This study found that transforming growth factor beta 1 (TGF-β1) induces desmoplasia, the proliferation of fibrotic tissue, in experimental models of human pancreatic carcinoma. The researchers transfected pancreatic tumor cells (PANC-1) that do not normally induce desmoplasia or express TGF-β1 with the TGF-β1 gene. They found that the TGF-β1-transfected cells gained the ability to induce fibroblast growth in culture and in vivo in nude mice. This effect was due both to the direct effects of TGF-β1 and its upregulation of other growth factors and matrix proteins. Therefore, TGF-β1 plays an important role in the desm

1. [CANCER RESEARCH 61, 550–555, January 15, 2001]
Transforming Growth Factor-␤1 Induces Desmoplasia in an Experimental
Model of Human Pancreatic Carcinoma1
Matthias Lo¨hr,2
Christian Schmidt, Jo¨rg Ringel, Mario Kluth, Petra Mu¨ller, Horst Nizze, and Ralf Jesnowski
Division of Gastroenterology, Departments of Medicine [M. L., C. S., J. R., M. K., P. M., R. J.] and Pathology [H. N.], University of Rostock, D-18055 Rostock, Germany
ABSTRACT
Proliferation of fibrotic tissue (desmoplasia) is one of the hallmarks of
several epithelial tumors including pancreatic adenocarcinoma. This tis-
sue reaction may be deleterious or advantageous to the host or tumor. In
a systematic analysis, we identified two growth factors expressed by
human pancreatic carcinoma cells that are positively correlated with the
ability to induce fibroblast proliferation both in vitro and in vivo, i.e.,
transforming growth factor (TGF)-␤1 and fibroblast growth factor-2.
Here we demonstrate that the overexpression of TGF-␤1 induced up-
regulation of matrix proteins and growth factors in the TGF␤1-trans-
fected pancreatic tumor cells. Furthermore, transfection of PANC-1 cells
induces the same change in fibroblasts in either cocultivation experiments
or when they are grown in conditioned medium from TGF-␤1-transfected
PANC-1 cells. TGF-␤1-transfected pancreatic tumor cells induced a rich
stroma after orthotopical transplantation in the nude mouse pancreas.
The transfer of a single growth factor, TGF-␤1, conveys the ability to
induce a fibroblast response similar to that seen in desmoplasia in human
pancreatic adenocarcinoma. This effect cannot only be attributed to direct
effects of TGF-␤1 but also results from the up-regulation of several other
factors including collagen type I, connective tissue growth factor, and
platelet-derived growth factor.
INTRODUCTION
Desmoplasia is a characteristic feature of the growth of some
carcinomas (1). To date, it is not clear whether this process is a
mechanism to protect the tumor from the host or represents a defense
mechanism by the host (2), although there are hints that this stroma is
beneficial for the tumor (3). To tackle desmoplasia therapeutically by
either supporting or suppressing this development, it becomes neces-
sary to study the etiology and to attribute this feature to either the
tumor cells themselves or the host. Desmoplasia is of particular
predominance in ductal adenocarcinomas of the pancreas exhibiting a
strong stromal reaction (4). Therefore, pancreatic carcinoma has be-
come a model system to study the interrelation of epithelial tumor
cells, matrices, fibroblasts, and growth factors (5–8).
Desmoplastic tissue consists of fibroblasts, as the main cellular
component, and extracellular matrix proteins (9). The pancreatic tu-
mor cells themselves are able to produce ECM3
proteins (10–13) and
interact with ECM by expressing functionally active integrins (6, 14, 15).
To test the hypothesis of desmoplasia induction by a tumor-derived
growth factor, we conducted a deductive analysis correlating the
ability to induce desmoplasia with the expression of certain growth
factors. Furthermore, we reasoned that the overexpression of such a
growth factor, e.g., TGF-␤1 in a pancreatic tumor cell line known
neither to induce desmoplasia nor to express substantial amounts of
TGF-␤1 and FGF-2, should result in the gain of the ability to induce
fibroblast growth and in an induction of desmoplasia in a xenografted
nude mouse model by virtue of direct and indirect effects of TGF-␤1.
MATERIALS AND METHODS
Cell Culture and Transfection. AsPC-1, BxPC-3, Capan-1, and PANC-1
cells, all from American Type Culture Collection, were cultivated in DMEM
with GlutaMAX I (Life Technologies, Inc.) supplemented with 10% heat-
inactivated FCS and antibiotics (100 units/ml penicillin, 100 ␮g/ml strepto-
mycinsulfate, and 250 ng/ml amphotericin B; Life Technologies, Inc.; Ref. 10).
Mature human recombinant TGF-␤1 was purchased from R&D Systems.
Full-length cDNA of TGF-␤1(16) was cut out of pRK5␤1E (BamHI) and
cloned into the pcDNA3 vector (Invitrogen) under the control of a cytomeg-
alovirus promoter. PANC-1 cells were transfected with this construct or with
the empty pcDNA3 plasmid (mock) by calcium phosphate coprecipitation in
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffered saline using
standard protocols as described (17). This plasmid also codes for the neo
resistance gene, enabling selection of transfectants with the antibiotic G418
(Sigma; 400 ␮g/ml). Resistant clones were expanded, and expression of the
transfected cDNA was confirmed by Northern blot, Western blot, and ELISA
(R&D).
Northern Blot and RT-PCR. Subconfluent layers of PANC-1/TGF-␤1
cells, mock transfected, untransfected PANC-1 cells, and AsPC-1 and BxPC-3
cells were lysed in ice-cold guanidine thiocyanate. RNA preparation was
performed as described (18). Ten ␮g of total RNA were subjected to standard
formamide gel electrophoresis as described. Gels were blotted to nylon mem-
branes (Qiagen) and hybridized with cDNA probes for TGF-␤1 (EcoRI/
HindIII digest of pcDNA3/TGF-␤1), type I collagen (pHCAL1U; Refs. 10 and
19), PDGF (Amersham), FGF-2,(20), and CTGF (21) using the nonradioactive
Dig labeling kit (Boehringer Mannheim, Mannheim, Germany). In addition,
RT-PCR was performed using published primers for TGF-␤1, PDGF-A, type
I collagen, and GAPDH. The primers were as follows: TGF-␤1 (22), sense
5Ј-CAG AAA TAC AGC AAC AAT TCC TGG-3Ј and antisense 5Ј-TTG
CAG TGT GTT ATC CCT GCT GTC-3Ј (190-bp product); PDGF-A (23),
sense 5ЈCAG TCA GAT CCA CAG CAT CC-3Ј and antisense 5Ј-AAT GAC
CGT CCT GGT CTT GC-3Ј (200-bp product); collagen type I (23), sense
5Ј-ACG TGA TCT GTG ACG AGA CC-3Ј and antisense 5Ј-AGC AAA GTT
TCC TCC GAG GC-3Ј (250-bp product); and GAPDH (24), sense 5Ј-ACC
ACA GTC CAT GCC ATC AC-3Ј and antisense 5Ј-TCC ACC ACC CTG
TTG CTG TA-3Ј (450-bp product). PCR conditions were the following:
denaturing for 30 s at 94°C; annealing for 60 s at 60°C (TGF-␤1) or at 64°C
(collagen, GAPDH, and PDGF); and extension for 60 s at 72°C. Amplified
DNA was sampled after 21, 24, 27, and 30 cycles, and the resulting PCR
products for TGF-␤1, collagen, and PDGF-A were loaded in the same gel
pockets as the GAPDH amplificate.
Reverse Slot Blot. Expression of genes of several growth factors, recep-
tors, and genes of ECM proteins was investigated by reverse slot blot. For this
purpose, plasmid DNA corresponding to 1 ␮g of cDNA insert was blotted onto
a nylon membrane (Qiagen) by use of a slot blot apparatus (Schleicher &
Schuell). Hybridization was performed according to standard procedures with
a probe obtained by Dig labeling (Boehringer Mannheim) of 7.5 ␮g of total
RNA in a reverse transcription reaction (25, 26). Hybrids were detected using
the chemiluminescent Dig detection system (Boehringer Mannheim) according
to the manufacturer’s instructions.
Cocultivation. PANC-1/TGF␤1 cells (5 ϫ 104
) were seeded onto Tran-
swell inserts (Costar) and were cocultivated with fibroblasts (5 ϫ 104
cells/
Received 1/28/00; accepted 11/14/00.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by Grant Lo 431/6 from the Deutsche Forschungsgemeinschaft as part of
the special topic program “Matrix in Biology and Medicine” (to M. L.). C. S. acknowl-
edges the support of the Bundesministerium fu¨r Bildung und Forschung.
2
To whom requests for reprints should be addressed, at Sektion Molekulare Gastro-
enterologie, Medizinische Klinik IV, Fakulta¨t fu¨r Klinische Medizin Mannheim, Univer-
sita¨t Heidelberg, Theodor Kutzer Ufer 1-3, D-68135 Mannheim, Germany. Phone: 49-
621-383-2900; Fax: 49-381-383-1986; E-mail: matthias.loehr@med4.ma.uni-heidelberg.de.
3
The abbreviations used are: ECM, extracellular matrix; TGF, transforming growth
factor; RT-PCR, reverse transcription-PCR; PDGF, platelet-derived growth factor; CTGF,
connective tissue growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
Dig, digoxygenin; FGF, fibroblast growth factor; Erk, extracellular signal-regulated
kinase.
550

2. well) seeded in six-well tissue culture plates. After 7 days of incubation in
DMEM/1% FCS, the cells were trypsinized and counted (trypan blue exclusion
test). Controls were cocultivated of fibroblasts with fibroblasts, PANC-1,
PANC-1 mock transfected, and BxPC-1 cells (American Type Culture Col-
lection). From respective parallel experiments, conditioned media (see below)
and RNA (see above) were prepared for subsequent analysis.
Conditioned Media. PANC-1/TGF␤1 cells were seeded in DMEM/10%
FCS. After 2 days of incubation, cells were washed three times with PBS (pH
Fig. 1. Desmoplastic potential of several human
pancreatic adenocarcinoma cell lines upon xeno-
transplantation on nude mice. Top, tissue culture.
Bottom, tumors established on nude mice. Left to
right: Panc-1, PaCa-44, Capan-1, and BxPC-3. The
two cell lines to the right develop a stroma on the
nude mouse. H&E stain, ϫ250.
Fig. 2. A, Northern blot of RNA from native Panc-1 cells (Lanes
1 and 2), TGF-␤1-transfected Panc-1 cells (Lanes 3 and 4), mock
transfected Panc-1 cells (Lanes 5 and 6), and fibroblasts (Lane 7) for
TGF-␤1 (top) and GAPDH (bottom). Lanes 1, 3, and 5, with FCS;
Lanes 2, 4, and 6, without FCS. B, Northern blot of Panc-1/TGF-␤1
and Panc-1 for collagen I and ethidium bromide gel (top). Northern
blot of Panc-1 ϩ/Ϫ FCS (1 ϩ 2); Panc-1/TGF␤1 ϩ/Ϫ FCS (2 ϩ 3);
Panc-1-mock ϩ/Ϫ FCS (5 ϩ 6) and fibroblasts for FGF-2 (bottom).
C, reverse slot blot with different cDNA probes hybridized with
Dig-labeled cDNA from Panc-1 mock and Panc-1/TGF-␤1.
551
TGF-〉1 INDUCES DESMOPLASIA IN PANCREATIC CARCINOMA

3. 7.4) to remove any FCS traces and refed with fresh medium containing no
FCS. After incubation for another 2 days, the supernatants were collected and
filter sterilized. Skin fibroblasts were incubated with serial dilutions of this
concentrated medium for 2 days, and induction of proliferation was investi-
gated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test
(Boehringer Mannheim). Controls included conditioned media of PANC-1 and
mock-transfected PANC-1 cells.
ELISA. 1.3 ϫ 104
cells of TGF-␤1-transfected and mock-transfected
PANC-1 were plated in six-well plates with DMEM and 10% FCS. After 2
days, cells were grown with DMEM without FCS (transfected cells all of the
time with 400 ␮g/ml G418) for 1, 2, or 3 days, after that the supernatant was
collected. TGF-␤1 and PDGF were quantified using the Quantikine TGF-␤1
and PDGF immunoassays (R&D) according to the instructions of the manu-
facturer.
Western Blot. Proteins were separated by SDS-PAGE, transferred to a
polyvinylidene difluoride membrane (Roche), and blocked for 1 h in Tris-
buffered saline (TBS; 10 mM Tris, 10 mM NaCl) containing 1% skim milk and
0.01% Tween 20. After incubation with the primary antibody for 1 h, blots
were developed using alkaline phosphatase-labeled secondary antibodies and
chemiluminescence (CDP-star; Roche). The following primary antibodies
were used in a dilution of 1:1000: PCNA (Santa Cruz; sc-56), TGF-␤-1
(sc-146), p21wafI
(sc-6246), p-Tyr (sc-7020), Erk 1 (sc-94-G), Erk 2 (sc-1647),
and Erk 3 (sc-6268). As detection antibodies, mouse-antigoat immunoglobulin
(Dako; 1:5000), rabbit-antimouse immunoglobulin (Dako; 1:5000), and swine-
antirabbit immunoglobulin-AP (Dako, 1:5000) were used (27).
Nuclear Extracts. Cells were scraped, washed with Tris-buffered saline,
resuspended in hypotonic buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2,
and 0.5 mM EDTA), and allowed to swell on ice for 20 min. The nuclei were
collected by centrifugation at 12,000 ϫ g for 5 min in a microcentrifuge and
analyzed by Western blotting (28).
Nude Mouse Model. A suspension of 1 ϫ 106
PANC-1/TGF␤1 cells or
mock-transfected PANC-1 cells were injected orthotopically into athymic nude
mice (29, 30). Nude mice were killed after solid tumors were palpable. Tumors
were removed, fixed in 4% formaldehyde, and examined after H&E or Mas-
son-Goldner trichrome staining. Immunocytochemistry was performed as de-
scribed before with antibodies against type I collagen (1:100; Calbiochem) and
fibronectin (1:400; Sigma; Ref. 10). Detection was performed using horserad-
ish peroxidase-conjugated rabbit-antimouse and swine-antirabbit IgGs (Dako
Diagnostika) as secondary and third antibodies and 3-amino-9-ethylcarbazole
as substrate.
RESULTS
Induction of Desmoplasia Is Associated with the Expression of
TGF-␤1 and FGF-2. In a deductive analysis on human pancreatic
carcinoma cells in vitro and in vivo (6, 31), using all of the published
information on the expression of various growth factors described in
pancreatic carcinoma (22, 32–42), the stromal reaction (Fig. 1) was
found to be positively correlated with the expression of TGF-␤1
and/or FGF-2 (Ref. 31; Table 1). We therefore chose PANC-1 cells
that did not express significant amounts of TGF-␤1 as a model for the
subsequent experiments investigating the role of TGF-␤1 in desmo-
plasia.
Stable Expression of Functional TGF-␤1 in PANC-1 Induces
Up-Regulation of Matrix Proteins and Growth Factors. Expres-
sion of the transfected TGF-␤1 cDNA in PANC-1/TGF-␤1 cells was
verified by Northern and Western blots (Figs. 2A and 3A). The
TGF-␤1 protein was released into the culture medium as demon-
strated by ELISA of serum-free supernatants; it was native, i.e.,
inactive, and had to be activated by acidification before quantification.
In the TGF-␤1-transfected PANC-1 cells, the expression of colla-
gen type I was increased (Fig. 2B). Also, PDGF-A was increased (Fig.
2C), whereas the expression of FGF-2 (data not shown), epidermal
growth factor, and the ␣5 integrin subunit (Fig. 2C) was similar in
TGF-␤1-transfected and mock-transfected PANC-1 cells by Northern
blot or reverse slot blot.
TGF-␤1 inhibits growth by acting on the cell cycle by modu-
lating, for example, p21wafI
and PCNA. TGF-␤1-transfected
PANC-1 cells exhibited a substantial increase in p21wafI
expres-
sion on the protein level on Western blot of nuclear extracts (Fig.
3B); on ELISA, p21wafI
was 5.4 units/mg protein in untransfected
and 16.3 units/mg protein in transfected cells. Conclusively, the
transfected cells demonstrated decreased nuclear levels of PCNA
on the protein level (Fig. 3B).
TGF-␤1-transfected PANC-1 Cells Induce Fibroblast Growth
and Up-Regulation of Matrix Proteins and TGF-␤1 in Fibro-
blasts. Cocultivation of fibroblasts with PANC-1/TGF-␤1 cells in
the Transwell system led to an increase in proliferation of both the
Table 1 Ability to induce desmoplasia as assessed by induction of stromal tissue upon
xenotransplantation in nude mice in human pancreatic carcinoma cell lines
Cell line Desmoplasiaa
EGFb
FGF-1b
FGF-2b
FGF-3b
TGF-␤1b
AsPC-2 ϩ (ϩ) Ϫ (ϩ) (ϩ) (ϩ)
BxPC-3 ϩ ϩϩ ϩϩ (ϩ) (ϩ) ϩ
Capan-1 ϩϩ Ϫ ϩϩϩc
ϩϩc
(ϩ) ϩϩ
Capan-2 ϩϩ Ϫ Ϫ ϩϩϩc
(ϩ) ϩϩ
PaCa-2 Ϫ Ϫ ϩc
(ϩ) (ϩ) ϩ
PaCa-3 Ϫ ϩ Ϫ Ϫ (ϩ) ϩ
PaCa-44 Ϫ Ϫ (ϩ) (ϩ)c
(ϩ) ϩ
PANC-1 Ϫ Ϫ (ϩ) ϩ (ϩ) ϩc
a
For example, see Fig. 1.
b
Assessment of growth factor expression on the RNA and protein levels as published
(33, 35, 38, 40, 41) and confirmed by us (52).
c
Only positive if cultivated in FCS-depleted medium.
EGF, epidermal growth factor.
Fig. 3. A, Western blot of whole-cell lysates from mock-transfected Panc-1 cells (Lane
1) and TGF-␤1-transfected Panc-1 cells (Lane 2) incubated with antibodies against
TGF-␤1 (top) and cytokeratin 19 (bottom). B, total cell lysates of pancreatic carcinoma
cell lines Panc-1 (Lane 1), mock-transfected Panc-1 (Lane 2), and TGF-␤1-transfected
Panc-1 (Lane 3) incubated with antibodies against p21waf1 (top) and PCNA (bottom).
552
TGF-〉1 INDUCES DESMOPLASIA IN PANCREATIC CARCINOMA

4. fibroblasts and the tumor cells (Fig. 4A), whereas cocultivation
with mock-transfected PANC-1 cells did not exhibit this effect. On
the RNA level, induction of collagen type I in the fibroblasts could
be demonstrated after incubation with conditioned media from
TGF-␤1-transfected PANC-1 cells (Fig. 4B); moreover, an up-
regulation of TGF-␤1 expression could be demonstrated by RT-
PCR under this conditions (Fig. 4B). Although CTGF expression
remained unchanged in the PANC-1 cells after TGF-␤1 transfec-
tion (data not shown), a significant increase in CTGF mRNA was
detectable in fibroblasts after cocultivation with TGF-␤1-trans-
fected PANC-1 cells (Fig. 4B).
Incubation of fibroblasts in conditioned media of TGF-␤1-trans-
fected PANC-1 cells resulted in more pronounced tyrosine phos-
phorylation of proteins in fibroblasts than incubation with super-
natants from mock-transfected and untransfected PANC-1 cells
(Fig. 5, top). Furthermore, mitogen-activated protein kinases were
activated as indicated by a mobility shift of Erk 1/2 (Fig. 5,
bottom). As mentioned with the tyrosine phosphorylation, the most
pronounced phosphorylation of Erk 1/2 and 3 could be demon-
strated after incubation with supernatants of PANC-1/TGF-␤1
(Fig. 5, bottom). Here, an increase in the activated, i.e., phospho-
rylated, kinases was evident.
TGF-␤1-transfected PANC-1 Cells Induce Desmoplasia with
Increase in Matrix Proteins in Vivo. PANC-1/TGF-␤1 trans-
fected cells and mock-transfected cells were injected orthotopi-
cally into the nude mouse pancreas. Tumors were harvested after 2
months. The tumors grown from TGF-␤1-transfected cells demon-
strated an increased desmoplasia surrounding the tumor cells as
compared with the mock-transfected cells (Fig. 6). This was evi-
dent both on the tumor margin toward the normal mouse pancreas
as well as within the tumor. In addition, collagen type I and
fibronectin could be detected in increased amounts surrounding the
tumor cells (Fig. 6).
DISCUSSION
The desmoplastic reaction is one of the morphological hallmarks of
several human tumors (1) originating from solid epithelial glands,
such as pancreatic adenocarcinoma, that sets it apart from other
epithelial tumors. Beside the description and static expression analysis
of potential factors, no detailed analysis has been performed to dissect
this phenomenon. The pancreatic tumor cells themselves produce
matrix proteins (10) and express a variety of integrins (6, 15). Fur-
Fig. 4. A, cocultivation of mock-transfected Panc-1 cells and TGF-␤1-transfected
Panc-1 cells with fibroblasts in the TransWell system. Cultivation of the tumor cells on top
in the insert with the fibroblasts in the bottom well or vice versa is shown. The outer of
the four columns in each set represent the baseline of tumor cells (left/light gray) and
fibroblasts (right/white) grown without cocultivation. The inner columns represent the cell
counts for tumor cells (dark gray) and fibroblasts (black) under cocultivation for 3 days.
COL I, collagen I. B, Northern blot of RNA from fibroblasts cultivated with and without
FCS (Lanes 1 and 2); or with conditioned media from Panc-1 (Lane 3); Panc-1/TGF␤1
(Lane 4) and Panc-1-mock (Lane 5) hybridized for collagen I and 18S rRNA (loading
control. Middle: RT-PCR products for TGF-␤1 and GAPDH (control) of fibroblasts
incubated in DMEM ϩ FCS (Lane 1); or in conditioned media from Panc-1-mock for 1
or 3d (Lanes 2 and 3) and Panc-1/TGF-␤1 for 1 or 3 days (Lanes 4 and 5) after 27 (left)
and 30 (right) cycles. Bottom, Northern blot for CTGF in fibroblasts after cultivation alone
(F) or after cocultivation with mock-transfected (FM) and TGF-␤1-transfected (FT)
PANC-1 cells (loading control, 18S rRNA).
Fig. 5. Induction of tyrosine phosphorylation in fibroblasts after incubation in super-
natants from Panc-1, Panc-1 mock, and Panc-1/TGF-␤1; Lane 1, control (plain DMEM
medium; top). Activation of mitogen-activated protein kinases Erk 1 and Erk 2 after
incubation in supernatants (bottom). P, the activated, hence phosphorylated, kinase.
553
TGF-〉1 INDUCES DESMOPLASIA IN PANCREATIC CARCINOMA

5. thermore, the expression of growth factors and their receptors has
been demonstrated conclusively, however, mostly related to a dem-
onstration of the autocrine growth-promoting effect (35, 36, 43).
To test our hypothesis of a positive correlation of stroma induction
and TGF-␤1 expression, we successfully transfected the tumor cell
line PANC-1 with a TGF-␤1 expression vector.
For TGF-␤1, it has been suggested that the major regulatory step
controlling TGF-␤1 activity takes place extracellularly. The same was
true for the transfected PANC-1 cells; TGF-␤1 was released into the
culture medium in a latent, i.e., not activated, state. Recently, it was
demonstrated that latent TGF-␤1 can bind to and be activated by the
␣v␤6 integrin (44). These integrin subunits are also expressed by
pancreatic carcinomas (15), i.e., the PANC-1 cells (6). Thus, activa-
tion of the released TGF-␤1 may be accomplished in this way.
Expression of TGF-␤1 resulted in an up-regulation of the matrix
proteins collagen type I and fibronectin in the tumor cells themselves.
Furthermore, PDGF expression was increased in the transfected cells.
This altered gene expression resulted in several paracrine effects on
fibroblasts in cocultivation experiments. We could demonstrate an
increase in collagen type I synthesis in the fibroblasts after stimulation
with supernatants from TGF-␤1-transfected PANC-1 cells. Similarly,
the activation of collagen type VII regulatory elements by TGF has
been described recently (45). The fibroblasts themselves produced
more TGF-␤1 upon stimulation (cocultivation or conditioned media)
by the TGF-␤1-transfected PANC-1 cells. This is supported by the
observation that in pancreatic carcinoma tissue, TGF-␤1 is most
predominant in the stroma (46). Furthermore, collagen type I, the most
predominant basal membrane matrix protein in pancreatic carcinoma
(10), is also up-regulated, both in the tumor cells themselves and in
the fibroblasts upon cocultivation. This up-regulation, however, may
only be in part attributed to TGF-␤1 itself; it could also be the result
of the up-regulation of PDGF-A that has been shown to be a cofactor
in TGF-␤1-induced collagen type I stimulation (23). In the fibroblasts,
after cocultivation with TGF-␤1-transfected PANC-1 cells, CTGF,
one of the index TGF-␤1 response genes (47), was increased. Inhibi-
tion of CTGF abrogated the TGF-␤1-induced collagen gene up-
regulation, confirming the pivotal role of this growth factor (48). As
a result of these alterations in gene expression mentioned above, the
transfection of TGF-␤1 in the pancreatic tumor cell line PANC-1 led
to a gain of stromal tissue after orthotopic transplantation in the nude
mouse when compared with mock-transfected PANC-1 cells.
The influence of the matrix on signal transduction has long been
under debate (49, 50). We have shown that a single growth factor,
TGF-␤1, is capable of conferring the desmoplastic potential to tumor
cells not capable of these features. Some of the effects may be
attributed to a direct effect of TGF-␤1, whereas others, e.g., the
up-regulation of collagen type I (51), may be the result of indirect
effects of TGF-␤1 intimately associated with the signal transduction
pathway involved in TGF-␤1 activities.
ACKNOWLEDGMENTS
We thank Roland M. Schmid for assistance in subcloning the TGF-␤1
plasmid and Thomas Gress (both of University of Ulm, Ulm, Germany) for
supplying us with the CTGF plasmid.
Fig. 6. Orthotopic tumors after intrapancreatic
injection of TGF-␤1-transfected PANC-1 cells (B,
D, and F) and mock-transfected PANC-1 cells (A,
C, and E) into the nude mouse pancreas. A and B,
Masson-Goldner trichrome staining. Immunocyto-
chemistry for collagen type I (C and D) and fi-
bronectin (E and F) is shown.
554
TGF-〉1 INDUCES DESMOPLASIA IN PANCREATIC CARCINOMA

6. REFERENCES
1. van den Hooff, A. Connective tissue changes in cancer. Int. Rev. Connect. Tissue
Res., 10: 395–432, 1983.
2. Dvorak, H. F. Tumors: wounds that do not heal. Similarities between tumor stroma
generation and wound healing. N. Engl. J. Med., 315: 1650–1659, 1986.
3. Sethi, T., Rintoul, R. C., Moore, S. M., MacKinnon, A. C., Salter, D., Choo, C.,
Chilvers, E. R., Dransfield, I., Donnelly, S. C., Strieter, R., and Haslett, C. Extracel-
lular matrix proteins protect small cell lung cancer cells against apoptosis: a mech-
anism for small cell lung cancer growth and drug resistance in vivo. Nat. Med., 5:
662–668, 1999.
4. Klo¨ppel, G., Lingenthal, G., Bu¨low, M. V., and Kern, H. F. Histological and fine
structural features of pancreatic ductal adenocarcinomas in relation to growth and
prognosis: studies in xenografted tumours and clinico-histopathological correlation in
a series of 75 cases. Histopathol. (Oxf.), 9: 841–856, 1985.
5. Longnecker, D. S., Jamieson, J. D., and Asch, H. L. Regulation of growth and
differentiation in pancreatic cancer. Pancreas, 4: 256–275, 1989.
6. Lo¨hr, M., Trautmann, B., Peters, S., Zauner, I., Meier, A., Klo¨ppel, G., Liebe, S., and
Kreuser, E. D. Expression and function of receptors for extracellular matrix proteins
in human ductal adenocarcinomas of the pancreas. Pancreas, 12: 248–259, 1996.
7. Ingber, D. E., Madri, J. A., and Jamieson, J. D. Neoplastic disorganization of
pancreatic epithelial cell-cell relations. Am. J. Pathol., 121: 248–260, 1985.
8. Hall, P. A., and Lemoine, N. R. Models for pancreatic cancer. In: N. R. Lemoine and
N. A. Wright (eds.), Cancer Surveys, Vol. 16, The Molecular Pathology of Cancer,
pp. 135–155. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1993.
9. Liotta, L. A., Rao, C. N., and Barsky, S. H. Tumor cell interaction with the
extracellular matrix. In: L. A. Liotta (ed.), The Role of Extracellular Matrix in
Development, pp. 357–371. New York: Alan R. Liss, Inc., 1984.
10. Lo¨hr, M., Trautmann, B., Go¨ttler, M., Peters, S., Zauner, I., Maillet, B., and Klo¨ppel,
G. Human ductal adenocarcinomas of the pancreas express extracellular matrix
proteins. Br. J. Cancer, 69: 144–151, 1994.
11. Haberern-Blood, C., Liotta, L. A., Rao, C. N., and Kupchik, H. Z. Laminin expression
by human pancreatic carcinoma cells in the nude mouse and in culture. J. Natl. Cancer
Inst., 79: 891–898, 1987.
12. Haglund, C., Roberts, P. J., Nordling, S., and Ekblom, P. Expression of laminin in
pancreatic neoplasms and in chronic pancreatitis. Am. J. Surg. Pathol., 8: 669–676,
1984.
13. Tani, T., Lumme, A., Linnala, A., Kivilaakso, E., Kiviluoto, T., Burgeson, R. E.,
Kangas, L., Leivo, I., and Virtanen, I. Pancreatic carcinomas deposit laminin-5,
preferably adhere to laminin-5, and migrate on the newly deposited basement mem-
brane. Am. J. Pathol., 151: 1289–1302, 1997.
14. Weinel, R. J., Rosendahl, A., Neumann, K., Chaloupka, B., Erb, D., Rothmund, M.,
and Santoso, S. Expression and function of VLA-␣2, -␣3, -␣5, and ␣6-integrin
receptors in pancreatic carcinoma. Int. J. Cancer, 52: 827–833, 1992.
15. Hall, P. A., Coates, P., Lemoine, N. R., and Horton, M. A. Characterization of integrin
chains in normal and neoplastic human pancreas. J. Pathol., 165: 33–41, 1991.
16. Wrann, M., Bodmer, S., Martin, R. D., Siepl, C., Hofer-Warbinek, R., Frei, K., Hofer,
E., and Fontana, A. T cell suppressor factor from human glioblastoma cells is a 12.5
Kd protein closely related to transforming growth factor-␤. EMBO J., 6: 1633–1636,
1987.
17. Jesnowski, R., Liebe, S., and Lo¨hr, M. Increasing the transfection efficacy and
subsequent long-term culture of resting human pancreatic duct epithelial cells. Pan-
creas, 17: 262–265, 1998.
18. Sparmann, G., Ja¨schke, A., Mu¨ller, P., Lo¨hr, M., Liebe, S., and Emmrich, J. Tissue
homogenization as a key step extracting RNA from human and rat pancreatic tissue.
Biotechniques, 22: 408–412, 1997.
19. Ma¨kela, J. K., Raassina, M., and Vuorio, E. Human pro␣1(I) collagen: cDNA
sequence for the C-propeptide domain. Nucleic Acids Res., 16: 349, 1988.
20. Abraham, J. A., Mergia, A., Whang, J. L., Tumolo, A., Friedman, J., Hjerrild, K. A.,
Gospodarowicz, D., and Fiddes, J. C. Nucleotide sequence of a bovine clone encoding
the angiogenic protein, basic fibroblast growth factor. Science (Washington DC),
545: 545–548, 1986.
21. Wenger, C., Ellenrieder, V., Alber, B., Lacher, U., Menke, A., Hameister, H., Wilda,
M., Iwamura, T., Beger, H. G., Adler, G., and Gress, T. M. Expression and differ-
ential regulation of connective tissue growth factor in pancreatic cancer cells. Onco-
gene, 18: 1078–1080, 1999.
22. van Laethem, J. L., Resibois, A., Rickaert, F., Deviere, J., Gelin, M., Cremer, M., and
Robberecht, P. Different expression of transforming growth factor ␤1 in pancreatic
ductal adenocarcinoma and cystic neoplasms. Pancreas, 15: 41–47, 1997.
23. Halloran, B. G., So, B. J., and Baxter, B. T. Platelet-derived growth factor is a
cofactor in the induction of 1 ␣(I) procollagen expression by transforming growth
factor-␤ 1 in smooth muscle cells. J. Vasc. Surg., 23: 767–773, 1996.
24. Ning, Y., Roschke, A., Christian, S., Lesser, J., Sutcliffe, J. S., and Ledbetter, D. H.
Identification of a novel paternally expressed transcript adjacent to snRPN in the
Prader-Willi syndrome critical region. Genome Res., 6: 742–746, 1996.
25. von Heimendahl, G., Gebhardt, E., and Dingermann, T. Proto-oncogene activation in
surgical specimens of rectal carcinoma. Anticancer Res., 8: 805–812, 1988.
26. Jesnowski, R., Mu¨ller, P., Schareck, W., Liebe, S., and Lo¨hr, M. Immortalized
pancreatic duct cells in vitro and in vivo. Ann. NY Acad. Sci., 880: 50–65, 1999.
27. Schmidt, C., Pommerencke, H., Du¨rr, F., Nebe, B., and Rychly, J. Mechanical
stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskel-
etally anchored proteins. J. Biol. Chem., 273: 5081–5085, 1998.
28. Tilbrook, P. A., Bittorf, T., Callus, B., Busfield, S. J., Spadaccini, A., Ali, M., Ingley,
E., and Klinken, S. P. Regulation of the erythropoietin receptor and involvement of
JAK2 in differentiation of J2E erythroid cells. Cell Growth Differ., 7: 511–520, 1996.
29. Fu, X., Guadagni, F., and Hoffman, R. M. A metastatic nude-mouse model of human
pancreatic cancer constructed orthotopically with histologically intact patient speci-
mens. Proc. Natl. Acad. Sci. USA, 89: 5645–5649, 1992.
30. Reyes, G., Villanueva, A., Garcia´, C., Sancho, F. J., Piulats, J., Lluı´s, F., and Capella´,
G. Orthotopic xenografts of human pancreatic carcinomas acquire genetic aberrations
during dissemination in nude mice. Cancer Res., 56: 5713–5719, 1996.
31. Lo¨hr, M. Stromal-epithelial interactions in pancreatic carcinomas. In: N. R. Lemoine
and J. Neoptolemos (eds.), Cell Biology of Pancreatic Cancer, pp. 18–35. London:
Blackwell Science, 1996.
32. Korc, M., Meltzer, P., and Trent, J. Enhanced expression of epidermal growth factor
receptor correlates with alterations of chromosome 7 in human pancreatic cancer.
Proc. Natl. Acad. Sci. USA, 83: 5141–5144, 1986.
33. Leung, H. Y., Hughes, C. M., Klo¨ppel, G., Williamsons, R. C. N., and Lemoine, N. R.
Localization of expression of fibroblast growth factors and their receptors in pancre-
atic adenocarcinoma by in situ hybridization. Int. J. Oncol., 4: 1219–1223, 1994.
34. Estival, A., Clerc, P., Vayesse, N., Tam, J. P., and Clemente, F. Decreased expression
of transforming growth factor ␣ during differentiation of human pancreatic cancer
cells. Gastroenterology, 103: 1851–1859, 1992.
35. Leung, H. Y., Gullick, W. J., and Lemoine, N. R. Expression and functional activity
of fibroblast growth factors and their receptors in human pancreatic cancer. Int. J.
Cancer, 59: 667–675, 1994.
36. Kornmann, M., Beger, H. G., and Korc, M. Role of fibroblast growth factors and their
receptors in pancreatic cancer and chronic pancreatitis. Pancreas, 17: 169–175, 1998.
37. Friess, H., Yamanaka, Y., Bu¨chler, M., Ebert, M., Beger, H. G., Gold, L. I., and Korc,
M. Enhanced expression of transforming growth factor ␤ isoforms in pancreatic
cancer correlates with decreased survival. Gastroenterology, 105: 1846–1856, 1993.
38. Barton, C., Hall, P. A., Hughes, C. M., Gullick, W. J., and Lemoine, N. R. Trans-
forming growth factor ␣ and epidermal growth factor in human pancreatic cancer.
J. Pathol., 163: 111–116, 1991.
39. Beauchamp, R. D., Lyons, R. M., Yang, E. Y., Coffey, R. J., and Moses, H. L.
Expression of and response to growth regulatory peptides by two human pancreatic
carcinoma cell lines. Pancreas, 5: 369–380, 1990.
40. Kalthoff, H., Roeder, C., Humburg, I., Thiele, H. G., Greten, H., and Schmiegel, W.
Modulation of platelet-derived growth factor A- and B-chain/c-sis mRNA by tumor
necrosis factor and other agents in adenocarcinoma cells. Oncogene, 6: 1015–1021,
1991.
41. Kalthoff, H., Roeder, C., Gieseking, J., Humburg, I., and Schmiegel, W. Inverse
regulation of human ERBB2 and epidermal growth factor receptors by tumor necrosis
factor ␣. Proc. Natl. Acad. Sci. USA, 90: 8972–8976, 1993.
42. Schmiegel, W., Roeder, C., Schmielau, J., Rodeck, U., and Kalthoff, H. Tumor
necrosis factor ␣ and the epithelial growth factor receptor in human pancreatic cancer
cells. Proc. Natl. Acad. Sci. USA, 90: 863–867, 1993.
43. Lemoine, N. R., Leung, H. Y., Barton, C. M., Hughes, C. M., Klo¨ppel, G., and
Gullick, W. J. Autocrine growth control of pancreatic cancer. Int. J. Pancreatol., 14:
69–70, 1993.
44. Munger, J. S., Huang, X., Kawakatsu, H., Griffiths, M. J. D., Dalton, S. L., Wu, J.,
Pittet, J. F., Kaminski, N., Garat, C., Matthay, M. A., Rifkin, D. B., and Sheppard, D.
The integrin ␣v␤6 binds and activates latent TGF-␤ 1: a mechanism for regulating
pulmonary inflammation and fibrosis. Cell, 96: 319–328, 1999.
45. Rodeck, U., Nishiyama, T., and Mauviel, A. Independent regulation of growth and
SMAD-mediated transcription by transforming growth factor ␤ in human melanoma
cells. Cancer Res., 59: 547–550, 1999.
46. Satoh, K., Shimosegawa, T., Hirota, M., Koizumi, M., and Toyota, T. Expression of
transforming growth factor ␤1 (TGF␤1) and its receptors in pancreatic duct cell
carcinoma and in chronic pancreatitis. Pancreas, 16: 468–474, 1998.
47. Geng, M. M., Ellenrieder, V., Wallrapp, C., Mu¨ller-Pilasch, F., Sommer, G., Adler,
G., and Gress, T. M. Use of representational difference analysis to study the effect of
TGF␤ on the expression profile of a pancreatic cancer cell line. Genes Chromosomes
Cancer, 26: 70–79, 1999.
48. Duncan, M. R., Frazier, K. S., Abramson, S., Williams, S., Klapper, H., Huang, X.,
and Grotendorst, G. R. Connective tissue growth factor mediates transforming growth
factor ␤-induced collagen synthesis: down-regulation by cAMP. FASEB J., 13:
1774–1786, 1999.
49. Bissell, M. J., Hall, H. G., and Parry, G. How does the extracellular matrix direct gene
expression? J. Theor. Biol., 99: 31–68, 1982.
50. Bissell, M. J., and Barcellos-Hoff, M. H. The influence of extracellular matrix on
gene expression: is structure the message? J. Cell Sci. (Suppl.), 8: 327–343, 1987.
51. Sparmann, G., Merkord, J., Ja¨schke, A., Nizze, H., Jonas, L., Lo¨hr, M., Liebe, S., and
Emmrich, J. Pancreatic fibrosis in experimental pancreatitis induced by dibutylin
dichloride. Gastroenterology, 112: 1664–1672, 1997.
52. Kalthoff, H., Lo¨hr, M., Roeder, C., and Schmiegel, W. Das Pankreaskarzinom.
Zellbiologie, Matrixproteine und Wachstumsregulation. In: G. Adler, U. R. Fo¨lsch, J.
Mo¨ssner, and M. V. Singer (eds.), Erkrankungen des exkretorischen Pankreas, Ed. 2,
pp. 385–404. Jena: Fischer, 1995.
555
TGF-〉1 INDUCES DESMOPLASIA IN PANCREATIC CARCINOMA