Pro-Inflammatory Cytokines Affect Pancreatic

Miranda ten Kate1, Leo J Hofland2, Peter M van Koetsveld2,
Johannes Jeekel1, Casper HJ van Eijck1
1Department of Surgery and 2Department of Internal Medicine, Erasmus Medical Center.
Rotterdam, the Netherlands
ABSTRACT
Objectives The potential role of surgery-
induced pro-inflammatory cytokines on the
development of tumor recurrence in
pancreatic cancer was investigated.
Main outcome measures The adhesion of 3
human pancreatic carcinoma cell lines,
PanC1, MiaPaCa and BxPC3 to monolayers
of microvascular endothelial cells after pre-
incubation with 0.1 or 10 ng/mL IL-1beta,
TNF-alpha or IL-6 was assessed in a
reproducible human in vitro assay. Untreated
monolayers served as controls.
Results Pre-incubation of microvascular
endothelial cells with IL-1beta or TNF-alpha,
but not IL-6, increased adhesion of all three
tumor cell lines as compared to adhesion in
the control group. Maximally stimulated
adhesion for PanC1 reached 159%, for
MiaPaCa 204% and for BxPC3 155% (all vs.
the control, P<0.001). Pre-incubation of
microvascular endothelial cells with IL-1beta
or TNF-alpha resulted in a significant up-
regulation of E-selectin, ICAM-1 and
VCAM-1 expression. The addition of anti-E-
selectin, anti-ICAM-1 or anti-VCAM-1
monoclonal antibodies did not decrease
adhesion to microvascular endothelial cells
pre-incubated with IL-1beta. Therefore,
enhanced tumor cell binding seems to be
independent of these adhesion molecules.
Conclusions Pro-inflammatory cytokines
derived from surgical trauma may enhance
tumor cell adhesion to microvascular
endothelial cells and thus bring about more
successful tumor cell implantation resulting in
an increased risk of metastasis formation.
INTRODUCTION
In the western world, pancreatic cancer leads
to approximately 150,000 deaths each year,
making it one of the five leading causes of
cancer-related deaths [1]. Potential curative
resection is still the only option that offers a
chance for cure in pancreatic cancer patients,
but this can only be performed in about 10-
15% of pancreatic cancer patients [2]. the
prognosis for these patients is still poor, since
more than 80% die within 5 years after
surgery. Recurrences are found locally, in the
intra-abdominal cavity (peritoneal and
hepatic) and to a lesser extent at extra-
abdominal sites [3].
Hematogenous metastasis develops from
circulating tumor cells. These cells can be
detected in 60-80% of pancreatic cancer
patients and this percentage increases during
surgical procedures [4, 5, 6].

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Operative trauma in the tissue itself may favor
development of tumor recurrence. An
association between surgical trauma and
tumor recurrence has been supported by
previous in vivo and in vitro studies [7, 8, 9,
10, 11, 12]. These studies showed that
surgical trauma brings about enhanced
locoregional tumor recurrence by the
inflammatory reaction it provokes. The
inflammatory reaction is not confined locally,
but spreads out systemically as well, yet to a
somewhat lesser degree [13, 14, 15, 16, 17].
Indeed, in several in vivo studies, surgical
trauma enhanced tumor recurrence at distant
sites as well [18, 12].
The inflammatory reaction provoked by
surgical trauma leads to the activation of
leukocytes and monocytes with the release of
pro-inflammatory cytokines and reactive
oxygen species. Thus, it has been found that,
after major abdominal surgery, the pro-
inflammatory cytokines interleukin-6 (IL-6),
interleukin-1beta (IL-1beta) and tumor
necrosis factor-alpha (TNF-alpha) in the
peripheral blood are elevated [13, 14, 15, 16,
17].
The adhesion of circulating tumor cells to the
microvascular endothelium of organs at
distant sites - like the liver and the lungs - is
an important step in blood-borne metastasis.
In previous in vitro experiments we showed
that pro-inflammatory cytokines are capable
of enhancing the adhesion of human colon
carcinoma cells to the microvascular
endothelium, most likely by the up-regulation
of adhesion molecules to the endothelium
[11].
In this study, we investigated the influence of
the pro-inflammatory cytokines IL-1beta,
TNF-alpha and IL-6 on the adhesion of
human pancreatic carcinoma cells to
microvascular endothelial cells. Therefore, a
reproducible human in vitro model was
developed. Moreover, the expression of cell
adhesion molecules on both endothelial and
tumor cells was assessed as well as the
influence of blocking antibodies to cell
adhesion molecules in tumor cell adhesion
assays.
MATERIALS AND METHODS
Cells
Human microvascular endothelial cells
(MECs) at passage 4 were purchased from
Cambrex (Verviers, Belgium) and maintained
in EGM-2-MV Bullet kit according to the
manufacturer’s instructions at 37°C, 95%
relative humidity and 5% CO2. Confluent
monolayers were passaged by trypsin/EDTA
(0.025%/0.01%) and cells were used up to
passage 8.
The human pancreatic carcinoma cell lines
PanC1, MiaPaCa and BxPC3 were also grown
in an EGM-2-MV Bullet kit in order to create
similar culture conditions to the endothelial
cells, which was necessary for the
experiments. The tumor cells were maintained
by serial passage after trypsinization using
trypsin/EDTA (0.05% 0.02%) (all, except
penicillin, obtained from Gibco, Breda, the
Netherlands; penicillin from Yamanouchi,
Leiderdorp, the Netherlands). Before the
adhesion assay, tumor cells were harvested
using trypsin and maintained in suspension
culture for two hours to regenerate cell-
surface proteins.
Adhesion Assay
To quantify tumor cell adhesion to MECs, a
standardized cell adhesion assay was
developed according to the methods of
Catterall et al . [19]. Briefly, endothelial
monolayers were established in 96 well
microtiter plates (Perkin Elmer, Groningen,
the Netherlands). To do this, confluent cells
were trypsinized and 2x10endothelial cells
were added to each well.
The plates were incubated at 37°C, 95%
relative humidity, 5% COand the medium
was replaced daily by fresh medium. The
MECs reached confluence in 3 to 4 days as
determined by light microscopy.
To determine the effect of cytokines on tumor
cell adhesion, endothelial monolayers were
pre-incubated with 0.1 or 10 ng/mL
recombinant human IL-1beta, TNF-alpha and

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IL-6 (R&D Systems, Uithoorn, The
Netherlands) for 4 or 12 hours. Untreated
monolayers served as controls. Not only was
the effect of the endothelial pre-incubation
investigated but the effect of tumor cell pre-
incubation as well. Therefore, BxPC3 cells
were pre-incubated with 10 ng/mL IL-1beta
for 12 hours before the adhesion assay.
To quantify tumor cell adhesion, the tumor
cells were labeled with calcein-AM
(Molecular Probes, Leiden, the Netherlands)
for 45 minutes, washed 3 times and added to
the wells (3x10per well). The plates were
centrifuged for 1 minute at 80 in a Heraeus
centrifuge (Etten Leur, the Netherlands) and
incubated at 37°C for 1 hour. Thereafter, the
wells were washed twice with medium to
remove non-adherent tumor cells. The
remaining fluorescence per well was
measured on a Perkin Elmer (Gouda, the
Netherlands) plate reader using 485 nm
excitation and 530 nm emission filters.
Immunocytochemistry
Endothelial and tumor cells were prepared for
staining by cytospin preparation, fixed in
acetone for 10 minutes and stored at -20°C
until used.
The cytospins were incubated for 30 minutes
at room temperature with the following
primary antibodies: mouse anti-human
monoclonal antibodies to E-selectin (R&D
Systems, Uithoorn, The Netherlands), ICAM-
1, VCAM-1 (Dako Cytomation Heverlee,
Belgium), sialyl Lewis a (sLea), sialyl Lewis
x (sLex) (Sanbio, Uden, the Netherlands),
lymphocyte function-associated antigen-1
(LFA-1) (alphaLbeta2) and very late
activation antigen-4 (VLA-4) (alpha4beta1)
(Becton Dickinson, Alphen a/d Rijn, the
Netherlands). Negative controls were
incubated with PBS. As secondary antibodies,
biotinylated goat anti-mouse antibodies were
used followed by incubation with
streptavidin-biotinylated alkalin-phosphatase
complex. Substrate development was carried
out with new fuchsin 4%. The cytospins were
counterstained with hematoxylin.
The expression of cell adhesion molecules
was quantified by two different observers
using semi-quantitative scoring system
ranging from no expression (-), weakly
positive (±) to positive expression (+).
Enzyme Immuno Assay (EIA)
Endothelial cells and tumor cells were grown
to confluence as described for the adhesion
assays in 96-well flat-bottomed multititer
plates (Becton Dickinson, Alphen a/d Rijn,
the Netherlands). The endothelial cells were
pre-incubated with either cell culture media
alone or with media containing IL-1beta,
TNF-alpha or IL-6 (0.1 and 10 ng/mL). The
tumor cells were pre-incubated with either
cell culture media alone or with media
containing IL-1beta or TNF-alpha (10
ng/mL). Following this pre-incubation, the
cells were washed with phosphate buffered
saline (room temperature, pH 7.4), fixed in
ethanol/methanol for 45 minutes and then
washed again. Subsequently, the wells were
incubated for 10 minutes with 1% goat serum
to block unspecific binding sites. Mouse
monoclonal antibody to E-selectin, ICAM-1,
VCAM-1 (R&D Systems, Uithoorn, The
Netherlands) sLea, sLe(Sanbio, Uden, the
Netherlands), LFA-1 (alphaLbeta2) or VLA-4
(alpha4beta1) (Becton Dickinson, Alphen a/d
Rijn, the Netherlands) was added for 1 hour,
followed by the addition of a second
antibody, biotinylated goat anti-mouse
antibody
(Sigma
Zwijndrecht,
the
Netherlands) in a dilution of 1:250. Increased
sensitivity was obtained using the ExtrAvidin-
Peroxidase system (Sigma, Zwijndrecht, the
Netherlands). Adding 2,2'-azino-bis(3-
ethylbenzothiazoline-6-sulfonic
acid)
diammonium salt in citrate-phosphate buffer
with urea hydrogen peroxide-developed
substrate. Incubation of the endothelial cells
without the primary antibody served as a
negative control. As a positive control, the
ExtrAvidin-Peroxidase (Sigma, Zwijndrecht,
the Netherlands) system was added followed
by substrate development without washing
away the peroxidase. After 40 minutes, the

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reaction was stopped with sodium fluoride
and photometrical evaluation was performed
with computer-controlled ELISA reader at a
lambda equal to 405 nm.
Function Blocking Assay
Endothelial monolayers were pre-incubated
with 10 ng/mL IL-1beta for 12 hours. One
hour before the adhesion assay was
performed, 50 μg/mL of function blocking
monoclonal mouse antibodies to human E-
selectin (R&D Systems, Uithoorn, The
Netherlands) were then added to the
endothelial monolayers. The same inhibition
assays were carried out with monoclonal
mouse antibodies to human ICAM-1 (25
μg/mL) and VCAM-1 (60 μg/mL) (R&D
Systems, Uithoorn, The Netherlands) (the
concentration of the antibody was in
accordance with the instructions of the
manufacturer).
STATISTICS
Data are reported as mean±SD values. The
data were analyzed using one- or two-way
analysis of variance (ANOVA) according to
the experimental design applied. The simple
and the repeated contrasts were applied to
ANOVA in order to compare the various
experiments with the control experiment as
well as adjacent categories, respectively. The
effects within specific categories were
evaluated using nested designs. The SPSS
version 13.0 for Windows was used to
analyze the data. Two-tailed P values less
than 0.05 were considered to be statistically
significant. Experiments (n=6) were
performed at least twice with comparable
results while quadriplate wells were used in
the EIA assays.
RESULTS
Validation of Assay
Labeling tumor cells with calcein-AM did not
decrease their viability (greater than 95%
using trypan blue). To determine the stability
of calcein labeling, the fluorescence of the
labeled cells and of the supernatant of the
labeled cells was measured. The fluorescence
of the labeled cells remained constant for at
least 60 minutes indicating retention of the
dye within the cells (data not shown). This
Comparisons between 4- and 12-hour pre-incubation times (the time factor was evaluated within the various categories using a nested design):
aP=0.352, bP<0.001, cP=0.003,
dP=0.023, eP=0.191, fP=0.129.
aP=0.712, bP=0.001, cP=0.420,
dP=0.139, eP=0.441, fP=0.302.
aP=0.577, bP<0.001, cP=0.065,
dP=0.127, eP=0.079, fP=0.001.
Figure 1. Adhesion of PanC1, MiaPaCa and BxPC3 to MECs after 4 and 12 hours of pre-incubation of MECs with 0.1
or 10 ng/mL IL-1beta, TNF-alpha or IL-6. Mean±SD values are shown. Generally, n=6 samples were evaluated for
each experiment. (Two-way ANOVA). Black P values refer to the comparison with the control group (untreated
MECs): simple contrast was applied within the two time evaluations by a nested design. Red P values refer to the
comparison between 0.1 and 10 ng/mL; repeated contrast was applied within the two time evaluations using a nested
design.

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result was also seen in the adhesion assay,
where maximal tumor cell adhesion was
reached after a one hour incubation followed
by a decrease at longer incubation times (data
not shown). Therefore, for all subsequent
experiments, the incubation time was 1 hour.
Dilution series with labeled tumor cells on
MEC monolayers showed a linear correlation
between cell number and measured
fluorescence (data not shown) which was
used as a standard to calibrate the
fluorescence measured. In this way, the
amount of adhered tumor cells in the
experimental wells could be determined.
Adhesion to Endothelial Cells
In all assays, PanC1 cells adhered to untreated
MEC in a percentage ranging from 10 to 20%,
i.e. the basal or control adhesion. The basal
adhesion of BxPC3 ranged from 20 to 30% in
all assays and that of MiaPaCa ranged from 5
to 20%
Pre-incubation of MEC with IL-1beta or
TNF-alpha, but not with IL-6 resulted in time-
and concentration-dependent-enhanced tumor
cell adhesion (Figure 1). Four-hour pre-
incubation with 0.1 ng/mL IL-1beta resulted
in a significant enhancement of BxPC3 only
(113% vs. control, P=0.016), while 4-hour
pre-incubation with 10 ng/mL IL-1beta
resulted in a significant enhancement for
MiaPaCa (144% vs. control, P=0.011) and
BxPC3 (127% vs. control, P<0.001). After a
4-hour pre-incubation of MEC with TNF-
alpha, adhesion for all 3 cell lines was
significantly enhanced (PanC1: P=0.004 and
P=0.010 for 0.1 and 10 ng/mL, respectively;
MiaPaCa and BxPC3: P<0.001 at each
concentration). As far as 4-hour pre-
incubation with IL-6 was concerned, only a
slight enhancement adhesion of MiaPaCa
(135% vs. control, P=0.039) was obtained
with 10 ng/mL IL-6. A twelve-hour pre-
incubation with IL-6 did not show any
significant enhancement of adhesion. The
enhancement of adhesion for all cell lines was
significant after 12 hours of pre-incubation
with 0.1 ng/mL of IL-1beta and the adhesion
for all cell lines significantly increased by
increasing the IL-1beta concentration to 10
ng/mL; for PanC1, adhesion reached 159%
vs. control (P<0.001), for MiaPaCa it was
204% (P<0.001) and for BxPC3 it was 127%
(P<0.001). TNF-alpha resulted in enhanced
adhesion for all cell lines at 0.1 ng/mL as well
as at 10 ng/mL, but a significant increase with
the concentration was observed only for
BxPC3; for PanC1 it was 155% (P<0.001)
and 144%(P<0.001), for MiaPaCa 178%
(P=0.001) and 203% (P<0.001) and for
BxPC3 144% (P<0.001) and 137% (P<0.001)
vs. control at 01. and 10 ng/mL TNF-alpha,
respectively.
A time-dependent increase in adhesion was
observed with pre-incubation with 10 ng/mL
IL-1beta for all three cell lines (P<0.001) as
well as at the two concentrations (0.1 ng/mL:
P=0.003; 10 ng/mL: P=0.023) of TNF-alpha
for PanC1 cells only while a significant
decrease in adhesion was observed for the
BxPC3 cell line from 4- to 12-hour pre-
incubation with 10 ng/mL IL-6 (P=0.001).
Pre-incubation with higher concentrations of
the pro-inflammatory cytokines (50 and 100
ng/mL) did not result in a more pronounced
enhancement of adhesion (data not shown).
The Role of Cell Adhesion Molecules
It is known that both sialyl Lewis a (sLea) and
sialyl Lewis x (sLex) are ligands for E-
selectin; LFA-1 (alphaLbeta2) is a ligand for
ICAM-1, and VLA-4 (alpha4beta1) for
VCAM-1.
Table 1 shows the expression of cell adhesion
molecules on untreated MECs, MECs pre-
incubated with 10 ng/mL IL-1beta for 12
hours, as well as on PanC1, MiaPaCa and
BxPC3. Untreated MECs express ICAM-1, as
well as LFA-1 and VLA-4, whereas E-
selectin and VCAM-1 are not expressed. Both
these adhesion molecules are up-regulated by
exposure of MECs to IL-1beta. BxPC3 cells
express all tested adhesion molecules, PanC1
cells do not express VCAM-1 only, whereas
MiaPaCa cells selectively express sLea.
This difference in the adhesion molecule
pattern is displayed in the percentage of
adherent cells to MECs, which is highest for

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BxPC3 and lowest for MiaPaCa (data not
shown).
We further evaluated the endothelial cell
adhesion molecules by EIA (a semi-
quantitative assay) (Figure 2). The staining
intensity (in optical density units measured at
405 nm) of E-selectin expression on
unstimulated MECs was 0.2. Following pre-
incubation with 0.1 and 10 ng/mL IL-1beta
for 12 hours the staining intensities reached
0.19 (P=0.739) and 0.50 (P<0.001),
respectively. Pre-incubation with 0.1 and 10
ng/mL TNF-alpha significantly up-regulated
E-selectin expression to a staining intensity of
0.47 and 0.59, respectively (both P<0.001).
IL-6 did not up-regulate E-selectin
expression. ICAM-1 expression was up-
regulated from 0.2 to 0.27 (P<0.001) and to
0.40 (P<0.001) by pre-incubation with 0.1 and
10 ng/mL IL-1beta, respectively. Pre-
incubation with 0.1 and 10 ng/mL TNF-alpha
up-regulated expression to 0.48 (P<0.001) and
to 0.47 (P<0.001), respectively whereas once
again IL-6 did not influence expression. A
slight but significant enhancement in the
expression of VCAM-1 was induced in all but
one experiment (the pre-incubation with 0.1
ng/mL IL-6; P=0.062); maximal induction
was reached with 10 ng/mL TNF-alpha (from
0.23 to 0.31; P=0.001).
Next, we focused in more detail on
interactions in inhibition assays between
tumor cells and endothelial cells by using
monoclonal antibodies against adhesion
molecules on MECs. None of the function
blocking antibodies tested significantly
influenced basal adhesion apart from a slight
decrease in PanC1 cells with anti-E-selectin
(Figure 3). Pre-incubation with IL-1beta
Table 1. Cell adhesion molecules expressed by untreated MECs, MECs pre-incubated with 10 ng/mL IL-1beta (12 h) ,
PanC1, MiaPaCa and BxPC3 as determined by immunocytochemistry.
Cell adhesion
molecule
Untreated MECs
(control)
MECs +
IL-1beta
PanC1
MiaPaCa
BxPC3
E-selectin
-
+
±
-
±
ICAM-1
+
+
+
-
+
VCAM-1
-
+
-
-
+
sLea
-
-
+
+
+
sLex
-
-
+
-
+
LFA-1
+
+
±
-
+
VLA-4
+
+
±
-
+
Figure 2. Anti-E-selectin, anti-ICAM-1 and anti-
VCAM-1 expression on MECs pre-incubated for 12
hours with 0.1 and 10 ng/mL TNF-alpha, IL-1beta or
IL-6 assessed by EIA. Bars represent the mean±SD
absorbance values (OD 405 nm). Generally, n=4 wells
were evaluated for each experiment. (One-way
ANOVA). Black P values refer to the comparison with
the control group (untreated MECs); simple contrast
was applied. Red P values refer to the comparison
between 0.1 and 10 ng/mL; repeated contrast was
applied.

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significantly (P<0.001) enhanced adhesion of
all three cell lines both in the absence or the
presence of all the antibodies tested. Anti-E-
selectin was not capable of inhibiting the
enhanced adhesion of all 3 tumor cell lines to
IL-1beta pre-incubated MECs while slight
increases of adhesion were observed in PanC1
cells with anti-ICAM-1 and anti-VCAM-1
antibodies. No significant modifications were
found in MiaPaCa cells, while slight
decreases of adhesion were obtained in
BxPC3 with anti-ICAM-1 (with and without
the addition of anti-E-selectin) and anti-
VCAM-1 antibodies. (Figure 3). Furthermore,
pre-incubation of MECs with TNF-alpha
instead of IL-1beta gave similar results (data
not shown). The cell adhesion molecules sLea,
sLex, LFA-1 and VLA-4 are probably not the
ligands on MECs responsible for increased
tumor cell adhesion after exposure to the pro-
inflammatory cytokines. First of all, sLeand
sLeare neither expressed on MECs or on
activated MECs and its counterpart E-selectin
is not expressed on MiaPaCa and only weakly
expressed on PanC1 and BxPC3 (Table 1).
Furthermore, although both LFA-1 and VLA-
4 are expressed on MECs and are slightly up-
regulated after exposure to pro-inflammatory
cytokines (data not shown), their counterparts
are not expressed on MiaPaCa. However, the
adhesion of MiaPaCa clearly increased to
cytokine pre-incubated MECs and, therefore,
LFA-1 and VLA-4 are unlikely to be the
adhesion molecules on MEC which induce
enhanced adhesion.
Exposure of Tumor Cells
Since not only the endothelium but the
circulating tumor cells as well are exposed to
factors released during surgery, the influence
of pro-inflammatory cytokines on adhesion
molecule expression of the tumor cells was
investigated subsequently by EIA (Figure 4).
For PanC-1 the expression of VLA-4 shows a
slight but significant decrease after IL-1beta
pre-incubation (P=0.003). The expression of
LFA-1 on MiaPaCa after IL-1beta pre-
incubation is also slightly decreased
(P=0.016). On BxPC3 however, a slight but
significant increase in ICAM-1 (P<0.001) and
LFA-1 (P=0.049) was observed. Comparable
results were obtained after pre-incubation of
the tumor cells with TNF-alpha (data not
shown).
Therefore, the influence of exposing BxPC3
to IL-1beta on tumor cell adhesion was
Figure 3. Adhesion of PanC1, MiaPaCa and BxPC3 to
MECs. Prior to tumor cell adhesion, the MECs were
pre-incubated for 12 hours with 10 ng/mL IL-1beta
and/or for 1 hour with anti-E-selectin (E-sel), anti-
ICAM-1 (ICAM-1), both anti-E-selectin and anti-
ICAM-1 (E-sel+ICAM-1) or anti-VCAM-1 (VCAM-
1). Mean±SD values are shown. Generally, n=6
samples were evaluated for each experiment. (One-way
ANOVA). Black P values refer to the comparisons
with values observed without added antibodies (either
untreated MAC or IL-1beta pre-incubated MECs): the
simple contrast was applied. Red P values refer to the
comparisons between untreated MAC and IL-1beta
pre-incubated MEC; repeated contrast was applied.

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461
investigated (Figure 5). The overall analysis
showed that pre-incubation with IL-1beta
significantly enhanced adhesion (P<0.001)
while BxPC3 did not (P=0.241). The effect of
incubation with BxPC3 was not significantly
related to the presence or absence of pre-
incubation with IL-1beta (interaction term of
two-way ANOVA: P=0.096). In particular,
the effect of pre-incubation with BxPC3 was
not significant either in control MECs
(P=0.050) or in IL-1beta pre-incubated cells
(P=0.706) while the effect of pre-incubation
with IL-1beta was equally significant in
control (P<0.001) and pre-incubated
(P<0.001) BxPC3 cells.
DISCUSSION
Previous in vivo studies suggested a tumor-
promoting effect from surgical trauma, not
only locally, but systemically as well. This
theory was further explored in a human in
vitro model, showing that pro-inflammatory
cytokines, released during surgical trauma,
enhance the adhesion of human colon
carcinoma cells to mesothelial cells and
microvascular endothelial cells [11].
Therefore, surgical trauma may enhance
tumor recurrence loco-regionally as well as at
distant sites.
In this human in vitro study, we demonstrated
that the pro-inflammatory cytokines IL-1beta
and TNF-alpha significantly enhance the
adhesion of 3 human pancreatic carcinoma
cell lines to the microvascular endothelium.
IL-6, which is distinctly elevated during
surgical trauma and is an activator of several
inflammatory mechanisms, did not influence
Figure 4. Adhesion molecule expression on PanC1,
MiaPaCa and BxPC3 obtained with EIA. Orange bars
represent basal expression; green bars represent
expression after 12 hours pre-incubation with 10
ng/mL IL-1beta. Bars represent the mean±SD
absorbance values (OD 405 nm). Generally, n=4 wells
were evaluated for each experiment. (Two-way
ANOVA; the effect of the pre-incubation with IL-1beta
was evaluated within the various categories using a
nested design).
Figure 5. The effect of the pre-incubation of MECs
with 10 ng/mL IL-1beta and/or BxPC3. Bars represent
the mean±SD absorbance values (OD 405 nm).
Generally, n=6 samples were evaluated for each
experiment. (Two-way ANOVA: the effects of pre-
incubation with IL-1beta and BxPC3 were evaluated
within the various categories using a nested design).
Black P values refer to the comparisons with control
MECs (without IL-1beta pre-incubation). Red P values
refer to the comparisons between control BxPC3 and
pre-incubated BxPC3 cells.

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JOP. J Pancreas (Online) 2006; 7(5):454-464.
JOP. Journal of the Pancreas - http://www.joplink.net - Vol. 7, No. 5 - September 2006. [ISSN 1590-8577]
462
interactions between tumors and endothelial
cells.
In previous studies [11], we excluded the
possibility that enhanced adhesion after
cytokine exposure was caused by enhanced
growth of endothelial cells resulting in a
higher number of MEC and, consequently,
more binding receptors since proliferation
assays did not show enhanced growth of the
exposed MECs as compared to normal MECs
during the 12 hour pre-incubation period.
Interactions between tumor cells and
endothelial cells are accomplished by
adhesion molecules. The results of the EIA
showed that the endothelial adhesion
molecules E-selectin and ICAM-1 are
significantly up-regulated after pre-incubation
with TNF-alpha or IL-1beta. The question
then arises as to whether the increased tumor
cell binding is dependent on the up-regulation
of E-selectin or ICAM-1. VCAM-1 is not
likely to be the adhesion molecule responsible
since no significant enhancement in MEC
expression was found after cytokine pre-
incubation, except for a slight increase after
pre-incubation with 10 ng/mL TNF-alpha. In
previous studies, the adhesion of several
pancreatic carcinoma cell lines to human
umbilical vein endothelial cells (HUVECs),
which are macrovascular cells, could be
inhibited by antibodies against the E-selectin
adhesion molecule [20, 21]. In these studies
no inhibition in adhesion to activated
endothelium was observed using monoclonal
antibodies against E-selectin. The absence of
inhibition could not be attributed to the
malfunctioning of antibodies or technical
problems since we brought about strong
inhibition in the adhesion of HT29 colon
carcinoma cells to activated HUVECs using
the same protocol and antibodies [11].
However, in that particular study, we were not
able to block the adhesion of HT29 cells to
MECs. Therefore, it was concluded that
another adhesion molecule or a complex of
adhesion molecules was responsible for HT29
cell interactions with MECs. Again, in this
study no inhibition could be reached using E-
selectin antibodies in the adhesion of tumor
cells to MECs. Even for MiaPaCa, with
limited adhesion options according to
immunohistochemistry results concerning the
adhesion molecules tested, i.e. only binding
through E-selectin on activated MECs with
sLea
on MiaPaCa seemed possible, no
inhibition was observed using the E-selectin
antibody, suggesting that another adhesion
molecule or a complex of adhesion molecules
seems responsible for the enhanced adhesion
to MEC.
Antibodies against other major adhesion
molecules on MECs, ICAM-1 and VCAM-1,
did not lead to a reduction in tumor cell
adhesion to activated MECs either. Thus, both
colon carcinoma cells and pancreatic
carcinoma cells seem to exhibit different
adhesion patterns to macrovascular
endothelial cells as compared to
microvascular endothelial cells. In contrast to
the E-selectin dependent adhesion to
HUVECs, adhesion to MECs is E-selectin
independent and formed by another novel
adhesion molecule or a complex of adhesion
molecules consisting of more than E-selectin
and ICAM-1.
In the systemic inflammatory response during
and after surgery, the circulating tumor cells -
like endothelial cells - are exposed to pro-
inflammatory cytokines as well. Therefore,
the influence of pro-inflammatory cytokines
regarding adhesion molecule expression on
the tumor cells was evaluated as well. Only
ICAM-1 expression on BxPC3 showed a
slight enhancement after exposure to IL-1beta
and TNF-alpha; the expression of the other
adhesion molecules was not affected. These
rather moderate changes in adhesion molecule
expression after IL-1beta or TNF-alpha
exposure might be caused by the already high
basal adhesion molecule expression on tumor
tissue as compared to normal tissue [22].
Since only BxPC3 showed a difference after
IL-1beta or TNF-alpha exposure, we
performed adhesion assays with this cell line.
However, pre-incubation of BxPC3 with IL-
1beta gave comparable results when
compared to untreated BxPC3; therefore, in
this study, no effect of tumor cell exposure to
pro-inflammatory cytokines was detected. It
is interesting that tumor cells are capable of

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JOP. J Pancreas (Online) 2006; 7(5):454-464.
JOP. Journal of the Pancreas - http://www.joplink.net - Vol. 7, No. 5 - September 2006. [ISSN 1590-8577]
463
producing pro-inflammatory cytokines
themselves and that these tumoral cytokines
may influence interactions with the
microvascular endothelium [23].
In conclusion, pro-inflammatory cytokines
derived from surgical trauma enhance the
adhesion of these 3 tumor cell lines to MECs.
In addition, these cytokines increase the
expression level of several adhesion
molecules on the cell surface of MECs. The
inability of function blocking antibodies to E-
selectin, ICAM-1 and VCAM-1 to block
increased tumor cell adhesion to MECs,
suggests that other adhesion molecules or
even a complex of adhesion molecules
determine this enhanced tumor cell adhesion
by the pro-inflammatory cytokines which
were evaluated.
Although surgery is the cornerstone in the
treatment of pancreatic cancer and the only
chance of cure, it may bring about more
successful tumor cell implantation locally and
in distant organs resulting in an increased risk
of metastasis formation. Further studies,
including in vivo studies, are required to
unravel the precise mechanisms by which
surgery enhances the development of distant
metastases. Ultimately, this may lead to new
treatment
modalities
targeting
the
development of post-operative tumor
development.
Received May 15th, 2006 - Accepted June
28th, 2006
Keywords
Cell Adhesion; Cytokines;
Endothelium; Pancreatic Neoplasms; Surgery
Abbreviations
MEC:
microvascular
endothelial cell; HUVEC: human umbilical
vein endothelial cell
Acknowledgement We would like to
acknowledge the valuable insights and
observations contributed by the consulting
biostatistician of JOP. Journal of the Pancreas
Correspondence
Casper HJ van Eijck
Department of Surgery
Erasmus Medical Centre
Dr. Molewaterplein 40
3015 GD Rotterdam
The Netherlands
 
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