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J. Exp. Med. Vol. 208 No. 3 469-478
Pancreatic cancer is a very aggressive disease
with dismal prognosis (Hidalgo, 2010). Desmo-
plasia/fibrosis, which is not present around nor-
mal pancreatic ducts, is a hallmark in pancreatic
cancer and it is believed to play an active role in
disease progression and aggressiveness (Kleeff
et al., 2007; Mahadevan and Von Hoff, 2007).
Tumor stroma is predominantly infiltrated by
Th2 (GATA-3+) over Th1 (T-bet+) cells (Tassi
et al., 2008). This immune infiltrate correlates
with the presence in the blood of pancreatic
cancer patients of tumor-specific CD4+ T cells
producing mostly IL-5 and IL-13 (Tassi et al.,
2008). Th2 cytokines and IL-13 in particular are
strongly linked to fibrogenesis (Wynn, 2004).
Open questions are what leads to the Th2 im-
mune deviation in pancreatic cancer and whether
Th2 cells present at the tumor site have a role in
disease progression. We hypothesized that tumor-
resident DCs are conditioned by factors released
by tumor cells or tumor stroma to favor, in the
draining LNs, differentiation of tumor-specific
Th2 cells, which then home to the tumor and
possibly contribute to disease progression by inter-
acting with other immune and nonimmune cells
(Joyce and Pollard, 2009) and, through Th2 cyto-
kines secretion, to fibrosis (Wynn, 2004).
The thymic stromal lymphopoietin (TSLP;
i.e., an IL-7–like cytokine) has been recently
Maria Pia Protti:
Abbreviations used: CAF, cancer-
associated fibroblast; CI,
confidence interval; G/T,
human dermal fibroblast; LCM,
laser capture microdissection;
derived chemokine; SMA,
smooth muscle actin; TARC/
CCL17, thymus and activation-
regulated chemokine; TSLP,
thymic stromal lymphopoietin;
TSLPR, TSLP receptor.
Intratumor T helper type 2 cell infiltrate
correlates with cancer-associated fibroblast
thymic stromal lymphopoietin production
and reduced survival in pancreatic cancer
Lucia De Monte,1,6 Michele Reni,2,7 Elena Tassi,1,6 Daniela Clavenna,3,7
Ilenia Papa,3,7 Helios Recalde,4 Marco Braga,5,7,8 Valerio Di Carlo,5,7,8
Claudio Doglioni,3,7,8 and Maria Pia Protti1,6
1Tumor Immunology Unit, 2Clinical Oncology Unit, 3Pathology Unit, 4Blood Bank, 5Pancreas Unit, 6Division of Immunology,
Transplantation and Infectious Diseases, and 7Division of Molecular Oncology, San Raffaele Scientific Institute, 20132 Milan, Italy
8San Raffaele Vita-Salute University, 20132 Milan, Italy
Pancreatic cancer is a very aggressive disease characterized by a marked desmoplasia with a
predominant Th2 (GATA-3+) over Th1 (T-bet+) lymphoid infiltrate. We found that the ratio
of GATA-3+/T-bet+ tumor-infiltrating lymphoid cells is an independent predictive marker of
patient survival. Patients surgically treated for stage IB/III disease with a ratio inferior to
the median value had a statistically significant prolonged overall survival, implying an
active role for Th2 responses in disease progression. Thymic stromal lymphopoietin (TSLP),
which favors Th2 cell polarization through myeloid dendritic cell (DC) conditioning, was
secreted by cancer-associated fibroblasts (CAFs) after activation with tumor-derived tumor
necrosis factor and interleukin 1. TSLP-containing supernatants from activated CAFs
induced in vitro myeloid DCs to up-regulate the TSLP receptor (TSLPR), secrete Th2-
attracting chemokines, and acquire TSLP-dependent Th2-polarizing capability in vitro.
In vivo, Th2 chemoattractants were expressed in the tumor and in the stroma, and TSLPR-
expressing DCs were present in the tumor stroma and in tumor-draining but not in nond-
raining lymph nodes. Collectively, this study identifies in pancreatic cancer a cross talk
between tumor cells and CAFs, resulting in a TSLP-dependent induction of Th2-type in-
flammation which associates with reduced patient survival. Thus, blocking TSLP production
by CAFs might help to improve prognosis in pancreatic cancer.
© 2011 De Monte et al. This article is distributed under the terms of an
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The Journal of Experimental Medicine
TSLP-driven Th2 polarization in pancreatic cancer | De Monte et al.
Th2 cells varied among the samples (Fig. 1 B). To verify pos-
sible quantitative differences among samples, we then calcu-
lated the G/T ratio for each patient (Fig. 1 C). Indeed, Cox
regression model showed no significant correlation between
overall survival and the absolute number of either GATA-3+
(hazard ratio = 1.00; 95% confidence interval [CI] 1.00–1.00;
P = 0.73) or T-bet+ (hazard ratio = 1.00; 95% CI 1.00–1.00;
P = 0.52) cells. Conversely, a significant correlation between
G/T ratio and overall survival was detected (hazard ratio =
1.04; 95% CI 1.01–1.04; P = 0.038). The median value of
G/T ratio was 5.2; 35 patients had a G/T ratio ≤ 5.2 (group A)
and 34 patients had a G/T ratio > 5.2 (group B; patient
characteristics grouped by the G/T ratio are reported in
Table I). After a median followup of 41 mo (range 22–134),
58 patients had recurrence and 53 died from the disease. Me-
dian 1-yr and 2-yr disease-free survival was 15.2 mo, 71 and
43% for group A, and 11.0 mo, 47 and 18% for group B (P =
0.02). Median 2-yr and 3-yr overall survival was 32.7 mo, 66
and 45% for group A, and 20.2 mo, 44 and 26% in group B
(P = 0.008; Fig. 1 D and Fig. S1). Multivariate analysis strati-
fying for tumor stage, grading, size, site, patient performance
status, gender, age, surgical resection margins, postoperative
CA19.9 value, and postoperative treatment confirmed that the
G/T ratio was independently predictive of both disease-free
survival (P = 0.002, hazard ratio = 1.06, 95% CI 1.03–1.10)
and overall survival (P = 0.005, hazard ratio = 1.06, 95% CI
1.02–1.09). Hence, the GATA-3+ cells present in the tumor
stroma associate with disease progression, and we identified a
parameter (i.e., the G/T ratio) that is independently predic-
tive of patients’ survival.
TSLP is produced by cancer-associated fibroblasts (CAFs)
after activation by tumor-derived TNF and IL-1
To investigate the potential role of
TSLP in driving Th2 immune deviation
associated with induction of Th2 responses through DC acti-
vation (Liu et al., 2007). Hence, in this paper we evaluated,
first, the prognostic significance of Th2-infiltrating lymphoid
cells in surgical specimens of patients who had resection of
stage IB/III pancreatic cancer and, second, the potential im-
plication of TSLP in inducing the Th2 immune deviation
present in pancreatic cancer.
The ratio of GATA-3+/T-bet+ (G/T) tumor-infiltrating
lymphoid cells predicts survival after surgery in patients
with stage IB/III pancreatic cancer
To determine the possible association between Th2 cells and
disease progression, we enumerated by immunohistochemis-
try the GATA-3+ and T-bet+ lymphoid cell–infiltrating tumor
samples from 69 patients who underwent surgical resection
(Fig. 1 A, left). GATA-3 was also expressed in the cytoplasm
of epithelial cells as already shown in Tassi et al. (2008; Fig. 1 A,
top). Lymphoid cell infiltrate was mostly present exclusively
in the tumor stroma and varied among samples. Pancreatic
tissue from surgical samples of patients who underwent sur-
gery for benign lesions is also shown as normal control. Com-
pared with the tumor, in which the stromal component is
very represented, normal pancreatic tissue is composed by a
compact acinar structure that contains rare and equal num-
bers of lymphoid cells positive for GATA-3 and T-bet (Fig. 1 A,
right). Because the amount of lymphoid cells in the tumor
varied among samples, we then calculated for each patient
the percentage of positive lymphoid cells and found that in
all but one sample the percentage of GATA-3+ cells was sig-
nificantly higher than that of T-bet+ (Fig. 1 B), demonstrating
that Th2 immune deviation in pancreatic cancer is a gener-
alized phenomenon. However, the percentage of intratumor
Figure 1. The ratio of G/T tumor-
infiltrating lymphoid cells predicts survival
after surgery in patients with stage IB/III
pancreatic cancer. (A) Representative immuno-
histochemical analysis for lymphoid
GATA-3 and T-bet staining in the tumor (n = 69;
left) and normal pancreatic tissue from
benign lesions (n = 3; right). The arrows indi-
cate rare positive cells in normal tissue. Bars,
100 µm. (B) Percentage of GATA-3+ (circles)
and T-bet+ (triangles) lymphoid cells for each
of the analyzed tumor samples (n = 69). The
values are significantly different and indicated
as: ***, P < 0.001 (determined by paired one-
tailed Student’s t test). (C) Waterfall plot of
the G/T ratio for each tumor sample. The
dashed line indicates the G/T ratio 5.2, which
is the median value. (D) Kaplan-Meier curve
for overall survival by median G/T ratio (5.2).
Survival significantly decreased (P = 0.008) as
a function of a G/T ratio ≥ 5.2.
JEM VOL. 208, March 14, 2011
of TSLP (Fig. 2 A) and proinflammatory cytokines (Fig. 2 D)
in the surrounding tissue is possibly related to its morpholog-
ical characteristics that could vary among samples from
conserved normal parenchyma to preexisting or associated
To identify cells producing the proinflammatory cyto-
kines, we tested their expression in tumor epithelial and stro-
mal cells collected by LCM and in cultured paired tumor cells
and CAFs from the same patient (Fig. 2 E). TNF was mainly
expressed in tumor epithelial cells, compared with stroma
cells, and not in CAFs (Fig. 2 E, left). IL-1 was expressed in
both tumor compartments and, although at low levels, in
CAFs (Fig. 2 E, right and inset). IL-1 secretion was con-
firmed by immunohistochemistry (unpublished data). As the
two proinflammatory cytokines were expressed in tumor
cells, we further tested whether treatment of CAFs with
tumor cell supernatant induced TSLP secretion. CAFs, but
not HDFs, treated with the tumor cell supernatant produced
TSLP that was significantly inhibited by anti-TNF (34% in-
hibition) and anti–IL-1 (38% inhibition) Abs but not by an
isotype control (Fig. 2 F). Collectively, these experiments
demonstrate that TSLP expression is up-regulated in pancre-
atic cancer and released by CAFs under the influence of
TNF and IL-1 that are expressed in pancreatic cancer and
secreted by tumor cells.
Myeloid DCs are activated by the supernatant
of proinflammatory cytokine-treated CAFs and acquire
We next evaluated, in vitro, the effects of the supernatant of
activated CAFs on myeloid DC maturation and function
(Fig. 3). DCs conditioned by CAF supernatant strongly up-
regulated activation and maturation markers at levels compa-
rable to or even higher than those of TSLP and Poly I:C (i.e.,
controls for DCs maturation; Fig. 3 A).
The potential role of CAF-released TSLP in DC activation/
maturation was then investigated by looking at the expression of
in pancreatic cancer, we evaluated, by real-time PCR, TSLP
mRNA expression in tumor and macroscopically uninvolved
surrounding tissues from surgical specimens, and in isolated
and in vitro–cultured tumor cells and CAFs, as described in
Materials and methods. CAF cell lines were obtained from
surgical samples of 15 patients, and corresponding tumor cell
lines from the same patients were obtained in five cases. Because
normal human pancreatic fibroblasts were not available, human
dermal fibroblasts (HDFs) from normal skin were used as
control. All cell lines were tested after few passages in culture
and their characterization is reported in Fig. S2. Although
tumor cell lines, when established, could then be kept in
long-term culture as stabilized lines, usually CAFs could not
grow longer than nine passages. We found that TSLP was ex-
pressed in the tumor and, to a much lower extent, in the sur-
rounding tissue (Fig. 2 A). Moreover, TSLP was expressed in
CAFs but not in tumor cells and HDFs (Fig. 2 A). Expression
of TSLP in the stroma, and not in tumor epithelial cells, was
confirmed by laser capture microdissection (LCM; Fig. 2 B).
We then measured, by ELISA, TSLP secretion by CAFs
after activation with proinflammatory cytokines known to
induce TSLP production in epithelial cells (Lee and Ziegler,
2007), smooth muscle cells (Zhang et al., 2007), and skin
keratinocytes (Bogiatzi et al., 2007). TSLP secretion, in the
absence of activation, was observed in a limited number of
CAFs and never in tumor cells and HDFs (unpublished data).
TNF and IL-1 used as single agents or in combination
strongly increased or induced a significantly higher TSLP se-
cretion in CAFs than in HDFs. However, in agreement with
the mRNA expression data, no TSLP production by tumor
cells was observed (Fig. 2 C).
We then confirmed the presence of the proinflammatory
cytokines in tumor samples (Fig. 2 D). TNF was expressed in
the tumor at higher levels than in the corresponding sur-
rounding tissue (Fig. 2 D, top), whereas IL-1 was expressed
in both tissues with prevalence in one or the other depending
on the sample (Fig. 2 D, bottom). The variability of expression
Table I. Characteristics of the 69 patients grouped by the G/T ratio of tumor-infiltrating lymphoid cells
Variable G/T ≤ 5.2 (n = 35)G/T > 5.2 (n = 34)
Tumor site: head
Tumor size > 3 cm
Surgical margin R1
Median preoperative CA19.9
Median postoperative CA19.9
217 ± 3453.8 range (1–18,470)
16 ± 88.8 range (1–416)
94 ± 1342.6 range (1–6,832)
23 ± 678.6 range (0.1–3,745)
aPatients who had R0 or R1 resection of a stage IB-III pancreatic cancer, aged 18–75 yr and Karnofsky Performance Status (KPS) > 60, were eligible for adjuvant therapy. The
patients were required to have postoperative treatment initiation within 2 mo from surgery, no previous chemotherapy or radiotherapy for pancreatic cancer, and adequate
bone marrow, liver, and kidney. After tumor resection, 31 patients were treated with gemcitabine (Burris et al., 1997) and 38 patients received the PEFG regimen consisting of
cisplatin, epirubicin, gemcitabine, and 5-fluorouracil (Reni et al., 2005). In both cases, chemotherapy was administered for 3 mo followed by chemoradiation (Reni et al., 2005).
TSLP-driven Th2 polarization in pancreatic cancer | De Monte et al.
Figure 2. TSLP is expressed by CAFs, and its secretion is induced by proinflammatory cytokines released by tumor cells. (A) TSLP mRNA ex-
pression in tumor and surrounding tissues, tumor cell lines, CAFs, and HDFs. Each dot represents a different surgical sample or a different cell line.
(B) TSLP is expressed in vivo in the stroma. LCM (left) was used to collect stromal (top) and epithelial (bottom) tumor cells from surgical specimens and
TSLP mRNA expression analyzed (right; representative of tumor samples from three patients). The mRNA expression levels were normalized to the expres-
sion of GAPDH. TSLP mRNA expression of Caco2 cell line was used as calibrator, as in Rimoldi et al. (2005). (C) TSLP protein secretion by CAFs, HDFs, and
tumor cell lines treated with proinflammatory cytokines as single agent or in combination. Left, 20 ng/ml TNF; middle, 10 ng/ml IL-1; right, TNF plus
IL-1. Each dot represents a different cell line. (D) TNF (top) and IL-1 (bottom) mRNA expression in tumor and the corresponding surrounding tissue
(each dot and corresponding triangle represent surgical samples from single patients). (E) TNF (left) and IL-1 (right) mRNA expression in tumor epithelial
and stromal cells collected by LCM (top; representative of tumor samples from three patients) and in isolated and in vitro–cultured paired tumor cell lines
and CAFs from the same patient (bottom; representative of tumor samples from three patients). Inset magnifies IL-1 expression in CAFs. (F) TSLP protein
secretion by CAFs treated with supernatant from tumor cell lines (tumor cells sup) in the absence and presence of anti-TNF, anti–IL-1, and isotype con-
trol Abs (representative of experiments performed with three different tumor cell lines and three CAFs). Data in A–F are means of at least duplicate deter-
minations ± SD. Responses significantly different in A, C, D, and F are indicated as: *, P < 0.05; **, 0.001< P < 0.01 (determined by paired or unpaired
one-tailed Student’s t test).
JEM VOL. 208, March 14, 2011
thymus and activation-regulated chemokine (TARC/CCL17)
and macrophage-derived chemokine (MDC/CCL22) were
secreted as expected by TSLP-treated DCs and, importantly,
by DCs conditioned with the supernatant of activated CAFs.
In contrast, DCs conditioned with the supernatant of acti-
vated HDFs secreted MDC/CCL22 but not TARC/CCL17
(Fig. 3 D). Th1 chemoattractants RANTES/CCL5 and
IP-10/CXCL10 were secreted exclusively by DCs conditioned
with the HDF supernatant (Fig. 3 D).
To test their polarizing capability, DCs conditioned with
the supernatant of TNF-treated CAFs were co-cultured with
naive CD4+CD45RA+ T cells, purified from cord blood, in
the absence or in the presence of neutralizing Abs. After 6 d,
release of IFN- and IL-13 (as prototypic Th1 and Th2 cyto-
kines, respectively) by activated CD4+ T cells was assessed by
ELISA (Fig. 3 E). TSLP-treated DCs, used as positive control,
induced IL-13 as expected but not IFN-–producing CD4+
T cells. Notably, DCs conditioned with CAF supernatant also
induced secretion of IL-13 that was inhibited by an anti-TSLP
(47% inhibition) but not an anti-TNF Ab. CD4+
T cells activated with DCs cultured with medium alone
or TNF or the supernatant of TNF-treated HDFs re-
mained unpolarized (Fig. 3 E). Collectively, these data
demonstrate that CAFs, via the release of TSLP, in-
duce DCs with features of TSLP-treated DCs.
the TSLP receptor (TSLPR), which is up-regulated in TSLP-
treated DCs (Soumelis et al., 2002; Ito et al., 2005; Lu et al.,
2009). We found that DCs treated with CAF supernatant ex-
pressed the TSPLR at higher levels than those treated with
HDF supernatant (Fig. 3 B), indicating that a maturation
stimulus for DC activation present in the CAF supernatant is
TSLP. We further demonstrated that TSLP present in the su-
pernatant was responsible for DC activation by experiments
in which CD80 expression, whose up-regulation is particularly
influenced by TSLP (Soumelis et al., 2002; Bogiatzi et al., 2007),
was inhibited by the addition of an anti-TSLP Ab (Fig. 3 C).
We then measured the secretion of cytokines and chemo-
kines by DCs either left untreated or treated with the super-
natants of TNF-treated CAFs and HDFs. TNF and TSLP were
used as controls. After 24 h, DCs were extensively washed to
remove any exogenous or fibroblast-produced cytokine and
left for a further 48 h in culture before collecting the super-
natant. IL-12, IL-10, TNF, IL-1, and IL-6 were not produced
in any condition (unpublished data). The Th2 chemoattractants
Figure 3. Supernatant of TNF-treated CAF (CAF sup)
activates in vitro myeloid DCs with features of TSLP-treated
DCs. (A) FACS analysis of DCs after 24-h incubation with the
indicated stimuli (representative of independent experiments,
n = 3). Experiments were performed with supernatants from
three different CAFs. Open histograms represent staining of DC
activation markers; filled histograms represent the isotype control.
(B) FACS analysis of TSLPR expression by DCs activated in the
presence of medium plus TNF, supernatant of TNF-treated HDF
(HDF sup; obtained from two different HDFs), and CAF sup (ob-
tained from three different CAFs; representative of independent
experiments n = 3). (C) FACS analysis of CD80 expression by DCs
incubated with CAF sup (obtained from three different CAFs) in
the absence and in the presence of an anti-TSLP Ab (representa-
tive of independent experiments, n = 4). Experiments with me-
dium plus TNF and TSLP, in the absence and in the presence of
the anti-TSLP Ab, are shown as negative and positive controls,
respectively. (D) Chemokine production by DCs activated with
the following stimuli: medium alone, 20 ng/ml TNF; 15 ng/ml
TSLP; HDF sup and CAF sup (representative of three experiments
performed with three different CAFs and two different HDFs).
(E) CAF sup endows DCs with Th2 polarizing capability that de-
pends on TSLP. CD4+CD45RA+ naive T cells were cultured with
DCs previously activated with the indicated stimuli. At day 6,
IFN- and IL-13 secreted by CD4+ T cells were tested by ELISA.
When indicated, anti-TSLP and anti-TNF Abs were added in cul-
ture. Data are means of duplicate determinations ± SD and rep-
resent one of five experiments (performed with four different
CAFs and two different HDFs). Release of IL-13 significantly
lower in the presence of an anti-TSLP Ab are indicated as:
*, P < 0.05; **, 0.001 < P < 0.01 (determined by unpaired one-
tailed Student’s t test).
TSLP-driven Th2 polarization in pancreatic cancer | De Monte et al.
DCs with features of TSLP-treated DCs and Th2-attracting
chemokines are present in vivo in pancreatic cancer patients
Finally, we investigated, by immunohistochemistry and flow
cytometry, the presence in vivo of TSLP-conditioned DCs,
identified as CD11c+TSLPR+ cells. Myeloid DCs in the
steady state express very low levels of TSLPR that is up-
regulated in the presence of TSLP (Reche et al., 2001). In-
deed, we found CD11c+TSLPR+ cells in the tumor (Fig. 4 A)
and in draining but not in nondraining LNs (Fig. 4 B). These
findings witness DC activation in vivo by a stimulus able to
up-regulate the TSLPR. This evidence, along with the in vitro
demonstration that CD80 expression by DCs conditioned by
CAF supernatant depends on TSLP secretion (shown in
Fig. 3 C), validates TSLP as the cytokine responsible for activa-
tion in the tumor of DCs with features of TSLP-treated DCs.
In our hypothesis, TSLP-conditioned DCs present in the
draining LNs activate Th2 cells that then home to the tumor
under the influence of Th2 attractant chemokines. Thus, we
evaluated, by real-time PCR, mRNA expression of TARC/
CCL17 and MDC/CCL22 in tumor samples and found ex-
pression in the tumor but not in the corresponding surround-
ing tissue (Fig. 4 C). We then measured expression of the two
cytokines in tumor epithelial and stromal cells collected by
LCM and in cultured paired tumor cells and CAFs from the
same patient (Fig. 4 D). We found expression of both cyto-
kines in the stroma (Fig. 4 D, top). In addition, MDC/CCL22,
as already described in ovarian cancer (Curiel et al., 2004),
was also expressed in tumor epithelial cells (Fig. 4 D, right).
As for TNF (Fig. 2 E), TARC/CCL17 and MDC/CCL22
expression in the stroma was not attributable to CAFs (Fig. 4 D,
bottom) but, in agreement with our previous in vitro results
(Fig. 3 D), was most likely attributable to inflammatory cells
(e.g., DCs). Collectively, these results support our hypothesis
that TSLP-conditioned DCs induced by CAF-secreted TSLP
migrate to the draining LNs to prime Th2 cells that home to the
tumor under the influence of Th2-attracting chemokines.
In this study, we first show that in pancreatic cancer the Th2
immune deviation has an active role in tumor progression, in
that the quantity of Th2 with respect to Th1 cells present
in the tumor stroma has a direct correlation with prognosis in
surgically resected patients. Although tumor antigen–specific
CD4+ Th2 cells have been already described in the blood
of patients with different neoplastic diseases (Tatsumi et al.,
2002, 2003; Slager et al., 2003; Marturano et al., 2008), to
our knowledge this is the first demonstration of a statisti-
cally significant correlation between prevalent Th2 over
Th1 tumor immune infiltrate and poor prognosis. Notably,
statistical analysis proved that the ratio, rather than the
absolute number, of GATA-3+ cells correlate with poor
prognosis, pointing to the importance for the clinical out-
come of the balance between Th2 and Th1 cells present in the
Second, we addressed what leads to the Th2 immune de-
viation in pancreatic cancer and identified a central role for
Figure 4. TSLP-conditioned DCs and Th2-attractant chemokines are
present in vivo. (A and B) TSLP-conditioned DCs are present in the tumor
and in draining but not in nondraining LNs. (A) Immunohistochemical analy-
sis for CD11c+TSLPR+ cells in the tumor representative of tumor samples
from 10 patients. Bars, 100 µm. (B) FACS analysis of CD11c+TSLPR+CD14
cells in draining (top) and corresponding nondraining (bottom) LNs repre-
sentative of paired samples from four patients. Open histograms represent
TSLPR staining; filled histograms represent the isotype control. Num-
bers indicate the percentage of gated (left) and positive (right) cells.
(C and D) TARC/CCL17 and MDC/CCL22 mRNA expression in tumor and the
corresponding surrounding tissue (C; each dot and corresponding triangle
represent surgical samples from single patients), in tumor epithelial and
stromal cells collected by LCM (D, top; representative of tumor samples
from three patients), and in isolated and in vitro–cultured paired tumor cell
lines and CAFs from the same patient (D, bottom; representative of tumor
samples from three patients). Data in C and D are means of at least dupli-
cate determinations ± SD. Responses significantly different are indicated as:
*, P < 0.05 (determined by paired one-tailed Student’s t test).
CAFs, which, through TSLP secretion, activate mDCs with
features of TSLP-conditioned DCs with Th2-polarizing ca-
pability. Notably, DCs with the feature of TSLP-activated
DCs (i.e., CD11c+TSLPR+) were found in vivo in the tumor
stroma and in draining, but not in nondraining, LNs.
JEM VOL. 208, March 14, 2011
macrophages in pancreatic cancer has been reported (Kurahara
et al., 2009), and we also have preliminary evidence of tumor
stroma infiltration by CD68+CD163+ cells (unpublished data).
Clinical outcome in pancreatic cancer patients with re-
sectable tumor is still disappointing with a median survival of
20–22 mo (Hidalgo, 2010). However, time to relapse and
overall survival in this patient population may greatly vary,
and few risk factors for recurrent disease have been defined so
far (Hidalgo, 2010). In this paper, we identified the ratio of
Th2/Th1 tumor-infiltrating lymphoid cells as an indepen-
dent prognostic marker of survival that might be used for pa-
tient stratification in future prospective studies.
Notably, elucidation of the cytokine/chemokine network
implicated in the Th2 immune deviation may allow for the
design of innovative therapeutic strategies to complement
currently available therapies (including therapeutic vaccina-
tion or adoptive immunotherapy) for pancreatic cancer based
on proinflammatory cytokines and TSLP blocking strategies.
Toward the feasibility of these approaches, clinical trials of
TNF antagonists in advanced cancer patients have resulted in
disease stabilization and some partial responses (Harrison et al.,
2007; Mantovani et al., 2008). Clinical grade anti–IL-1 Abs
are available to treat autoimmune and autoinflammatory dis-
eases (van den Berg, 2000; Atzeni and Sarzi-Puttini, 2009;
Lachmann et al., 2009) and, recently, treatment with an IL-1
inhibitor in patients with smoldering or indolent multiple
myeloma at risk of progression to active myeloma was responsi-
ble for improved progression-free survival (Lust et al., 2009).
Finally, concerning possible ways to silence TSLP, as a result of
its role in allergy, several companies are already involved in devel-
oping clinical grade neutralizing Abs, which should be available
for clinical applications in the near future (Edwards, 2008).
Future studies will address the mechanisms by which Th2
cells promote tumor progression and, specifically, which cel-
lular and molecular interactions they use to do so within the
tumor microenvironment. These studies will also possibly be
conducted in animal models of spontaneous pancreatic can-
cer development recapitulating the different steps of pancre-
atic carcinogenesis from inception to invasive cancer paralleling
the human disease (Hingorani et al., 2003; Leach, 2004). Further-
more, whether the mechanisms of Th2-mediated inflammation
constitute a common phenomenon operative in multiple human
tumors should also be investigated. Specifically, the role of
TSLP and the ratio of G/T-infiltrating lymphocytes in other
tumor types, either gastrointestinal or other epithelial tumors
with important fibrotic component, should be addressed.
In summary, we identified in human pancreatic cancer a
complex cross talk among tumor cells, CAFs, DCs, and T cells
leading to Th2 inflammation, which significantly correlates
with poor survival. Thus, therapeutic intervention aimed at
interfering with this negative cross talk may prove to be ben-
eficial for improving prognosis in pancreatic cancer patients.
MATERIALS AND METHODS
Patient population enrolled for survival analysis. 69 patients who had R0
or R1 resection of a stage IB-III pancreatic cancer, according to the 2002 stag-
ing criteria of the American Joint Commission on Cancer (Evans et al., 2002),
Previous studies addressed the presence of DCs in pancre-
atic cancer (Dallal et al., 2002; Fukunaga et al., 2004) with
somewhat conflicting results. DCs were identified by anti-
S100 staining in Fukunaga et al. (2004) and by anti-S100 and
CD1a in Dallal et al. (2002). Dallal et al. (2002) found signifi-
cant numbers of S100+ or CD1a+ cells in a very small per-
centage of patients. Compared with this paper, Fukunaga
et al. (2004) identified patients with different levels of im-
mune infiltration and found that infiltration of S100+ cells
paralleled infiltration by CD4+ and CD8+ T cells. The DC
marker used in our study is different from the ones previously
used. Because CD11c expression is not limited to DCs, it is
possible that neutrophils and monocytes, which are possibly
present in the tumor stroma, might also have been stained.
Cancer-related inflammation has been proposed as the sev-
enth hallmark of cancer (Dunn et al., 2004; Grivennikov et al.,
2010), and recently proinflammatory CAFs from a trans-
genic mouse tumor model were shown to orchestrate tumor-
promoting inflammation in a NF-B–dependent manner (Erez
et al., 2010). In this scenario, our data identify a new molecule
(i.e., TSLP) and a new function for CAFs in driving, under
the influence of tumor cells, Th2-mediated inflammation that
correlates with reduced survival in pancreatic cancer.
Collectively, based on our in vitro and in vivo data we
propose a model of a complex cross talk among tumor cells,
CAFs, Th2 cells, and possibly other immune cells favoring
tumor promotion (Fig. S3). First, proinflammatory cytokines
(TNF and IL-1) are released by pancreatic tumor cells and
elicit the release of TSLP by CAFs (Fig. S3 A). Secretion of
these cytokines by pancreatic tumor cells has been previously
reported (Arlt et al., 2002; Müerköster et al., 2004; Egberts
et al., 2008). However, it is still unknown which signals are driv-
ing their secretion. Endogenous factors released by dead and
dying tumor cells or present in the tumor microenvironment
might serve as endogenous stimuli for NF-kB and inflamma-
some activation required for proinflammatory cytokine acti-
vation and release (Tschopp and Schroder, 2010; Fig. S3 A).
Second, TSLP released by activated CAFs induces activation/
maturation of tumor antigen–loaded resident DCs (Fig. S3,
B and C). Third, activated DCs migrate to the draining LNs
where they activate tumor antigen–specific CD4+ Th2 cells
(Fig. S3 D). Fourth, CD4+ Th2 cells primed in the draining
LNs home to the tumor under the influence of tumor-
derived Th2 chemoattractants (TARC and MDC; Fig. S3 E).
Fifth, recruited CD4+GATA-3+ Th2 cells may exert tumor-
promoting effector functions (Fig. S3 F). Indeed, an altered
balance between Th2 and Th1 cytokines present in the tumor
microenvironment might further contribute to fibrosis. It has
been shown that fibrogenesis is strongly linked with develop-
ment of Th2 responses, and Th1 and Th2 cytokines exert op-
posing roles by promoting collagen degradation and synthesis,
respectively (Wynn, 2004). Th2 cells might also promote differ-
entiation of M2 macrophages both directly by tumor antigen–
specific recognition of peptide–MHC class II complexes
at the surface of differentiating monocytes and indirectly
through Th2 cytokines release. The presence of M2-polarized
TSLP-driven Th2 polarization in pancreatic cancer | De Monte et al.
the following stimuli: 20 ng/ml TNF, 10 ng/ml IL-1, and supernatant from
tumor cells. In inhibition experiments, 2 µg/ml anti-TNF, anti–IL-1, or
isotype-matched Abs (BD) were added. TSLP release was quantified by
ELISA (R&D Systems).
Myeloid DC isolation and activation. Myeloid DCs were isolated from
blood buffy coats of adult healthy donors after separation of mononuclear
cells and enrichment in monocytes using density gradients as described in
Recalde (1984). Monocytes were stained with anti-CD3, anti-CD19, anti-
CD16, anti-CD14, anti-CD56 fluorescein (FITC), CD4-PE, and CD11c-
APC–conjugated Abs. LineageCD4+CD11c+ cells were then isolated by
sorting (MoFlo cell sorter; Beckman Coulter) with a purity of 97–99%. DCs
were cultured at 106/ml in 96-well plates in 2% FBS IMDM with or without
the following stimuli: 15 ng/ml TSLP (R&D Systems), 25 ng/ml poly I:C
(Sigma-Aldrich), and supernatants of untreated or TNF-treated CAFs and HDFs.
In inhibition experiments, 1 µg/ml anti-TSLP (R&D Systems) or 10 µg/ml
anti-TNF (BD) Abs were added. After 24 h, DCs were washed and left for a
further 48 h in culture. Cytokine and chemokines release in DC supernatants
was measured by human chemokines and the inflammatory cytokines CBA
(BD) or ELISA (TARC/CCL17 and MDC/CCL22; R&D Systems).
FACS analysis. DCs were stained with anti-CD80, anti-CD83, anti-HLA-DR,
anti-CD86, and anti-CD40 Abs conjugated with either FITC or PE (BD)
and anti-TSLPR, followed by biotinylated anti–mouse IgG, and then with
streptavidin-PE or –PE-Cy5 (BioLegend). Samples were acquired with
DC/T cell co-culture. Naive CD4+ T cells were purified from cord
blood with anti-CD4–coated beads (Miltenyi Biotec), obtaining ≥90%
CD4+CD45RA+ cells. Activated DCs were added to naive CD4+ T cells
(5 × 104/well) at a 1:5 ratio in 96-well plates. After 6 d, supernatants were
tested for IL-13 and IFN- release by ELISA (Mabtech).
Immunohistochemical analysis. Immunohistochemistry was performed
on tissue sections from surgical specimens, as detailed in Tassi et al. (2008).
The following Abs were used: anti–T-bet and anti–IL-1 (Santa Cruz Bio-
technology, Inc.), anti–GATA-3 (R&D Systems), anti-CD11c (Novocastra),
and anti-TSLPR (BioLegend). For GATA-3 and T-bet evaluation, immuno-
stained slides were digitalized with the Aperio slide scanner and correspond-
ing tumor areas on adjacent sections were selected. Lymphocytes with nuclear
staining were counted using the IHC Nuclear Image Analysis algorithm of
the Spectrum Plus software (Aperio) and normalized to a 1-mm2 area.
CD11c and TSLPR coexpression was visualized using a sequential immuno-
peroxidase labeling and erasing technique (Glass et al., 2009) with the alco-
hol-soluble peroxidase substrate 3-amino-9-ethylcarbazole, combined with
an antibody-antigen elution after the first antibody reaction.
Statistical analysis. The survival curves were estimated with univariate
analyses according to the Kaplan-Meier method and compared using the
log-rank test. Univariate and multivariate analyses by the Cox proportional
hazard model were performed to estimate the independent potential risk
factors that influence disease-free survival and overall survival. All the proba-
bility values were from two-sided tests. Analyses were performed using the
Statistica 4.0 statistical package for Windows (Statsoft).
Online supplemental material. Fig. S1 shows a scatter plot analy-
sis of overall survival and G/T ratio. Fig. S2 shows the characteristics of
cell lines used in the study. Fig. S3 depicts a model of cross talk among
tumor cells, CAFs, and DCs driving Th2 inflammation in pancreatic can-
cer. Online supplemental material is available at http://www.jem.org/cgi/
We thank M. Bellone, P. Dellabona, R. Pardi, V. Russo, and C. Traversari for helpful
discussions and critical review of this manuscript; S. Grassi for helping with the
LCM experiments; M. Alessio for providing reagents for Western blot analyses;
followed by different chemotherapeutic regimens and radiotherapy, were
enrolled in the survival study. The characteristics of the patients are reported
in Table I.
Tissue specimens and establishment of cell lines. Tumor and LN
samples were collected at surgery. Surrounding tissue was sampled at least at
a 1-cm distance from macroscopically evident neoplastic tissue. However,
morphological characteristics varied among samples from conserved normal
parenchyma to preexisting or associated obstructive pancreatitis. Normal
pancreatic tissue was obtained from surgical samples of patients who under-
went surgery for benign pancreatic lesions. The Institutional Ethics Commit-
tee (Comitato Etico Fondazione Centro San Raffaele del Monte Tabor,
Istituto Scientifico Ospedale San Raffaele) had approved the study protocol,
and written informed consent was obtained from all donors. Tumor speci-
mens were in part frozen for RNA extraction and in part used for cell cul-
ture. Tumor pieces were cultured in IMDM medium (Lonza) plus 10% FBS,
and after a few passages tumor cells and CAFs were separated with anti-fibro-
blast Ab-coated beads (Miltenyi Biotec). Draining and nondraining LNs were
mashed in 2% FBS RPMI, 10 mM Hepes, and 400 U/ml collagenase D
Western blotting. Expression of smooth muscle actin (SMA), pan-
cytokeratin, and p53 by cultured CAFs and tumor cells was evaluated by Western
blotting, as described in De Monte et al. (2008). Abs used were mouse anti-
pan cytokeratin (Sigma-Aldrich), rabbit polyclonal anti–-SMA (Abcam),
mouse anti-p53 (DAKO), and mouse anti–-actin (Sigma-Aldrich), followed
by goat anti–mouse HRP or swine anti–rabbit HRP (DAKO), respectively.
Immunodetection was performed by enhanced chemiluminescence (GE
Healthcare), followed by autoradiography on Biomax films (Kodak).
Detection of K-ras mutations. Genomic DNA was extracted from 2 × 106
pancreatic cancer cells or CAFs with the QIAamp DNA mini kit (QIAGEN),
and 30 ng of each sample was amplified for 40 cycles (58°C annealing tem-
perature) with PFU-Turbo DNA polymerase (Agilent Technologies) with
the following primers: Kras forward, 5-GGTGGAGTATTTGATAGTG-
TATTAAACC-3; Kras reverse, 5-TCATGAAAATGGTCAGAGA-
AACC-3. 283-bp PCR products containing k-ras codon 12 and 13 purified
with QIAquick kit (QIAGEN) were then sequenced on both strands and
analyzed with the FinchTv software (Geospiza).
LCM. Epithelial and stromal tumor cells were collected from frozen samples
using the Arcturus LCM system, according to the manufacturer’s instructions.
The two tumor components, identified under microscopy, were cut and
transferred on capsure HS caps and total RNA was extracted as described in
the next section.
Real-time PCR. Total RNA was extracted using the PicoPure RNA isola-
tion kit (Arcturus) or the RiboPure kit (Ambion), according to the manufac-
turers’ instructions. 1 µg RNA was retro-transcribed with the High-Capacity
cDNA reverse transcription kit (Applied Biosystems), and 10–50 ng cDNA
was used for real-time PCR. To analyze the expression of different genes
from RNA obtained by LCM a preamplification step was performed, accord-
ing to the manufacturers’ instructions. Assays on demand specific for human
TSLP (Hs00263639-m1), IL-1 (Hs00174097-m1), TNF (Hs00174128),
TARC/CCL17 (Hs00171074-m1), MDC/CCL22 (Hs99999075-m1), and
GAPDH (Hs99999905-m1; Applied Biosystems) were used. Real-time PCR
was performed on an AB7900HT machine (Applied Biosystems) using the
S.D.S2.1 program for the analysis. Fold induction among samples was calcu-
lated by 2Ct method. The target gene values were normalized with
Fibroblasts stimulation and TSLP quantification. Fibroblasts were cul-
tured overnight at 150.000–300.000 cells/ml in IMDM with 10% FBS, fol-
lowed by 5–8 h of starving without serum. Medium was then replaced with
IMDM with 2% FBS, and fibroblasts were cultured for 48 h with or without
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