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Transforming Growth Factor-ß1 Induces Desmoplasia in an Experimental Model of Human Pancreatic Carcinoma¹

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

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Proliferation of fibrotic tissue (desmoplasia) is one of the hallmarks of several epithelial tumors including pancreatic adenocarcinoma. This tissue 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)-ß1 and fibroblast growth factor-2. Here we demonstrate that the overexpression of TGF-ß1 induced up-regulation of matrix proteins and growth factors in the TGFß1-transfected 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-ß1-transfected PANC-1 cells. TGF-ß1-transfected pancreatic tumor cells induced a rich stroma after orthotopical transplantation in the nude mouse pancreas. The transfer of a single growth factor, TGF-ß1, 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-ß1 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

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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 necessary 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 become a model system to study the interrelation of epithelial tumor cells, matrices, fibroblasts, and growth factors (5, 6, 7, 8) .

Desmoplastic tissue consists of fibroblasts, as the main cellular component, and extracellular matrix proteins (9) . The pancreatic tumor cells themselves are able to produce ECM³ proteins (10, 11, 12, 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-ß1 in a pancreatic tumor cell line known neither to induce desmoplasia nor to express substantial amounts of TGF-ß1 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-ß1.

	MATERIALS AND METHODS

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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 µg/ml streptomycinsulfate, and 250 ng/ml amphotericin B; Life Technologies, Inc.; Ref. 10 ). Mature human recombinant TGF-ß1 was purchased from R&D Systems. Full-length cDNA of TGF-ß1(16) was cut out of pRK5ß1E (BamHI) and cloned into the pcDNA3 vector (Invitrogen) under the control of a cytomegalovirus 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 µg/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-ß1 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 µg of total RNA were subjected to standard formamide gel electrophoresis as described. Gels were blotted to nylon membranes (Qiagen) and hybridized with cDNA probes for TGF-ß1 (EcoRI/HindIII digest of pcDNA3/TGF-ß1), 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-ß1, PDGF-A, type I collagen, and GAPDH. The primers were as follows: TGF-ß1 (22) , sense 5'-CAG AAA TAC AGC AAC AAT TCC TGG-3' and antisense 5'-TTG CAG TGT GTT ATC CCT GCT GTC-3' (190-bp product); PDGF-A (23) , sense 5'CAG TCA GAT CCA CAG CAT CC-3' and antisense 5'-AAT GAC CGT CCT GGT CTT GC-3' (200-bp product); collagen type I (23) , sense 5'-ACG TGA TCT GTG ACG AGA CC-3' and antisense 5'-AGC AAA GTT TCC TCC GAG GC-3' (250-bp product); and GAPDH (24) , sense 5'-ACC ACA GTC CAT GCC ATC AC-3' and antisense 5'-TCC ACC ACC CTG TTG CTG TA-3' (450-bp product). PCR conditions were the following: denaturing for 30 s at 94°C; annealing for 60 s at 60°C (TGF-ß1) 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-ß1, 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, receptors, and genes of ECM proteins was investigated by reverse slot blot. For this purpose, plasmid DNA corresponding to 1 µg 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 µg 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/TGFß1 cells (5 x 10⁴) were seeded onto Transwell inserts (Costar) and were cocultivated with fibroblasts (5 x 10⁴ cells/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 Collection). From respective parallel experiments, conditioned media (see below) and RNA (see above) were prepared for subsequent analysis.

Conditioned Media.
PANC-1/TGFß1 cells were seeded in DMEM/10% FCS. After 2 days of incubation, cells were washed three times with PBS (pH 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 investigated 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 x 10⁴ cells of TGF-ß1-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 µg/ml G418) for 1, 2, or 3 days, after that the supernatant was collected. TGF-ß1 and PDGF were quantified using the Quantikine TGF-ß1 and PDGF immunoassays (R&D) according to the instructions of the manufacturer.

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-ß-1 (sc-146), p21^wafI (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 x g for 5 min in a microcentrifuge and analyzed by Western blotting (28) .

Nude Mouse Model.
A suspension of 1 x 10⁶ PANC-1/TGFß1 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 Masson-Goldner trichrome staining. Immunocytochemistry was performed as described before with antibodies against type I collagen (1:100; Calbiochem) and fibronectin (1:400; Sigma; Ref. 10 ). Detection was performed using horseradish peroxidase-conjugated rabbit-antimouse and swine-antirabbit IgGs (Dako Diagnostika) as secondary and third antibodies and 3-amino-9-ethylcarbazole as substrate.

	RESULTS

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Induction of Desmoplasia Is Associated with the Expression of TGF-ß1 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, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) , the stromal reaction (Fig. 1) was found to be positively correlated with the expression of TGF-ß1 and/or FGF-2 (Ref. 31 ; Table 1 ). We therefore chose PANC-1 cells that did not express significant amounts of TGF-ß1 as a model for the subsequent experiments investigating the role of TGF-ß1 in desmoplasia.

View larger version (119K): [in this window] [in a new window]   Fig. 1. Desmoplastic potential of several human pancreatic adenocarcinoma cell lines upon xenotransplantation 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, x250.

View larger version (64K): [in this window] [in a new window]   Fig. 2. A, Northern blot of RNA from native Panc-1 cells (Lanes 1 and 2), TGF-ß1-transfected Panc-1 cells (Lanes 3 and 4), mock transfected Panc-1 cells (Lanes 5 and 6), and fibroblasts (Lane 7) for TGF-ß1 (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-ß1 and Panc-1 for collagen I and ethidium bromide gel (top). Northern blot of Panc-1 +/- FCS (1 + 2); Panc-1/TGFß1 +/- FCS (2 + 3); Panc-1-mock +/- FCS (5 + 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-ß1.

View larger version (60K): [in this window] [in a new window]   Fig. 3. A, Western blot of whole-cell lysates from mock-transfected Panc-1 cells (Lane 1) and TGF-ß1-transfected Panc-1 cells (Lane 2) incubated with antibodies against TGF-ß1 (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-ß1-transfected Panc-1 (Lane 3) incubated with antibodies against p21waf1 (top) and PCNA (bottom).

TGF-ß1 inhibits growth by acting on the cell cycle by modulating, for example, p21^wafI and PCNA. TGF-ß1-transfected PANC-1 cells exhibited a substantial increase in p21^wafI expression on the protein level on Western blot of nuclear extracts (Fig. 3B) ; on ELISA, p21^wafI 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-ß1-transfected PANC-1 Cells Induce Fibroblast Growth and Up-Regulation of Matrix Proteins and TGF-ß1 in Fibroblasts.
Cocultivation of fibroblasts with PANC-1/TGF-ß1 cells in the Transwell system led to an increase in proliferation of both the 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-ß1-transfected PANC-1 cells (Fig. 4B) ; moreover, an up-regulation of TGF-ß1 expression could be demonstrated by RT-PCR under this conditions (Fig. 4B) . Although CTGF expression remained unchanged in the PANC-1 cells after TGF-ß1 transfection (data not shown), a significant increase in CTGF mRNA was detectable in fibroblasts after cocultivation with TGF-ß1-transfected PANC-1 cells (Fig. 4B) .

View larger version (40K): [in this window] [in a new window]   Fig. 4. A, cocultivation of mock-transfected Panc-1 cells and TGF-ß1-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/TGFß1 (Lane 4) and Panc-1-mock (Lane 5) hybridized for collagen I and 18S rRNA (loading control. Middle: RT-PCR products for TGF-ß1 and GAPDH (control) of fibroblasts incubated in DMEM + FCS (Lane 1); or in conditioned media from Panc-1-mock for 1 or 3d (Lanes 2 and 3) and Panc-1/TGF-ß1 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-ß1-transfected (FT) PANC-1 cells (loading control, 18S rRNA).

View larger version (46K): [in this window] [in a new window]   Fig. 5. Induction of tyrosine phosphorylation in fibroblasts after incubation in supernatants from Panc-1, Panc-1 mock, and Panc-1/TGF-ß1; 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.

View larger version (177K): [in this window] [in a new window]   Fig. 6. Orthotopic tumors after intrapancreatic injection of TGF-ß1-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. Immunocytochemistry for collagen type I (C and D) and fibronectin (E and F) is shown.

	DISCUSSION

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To test our hypothesis of a positive correlation of stroma induction and TGF-ß1 expression, we successfully transfected the tumor cell line PANC-1 with a TGF-ß1 expression vector.

For TGF-ß1, it has been suggested that the major regulatory step controlling TGF-ß1 activity takes place extracellularly. The same was true for the transfected PANC-1 cells; TGF-ß1 was released into the culture medium in a latent, i.e., not activated, state. Recently, it was demonstrated that latent TGF-ß1 can bind to and be activated by the _vß₆ integrin (44) . These integrin subunits are also expressed by pancreatic carcinomas (15) , i.e., the PANC-1 cells (6) . Thus, activation of the released TGF-ß1 may be accomplished in this way. Expression of TGF-ß1 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-ß1-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-ß1 upon stimulation (cocultivation or conditioned media) by the TGF-ß1-transfected PANC-1 cells. This is supported by the observation that in pancreatic carcinoma tissue, TGF-ß1 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-ß1 itself; it could also be the result of the up-regulation of PDGF-A that has been shown to be a cofactor in TGF-ß1-induced collagen type I stimulation (23) . In the fibroblasts, after cocultivation with TGF-ß1-transfected PANC-1 cells, CTGF, one of the index TGF-ß1 response genes (47) , was increased. Inhibition of CTGF abrogated the TGF-ß1-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-ß1 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-ß1, 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-ß1, whereas others, e.g., the up-regulation of collagen type I (51) , may be the result of indirect effects of TGF-ß1 intimately associated with the signal transduction pathway involved in TGF-ß1 activities.

	ACKNOWLEDGMENTS

 
We thank Roland M. Schmid for assistance in subcloning the TGF-ß1 plasmid and Thomas Gress (both of University of Ulm, Ulm, Germany) for supplying us with the CTGF plasmid.

	FOOTNOTES

 
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. acknowledges the support of the Bundesministerium für Bildung und Forschung.

² To whom requests for reprints should be addressed, at Sektion Molekulare Gastroenterologie, Medizinische Klinik IV, Fakultät für Klinische Medizin Mannheim, Universität Heidelberg, Theodor Kutzer Ufer 1-3, D-68135 Mannheim, Germany. Phone: 49-621-383-2900; Fax: 49-381-383-1986; E-mail: matthias.loehr{at}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.

Received 1/28/00. Accepted 11/14/00.

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