transforming growth factor-b1 induces desmoplasia in an...

[CANCER RESEARCH 61, 550–555, January 15, 2001]

Transforming Growth Factor- b1 Induces Desmoplasia in an ExperimentalModel of Human Pancreatic Carcinoma1

Matthias Lohr, 2 Christian Schmidt, Jorg Ringel, Mario Kluth, Petra Muller, Horst Nizze, and Ralf JesnowskiDivision 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 ofseveral epithelial tumors including pancreatic adenocarcinoma. This tis-sue reaction may be deleterious or advantageous to the host or tumor. Ina systematic analysis, we identified two growth factors expressed byhuman pancreatic carcinoma cells that are positively correlated with theability to induce fibroblast proliferation both in vitro and in vivo, i.e.,transforming growth factor (TGF)- b1 and fibroblast growth factor-2.Here we demonstrate that the overexpression of TGF-b1 induced up-regulation of matrix proteins and growth factors in the TGFb1-trans-fected pancreatic tumor cells. Furthermore, transfection of PANC-1 cellsinduces the same change in fibroblasts in either cocultivation experimentsor when they are grown in conditioned medium from TGF-b1-transfectedPANC-1 cells. TGF-b1-transfected pancreatic tumor cells induced a richstroma after orthotopical transplantation in the nude mouse pancreas.The transfer of a single growth factor, TGF-b1, conveys the ability toinduce a fibroblast response similar to that seen in desmoplasia in humanpancreatic adenocarcinoma. This effect cannot only be attributed to directeffects of TGF-b1 but also results from the up-regulation of several otherfactors including collagen type I, connective tissue growth factor, andplatelet-derived growth factor.

INTRODUCTION

Desmoplasia is a characteristic feature of the growth of somecarcinomas (1). To date, it is not clear whether this process is amechanism to protect the tumor from the host or represents a defensemechanism by the host (2), although there are hints that this stroma isbeneficial for the tumor (3). To tackle desmoplasia therapeutically byeither supporting or suppressing this development, it becomes neces-sary to study the etiology and to attribute this feature to either thetumor cells themselves or the host. Desmoplasia is of particularpredominance in ductal adenocarcinomas of the pancreas exhibiting astrong stromal reaction (4). Therefore, pancreatic carcinoma has be-come a model system to study the interrelation of epithelial tumorcells, matrices, fibroblasts, and growth factors (5–8).

Desmoplastic tissue consists of fibroblasts, as the main cellularcomponent, and extracellular matrix proteins (9). The pancreatic tu-mor cells themselves are able to produce ECM3 proteins (10–13) andinteract with ECM by expressing functionally active integrins (6, 14, 15).

To test the hypothesis of desmoplasia induction by a tumor-derivedgrowth factor, we conducted a deductive analysis correlating theability to induce desmoplasia with the expression of certain growth

factors. Furthermore, we reasoned that the overexpression of such agrowth factor,e.g., TGF-b1 in a pancreatic tumor cell line knownneither to induce desmoplasia nor to express substantial amounts ofTGF-b1 and FGF-2, should result in the gain of the ability to inducefibroblast growth and in an induction of desmoplasia in a xenograftednude mouse model by virtue of direct and indirect effects of TGF-b1.

MATERIALS AND METHODS

Cell Culture and Transfection. AsPC-1, BxPC-3, Capan-1, and PANC-1cells, all from American Type Culture Collection, were cultivated in DMEMwith GlutaMAX I (Life Technologies, Inc.) supplemented with 10% heat-inactivated FCS and antibiotics (100 units/ml penicillin, 100mg/ml strepto-mycinsulfate, and 250 ng/ml amphotericin B; Life Technologies, Inc.; Ref. 10).Mature human recombinant TGF-b1 was purchased from R&D Systems.Full-length cDNA of TGF-b1(16) was cut out of pRK5b1E (BamHI) andcloned into the pcDNA3 vector (Invitrogen) under the control of a cytomeg-alovirus promoter. PANC-1 cells were transfected with this construct or withthe empty pcDNA3 plasmid (mock) by calcium phosphate coprecipitation inN,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffered saline usingstandard protocols as described (17). This plasmid also codes for theneoresistance gene, enabling selection of transfectants with the antibiotic G418(Sigma; 400mg/ml). Resistant clones were expanded, and expression of thetransfected cDNA was confirmed by Northern blot, Western blot, and ELISA(R&D).

Northern Blot and RT-PCR. Subconfluent layers of PANC-1/TGF-b1cells, mock transfected, untransfected PANC-1 cells, and AsPC-1 and BxPC-3cells were lysed in ice-cold guanidine thiocyanate. RNA preparation wasperformed as described (18). Tenmg of total RNA were subjected to standardformamide gel electrophoresis as described. Gels were blotted to nylon mem-branes (Qiagen) and hybridized with cDNA probes for TGF-b1 (EcoRI/HindIII digest of pcDNA3/TGF-b1), type I collagen (pHCAL1U; Refs. 10 and19), PDGF (Amersham), FGF-2,(20), and CTGF (21) using the nonradioactiveDig labeling kit (Boehringer Mannheim, Mannheim, Germany). In addition,RT-PCR was performed using published primers for TGF-b1, PDGF-A, typeI collagen, and GAPDH. The primers were as follows: TGF-b1 (22), sense59-CAG AAA TAC AGC AAC AAT TCC TGG-39 and antisense 59-TTGCAG TGT GTT ATC CCT GCT GTC-39(190-bp product); PDGF-A (23),sense 59CAG TCA GAT CCA CAG CAT CC-39and antisense 59-AAT GACCGT CCT GGT CTT GC-39(200-bp product); collagen type I (23), sense59-ACG TGA TCT GTG ACG AGA CC-39and antisense 59-AGC AAA GTTTCC TCC GAG GC-39(250-bp product); and GAPDH (24), sense 59-ACCACA GTC CAT GCC ATC AC-39and antisense 59-TCC ACC ACC CTGTTG CTG TA-39 (450-bp product). PCR conditions were the following:denaturing for 30 s at 94°C; annealing for 60 s at 60°C (TGF-b1) or at 64°C(collagen, GAPDH, and PDGF); and extension for 60 s at 72°C. AmplifiedDNA was sampled after 21, 24, 27, and 30 cycles, and the resulting PCRproducts for TGF-b1, collagen, and PDGF-A were loaded in the same gelpockets 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 thispurpose, plasmid DNA corresponding to 1mg of cDNA insert was blotted ontoa nylon membrane (Qiagen) by use of a slot blot apparatus (Schleicher &Schuell). Hybridization was performed according to standard procedures witha probe obtained by Dig labeling (Boehringer Mannheim) of 7.5mg of totalRNA in a reverse transcription reaction (25, 26). Hybrids were detected usingthe chemiluminescent Dig detection system (Boehringer Mannheim) accordingto the manufacturer’s instructions.

Cocultivation. PANC-1/TGFb1 cells (53 104) were seeded onto Tran-swell inserts (Costar) and were cocultivated with fibroblasts (53 104 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 markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grant Lo 431/6 from the Deutsche Forschungsgemeinschaft as part ofthe special topic program “Matrix in Biology and Medicine” (to M. L.). C. S. acknowl-edges the support of the Bundesministerium fur Bildung und Forschung.

2 To whom requests for reprints should be addressed, at Sektion Molekulare Gastro-enterologie, Medizinische Klinik IV, Fakulta¨t fur Klinische Medizin Mannheim, Univer-sitat Heidelberg, Theodor Kutzer Ufer 1-3, D-68135 Mannheim, Germany. Phone: 49-621-383-2900; Fax: 49-381-383-1986; E-mail: [email protected].

3 The abbreviations used are: ECM, extracellular matrix; TGF, transforming growthfactor; 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-regulatedkinase.

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well) seeded in six-well tissue culture plates. After 7 days of incubation inDMEM/1% FCS, the cells were trypsinized and counted (trypan blue exclusiontest). 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/TGFb1 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 humanpancreatic adenocarcinoma cell lines upon xeno-transplantation on nude mice.Top, tissue culture.Bottom, tumors established on nude mice.Left toright: Panc-1, PaCa-44, Capan-1, and BxPC-3. Thetwo cell lines to theright develop a stroma on thenude mouse. H&E stain,3250.

Fig. 2.A, Northern blot of RNA from native Panc-1 cells (Lanes1 and 2), TGF-b1-transfected Panc-1 cells (Lanes 3and 4), mocktransfected Panc-1 cells (Lanes 5and6), and fibroblasts (Lane 7) forTGF-b1 (top) and GAPDH (bottom).Lanes 1, 3,and5, with FCS;Lanes 2, 4,and6, without FCS.B, Northern blot of Panc-1/TGF-b1and Panc-1 for collagen I and ethidium bromide gel (top). Northernblot of Panc-11/2 FCS (11 2); Panc-1/TGFb1 1/2 FCS (21 3);Panc-1-mock1/2 FCS (51 6) and fibroblasts for FGF-2 (bottom).C, reverse slot blot with different cDNA probes hybridized withDig-labeled cDNA from Panc-1 mock and Panc-1/TGF-b1.

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TGF-B1 INDUCES DESMOPLASIA IN PANCREATIC CARCINOMA



7.4) to remove any FCS traces and refed with fresh medium containing noFCS. After incubation for another 2 days, the supernatants were collected andfilter sterilized. Skin fibroblasts were incubated with serial dilutions of thisconcentrated 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 andmock-transfected PANC-1 cells.

ELISA. 1.3 3 104 cells of TGF-b1-transfected and mock-transfectedPANC-1 were plated in six-well plates with DMEM and 10% FCS. After 2days, cells were grown with DMEM without FCS (transfected cells all of thetime with 400mg/ml G418) for 1, 2, or 3 days, after that the supernatant wascollected. TGF-b1 and PDGF were quantified using the Quantikine TGF-b1and PDGF immunoassays (R&D) according to the instructions of the manu-facturer.

Western Blot. Proteins were separated by SDS-PAGE, transferred to apolyvinylidene difluoride membrane (Roche), and blocked for 1 h in Tris-buffered saline (TBS; 10 mM Tris, 10 mM NaCl) containing 1% skim milk and0.01% Tween 20. After incubation with the primary antibody for 1 h, blotswere developed using alkaline phosphatase-labeled secondary antibodies andchemiluminescence (CDP-star; Roche). The following primary antibodieswere used in a dilution of 1:1000: PCNA (Santa Cruz; sc-56), TGF-b-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 werecollected by centrifugation at 12,0003 g for 5 min in a microcentrifuge andanalyzed by Western blotting (28).

Nude Mouse Model. A suspension of 13 106 PANC-1/TGFb1 cells ormock-transfected PANC-1 cells were injected orthotopically into athymic nudemice (29, 30). Nude mice were killed after solid tumors were palpable. Tumorswere 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) andfibronectin (1:400; Sigma; Ref. 10). Detection was performed using horserad-ish peroxidase-conjugated rabbit-antimouse and swine-antirabbit IgGs (DakoDiagnostika) as secondary and third antibodies and 3-amino-9-ethylcarbazoleas substrate.

RESULTS

Induction of Desmoplasia Is Associated with the Expression ofTGF-b1 and FGF-2. In a deductive analysis on human pancreaticcarcinoma cellsin vitro andin vivo (6, 31), using all of the publishedinformation on the expression of various growth factors described inpancreatic carcinoma (22, 32–42), the stromal reaction (Fig. 1) wasfound to be positively correlated with the expression of TGF-b1and/or FGF-2 (Ref. 31; Table 1). We therefore chose PANC-1 cellsthat did not express significant amounts of TGF-b1 as a model for the

subsequent experiments investigating the role of TGF-b1 in desmo-plasia.

Stable Expression of Functional TGF-b1 in PANC-1 InducesUp-Regulation of Matrix Proteins and Growth Factors. Expres-sion of the transfected TGF-b1 cDNA in PANC-1/TGF-b1 cells wasverified by Northern and Western blots (Figs. 2Aand 3A). TheTGF-b1 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-b1-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), epidermalgrowth factor, and thea5 integrin subunit (Fig. 2C) was similar inTGF-b1-transfected and mock-transfected PANC-1 cells by Northernblot or reverse slot blot.

TGF-b1 inhibits growth by acting on the cell cycle by modu-lating, for example, p21wafI and PCNA. TGF-b1-transfectedPANC-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 untransfectedand 16.3 units/mg protein in transfected cells. Conclusively, thetransfected cells demonstrated decreased nuclear levels of PCNAon the protein level (Fig. 3B).

TGF-b1-transfected PANC-1 Cells Induce Fibroblast Growthand Up-Regulation of Matrix Proteins and TGF-b1 in Fibro-blasts. Cocultivation of fibroblasts with PANC-1/TGF-b1 cells inthe Transwell system led to an increase in proliferation of both the

Table 1 Ability to induce desmoplasia as assessed by induction of stromal tissue uponxenotransplantation in nude mice in human pancreatic carcinoma cell lines

Cell line Desmoplasiaa EGFb FGF-1b FGF-2b FGF-3b TGF-b1b

AsPC-2 1 (1) 2 (1) (1) (1)BxPC-3 1 11 11 (1) (1) 1Capan-1 11 2 111c 11c (1) 11Capan-2 11 2 2 111c (1) 11PaCa-2 2 2 1c (1) (1) 1PaCa-3 2 1 2 2 (1) 1PaCa-44 2 2 (1) (1)c (1) 1PANC-1 2 2 (1) 1 (1) 1c

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 (Lane1) and TGF-b1-transfected Panc-1 cells (Lane 2) incubated with antibodies againstTGF-b1 (top) and cytokeratin 19 (bottom).B, total cell lysates of pancreatic carcinomacell lines Panc-1 (Lane 1), mock-transfected Panc-1 (Lane 2), and TGF-b1-transfectedPanc-1 (Lane 3) incubated with antibodies against p21waf1 (top) and PCNA (bottom).

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fibroblasts and the tumor cells (Fig. 4A), whereas cocultivationwith mock-transfected PANC-1 cells did not exhibit this effect. Onthe RNA level, induction of collagen type I in the fibroblasts couldbe demonstrated after incubation with conditioned media fromTGF-b1-transfected PANC-1 cells (Fig. 4B); moreover, an up-regulation of TGF-b1 expression could be demonstrated by RT-PCR under this conditions (Fig. 4B). Although CTGF expressionremained unchanged in the PANC-1 cells after TGF-b1 transfec-tion (data not shown), a significant increase in CTGF mRNA wasdetectable in fibroblasts after cocultivation with TGF-b1-trans-fected PANC-1 cells (Fig. 4B).

Incubation of fibroblasts in conditioned media of TGF-b1-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 wereactivated as indicated by a mobility shift of Erk 1/2 (Fig. 5,bottom). As mentioned with the tyrosine phosphorylation, the mostpronounced phosphorylation of Erk 1/2 and 3 could be demon-strated after incubation with supernatants of PANC-1/TGF-b1(Fig. 5, bottom). Here, an increase in the activated,i.e., phospho-rylated, kinases was evident.

TGF-b1-transfected PANC-1 Cells Induce Desmoplasia withIncrease in Matrix Proteins in Vivo. PANC-1/TGF-b1 trans-fected cells and mock-transfected cells were injected orthotopi-cally into the nude mouse pancreas. Tumors were harvested after 2months. The tumors grown from TGF-b1-transfected cells demon-strated an increased desmoplasia surrounding the tumor cells ascompared with the mock-transfected cells (Fig. 6). This was evi-dent both on the tumor margin toward the normal mouse pancreasas well as within the tumor. In addition, collagen type I andfibronectin could be detected in increased amounts surrounding thetumor cells (Fig. 6).

DISCUSSION

The desmoplastic reaction is one of the morphological hallmarks ofseveral human tumors (1) originating from solid epithelial glands,such as pancreatic adenocarcinoma, that sets it apart from otherepithelial tumors. Beside the description and static expression analysisof potential factors, no detailed analysis has been performed to dissectthis phenomenon. The pancreatic tumor cells themselves producematrix proteins (10) and express a variety of integrins (6, 15). Fur-

Fig. 4. A, cocultivation of mock-transfected Panc-1 cells and TGF-b1-transfectedPanc-1 cells with fibroblasts in the TransWell system. Cultivation of the tumor cells on topin the insert with the fibroblasts in the bottom well orvice versais shown. Theouter ofthe four columnsin each set represent the baseline of tumor cells (left/light gray) andfibroblasts (right/white) grown without cocultivation. Theinner columnsrepresent the cellcounts 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 withoutFCS (Lanes 1and2); or with conditioned media from Panc-1 (Lane 3); Panc-1/TGFb1(Lane 4) and Panc-1-mock (Lane 5) hybridized for collagen I and 18S rRNA (loadingcontrol. Middle: RT-PCR products for TGF-b1 and GAPDH (control) of fibroblastsincubated in DMEM1 FCS (Lane 1); or in conditioned media from Panc-1-mock for 1or 3d (Lanes 2and3) and Panc-1/TGF-b1 for 1 or 3 days (Lanes 4and5) 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-b1-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-b1; Lane 1,control (plain DMEMmedium; top). Activation of mitogen-activated protein kinases Erk 1 and Erk 2 afterincubation in supernatants (bottom).P, the activated, hence phosphorylated, kinase.

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thermore, the expression of growth factors and their receptors hasbeen 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 inductionand TGF-b1 expression, we successfully transfected the tumor cellline PANC-1 with a TGF-b1 expression vector.

For TGF-b1, it has been suggested that the major regulatory stepcontrolling TGF-b1 activity takes place extracellularly. The same wastrue for the transfected PANC-1 cells; TGF-b1 was released into theculture medium in a latent,i.e., not activated, state. Recently, it wasdemonstrated that latent TGF-b1 can bind to and be activated by theavb6 integrin (44). These integrin subunits are also expressed bypancreatic carcinomas (15),i.e., the PANC-1 cells (6). Thus, activa-tion of the released TGF-b1 may be accomplished in this way.Expression of TGF-b1 resulted in an up-regulation of the matrixproteins 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 onfibroblasts in cocultivation experiments. We could demonstrate anincrease in collagen type I synthesis in the fibroblasts after stimulationwith supernatants from TGF-b1-transfected PANC-1 cells. Similarly,the activation of collagen type VII regulatory elements by TGF hasbeen described recently (45). The fibroblasts themselves producedmore TGF-b1 upon stimulation (cocultivation or conditioned media)by the TGF-b1-transfected PANC-1 cells. This is supported by theobservation that in pancreatic carcinoma tissue, TGF-b1 is mostpredominant in the stroma (46). Furthermore, collagen type I, the mostpredominant basal membrane matrix protein in pancreatic carcinoma

(10), is also up-regulated, both in the tumor cells themselves and inthe fibroblasts upon cocultivation. This up-regulation, however, mayonly be in part attributed to TGF-b1 itself; it could also be the resultof the up-regulation of PDGF-A that has been shown to be a cofactorin TGF-b1-induced collagen type I stimulation (23). In the fibroblasts,after cocultivation with TGF-b1-transfected PANC-1 cells,CTGF,one of the index TGF-b1 response genes (47), was increased. Inhibi-tion of CTGF abrogated the TGF-b1-induced collagen gene up-regulation, confirming the pivotal role of this growth factor (48). Asa result of these alterations in gene expression mentioned above, thetransfection of TGF-b1 in the pancreatic tumor cell line PANC-1 ledto a gain of stromal tissue after orthotopic transplantation in the nudemouse when compared with mock-transfected PANC-1 cells.

The influence of the matrix on signal transduction has long beenunder debate (49, 50). We have shown that a single growth factor,TGF-b1, is capable of conferring the desmoplastic potential to tumorcells not capable of these features. Some of the effects may beattributed to a direct effect of TGF-b1, whereas others,e.g., theup-regulation of collagen type I (51), may be the result of indirecteffects of TGF-b1 intimately associated with the signal transductionpathway involved in TGF-b1 activities.

ACKNOWLEDGMENTS

We thank Roland M. Schmid for assistance in subcloning the TGF-b1plasmid and Thomas Gress (both of University of Ulm, Ulm, Germany) forsupplying us with the CTGF plasmid.

Fig. 6. Orthotopic tumors after intrapancreaticinjection of TGF-b1-transfected PANC-1 cells (B,D, andF) and mock-transfected PANC-1 cells (A,C, andE) into the nude mouse pancreas.A andB,Masson-Goldner trichrome staining. Immunocyto-chemistry for collagen type I (Cand D) and fi-bronectin (EandF) is shown.

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2001;61:550-555. Cancer Res Matthias Löhr, Christian Schmidt, Jörg Ringel, et al. Experimental Model of Human Pancreatic Carcinoma

1 Induces Desmoplasia in anβTransforming Growth Factor-

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