TNF, which are the two main cytokines measured in
primary breast tumor supernatants with an activating potential on
pDC, we measured the expression of GM-CSF receptor (CD116)
and TNF receptor (TNF-RII) by flow cytometry in tumor-infiltrating pDC using the gate strategy showed in Supplementary Fig. S5.
As for blood pDC (Fig. 4, right) tumor pDC expressed high levels
of CD116 (Fig. 4, left), while lower levels were detected on
juxtatumor pDC (Fig. 4, middle). On the contrary, tumor pDC
did not express TNF-RII, similar to blood pDC, while juxtatumor
pDC showed a small increase in the specific staining for this
OF6 Cancer Res; 75(14) July 15, 2015
receptor (Fig. 4, left, right, and middle, respectively). These data
indicate that the expression of GM-CSF receptor is not affected by
the tumor microenvironment, and that tumor pDC have the
ability to respond to tumor-derived GM-CSF.
Tumor-derived GM-CSF promotes pDC viability and
maturation
To address the function of primary tumor-derived GM-CSF, we
analyzed the effect of tumor supernatants containing a broad range
of GM-CSF concentrations, on the viability and maturation of pDC
purified from the blood of healthy donors. We observed a significant positive correlation between GM-CSF levels in tumor supernatants, and the viability of pDC after 48 hours of culture (Fig. 4B,
top). This was not the case for TNF (Fig. 4B, bottom), which,
together with the lack of TNF receptor on tumor pDC (Fig. 4A, left),
suggested that GM-CSF rather than TNF might be involved in
modulating pDC viability and function in the primary tumor
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
GM-CSF Activates pDC for Th2 Priming in Aggressive Breast Cancer
Juxtatumor pDC
Tumor pDC
GM-CSF-Ra
B
GM-CSF-Ra
TNF-R II
4821
0
Tum sup. GM-CSF
(ng/mL; 1/10 dilution)
Spearman P = 0.0004
***
0.2
0.1
0.0
0
20
40
60
GM-CSF-Ra
260
80
100
0.3
Spearman P < 0.0001
***
0.2
0.1
0.0
0
0.0
20
40
60
80
100
% of viable pDC
Tum sup. TNF
(ng/mL; 1/10 dilution)
Tum sup. TNF
(ng/mL; 1/10 dilution)
0.1
0
500
1,000
1,500
2,000
2,500
CD80-specific MFI
Spearman P = 0.0262
ns
0.3
0.2
0
CD80
% of viable pDC
0.8
TNF-R II
3456
C
Viability
0.3
Blood pDC
TNF-R II
4109
Tum sup. GM-CSF
(ng/mL; 1/10 dilution)
A
0.4
Spearman P = 0.9952
ns
0.3
0.2
0.1
0.0
0
2,000
4,000
6,000
CD80-specific MFI
Figure 4.
Breast tumor-derived GM-CSF promotes pDC survival and CD80 expression. A, primary tumor and juxtatumor tissues were digested and the levels of GM-CSF-Ra
and TNFRII expression on pDC were quantified by FACS. Tumor, juxtatumor, and blood pDC were identified using the gate strategy shown in Supplementary
Fig. S5. Gray histograms represent unstained cells. Black histograms represent specific staining for the receptors. Inset values indicate specific MFI on over all viable
pDC. Data are from one representative breast tumor and juxtatumor tissue out of 5. B, correlation between the percentage of viable pDC and the amounts of
GM-CSF and TNF obtained after 1/10 dilution of tumor supernatants before pDC stimulation in culture. C, correlation between CD80-specific MFI and amounts
of GM-CSF and TNF obtained after 1/10 dilution of tumor supernatants before pDC stimulation in culture. Each dot represents an independent experiment
performed on pDC stimulated with a different tumor supernatant. ns, nonsignificant.
microenvironment. Tumor-derived GM-CSF was able to upregulate
costimulatory molecules on purified primary pDC. We analyzed
pDC maturation in the same experimental settings as Fig. 4B. As for
pDC viability (Fig. 6B), we observed a significant positive correlation between GM-CSF concentration and pDC maturation based
on the measurement of surface CD80 expression after 48 hours of
culture (Fig. 4C, top). CD80 expression on pDC did not correlate
with TNF concentration (Fig. 4C, bottom), suggesting that pDC
maturation with tumor supernatant, as well as pDC viability, are
driven by GM-CSF, and not TNF. To directly assess and quantify the
ability of tumor-derived GM-CSF to promote pDC viability, we
cultured purified primary healthy pDC with tumor supernatant in
the absence or presence of a blocking antibody against GM-CSF
(aGM-CSF; Fig. 5). Tumor supernatants were able to maintain pDC
viability to levels similar to culture medium alone (Fig. 5A).
Blocking GM-CSF induced an important and statistically significant
drop in pDC viability of about 50% (Fig. 5A). Thus, the secreted
tumor microenvironment is able to maintain pDC viability through
GM-CSF. Breast tumor supernatants were able to upregulate not
only CD80 (Figs. 4C and 5B), but also CD86 and ICOSL expression
www.aacrjournals.org
on pDC compared with unstimulated pDC (Fig. 5B). Blocking GMCSF in the context of tumor supernatants decreased CD80 and
ICOSL upregulation by 62% and 37%, respectively, although not
affecting CD86 expression (Fig. 5B).
Breast cancer-derived GM-CSF converts pDC into mature
dendritic cells that are able to stimulate na€ve CD4 T cells
It was previously shown that pDC did not stimulate na€ve CD4
T cells unless properly activated (10). The activated phenotype of
pDC stimulated with breast tumor and juxtatumor supernatants
was associated with a lack of detectable amounts of IFNa in pDC
supernatants (Supplementary Fig. S7), as was observed in other
systems of cytokine-induced pDC activation (10). This further
supported a TLR-independent pDC activation in the context of the
breast cancer microenvironment.
Next, we asked whether pDC matured by the breast tumor
supernatant were able to induce the expansion of allogeneic
primary na€ve CD4 T cells. To do so, we selected a panel of
GM-CSF–positive breast tumor supernatants to stimulate pDC.
We found that pDC activated with these breast tumor supernatants
Cancer Res; 75(14) July 15, 2015
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
OF7
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
Ghirelli et al.
A
Viability
ns
**
% of DAPI- pDC
100
80
60
40
20
0
Tum sup
Isotype
a-GM-CSF
-
+
+
-
B
+
+
CD80
***
Specific M FI
10,000
ICOSL
**
***
25,000
CD86
*
4,500
4,000
3,500
3,000
ns
***
20,000
7,000
4,000
3,000
2,000
2,000
1,000
0
Tum sup
Isotype
a-GM-CSF
0
0
-
+
+
-
+
+
-
+
+
-
+
+
-
+
+
-
+
+
Figure 5.
Breast tumors promote pDC viability and maturation through GM-CSF. A, viability of pDC after 48 hours of stimulation with tumor supernatants (Tum sup). Tumor
supernatants were diluted 1/10 and they were incubated with blocking antibodies against GM-CSF (a-GM-CSF) 30 minutes before adding pDC to the culture.
Isotype-matched control was used. pDC viability was measured by FACS and it is expressed as percentage of DAPI . Each dot represents an independent
experiment performed on pDC stimulated with a different tumor supernatant. Bars represent median. B, pDC maturation was assessed by FACS in the same
experimental settings as A. The specific MFI of CD80, ICOSL, and CD86 is expressed on overall viable pDC. Each dot represents an independent experiment
performed on pDC stimulated with a different tumor supernatant. Bars represent median. , P < 0.05; , P < 0.005; , P < 0.001. ns, nonsignificant.
induced the highest CD4 T-cell expansion, as compared with
medium alone and exogenous GM-CSF (Fig. 6A). In this system,
we could not block endogenous GM-CSF to address its involvement in T-cell priming because of the strong decrease in pDC
viability (Fig. 5A), leading to insufficient pDC numbers to perform
T-cell coculture assay. However, we could correlate the concentration of tumor-derived GM-CSF with the ability of each supernatant
to promote pDC-mediated CD4 T-cell expansion. This revealed a
strong and significant positive correlation between GM-CSF levels
and T-cell expansion (Fig. 6B). Thus, breast cancer-derived GM-CSF
confers pDC a na€ve CD4 T-cell stimulatory capacity.
pDC primed with GM-CSF containing tumor supernatants
induce effector CD4 T cells with a regulatory Th2-biased
cytokine profile
To evaluate the effector functions of CD4 T cells primed
with tumor-activated pDC, we measured an extended panel of
T-cell–derived cytokines. When compared with medium-pDC, T
cells activated with GM-CSF–positive tumor-pDC produced
higher amounts of IL4, IL5, IL10, IL13, and TNF, but lower
OF8 Cancer Res; 75(14) July 15, 2015
amounts of IFNg (Fig. 6C). This higher IL4, IL5, and IL13,
combined to lower IFNg production, together with lower expression of TBX21 (Tbet; Supplementary Fig. S8A, left) indicated a
global Th2 bias. The transcription factor GATA3 was less expressed
in CD4 T cells activated with tumor-primed pDC as compared
with medium-pDC and GM-CSF-pDC (Supplementary Fig. S8A,
middle), suggesting that the Th2 bias observed might be GATA3
independent.
We did not observe any differences in FOXP3 and TGFb
expression (Supplementary Fig. S8A, right and Supplementary
Fig. S8B, left) by CD4 T cells primed with differentially activated
pDC. Interestingly, GM-CSF–positive tumor-pDC induced higher
IL10 production by CD4 T cells, as compared with GM-CSF-pDC
(Fig. 6C). This suggested that other factors within the secreted
tumor microenvironment might act in synergy with tumorderived GM-CSF to further increase IL10 priming, and promote
a regulatory Th2 phenotype (19).
Of particular interest is the bi-modal distribution of IL5
(Fig. 6C). A group of GM-CSF–positive tumor supernatants
primed pDC to activate CD4 T cells to secrete high amounts of
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
GM-CSF Activates pDC for Th2 Priming in Aggressive Breast Cancer
B
**
***
9
Spearman P = 0,0002
**
6
3
0
Med
Tum
GM-CSF
T-cell fold expansion
T-cell fold expansion
A
***
9
6
3
0
0.0
0.1
0.2
0.3
Tum GM-CSF (ng/mL;1/10 dilution)
C
4
IL4
*
**
IL5
ns
ns
ng/mL
3
*
12
4.5
4
8
2
4
8
0
D
IL13
*
ns
**
6
6
TNF
**
ns
**
Med Tum GM-CSF
0
0
ns
*
0.6
Med Tum GM-CSF
0.2
0.3
0.1
0
***
5
0.6
1
0.5
0.3
0.1
ns
8
*
*
4
2
2
Med Tum GM-CSF
IFNg
6
4
4
2
1
ng/mL
9.0
*
IL10
**
ns
**
Med Tum GM-CSF
**
0
8
2
Med Tum GM-CSF
***
0
6
5
4
3
2
2
1
Med Tum GM-CSF
***
0.1
0
0
Unst αCD3/28
0
Unst αCD3/28
0
Unst αCD3/28
Unst αCD3/28
0
Unst αCD3/28
0
Unst αCD3/28
Figure 6.
GM-CSF–positive breast tumors convert pDC into mature DC, inducing a regulatory Th2-biased CD4 T-cell differentiation. pDC were cultured for 48 hours without
stimulation (Med), with breast tumor supernatants containing different amounts of GM-CSF (Tum) and with recombinant GM-CSF. Matured pDC were then
cocultured with allogeneic na€ve CD4 T cells. After 6 days of coculture, CD4 T cells were restimulated with anti-CD3/CD28 (aCD3/28) for 24 hours. A, CD4 T-cell fold
expansion was calculated after 6 days of coculture. B, correlation between T-cell fold expansion and the amounts of GM-CSF obtained after 1/10 dilution of
tumor supernatants before pDC stimulation. C, CD4 T-cell cytokines were measured 24 hours after aCD3/CD28 T-cell restimulation in the same experimental
settings as A and B. For A, B, and C, each dot represents an independent experiment performed on pDC stimulated with a different tumor supernatant
containing GM-CSF. Bars represent median. N ¼ 15. D, digested cells from breast tumor tissues were put in culture without stimulation (Unst) or with aCD3/28.
Cytokines were measured in supernatants collected after 24 hours. Each dot represents the measurement performed on a different tumor sample. N ¼ 24.
, P < 0.05; , P < 0.005; , P < 0.001. ns, nonsignificant.
IL5 (1.45–8.75 ng/mL), whereas a second group did not. This may
be due to other soluble factors present in the tumor microenvironment or to different costimulatory molecules expressed by
pDC.
We did not observe significant differences in IL22 production
between CD4 T cells stimulated with tumor-pDC and mediumpDC or GM-CSF-pDC (Supplementary Fig. S8B, right).
In summary, CD4 T cells primed by GM-CSF–positive tumoractivated pDC produced a large panel of T helper cytokines,
suggesting efficient effector functions. This cytokine profile was
qualitatively biased toward a regulatory Th2 phenotype, characterized by Th2 cytokines, and high levels of IL10.
To assess the in vivo relevance of these results, we digested
primary breast cancer tissues, and we stimulated the single cell
suspension ex vivo in a polyclonal manner with a CD3/CD28
(aCD3/CD28) microbeads for 24 hours (Fig. 6D). The same
panel of Th cytokines as for in vitro-primed CD4 T cells was
www.aacrjournals.org
measured in supernatants of restimulated tumor-infiltrating T
cells. In unstimulated conditions, we never found detectable
amounts of IL4, IL5, and IL13, and only in a minority of
supernatants we detected low amounts of IL10, TNF, and IFNg
(Fig. 6D and Supplementary Table S4). After anti-CD3/CD28
stimulation, we observed a significant increase in IL4, IL5, and
IL13, respectively, in 30%, 12%, and 39% of supernatants
(Fig. 6D and Supplementary Table S4); 91% of the samples
were positive for TNF and IFNgafter aCD3/CD28 stimulation,
and 83% of them showed IL10 production with levels up to
4.7 ng/mL (Fig. 6D and Supplementary Table S4). These results
indicated a good match between the cytokine profile of in vitroprimed na€ve T cells with tumor-activated pDC, and ex vivo
restimulated tumor-infiltrating T cells. This strengthens the in
vivo relevance of the cytokine profile that we observed with
allogeneic na€ve CD4 T cells cocultured with tumor-activated
pDC (Fig. 6C).
Cancer Res; 75(14) July 15, 2015
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
OF9
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
Ghirelli et al.
P = 0.038
*
60
40
20
0
Aggr.
P = 0.13
80
ns
60
40
20
0
Non aggr.
Aggr.
Non aggr.
% of tumors with
high pDC and high GM-CSF
% of tumors with
high pDC
80
% of tumors with
high GM-CSF
A
nal B). Nonaggressive group includes luminal A breast cancer
types, which are hormone receptor–positive and HER2-negative.
By performing a c2 test, we showed that the group of aggressive
breast cancer (20) was enriched in high pDC compared with
nonaggressive tumors (P ¼ 0.038; Fig. 7A, top left). This significance was stronger if the comparison between aggressive and
nonaggressive breast cancer was calculated taking into account
samples showing concomitant high levels of pDC and GM-CSF
(P ¼ 0.0012), which were 30.8% and 6.5%, respectively (Fig. 7A,
top right), and it was lost when the same analysis was performed
taking in account only GM-CSF high tumors (Fig. 7A, top middle).
High levels of TNF, either alone or in combination with high pDC
levels, were not associated to breast cancer aggressiveness (Fig. 7A,
bottom). Figure 7B shows an example of an aggressive breast
cancer (triple-negative) stained for GM-CSF and pDC in two
consecutive slides. Dashed lines delimitate the tumor area positive for GM-CSF, and the black arrows show the localization of a
pDC cluster next to a GM-CSF–positive area.
P = 0.5
80
60
40
20
0
B
GM-CSF
% of tumors with
% of tumors with
high TNF
ns
Aggr.
pDC
Non aggr.
high pDC and high TNF
Concomitant high levels of pDC and GM-CSF are characteristic
of aggressive breast cancer
Because of the short time delay between the enrolment of the
last patient in this study and the current time (less than 5 years),
we decided to follow a new approach to assess the physiopathological implication of pDC and GM-CSF in breast cancer. Samples were stratified based on the distribution of pDC and GM-CSF
amounts, or pDC and TNF amounts, as quantified by FACS and
ELISA, respectively (Fig. 3C, middle and Fig. 3A, top left and
middle). We defined tumors with high amounts of pDC respective
to high levels of GM-CSF or TNF as the tumors for which the
percentage of pDC respective to the level of GM-CSF or TNF were
greater than the 66th percentile of the distribution across all
tumors. After histopathological examination, tumors were classified in aggressive and nonaggressive groups. Aggressive tumors
include hormone receptor/HER2-negative samples (triple-negative, TN), hormone receptor-negative and HER2-positive samples
(HER2þ), and hormone receptor/HER2-positive samples (lumi-
80
P = 0.001
**
60
Aggressive breast cancer
TN, HER2+, LB (N = 26)
Non-aggressive breast cancer
LA (N = 92)
40
20
0
80
Aggr.
Non aggr.
P = 0.1
ns
60
40
20
0
Aggr.
GM-CSF
Non aggr.
pDC
Figure 7.
High levels of pDC and GM-CSF are characteristic of aggressive breast cancer. A, bar graphs represent the percentage of tumors characterized by high pDC
(top left), high GM-CSF (top middle), high pDC and high GM-CSF (top right), high TNF (bottom left), and high pDC and high TNF (bottom right). Black
þ
histograms represent triple-negative (TN), HER2 , and luminal B (LB) breast tumors; white histograms represent luminal A (LA) breast tumors. TN, hormone
þ
receptor and HER2-negative tumors; HER2 , hormone receptor–negative and HER2-positive tumors; LB, hormone receptor and HER2-positive tumors; LA,
hormone receptor–positive and HER2-negative tumors. This analysis was performed on the whole cohort of 118 breast cancer samples collected for this study. B, GMCSF and pDC (BDCA2-positive cells) staining in two consecutive sections of the same triple-negative breast cancer sample. Dashed lines show the tumor
area where GM-CSF is produced and black arrows show the localization of pDC. Bars represent a length of 50 mm. Pictures were taken with a CFW-1308C
color digital camera (Scion Corporation) on a Leica DM 4000 B microscope. ns, nonsignificant.
OF10 Cancer Res; 75(14) July 15, 2015
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
GM-CSF Activates pDC for Th2 Priming in Aggressive Breast Cancer
Overall these data indicate that high amounts of pDC and GMCSF were associated with the tumor subgroups of worst prognosis.
This suggests a role for pDC and GM-CSF in the generation and
maintenance of an immunoregulatory environment, which may
favor a bad outcome for breast cancer patients.
Discussion
In this study, we investigated the role of pDC as players at the
interface between innate and adaptive immunity and their behavior in shaping immune response after activation with breast
tumor-derived soluble factors. pDC link innate and adaptive
immunity, and produce type I IFN following activation through
TLR-7 and -9 by microbes and microbial products, thus participating in the protection against infections (10, 21). However,
pDC were also implicated in a variety of sterile inflammatory
conditions (10, 22, 23), and infiltrate a broad range of solid
tumors such as head and neck cancer (24), ovarian cancer (25),
cervical carcinoma (26), lung cancer (27), thyroid cancer (28),
breast cancer (29), and melanoma (30). Despite the increasing
number of observations about pDC presence in solid tumors, the
nature of the signals they receive within the tumor microenvironment is not known (221). This question is crucial because pDC
harbor a great level of functional plasticity depending on the
microenvironmental stimuli they receive (10). Our data now
provide a physiopathological relevance to the GM-CSF pathway
for pDC activation within the breast tumor microenvironment.
As key sensors at the interface between innate and adaptive
immunity, pDC can efficiently prime CD4 na€ve T cells in the
lymph nodes (31), and induce Th1 or Th2 polarization depending
on the signals present in their microenvironment (10, 32). In our
study, the cytokine profile of na€ve CD4 T cells primed with GMCSF–positive breast tumor-activated pDC was characterized by
higher production of IL4, IL5, IL13, IL10, and TNF, accompanied
by a lower IFNg production, as compared with unstimulated pDC.
This profile was different from that of CD4 T cells stimulated with
GM-CSF-pDC, which was characterized by more IL13 and IFNg,
and less IL5, as compared with tumor-pDC, suggesting that other
tumor-derived factors than GM-CSF may act concomitantly to
shape the ability of pDC to prime CD4 T cells. Production of TNF
by CD4 T cells primed with tumor-activated pDC is puzzling given
the differential regulation of IL10 and TNF described in vitro (19).
Tumor T-cell–derived TNF has been shown to participate to tumor
progression (8). CD4 T cells cocultured with tumor-primed pDC
produced high levels of IL10, which was consistent with the high
ICOSL expression on pDC stimulated with tumor supernatants.
ICOSL is known to be required for pDC to prime CD4 T cells to
produce IL10 (12). This cytokine profile was confirmed in independent experiments using tumor-infiltrating T cells following
polyclonal restimulation. This provides a strong basis for the
implication of a GM-CSF/pDC/Th2 axis in breast cancer.
Th2 cytokines were shown to contribute to tumorigenesis in
several models. IL13 produced by NKT cells induced myeloid cells
to make TGFb, which inhibited CTL functions in several mouse
models (33). A study performed on a spontaneous mouse breast
cancer model underlined the role of Th2 cells in facilitating the
development of lung metastasis through macrophage activation
(34), supporting a previous work demonstrating the role of IL4 in
mediating lung metastasis (35). On the other side a more recent
work, showed that adaptive immune cells do not play any role in
the development of a metastatic HER2þ breast cancer (36). Th2
www.aacrjournals.org
and TGFb activation was associated with poor prognosis in
human breast cancer (37). Overall, Th2 cells and Th2 cytokines
have been related to induction of T-cell anergy and loss of T-cell–
mediated cytotoxicity leading to a downregulation of cell-mediated antitumor immunity (13).
Human breast cancer has also been associated to a type II
inflammation (38) that was shown to be mediated by TSLP action
on tumor-infiltrating dendritic cells. However, a large proportion
of tumors (61.4%) was TSLP-negative, raising the question of
alternative or parallel pathways to induce Th2 responses. Our
results describe a novel pDC/GM-CSF/Th2 axis as a new mechanism to explain the development of a regulatory type II inflammation characteristic of human breast cancer. Further studies will
be needed to evaluate the relative role of these pathways within
the breast cancer microenvironment.
pDC were suggested to have properties in driving immune
tolerance to tumors. Tumor-infiltrating pDC drive CD4 and CD8
T cells to produce IL10 and acquire a regulatory phenotype in
ovarian cancer (25, 39). pDC can induce regulatory T cells in vitro
as well as in vivo in melanoma and breast cancer (40, 41) to help
maintaining a tolerogenic microenvironment. pDC were identified as an adverse prognostic factor for breast cancer patient
survival (29) as well as melanoma cancer patients (42), which
suggest their regulatory rather than antitumor properties. A recent
work has shown that pDC accumulate in aggressive breast tumors
contributing to tumor immune tolerance and poor clinical outcome (41).
GM-CSF can be produced by a large variety of cell types, such as
fibroblasts, endothelial cells, T cells, macrophages, mesothelial
cells, and epithelial cells (43). Previous studies have shown the
presence of GM-CSF in diverse type of tumors, including breast
cancer (17, 44–47). GM-CSF can function as an immune adjuvant
by activating NK cells and myeloid cells (48), and is widely used in
antitumor therapy (49, 50). However, GM-CSF is known to be
able to suppress the immune response since decades (51, 52), and
some clinical studies have shown that GM-CSF as anticancer
vaccine adjuvant can have adverse outcome in term of relapsefree and overall survival (53, 54). It was recently shown that breast
cancer-derived GM-CSF has a protumorigenic role, and a positive
correlation between high levels of endogenous GM-CSF,
increased metastasis, and reduced survival was observed in breast
cancer patients (47).
To the best of our knowledge, there are no studies available on
the combined role of pDC and GM-CSF in breast cancer. Our data
provide evidence for pDC as a novel cellular target of tumorderived GM-CSF. The clinical relevance of pDC and GM-CSF as
coordinated immunoregulatory players within the breast cancer
microenvironment is of high importance. We found that high
levels of pDC and GM-CSF were characteristic of aggressive breast
cancer reflecting their role in shaping the immune response
toward a regulatory Th2 phenotype.
In our current study, we had to pool various types of aggressive
breast cancer (TN, HER2-positive, and luminal B) because of the
relative low number patients in each of these subgroups. Future
studies on larger aggressive breast cancer cohorts may be important to delineate the specific impact of the tumor/GM-CSF/pDC
axis in their respective clinical outcome.
Given the complexity of the tumor microenvironment, several
factors probably act in combination to activate innate immunity.
It will be important to continue dissecting the breast cancer
microenvironment complexity, identify other endogenous factors
Cancer Res; 75(14) July 15, 2015
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
OF11
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
Ghirelli et al.
that can shape the immune response, and understand their
mutual interactions. Additional clinical studies will be important
to define whether breast cancer-derived GM-CSF, and its downstream functional impact on pDC, can serve as novel prognostic
biomarkers or therapeutic targets to improve the clinical care of
breast cancer patients.
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): M.-H. Donnadieu, M. Caly
Study supervision: X. Sastre-Garau, V. Soumelis
Acknowledgments
The authors thank Zofia Maciorowski and Annick Viguier for the cytofluorimetric cell sorting, and Philippe Benaroch and Elisabetta Volpe for helpful
suggestions and critical reading of the article.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: C. Ghirelli, C. Caux, V. Soumelis
Development of methodology: C. Ghirelli, C. Caux
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): C. Ghirelli, F. Reyal, R. Zollinger, P. Sirven, P. Michea,
N. Bendriss-Vermare, V. Fourchotte, A. Vincent-Salomon, B. Sigal-Zafrani,
X. Sastre-Garau, X. Sastre-Garau
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): C. Ghirelli, F. Reyal, M. Jeanmougin, R. Zollinger,
P. Sirven, P. Michea
Writing, review, and/or revision of the manuscript: C. Ghirelli, F. Reyal,
M. Jeanmougin, C. Caux, N. Bendriss-Vermare, V. Soumelis
Grant Support
This work was supported by Leonardo da Vinci Unipharma Graduates
Program (C. Ghirelli), Association pour la Recherche Contre le Cancer
(C. Ghirelli), European Marie Curie Excellence Grant (R. Zollinger and
V. Soumelis), Fondation pour la Recherche Medicale (R. Zollinger), and Curie
Institute "PIC Tumor Microenvironment" funding (M. Jeanmougin, P. Sirven,
and V. Soumelis).
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 August 13, 2014; revised March 24, 2015; accepted March 27, 2015;
published OnlineFirst May 14, 2015.
References
1. Pardoll D. Does the immune system see tumors as foreign or self? Annu Rev
Immunol 2003;21:807–39.
2. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420:
860–7.
3. de Visser KE, Korets LV, Coussens LM. De novo carcinogenesis promoted by
chronic inflammation is B lymphocyte dependent. Cancer Cell 2005;7:
411–23.
4. Condeelis J, Pollard JW. Macrophages: obligate partners for tumor cell
migration, invasion, and metastasis. Cell 2006;124:263–6.
5. Mantovani A, Romero P, Palucka AK, Marincola FM. Tumour immunity:
effector response to tumour and role of the microenvironment. Lancet
2008;371:771–83.
6. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer.
Cell 2010;140:883–99.
7. Moore RJ, Owens DM, Stamp G, Arnott C, Burke F, East N, et al. Mice
deficient in tumor necrosis factor-alpha are resistant to skin carcinogenesis.
Nat Med 1999;5:828–31.
8. Balkwill F. TNF-alpha in promotion and progression of cancer. Cancer
Metastasis Rev 2006;25:409–16.
9. Menetrier-Caux C, Montmain G, Dieu MC, Bain C, Favrot MC, Caux C, et al.
Inhibition of the differentiation of dendritic cells from CD34(þ) progenitors by tumor cells: role of interleukin-6 and macrophage colony-stimulating factor. Blood 1998;92:4778–91.
10. Liu YJ. IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu Rev Immunol 2005;23:275–306.
11. Kadowaki N, Antonenko S, Lau JY, Liu YJ. Natural interferon alpha/betaproducing cells link innate and adaptive immunity. J Exp Med 2000;
192:219–26.
12. Ito T, Yang M, Wang YH, Lande R, Gregorio J, Perng OA, et al. Plasmacytoid
dendritic cells prime IL-10-producing T regulatory cells by inducible
costimulator ligand. J Exp Med 2007;204:105–15.
13. DeNardo DG, Coussens LM. Inflammation and breast cancer. Balancing
immune response: crosstalk between adaptive and innate immune cells
during breast cancer progression. Breast Cancer Res 2007;9:212.
14. Ghirelli C, Zollinger R, Soumelis V. Systematic cytokine receptor profiling
reveals GM-CSF as a novel TLR-independent activator of human plasmacytoid predendritic cells. Blood 2010;115:5037–40.
15. Grouard G, Rissoan MC, Filgueira L, Durand I, Banchereau J, Liu YJ. The
enigmatic plasmacytoid T cells develop into dendritic cells with interleukin
(IL)-3 and CD40-ligand. J Exp Med 1997;185:1101–11.
16. Rissoan MC, Soumelis V, Kadowaki N, Grouard G, Briere F, de Waal Malefyt
R, et al. Reciprocal control of T helper cell and dendritic cell differentiation.
Science 1999;283:1183–6.
OF12 Cancer Res; 75(14) July 15, 2015
17. Wislez M, Fleury-Feith J, Rabbe N, Moreau J, Cesari D, Milleron B, et al.
Tumor-derived granulocyte-macrophage colony-stimulating factor and
granulocyte colony-stimulating factor prolong the survival of neutrophils
infiltrating bronchoalveolar subtype pulmonary adenocarcinoma. Am J
Pathol 2001;159:1423–33.
18. Tazi A, Bouchonnet F, Grandsaigne M, Boumsell L, Hance AJ, Soler P.
Evidence that granulocyte macrophage-colony-stimulating factor regulates
the distribution and differentiated state of dendritic cells/Langerhans cells
in human lung and lung cancers. J Clin Invest 1993;91:566–76.
19. Liu YJ. Thymic stromal lymphopoietin and OX40 ligand pathway in the
initiation of dendritic cell-mediated allergic inflammation. J Allergy Clin
Immunol 2007;120:238–44; quiz 45–6.
20. Voduc KD, Cheang MC, Tyldesley S, Gelmon K, Nielsen TO, Kennecke H.
Breast cancer subtypes and the risk of local and regional relapse. J Clin
Oncol 2010;28:1684–91.
21. Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang YH, Homey B, et al.
Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial
peptide. Nature 2007;449:564–9.
22. Lande R, Gilliet M. Plasmacytoid dendritic cells: key players in the initiation
and regulation of immune responses. Ann N Y Acad Sci 2010;1183:
89–103.
23. Charles J, Chaperot L, Salameire D, Di Domizio J, Aspord C, Gressin R, et al.
Plasmacytoid dendritic cells and dermatological disorders: focus on their
role in autoimmunity and cancer. Eur J Dermatol 2010;20:16–23.
24. Hartmann E, Wollenberg B, Rothenfusser S, Wagner M, Wellisch D, Mack B,
et al. Identification and functional analysis of tumor-infiltrating plasmacytoid dendritic cells in head and neck cancer. Cancer Res 2003;63:
6478–87.
25. Zou W, Machelon V, Coulomb-L'Hermin A, Borvak J, Nome F, Isaeva T, et al.
Stromal-derived factor-1 in human tumors recruits and alters the function of
plasmacytoid precursor dendritic cells. Nat Med 2001;7:1339–46.
26. Bontkes HJ, Ruizendaal JJ, Kramer D, Meijer CJ, Hooijberg E. Plasmacytoid
dendritic cells are present in cervical carcinoma and become activated by
human papillomavirus type 16 virus-like particles. Gynecol Oncol
2005;96:897–901.
27. Perrot I, Blanchard D, Freymond N, Isaac S, Guibert B, Pacheco Y, et al.
Dendritic cells infiltrating human non-small cell lung cancer are blocked at
immature stage. J Immunol 2007;178:2763–9.
28. Tsuge K, Takeda H, Kawada S, Maeda K, Yamakawa M. Characterization of
dendritic cells in differentiated thyroid cancer. J Pathol 2005;205:565–76.
29. Treilleux I, Blay JY, Bendriss-Vermare N, Ray-Coquard I, Bachelot T,
Guastalla JP, et al. Dendritic cell infiltration and prognosis of early stage
breast cancer. Clin Cancer Res 2004;10:7466–74.
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
GM-CSF Activates pDC for Th2 Priming in Aggressive Breast Cancer
30. Vermi W, Bonecchi R, Facchetti F, Bianchi D, Sozzani S, Festa S, et al.
Recruitment of immature plasmacytoid dendritic cells (plasmacytoid
monocytes) and myeloid dendritic cells in primary cutaneous melanomas.
J Pathol 2003;200:255–68.
31. Sapoznikov A, Fischer JA, Zaft T, Krauthgamer R, Dzionek A, Jung S. Organdependent in vivo priming of naive CD4þ, but not CD8þ, T cells by
plasmacytoid dendritic cells. J Exp Med 2007;204:1923–33.
32. Boonstra A, Asselin-Paturel C, Gilliet M, Crain C, Trinchieri G, Liu YJ, et al.
Flexibility of mouse classical and plasmacytoid-derived dendritic cells in
directing T helper type 1 and 2 cell development: dependency on antigen
dose and differential toll-like receptor ligation. J Exp Med 2003;197:101–9.
33. Berzofsky JA, Terabe M. A novel immunoregulatory axis of NKT cell subsets
regulating tumor immunity. Cancer Immunol Immunother 2008;57:
1679–83.
34. DeNardo DG, Barreto JB, Andreu P, Vasquez L, Tawfik D, Kolhatkar N, et al.
CD4(þ) T cells regulate pulmonary metastasis of mammary carcinomas by
enhancing protumor properties of macrophages. Cancer Cell 2009;16:
91–102.
35. Kobayashi M, Kobayashi H, Pollard RB, Suzuki F. A pathogenic role of Th2
cells and their cytokine products on the pulmonary metastasis of murine
B16 melanoma. J Immunol 1998;160:5869–73.
36. Ciampricotti M, Vrijland K, Hau CS, Pemovska T, Doornebal CW, Speksnijder EN, et al. Development of metastatic HER2(þ) breast cancer is
independent of the adaptive immune system. J Pathol 2011;224:56–66.
37. Teschendorff AE, Gomez S, Arenas A, El-Ashry D, Schmidt M, Gehrmann
M, et al. Improved prognostic classification of breast cancer defined by
antagonistic activation patterns of immune response pathway modules.
BMC Cancer 2010;10:604.
38. Pedroza-Gonzalez A, Xu K, Wu TC, Aspord C, Tindle S, Marches F, et al.
Thymic stromal lymphopoietin fosters human breast tumor growth by
promoting type 2 inflammation. J Exp Med 2011;208:479–90.
39. Wei S, Kryczek I, Zou L, Daniel B, Cheng P, Mottram P, et al. Plasmacytoid
dendritic cells induce CD8 þregulatory T cells in human ovarian carcinoma. Cancer Res 2005;65:5020–6.
40. Matta BM, Castellaneta A, Thomson AW. Tolerogenic plasmacytoid DC.
Eur J Immunol 2010;40:2667–76.
41. Sisirak V, Faget J, Gobert M, Goutagny N, Vey N, Treilleux I, et al. Impaired
IFN-a production by Plasmacytoid dendritic cells favors regulatory T cell
expansion and contributes to breast cancer progression. Cancer Res 2012;
72:5188–97.
42. Jensen TO, Schmidt H, Moller HJ, Donskov F, Hoyer M, Sjoegren P, et al.
Intratumoral neutrophils and plasmacytoid dendritic cells indicate poor
prognosis and are associated with pSTAT3 expression in AJCC stage I/II
melanoma. Cancer 2012;118:2476–85.
www.aacrjournals.org
43. Shi Y, Liu CH, Roberts AI, Das J, Xu G, Ren G, et al. Granulocytemacrophage colony-stimulating factor (GM-CSF) and T-cell responses:
what we do and don't know. Cell Res 2006;16:126–33.
44. Hensley C, Spitzler S, McAlpine BE, Lynn M, Ansel JC, Solomon AR, et al.
In vivo human melanoma cytokine production: inverse correlation of
GM-CSF production with tumor depth. Exp Dermatol 1998;7:335–41.
45. Zijlmans HJ, Fleuren GJ, Baelde HJ, Eilers PH, Kenter GG, Gorter A. Role of
tumor-derived proinflammatory cytokines GM-CSF, TNF-alpha, and IL-12
in the migration and differentiation of antigen-presenting cells in cervical
carcinoma. Cancer 2007;109:556–65.
46. Trutmann M, Terracciano L, Noppen C, Kloth J, Kaspar M, Peterli R, et al.
GM-CSF gene expression and protein production in human colorectal
cancer cell lines and clinical tumor specimens. Int J Cancer 1998;77:
378–85.
47. Su S, Liu Q, Chen J, Chen J, Chen F, He C, et al. A positive feedback loop
between mesenchymal-like cancer cells and macrophages is essential to
breast cancer metastasis. Cancer Cell 2014;25:605–20.
48. Pardoll DM. Paracrine cytokine adjuvants in cancer immunotherapy. Annu
Rev Immunol 1995;13:399–415.
49. Emens LA, Asquith JM, Leatherman JM, Kobrin BJ, Petrik S, Laiko M, et al.
Timed sequential treatment with cyclophosphamide, doxorubicin, and an
allogeneic granulocyte-macrophage colony-stimulating factor-secreting
breast tumor vaccine: a chemotherapy dose-ranging factorial study of
safety and immune activation. J Clin Oncol 2009;27:5911–8.
50. Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, et al.
Vaccination with irradiated tumor cells engineered to secrete murine
granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S A
1993;90:3539–43.
51. Tsuchiya Y, Igarashi M, Suzuki R, Kumagai K. Production of colonystimulating factor by tumor cells and the factor-mediated induction of
suppressor cells. J Immunol 1988;141:699–708.
52. Bronte V, Chappell DB, Apolloni E, Cabrelle A, Wang M, Hwu P, et al.
Unopposed production of granulocyte-macrophage colony-stimulating
factor by tumors inhibits CD8þ T cell responses by dysregulating antigen-presenting cell maturation. J Immunol 1999;162:5728–37.
53. Faries MB, Hsueh EC, Ye X, Hoban M, Morton DL. Effect of granulocyte/
macrophage colony-stimulating factor on vaccination with an allogeneic
whole-cell melanoma vaccine. Clin Cancer Res 2009;15:7029–35.
54. Filipazzi P, Valenti R, Huber V, Pilla L, Canese P, Iero M, et al. Identification
of a new subset of myeloid suppressor cells in peripheral blood of
melanoma patients with modulation by a granulocyte-macrophage colony-stimulation factor-based antitumor vaccine. J Clin Oncol 2007;25:
2546–53.
Cancer Res; 75(14) July 15, 2015
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.
OF13
Published OnlineFirst May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2386
Breast Cancer Cell−Derived GM-CSF Licenses Regulatory
Th2 Induction by Plasmacytoid Predendritic Cells in
Aggressive Disease Subtypes
Cristina Ghirelli, Fabien Reyal, Marine Jeanmougin, et al.
Cancer Res Published OnlineFirst May 14, 2015.
Updated version
Supplementary
Material
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
doi:10.1158/0008-5472.CAN-14-2386
Access the most recent supplemental material at:
http://cancerres.aacrjournals.org/content/suppl/2015/05/15/0008-5472.CAN-14-2386.DC1
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2015 American Association for Cancer
Research.