Myofibroblast-induced tumorigenicity of pancreatic ductal ...
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Myofibroblast-induced tumorigenicity of pancreatic ductal epithelial cells
is L1CAM-dependent
Heiner Schäfer1, Claudia Geismann1, Carola Heneweer2, Jan-Hendrik Egberts3, Olena Korniienko4, Helena
Kiefel5, Gerhard Moldenhauer5, Max G. Bachem6 Holger Kalthoff4, Peter Altevogt5, Susanne Sebens1,7
1Department of Internal Medicine I, Laboratory of Molecular Gastroenterology & Hepatology, UKSH-
Campus Kiel, Kiel, Germany; 2 Clinic for Diagnostic Radiology, UKSH-Campus Kiel, Kiel, Germany 3
Clinic of General Surgery and Thoracic Surgery, UKSH-Campus Kiel, Kiel, Germany; 4 Institute for
Experimental Cancer Research, UKSH-Campus Kiel, Kiel, Germany; 5German Cancer Research Center,
Department of Translational Immunology D015, Heidelberg, Germany; 6 Department of Clinical Chemistry
and Pathobiochemistry, University of Ulm, Ulm, Germany; 7 Institute for Experimental Medicine c/o
Department of Internal Medicine I, UKSH-Campus Kiel, Kiel, Germany
Short title: L1CAM-mediated tumorigenesis of pancreatic cancer
Keywords: CD171, L1CAM antibody, pancreatic cancer, myofibroblasts, tumorigenicity
Abbreviations: aPSC: activated pancreatic stellate cell; CAF: carcinoma asscociated fibroblast; EMT:
Epithelial-Mesenchymal Transition; MF: myofibroblast; PanIN: Pancreatic Intraepithelial Neoplasia;
PDAC: pancreatic ductal adenocarcinoma; PMF: pancreatic myofibroblast; SCID: severe combined
immunodeficiency; TGF-β1: Transforming Growth Factor-beta1; TRAIL: TNF-related apoptosis-inducing
ligand
Correspondence address:
Susanne Sebens, Ph.D.
Institute for Experimental Medicine
c/o Department of Internal Medicine I, UKHS-Campus Kiel
Arnold-Heller-Str. 3, Haus 6, 24105 Kiel, Germany
Phone: +49-431-597-3835/ Fax: +49-431-597-1427
Email: [email protected]
Carcinogenesis Advance Access published November 17, 2011 at Pennsylvania State U
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Abstract
Pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis, representing one risk factor for PDAC,
are characterized by a marked desmoplasia enriched of pancreatic myofibroblasts (PMFs). Thus, PMFs are
thought to essentially promote pancreatic tumorigenesis. We recently demonstrated that the adhesion
molecule L1CAM is involved in epithelial-mesenchymal transition of PMF-cocultured H6c7 human ductal
epithelial cells and that L1CAM is expressed already in ductal structures of chronic pancreatitis with even
higher elevation in primary tumors and metastases of PDAC patients. This study aimed at investigating
whether PMFs and L1CAM drive malignant transformation of pancreatic ductal epithelial cells by
enhancing their tumorigenic potential. Cell culture experiments demonstrated that in the presence of PMFs,
H6c7 cells exhibit a profound resistance against death ligand induced apoptosis. This apoptosis protection
was similarly observed in H6c7 cells stably overexpressing L1CAM. Intrapancreatic inoculation of H6c7
cells together with PMFs (H6c7co) resulted in tumor formation in 7/8 and liver metastases in 6/8 SCID mice
whereas no tumors and metastases were detectable after inoculation of H6c7 cells alone. Likewise, tumor
outgrowth and metastases resulted from inoculation of L1CAM overexpressing H6c7 cells in 5/7 and 3/7
SCID mice, respectively, but not from inoculation of mock transfected H6c7 cells. Treatment of H6c7co
tumor-bearing mice with the L1CAM antibody L1-9.3/2a inhibited tumor formation and liver metastasis in
100% and 50%, respectively, of the treated animals. Overall, these data provide new insights into the
mechanisms of how PMFs and L1CAM contribute to malignant transformation of pancreatic ductal
epithelial cells in early stages of pancreatic tumorigenesis.
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumor diseases exhibiting an
unfavorable prognosis. In Western countries, PDAC ranked 4th in the order of fatal tumor diseases with a
still increasing prevalence (1). Owing to the lack of specific symptoms, PDAC is mostly detected in an
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advanced stage when the tumor has already metastasized and therapeutic options are limited accounting for
an overall 5-year survival rate of 2 % (2).
Most PDACs are thought to arise from the ductal epithelium. One hallmark of PDAC is its pronounced
desmoplasia (tumor stroma) mainly consisting of activated fibroblasts (also designated as myofibroblasts
(MF), activated pancreatic stellate cells (aPCS) or carcinoma-associated fibroblasts (CAF)) and extracellular
matrix proteins. This marked desmoplasia is already present in chronic pancreatitis (3-5) which represents
one risk factor for PDAC (3,4). Besides genetic alterations e.g. in the k-ras, p53 or DPC4/Smad4 genes,
continuous interactions between ductal and, later on, carcinoma cells and the stromal environment are
important for PDAC tumorigenesis (6,7,8). Guerra et al. showed that expression of mutant k-RasG12V in
acinar and centroacinar cells of adult mice did not result in the development of Pancreatic Intraepithelial
Neoplasias (PanINs) and invasive PDAC unless the animals undergo a chronic pancreatitis (9) indicating
that genetic alterations in somatic cells alone do not necessarily lead to neoplastic formation. Moskovitz et
al. demonstrated that chronic exposure to oxidative stress which is likely to occur during chronic
inflammation can induce chromosomal alterations in cultured normal pancreatic ductal epithelial cells (10).
Accordingly, genomic instability in terms of chromosomal and gene abnormalities could be detected already
in earliest PanIN lesions and tissues of chronic pancreatitis (10,11). In addition, the epithelial-mesenchymal
transition (EMT) is supposed to promote tumorigenesis. Upon this process, epithelial and carcinoma cells,
respectively, lose their epithelial phenotype by acquiring a spindle-shaped morphology and exhibiting a
reduced expression of the epithelial specific surface molecule E-cadherin whereas the expression of
mesenchymal marker proteins such as vimentin and N-cadherin is elevated (12,13). These alterations are
thought to be prerequisite for cell dissociation from the epithelial tissue consequently leading to metastasis.
One important inducer of EMT is the transforming growth factor-beta 1 (TGF-β1) which can be released by
both stroma and tumor cells leading to a paracrine loop by which malignancy associated alterations of the
stroma and tumor are promoted. Thus, tumor cell derived TGF-β1 leads to activation of fibroblasts/stellate
cells and promotes desmoplastic reaction. Secretion of TGF-β1 by MFs (aPSCs/CAFs) fosters EMT in the
tumors cells and thereby tumor progression (14-17). Using the human pancreatic ductal cell line H6c7, we
recently demonstrated that coculture with pancreatic myofibroblasts (PMFs) led to TGF-β1-dependent EMT
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being associated with an upregulation of the adhesion molecule L1CAM (18). Moreover, as a result of this
elevated L1CAM expression, H6c7 cells acquire a chemoresistant and migratory phenotype (18). L1CAM
(CD171) is a member of the immunoglobulin superfamily and has been detected in a variety of tumors such
as melanoma, colon cancer as well as in PDAC (19-24) being associated with poor prognosis and short
survival times (21,24,25). Several studies demonstrated an involvement of L1CAM in various biological
processes of tumorigenesis such as tumor cell migration and invasion, chemoresistance as well as tumor
growth and survival (18,19,20,22,26) pointing to an important role of this protein in the development and
progression of tumors.
H6c7 cells do not harbor established mutations in oncogenes or tumor suppressor genes (27-29) regarded as
initial events in pancreatic tumorigenesis. Moreover, they are described to be non-tumorigenic and our
recent data support the hypothesis that i) malignant transformation of pancreatic ductal epithelial cells might
be initiated before genetic aberrations occur and ii) PMFs and L1CAM play an essential role in this scenario.
Based on these findings, the present study aimed at elucidating whether the PMF- and L1CAM-mediated
alterations of ductal epithelial cells relates to an increased tumor formation and metastasis in vivo.
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Material & Methods
Cell lines and cell culture. The human pancreatic ductal epithelial cell line H6c7 (kindly provided by Prof.
M.S. Tsao, Ontario Cancer Center, Toronto, Canada) was cultured as described (30). For some experiments,
H6c7 cells were retrovirally transduced either with an empty vector (mock) or with a vector encoding human
L1CAM (31). Before transduction, the L1CAM construct was verified by sequencing. The transduction
procedure with retroviral vectors and the selection of transduced clones with puromycin were described
previously (32).
Pancreatic fibroblasts. Murine myofibroblasts (PMFs) were isolated and cultured as described (8). The
myofibroblastic phenotype was confirmed by immunocytochemical staining for vimentin and α-smooth
muscle actin. Since the in vivo experiments were performed with PMFs, the in vitro experiments were
mainly done with murine PMFs and confirmative experiments were performed with human PMFs (see
supplementary data).
Coculture. For coculture, 1x105 H6c7 cells in 2 mL H6c7 medium were seeded into a well of a 6-well-plate,
and 2x105 PMFs in 1.5 mL PMF medium were seeded into a transwell insert (0.4 µm pore size, Greiner,
Frickenhausen, Germany) which was placed into a well with H6c7 cells. After 24 hours, medium was
exchanged. To continue the coculture for four weeks with PMFs (for human PMFs see supplementary data),
cells were seeded as described above. Then, every week cocultured H6c7 cells and cocultured PMFs were
detached, counted and freshly seeded at the respective cell numbers.
Antibodies and reagents. The mouse anti-human L1-9.3/2a antibody binding to the ectodomain of L1CAM
has been described earlier (33). Mouse IgG2a control antibodies (clone Cl.18) were purchased from
BioXCell (West Lebanon NH, USA). Killer TRAIL was purchased from Alexis Biochemicals (Grünberg,
Germany) and the agonistic anti-CD95/Fas antibody CH-11 was obtained from Coulter Immunotech
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(Hamburg, Germany). Both reagents were used at a final concentration of 0.1 µg/ml. Human TNF-α was
from Sigma-Aldrich Chemie (Taufkirchen, Germany) and used at a final concentration of 20 ng/ml.
Measurement of apoptosis by determination of caspase-3/-7 activity. For determination of apoptosis,
cells were either left untreated or were treated as indicated. Twenty-four hours after treatment, caspase-3/-7
activity was determined by using a kit from Promega (Mannheim, Germany) according to the
manufacturer’s instructions and as previously described (8,22). All assays were done in duplicates. Data are
expressed as n-fold treatment induced caspase-3/-7 activity (over basal) related to the cell number which
was determined in parallel.
Tumor model and therapy. All animal experiments have been approved by the local ministry and were
performed in the central animal facility of the University Hospital Schleswig-Holstein under pathogen-free
conditions. All experiments were assessed in four to six week old female SCID beige mice purchased from
Charles River Laboratories (Sulzfeld, Germany). For orthotopic cell inoculation, H6c7-mock, H6c7-
L1CAM, H6c7mono or H6c7co cells were cultured as described above, trypsinized and resuspended in 50 µl
Matrigel (BD Biosciences, Heidelberg, Germany) at a concentration of 2x106 cells. H6c7co cells that were
cocultured for 4 weeks in the presence of PMFs were inoculated together with 4x106 murine PMFs in 50 µl
Matrigel. All syringes containing Matrigel and cells were placed on ice until intra-pancreatic inoculation of
the mice (6-8 animals/group). For this purpose, animals were anesthetised and a median laparotomy was
performed. Then, cells were inoculated into the pancreatic body to the left of the organ midline. Tumor
growth was monitored as recently described (34) by high resolution ultrasound using a Vevo 770 small
animal micro-imaging system from VisualSonics (Toronto, Ontario, Canada).
To investigate whether tumor outgrowth of H6c7co cells can be inhibited by blockade of L1CAM, two
month after cell inoculation animals were randomized into two groups (n = 7) and treated either with 10
mg/kg body weight control IgG2a or L1-9.3/2a. All antibodies were diluted in 200 μL 0.9 % NaCl and intra-
peritoneally injected weekly for a time period of 3 months. In all experiments, the last ultrasound
examination was done after 5 months just before animals were sacrificed and pancreatic and liver tissues
were removed for final determination of the tumor size and preservation in liquid nitrogen.
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Immunohistochemistry. Six-µm thick consecutive cryostat sections were mounted on uncovered glass
slides, air-dried for at least 1 hour at room temperature (RT) and stored at -20°C. For staining procedure,
sections were dried again for 30 minutes (min) at RT, fixed in acetone (Merck, Darmstadt, Germany) for 10
min and air-dried again for 10 min. Then, slides were washed in PBS and incubated with 4 % bovine serum
albumine (BSA) in PBS (Serva, Heidelberg, Germany) for 20 min followed by incubation with the primary
antibody for 45 minutes at RT. Primary antibodies were diluted at a final concentration of 10 µg/mL
(L1CAM) or 2 µg/mL in PBS + 1 % BSA/PBS: mouse IgG1 anti-human L1CAM (clone UJ127; 35), mouse
IgG1 anti-human pan-Cytokeratin (DakoCytomation, Hamburg, Germany), mouse IgG1 anti-human
vimentin (Santra Cruz Biotechnology, Heidelberg, Germany), rabbit-anti-human E-Cadherin (Cell Signaling
via New England Biolabs, Frankfurt a.M., Germany). Primary antibodies were detected with EnVision
conjugated with peroxidase (DakoCytomation) for 30 min. The substrate reaction for peroxidase was
performed with the AEC substrate from DakoCytomation according to the instructions of the manufacturer.
Afterwards, sections were washed in water, counterstained in 50 % haemalaun (Merck) and mounted with
glycerol-gelatin (Merck). The same protocol was performed for negative controls in which an isotype-
matched control antibody was used, which revealed either no staining or only weak background staining.
Statistical analysis. Data are presented as mean ± standard deviation and were analyzed by student´s t-test.
A p-value < 0.05 was considered statistically significant.
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Results
H6c7 cells exhibiting elevated L1CAM expression and EMT-associated alterations during coculture
with PMFs show a decreased apoptosis sensitivity towards death ligands. Our previous results showed
that upon coculture with PMFs H6c7 cells acquire a migratory and chemoresistant phenotype (18) thus
pointing to the capability of PMFs to induce malignant transformation of H6c7 cells. In support of these
findings, H6c7 cells cocultured with PMFs for four weeks were characterized by EMT-associated
alterations. As shown in Figure 1A, H6c7 cells that were positively stained with a pan-cytokeratin antibody
indicating their epithelial origin, exhibited elevated expression levels of L1CAM when cocultured in the
presence of PMFs (H6c7co) in contrast to monocultured H6c7 cells (H6c7mono). Moreover, whereas
H6c7mono cells were characterized by a transmembraneous expression of E-cadherin and the absence of
vimentin, E-cadherin expression was lost in single H6c7co cells and numerous cells exhibited a strong
expression of vimentin (indicated by arrows). Coculture-mediated alterations of L1CAM, E-cadherin and
vimentin expression in H6c7co cells were confirmed by western blotting (Supplementary Figure 1). In
addition, numerous H6c7co cells already exhibited a spindle-like cell shape which is consistent with the
above mentioned alterations. To further elucidate this conversion process we investigated whether coculture
with PMFs also affects proliferation and death ligand-induced apoptosis. Whereas proliferation of H6c7co
cells was rather decelerated than accelerated upon coculture with PMFs (data not shown), the apoptotic
response towards death ligands was considerably altered. As shown in Figure 1B, H6c7mono cells showed a
clear apoptosis induction after stimulation with TRAIL (5.6-fold), TNF-α (2.6-fold) or the CD95 agonistic
antibody CH-11 (5.3-fold) as determined by caspase-3/-7 assay. In contrast, H6c7co cells became much less
apoptotic in response to these stimuli, in particular after treatment with TRAIL (1.9-fold) and CH-11 (2.2-
fold). Similar data were obtained upon coculture with human PMFs (Supplementary Figure 2). In order to
elucidate whether L1CAM might account for protection from death ligand-induced apoptosis, L1CAM
expression was suppressed by siRNA transfection in H6c7co cells during coculture and apoptosis induction
after treatment with TRAIL and CH11 was analyzed. As demonstrated in Figure 1C, caspase-3/-7 activity
was markedly increased in H6c7co cells after treatment with either apoptotic stimuli when L1CAM
expression was inhibited compared to control transfected cells. Overall these data demonstrate that H6c7
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cells undergo EMT-associated alterations in the presence of PMFs which is associated with an increased
expression of L1CAM and an L1CAM-dependent apoptosis resistant phenotype.
H6c7 cells exhibiting elevated L1CAM expression by stable transfection partially exhibit EMT-
associated alterations and apoptosis resistance towards death ligands.
To further confirm the importance of L1CAM in malignant transformation of H6c7 cells, stably L1CAM
overexpressing H6c7 cells were analyzed regarding EMT-associated alterations and death ligand-induced
apoptosis. Of the L1CAM transfectants (H6c7-L1CAM) exhibiting strong expression of L1CAM scattered
cells were characterized by an elongated cell shape and a loss of E-cadherin expression similarly to H6c7co
cells (Figure 1D). In contrast, H6c7-mock cells grown in a dense monolayer were all characterized by a
transmembraneous expression of E-cadherin and the absence of L1CAM (Figure 1D). In addition, the
number of vimentin expressing cells was slightly increased in the population of H6c7-L1CAM cells
compared to H6c7-mock cells albeit weaker than in H6c7co cells. These results were confirmed by western
blot analysis demonstrating strong L1CAM expression in H6c7-L1CAM cells. According to the small
differences seen in the staining, the expression of E-cadherin was slightly reduced and the expression of
vimentin was weakly increased in these cells compared to the mock-transfected cells (Supplementary Figure
3). As observed upon coculture with PMFs, L1CAM overexpressing H6c7 cells showed a reduced
sensitivity towards death ligand-induced apoptosis (Figure 1E). In particular, caspase-3-/-7 activity was
reduced after stimulation with TRAIL compared to control transfected cells (5,3-fold in mock versus 4,2-
fold in H6c7-L1CAM) and CH-11 (5,2-fold in H6c7-mock versus 3,1-fold in H6c7-L1CAM). Similar to the
observations upon coculture, L1CAM overexpression did not affect proliferation of H6c7-L1CAM cells
(data not shown). Overall, these data indicate that the presence of PMFs or elevated expression of L1CAM
confers a reduced apoptosis sensitivity towards death ligand-induced apoptosis in pancreatic ductal epithelial
cells.
PMFs increase tumorigenicity of H6c7 cells leading to tumor growth and metastasis in vivo. Since
PMFs induce EMT-associated alterations in H6c7 cells involving L1CAM dependent migration and
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aptoptosis resistance (see above and reference 18), we next analyzed whether the tumorigenic potential of
H6c7 cells is increased along with the malignant conversion of the cells. For this purpose, SCID beige mice
were orthotopically inoculated with either H6c7 cells alone (H6c7mono) or with a combination of PMFs and
cocultured H6c7cells (H6c7co). As shown in Table 1, H6c7mono cells were almost non-tumorigenic
confirming previous results (28), only in 2/6 animals tiny lesions of unknown specificity could be detected
by ultrasound examination in the pancreas. In contrast, 7/8 animals exhibited outgrowth of pancreatic tumors
ranging between 8-80 mm3 in size (mean ± SD: 28 ± 30.1 mm3) after inoculation of H6c7co cells. Although
no metastases were detectable in spleens, 6/8 animals inoculated with H6c7co cells exhibited liver
metastases as already detected by high-resolution ultrasound (Figure 2A) and confirmed by post-mortem
analysis (Table 1). As demonstrated by immunhistochemical stainings with human-specific antibodies,
H6c7co tumors were largely composed of cytokeratin expressing cells confirming their human and epithelial
origin thus indicating that tumors are mainly composed of H6c7 cells (Figure 2B). Moreover, the majority of
these H6c7 cells exhibited an elongated cell shape and a strong L1CAM expression. Important to note, E-
cadherin expression was less prominent in the cells than cytokeratin or L1CAM indicating the loss E-
cadherin expression in tumoral H6c7cells. Vimentin could be detected in numerous cells within the primary
tumor albeit not in the frequency of cytokeratin and L1CAM (Figure 2B). Pan-cytokeratin staining of livers
revealed that most cells within metastases were indeed H6c7 cells partially expressing L1CAM.
Interestingly, all metastasized H6c7 cells expressed E-cadherin while vimentin expression was
predominantly detectable in cells at the margins of the metastases (Figure 2C). These data suggest that H6c7
cells become tumorigenic in the presence of PMFs leading to tumor formation and metastasis in vivo.
L1CAM overexpressing H6c7 cells become tumorigenic in vivo. In order to elucidate whether tumor
formation of H6c7 cells depends on L1CAM expression, H6c7 cells stably overexpressing L1CAM (H6c7-
L1CAM) or the respective control transfectants (H6c7-mock) were orthotopically inoculated in SCID beige
mice. Similarly to H6c7co cells, inoculation of H6c7-L1CAM cells (Table 2) led to pancreatic tumor
formation detectable in 71 % of the animals (5/7). In contrast, H6c7mock cells were almost non-tumorigenic
(Table 2) besides one animal bearing a tiny pancreatic lesion of unknown specificity. H6c7-L1CAM tumor
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sizes ranged between 4-15 mm3 (mean ± SD: 6.7 ± 5.6 mm3) (Table 2). Thus, H6c7-L1CAM tumors were
much smaller than those arising from inoculation of H6c7co cells and PMFs but could be detected by high-
resolution ultrasound examination (Figure 3A). Inoculation of H6c7-L1CAM cells resulted in liver
metastases in 3/7 animals (Table 2) whereas only in 1/6 animals inoculated with H6c7-mock cells a liver
metastasis was found. The metastases were not detected by high-resolution ultrasound most probably due to
difficult scanning conditions because large parts of the liver are located underneath the rib cage but later on
after removal of the liver. Overall, these findings point to a role of L1CAM in increasing the tumorigenicity
and to a lesser extent the metastatic potential of human pancreatic ductal epithelial cells.
Treatment with L1CAM-antibodies inhibits tumor growth and metastasis of tumorigenic H6c7 cells.
To support the idea that L1CAM expression confers an increased tumorigenic phenotype of H6c7 cells,
H6c7co tumor-bearing animals were treated with the L1CAM-blocking antibody L1-9.3/2a or a control
antibody. Antibody treatment started two months after cell inoculation when small tumors were detectable
by ultrasound-examination. One animal treated with the L1-9.3/2a antibody for 9 weeks died due to
intestinal obstruction but exhibited neither a pancreatic tumor nor liver metastases. As seen in Table 3 and
Figure 3B, weekly administration of L1CAM-blocking antibodies over a period of three months resulted in a
complete remission of H6c7co tumor growth and reduced formation of liver metastases in 50 % of the
animals (Table 3). In contrast, 6/7 H6c7co tumor-bearing mice that were treated with the control antibody
exhibited formation of pancreatic tumors ranging between 6-468 mm3 in size (mean ± SD: 112.6 ± 180.4
mm3) (Table 3). In one animal, the pancreatic lesion was too small for further analysis and was therefore
excluded from the statistics. Metastases in the liver were not detectable by high-resolution ultrasound but
later on during the post-mortem analysis in 5/7 animals (Table 3) underscoring our previous results (Table 1,
Figure 2). Together with our findings from the H6c7-L1CAM cells, these data confirm that tumor growth
and metastasis of pancreatic ductal epithelial cells depends on L1CAM expression and that L1CAM-
blocking antibodies are an appropriate tool for anti-cancer therapy of pancreatic tumors.
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Discussion
To date, PDAC is mostly diagnozed in an advanced stage limiting curative therapeutic approaches and
PDAC patient’s prognosis. Thus, an improved understanding of the early steps of pancreatic tumorigenesis
is pivotal to improve diagnostic and therapeutic measures.
The substantial impact of PMFs/CAFs on processes involved in PDAC development such as EMT, cell
migration and chemoresistance has been demonstrated by us and other groups (5,6,8,15,16). In accordance,
expression of the PMF marker protein alpha-smooth muscle actin is highly elevated in the stroma of PDAC
tissues correlating with short survival of the patients (36). We recently showed that H6c7 cells cocultured
with PMFs acquire a migratory and chemoresistant phenotype through PMF-induced L1CAM expression.
Accordingly, elevated L1CAM expression was detected in carcinoma cells in PDAC tissues but also in
ductal structures in stroma-enriched chronic pancreatitis indicating that PMFs might be involved in
malignant transformation of pancreatic ductal epithelial cells (18). We are now showing that H6c7 cells
become tumorigenic in the presence of PMFs resulting in the outgrowth of pancreatic tumors and liver
metastases whereas H6c7 cells alone were non-tumorigenic confirming the results by Furukawa et al. (28).
H6c7co tumors were strongly stained by a human pan-cytokeratin antibody indicating that they are largely
composed of human cells of epithelial origin. In search for the mechanisms leading to pancreatic
tumorigenesis, mutational activation of the k-ras gene has gained much attention since mutant k-ras is
present already in early PanINs and later on in 90% of PDAC (2,4). Hence, Qian et al. could demonstrate
that expression of mutant k-ras in H6c7 cells that per se exhibit none of the mutations commonly detected in
PDAC (27,28) induced tumor formation in 50 % of subcutaneously inoculated SCID mice (37). Similar
results have been demonstrated in rat models (38,39). Moreover, development of human PDAC was
recapitulated in different transgenic mouse models e.g. by expression of endogenous k-rasG12D induced
during early stages of embryonic pancreatic development in the mouse model of Hingorani et al. (40).
However, expression of k-rasG12D in adult mice more closely reflecting the situation in humans did not result
in PanIN formation and invasive PDAC unless a mild chronic pancreatitis was induced (9). Altogether these
data suggest that genetic alterations alone do not necessarily lead to PDAC development and that context-
derived factors such as stromal cells and their secreted factors are likewise important. This is supported by
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our own findings showing that already an indirect coculture with PFMs leads to EMT in H6c7 and PDAC
cells along with a migratory and apoptosis resistant phenotype which highly depends on humoral factos such
as TGF-β1, IL-1β and nitric oxide (NO) (8,18,30). Moreover, all these factors have been described as
potential inducers of L1CAM (18,22). The observation that elevated L1CAM expression in H6c7 cells
essentially contributes to the PMF-induced migratory and apoptosis resistant phenotype in vitro points to an
important role of this molecule in the malignant transformation of pancreatic ductal epithelial cells.
In line with this, H6c7 cells in H6c7co tumors exhibited a high L1CAM expression along with a marked
expression of vimentin and a reduced expression of E-Cadherin in numerous cells pointing to a continuation
of EMT in H6c7co tumors. Interestingly, the majority of H6c7 cells in PDAC liver metastases expressed E-
cadherin whilst vimentin expression was mainly detected in cells located at the margins of the metastases.
Similarly, E-cadherin expression in metastases but not in primary tumors has been reported by various
groups (41-43). Overall, these data are consistent with the idea of a mesenchymal-epithelial transition by
which metastasized tumor cells re-express E-cadherin to seed in the new tissue and to establish metastatic
foci (44).
Gavert et al. already demonstrated that subcutaneous inoculation of L1CAM overexpressing NIH3T3 cells
into nude mice results into formation of larger tumors than inoculation of control transfected cells (20). Our
study shows that stable overexpression of L1CAM led to tumor formation of H6c7 cells in 5/7 animals,
similarly seen after coinoculation with PMFs, whereas no pancreatic tumors developed after inoculation of
the control transfectants. However, H6c7-L1CAM tumors were much smaller in size and the incidence of
metastases in the liver was lower compared to tumors and metastases in animals after incoculation of
H6c7co cells. The observation that inoculation of H6c7-mock cells resulted in the formation of metastases at
least in one animal might be explained by the fact that these cells had undergone a double retroviral
transduction procedure (1st time for immortalization and 2nd time for stable transfection with the control
vector for L1CAM) increasing the tumorigenic potential of the cells. Overall, these data indicate that
expression of L1CAM substantially increases the tumorigenicity of H6c7 cells but is not sufficient to start
metastasis in full extent as occurring under the influence of PMFs. Supporting this idea, expression of
vimentin was much less pronounced in H6c7-L1CAM cells compared to H6c7-mock cells. Gavert et al.
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recently showed that L1CAM confers a motile phenotype in colon cancer cells resulting in metastasis to the
liver but without inducing EMT and stem cell properties (45,46). These results suggest that the impact of
L1CAM on metastasis is i) either more prominent in the tumorigenesis of the colon than of the pancreas or
ii) is more pronounced in fully transformed cancer cells than in epithelial cells where transformation still
commences. Accordingly, treatment of H6c7co tumor-bearing mice with L1CAM-blocking antibodies
completely abolished formation of primary tumors but reduced liver metastasis only in 50 % of the animals.
Thus, L1CAM seemed to be sufficient for initial tumor formation and growth of pancreatic ductal epithelial
cells but the process of metastasis apparently requires additional alterations and/or signals which are
provided e.g. by the stromal microenvironment. Stromal cells such as PMFs (and later on CAFs) are known
to promote cell migration and metastasis by producing a plethora of cytokines, growth factors and proteases
which enhance the migratory potential of epithelial/carcinoma cells and help to digest the extracellular
matrix (6,8,16,47).
Bao et al. reported elevated L1CAM expression on CD133+ cancer stem cells in gliomas and targeting of
L1CAM in these cells significantly reduced tumor outgrowth in immunocompromised mice (48) supporting
the view that L1CAM is able to confer a tumorigenic phenotype. Moreover, this group showed that L1CAM
is essentially involved in the regulation of DNA damage checkpoint responses thereby determining
radiosensitivity of glioblastoma stem cells (49). Under physiological conditions, these checkpoints are
tightly regulated and exert cytoprotective functions to promote cell survival (50). However, dysregulation of
these checkpoints e.g. as a result of sustained L1CAM expression might promote cell survival of damaged
cells that actually would have been eliminated in order to prevent proliferation of genetically altered cells
and tumorigenesis. Besides enhanced DNA repair, the protection from apoptosis induction might be another
mechanism by which cells harbouring DNA mutations can survive and expand. Accordingly, we could
demonstrate that H6c7 cells either cocultured in the presence of PFMs or stably transduced with
recombinant L1CAM acquired a resistant phenotype towards death ligand-induced apoptosis in particular
after stimulation of CD95 and TRAIL. To underscore the importance of L1CAM in apoptosis resistance of
tumor cells and the suitability of L1CAM antibodies as therapeutic tool, a recent study demonstrated that
L1CAM-dependent chemoresistance can be overcome by antibody-mediated blockade of L1CAM in PDAC
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and ovarian carcinoma cells in vivo resulting in significantly reduced tumor growth (unpublished
observations).
In summary, the present study demonstrates that the tumorigenic potential of pancreatic ductal epithelial
cells is highly increased in the presence of PMFs promoting tumor growth and metastasis. Elevated
expression of L1CAM plays an essential role in this scenario probably by conferring a profound apoptosis
protection allowing survival and expansion of genetically altered cells. Hence, L1CAM expression in ductal
structures of chronic pancreatitis as reported previously (18) has to be seriously regarded as first steps of
tumorigenesis.
Funding
This work was supported by the German Research Society DFG [SE-1831/2-1 to S.S.] and the German
Cluster of Excellence “Inflammation at Interfaces”.
Acknowledgements
The authors thank Dagmar Leisner and Maike Witt-Ramdohr for excellent technical assistance.
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Figure legends
Fig. 1: H6c7 cells exhibiting elevated L1CAM expression and EMT-associated alterations show a
decreased apoptosis sensitivity towards death ligands. (A-C) H6c7 cells were cultured in the absence
(H6c7mono) or presence of PMFs for 4 weeks (H6c7co) or (D,E) were stably transfected with a control vector
(H6c7-mock) or a vector encoding for L1CAM (H6c7-L1CAM) (A,D) Representative images of the staining of
cytokeratin, L1CAM, E-cadherin and vimentin in (A) H6c7mono and H6c7co cells as well as in (D) H6c7-mock
and H6c7-L1CAM cells. Scale bar = 20 µm. Arrows indicate E-cadherin negative and vimentin positive cells,
respectively. (B) H6c7mono and H6c7co cells as well as in (E) H6c7-mock and H6c7-L1CAM were either left
untreated or treated with TRAIL, TNF-α or the CD95 agonistic antibody CH-11. (C) H6c7co cells were treated
with control siRNA or L1CAM siRNA for 48 hours. Then, cells were either left untreated or treated with TRAIL
or CH-11. (B,C,E) After 24 hours, cells were analyzed for caspase-3/-7 activity. Means ±SD of treatment
induced caspase-3/-7 activity (n-fold of basal) from four to six independent experiments are shown. * p<0.05.
Fig. 2: PMFs increase tumorigenicity of H6c7 cells leading to tumor growth and metastasis in vivo. SCID
mice were intra-pancreatically inoculated with H6c7 cells alone (H6c7mono) or with H6c7 cells that were
cocultured for 4 weeks and inoculated with PMFs (H6c7co). (A) Representative ultrasound images of the
pancreatic area are shown from H6c7mono inoculated animals exhibiting no tumor (A1) or a tumor (A2) as well
as liver metastases (A3) of H6c7co inoculated animals. The arrows indicate the primary tumor and metastases.
Black areas within the tumor are fluid filled cysts. (B+C) Representative images from immunohistochemical
stainings with either control IgG1 or an antibody detecting cytokeratin, L1CAM, E-cadherin or vimentin in
tumor (B) and liver (C) sections of an H6c7co inoculated animal. The frame in the left pictures indicates the area
shown in pictures with higher magnification (right). Scale bar = 20 µm.
Fig. 3: L1CAM overexpressing H6c7 cells become tumorigenic in vivo and treatment with L1CAM-
antibodies inhibits tumor growth and metastasis of tumorigenic H6c7 cells (A) SCID mice were
orthotopically inoculated with H6c7-mock or H6c7-L1CAM cells. Representative ultrasound images of the
pancreatic area are shown from (A1) H6c7-mock inoculated animals exhibiting no primary tumor and from
(A2) H6c7-L1CAM inoculated animals exhibiting pancreatic tumor growth. (B) SCID mice were
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orthotopically inoculated with H6c7 cells that were cocultured for 4 weeks and inoculated with PMFs
(H6c7co). Two months after inoculation, animals were randomized and weekly treated with 10 mg/kg body
weight control IgG2a or the L1CAM-antibody L1-9.3/2a. Representative ultrasound images of the
pancreatic area are shown from (B1) control IgG1 treated animals exhibiting tumor formation and from (B2)
L1-9.3/2a treated animals exhibiting no pancreatic tumor growth (w/o). The arrow indicates fluid filled cysts
within primary tumors (black areas).
Supplementary Fig. 1: H6c7 cells cocultured with PMFs exhibit elevated L1CAM expression and
EMT-associated alterations. H6c7 cells were cultured in the absence (H6c7mono) or presence of PMFs for
4 weeks (H6c7co). Whole cell lysates were analysed by western blotting for the expression of L1CAM, E-
cadherin and vimentin. HSP90 was analysed as loading control. One representative western blot is shown.
Supplementary Fig. 2: H6c7 cells cocultured with human PMFs show a decreased apoptosis sensitivity
towards death ligands. H6c7 cells were cultured in the absence (H6c7mono) or presence of human PMFs
(hPMFs) for 2 weeks (H6c7co). H6c7mono and H6c7co cells were either left untreated or treated with
TRAIL, TNF-α or the CD95 agonistic antibody CH-11. After 24 hours, cells were analysed for caspase-3/-7
activity. Means ±SD of treatment induced caspase-3/-7 activity (n-fold of basal) from three independent
experiments are shown. * p<0.05.
Supplementary Fig. 3: H6c7-L1CAM cells exhibit EMT-associated alterations. Whole cell lysates from
H6c7-mock and H6c7-L1CAM cells were analysed by western blotting for the expression of L1CAM, E-
cadherin and vimentin. HSP90 was analysed as loading control. One representative western blot is shown.
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Table 1: PMFs increase tumorigenicity of H6c7 cells leading to tumor growth and
metastasis in vivo. SCID mice were orthotopically inoculated with H6c7 cells alone
(H6c7mono) or with H6c7 cells that were cocultured for 4 weeks and inoculated with PMFs
(H6c7co). (A) Number of animals exhibiting primary tumors and liver metastases. (B)
Volumes (in mm3) of resected pancreatic tumors from H6c7mono and H6c7co inoculated
animals. * indicates lesion detected only by high-resolution ultrasound.
A
no. of animals H6c7mono (% of animals) H6c7co (% of animals)
primary tumor 0/6 (0%) 7/8 (87,5%)
liver metastases 0/6 (0%) 6/8 (75 %)
B
Tumor H6c7mono H6c7co
1 0 80
2 0 8
3 0 48
4 0 12
5 2* 0
6 2* 8
7 0 60
8 - 8
Mean 0,57 28,00
SD 0,98 31,39
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Table 2: L1CAM overexpressing H6c7 cells become tumorigenic in vivo. SCID mice were
intra-pancreatically inoculated with H6c7-mock or H6c7-L1CAM cells. (A) Number of
animals exhibiting primary tumors and liver metastases. (B) Volumes (in mm3) of resected
tumors from H6c7-mock and H6c7-L1CAM inoculated animals. * indicates lesion detected
only by high-resolution ultrasound.
A
no. of animals H6c7-mock (% of animals) H6c7-L1CAM (% of animals)
primary tumor 0/7 (0%) 5/7 (71,4 %)
liver metastases 1/7 (14,3%) 3/7 (42,9 %)
B
Tumor H6c7-mock H6c7-L1CAM
1 0 12
2 0 4
3 0 9
4 1* 15
5 0 6
6 0 0
7 0 1*
Mean 0,14 6,71
SD 0,37 5,58
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Table 3: Treatment with L1CAM-antibodies inhibits tumor growth and metastasis of
tumorigenic H6c7 cells. SCID mice were intra-pancreatically inoculated with H6c7 cells that
were cocultured for 4 weeks and inoculated with PMFs (H6c7co). Two months after
inoculation, animals were randomized and weekly treated with 10 mg/kg body weight control
IgG2a or the L1CAM-antibody L1-9.3/2a. (A) Number of animals exhibiting primary tumors
and liver metastases. (B) Volumes (in mm3) of resected tumors from IgG2a and L1-9.2/2a
treated animals.
A
no. of animals H6c7co + IgG2a
(% of animals)
H6c7co + L1-9.3/2a
(% of animals)
primary tumor 6/7 (85,7 %) 0/6 (0%)
liver metastases 5/7 (71,4 %) 3/6 (50%)
B
Tumor H6c7co + IgG2a H6c7co + L1-9.3/2a
1 468 0
2 12 0
3 252 0
4 30 0
5 19 0
6 1 0
7 6 -
Mean 112,57 0
SD 180,39 0
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