Journal of Histochemistry and Cytochemistry, Vol. 49, 539-549, April 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Comparative Analysis of Galectins in Primary Tumors and Tumor Metastasis in Human Pancreatic Cancer

Pascal O. Berberata, Helmut Friessa, Li Wanga, Zhaowen Zhua, Thorsten Bleya, Luciano Frigerib, Arthur Zimmermannc, and Markus W. Büchlera
a Department of Visceral and Transplantation Surgery, University of Bern, Inselspital, Bern, Switzerland
b The Scripps Research Institute, La Jolla, California,
c Institute of Pathology, University of Bern, Inselspital, Bern, Switzerland

Correspondence to: Helmut Friess, Dept. of Visceral and Transplantation Surgery, University of Bern, Inselspital, CH-3010 Bern, Switzerland. E-mail: helmut.friess@insel.ch


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Galectins are galactoside-binding proteins that exhibit an important function in tumor progression by promoting cancer cell invasion and metastasis formation. Using Northern blotting and Western blotting analysis, in situ hybridization (ISH), and immunohistochemistry (IHC), we studied galectin-1 and galectin-3 in tissue samples of 33 primary pancreatic cancers and in tumor metastases in comparison to 28 normal pancreases. Furthermore, the molecular findings were correlated with the clinical and histopathological parameters of the patients. Northern blotting and Western blotting analysis showed significantly higher galectin-1 and galectin-3 mRNA and protein levels in pancreatic cancer samples than in normal controls. For galectin-1, no ISH signals and immunoreactivity were observed in acinar or ductal cells in the normal pancreas and in pancreatic cancer cells, whereas fibroblasts and extracellular matrix cells around the cancer mass exhibited strong mRNA signals and immunoreactivity. Galectin-3 mRNA signals and immunoreactivity were strongly present in most pancreatic cancer cells, whereas in the normal controls only faint ISH and IHC signals were seen in some ductal cells. Metastatic pancreatic cancer cells exhibited moderate to strong galectin-3 immunoreactivity but were negative for galectin-1. No relationship between the galectin-1 and galectin-3 mRNA levels and the tumor stage or between the IHC staining score and the tumor stage was found. However, galectin-1 mRNA levels and the IHC staining score were significantly higher in poorly differentiated tumors compared with well/moderately differentiated tumors, whereas for galectin 3 no differences were found. The expression pattern of galectin-1 and galectin-3 in pancreatic cancer tissues indicates that galectin-1 plays a role in the desmoplastic reaction that occurrs around pancreatic cancer cells, whereas galectin-3 appears to be involved in cancer cell proliferation. High levels of galectin-3 in metastatic cancer cells suggest an impact on metastasis formation. (J Histochem Cytochem 49:539–549, 2001)

Key Words: galectins, pancreas, pancreatic carcinoma, metastasis


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IN THE SEARCH for genes that promote or suppress tumor spread, it has been reported that pancreatic cancer cells overexpress several growth factors and growth factor receptors which are associated with tumor aggressiveness and shorter patient survival (Almoguera 1988 ; Friess et al. 1999 ). In addition, oncogene mutations, e.g., of K-ras, and mutations in tumor suppressor genes such as p53 are also commonly found in pancreatic cancer (Lemoine et al. 1992 ; Perillo et al. 1998 ). These findings indicate that alterations in the expression of growth factors, growth factor receptors, and K-ras and p53 mutations are important pathophysiological mechanisms that appear to give pancreatic cancer cells a fundamental growth advantage (Almoguera 1988 ; Friess et al. 1999 ). However, at present only little is known about the mechanisms and factors that enable pancreatic cancer cells to metastasize even in early tumor stages when the primary tumor is still of limited size. Recently, we reported that the antimetastatic gene KAI1, a member of the transmembrane four family, is upregulated in early tumor stages compared with advanced tumor stages (Friess et al. 1998a , Friess et al. 1998b ). It was also shown that KAI1 expression is significantly increased in non-metastasic primary tumors and that low levels of KAI1 are present in metastasized tumor cells (Friess et al. 1998a , Friess et al. 1998b ). These findings suggested that KAI1 might influence the metastatic ability of pancreatic cancer cells in vivo. Its role as a regulator of metastasis in other GI tumors has also been reported.

A potentially new pro-metastatic functioning gene family named the galectins was recently discovered (Barondes et al. 1994 ; Perillo et al. 1998 ; Andre et al. 1999 ). Members of the galectin family were proposed to promote cell transformation to a metastatic phenotype, to stimulate tumor growth, and to protect the immune surveillance of several gastrointestinal and other tumors (Barondes et al. 1994 ; Perillo et al. 1998 ).

Galectins are a family of ß-galactoside-binding proteins, also known as S-type lectins. They have been proposed to regulate cell growth, to mediate cell adhesion, and to influence apoptosis. At least 10 different animal galectins have been isolated, and all of them recognize galactose-containing oligosaccharides. There appears to be a difference in the carbohydrate specificity of the various galectins, with the implication of different functional roles among the members of the family (Gitt and Barondes 1986 ; Caron et al. 1990 ). In general, galectins are present in the cytoplasm and in the nucleus as well as extracellularly on the cell surface and especially within the extracellular matrix (Cherayil et al. 1989 ; Cooper and Barondes 1990 ). The secretion of galectins appears to be strongly regulated and of high complexity. Recent studies demonstrated that these proteins most probably do not use the classical secretory compartments such as the endoplasmic reticulum and the Golgi. It appears more likely that, depending on the structure of the galectin and the cell type, different alternative pathways are used, such as direct translocation or ectocytosis (Hughes 1999 ). The two galectins studied in the greatest detail are galectin-1 and galectin-3 (Perillo et al. 1998 ). Galectin-1 (also named galaptin and L-14.5) is a homodimer of 14-kD subunits that possesses two galactoside-binding sites and is expressed at various levels in many tissues under normal and pathological conditions (Catt et al. 1987 ; Bourne et al. 1994 ; Perillo et al. 1998 ). The function of galectins depends on their binding to specific ligands, such as laminin, lysosome-associated membrane proteins (LAMPs), and fibronectin (Do et al. 1990 ; Ozeki et al. 1995 ), which are localized either on the cell surface or in the extracellular matrix.

Galectin-3 (also named Mac-2, {varepsilon}-BP, L-34, L-29, CBP-35, CBP-30, hL-31, or LBL) is a monomer with two functional domains (Hughes 1997 ). It is thus far unique in having an extra long N-terminal domain that can bind to RNA (Wang et al. 1995 ). Therefore, in contrast to galectin-1, galectin-3 is also able to bind intracellular ligands, such as RNA within the nucleus (Wang et al. 1995 ) or bcl-2, a cytoplasmic regulator of apoptosis (Yang et al. 1996 ). Moverover, galectin-3 also binds a variety of ligands, such as laminin, carcinoembryogenic antigen (CEA), and LAMP-1 and -2, which are located in the extracellular matrix (Ohannesian et al. 1995 ).

Galectin-1 and galectin-3 overexpression has been detected in several tumors (Lotan et al. 1994 ; Schoeppner et al. 1995 ; Gillenwater et al. 1996 , Gillenwater et al. 1998 ; Bresalier et al. 1997 ; Sanjuan et al. 1997 ; Hsu et al. 1999 ). Recently, Schaffert and co-workers (1998) studied galectin-3 for the first time in pancreatic cancer. By looking at different pancreatic cancer lines and human pancreatic carcinoma tissue, they demonstrated a uniform and strong overexpression of galectin-3 in pancreatic cancer cells compared to normal controls. A correlation with their differentiation status could not be shown. Furthermore, it was also demonstrated that these observed features are not unique to malignant lesions. Chronic pancreatitis tissue samples also showed overexpression of galectin-3 in a similar pattern (Schaffert et al. 1998 ). Looking at galectin-3 in other malignant lesions, it has been reported, in contrast, that it is inversely correlated with the metastatic potential in some tumors and is in this way directly associated with more advanced stages of the tumors (Lotz et al. 1993 ; Xu et al. 1995 ; Castronovo et al. 1996 ). In functional studies it has been shown that the introduction of recombinant human galectin-3 into non-tumorigenic galectin-3-null breast cancer cells leads to tumor formation in athymic mice. Furthermore, in colon cancer metastasis formation is increased by elevated galectin-3 expression in the cancer cells (Bresalier et al. 1998 ).

The aim of this study was to continue the work of Schaffert and co-workers (1998) by analyzing galectin-1 and galectin-3 expression in a large representative number of pancreatic cancers and to correlate the molecular findings with clinicopathological patient data to further evaluate galectin's influence on tumor growth behavior and metastasis formation.


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Patients
Normal human pancreatic tissue samples were obtained from 28 individuals (11 women, 17 men; median age 43 years; range 18–57 years) through a multiorgan donor program in which no recipients were available for pancreatic transplantation. Tissue samples of 33 patients with carcinoma of the pancreas (17 women and 16 men; median age 67 years; range 49–83 years) were obtained after a Whipple resection. The patients were operated on in the Department of Visceral and Transplantation Surgery, Inselspital. From the patients resected since November of 1993, the tissue samples taken for this study were randomly selected. The diagnosis of pancreatic adenocarcinoma was confirmed by histopathological analysis. According to the TNM classification (Wittekind and Wagner 1997 ), there were eight Stage I, five Stage II, and 20 Stage III tumors. Grading was well-differentiated in seven tumors, moderately differentiated in 12, and poorly differentiated in 14.

Tissue Sampling
For RNA and protein extraction, normal and tumor specimens were frozen in liquid nitrogen immediately after surgical removal and stored at -80C until use. In addition, freshly removed normal and cancerous tissue samples were immediately fixed in formaldehyde solution for 12–24 hr and paraffin-embedded for ISH and IHC. The studies were approved by the ethical committee of the University of Bern, Switzerland.

Northern Blotting Analysis
Total RNA was extracted by the single-step guanidinium isothiocyanate method, size-fractionated on 1.2% agarose/1.8 mol/liter formaldehyde gels, and stained with ethidium bromide for verification of RNA integrity and loading equivalency (Korc et al. 1992 ; Friess et al. 1993 ). The RNA was electro-transferred onto nylon membranes and crosslinked by UV irradiation. For hybridization, 32P-labeled antisense galectin-1 and galectin-3 cRNA probes and a 32P-labeled 7S cDNA probe were used.

For the cRNA probes, the blots were prehybridized overnight at 65C and hybridized for 18 hr at 65C in the presence of the 32P-labeled antisense galectin-1 and galectin-3 riboprobes, washed twice at 65C in a solution containing 2 x SSC (0.3 M NaCl, 0.03 M Na3 citrate, pH 7) and 0.5% SDS, then washed twice for 15 min at 65C in a solution containing 0.1 x SSC and 0.5% SDS (Korc et al. 1992 ; Friess et al. 1993 , Friess et al. 1998a , Friess et al. 1998b ).

To assess equivalent RNA loading, the membranes were rehybridized with the 32P-labeled mouse 7S cDNA probe that cross-hybridizes with human 7S RNA, as reported previously (Friess et al. 1998a , Friess et al. 1998b ). The blots were then exposed at -80C to Fuji X-ray film with intensifying screens for 24–48 hr. The intensity of the galectin-1 and galectin-3 signals was quantified by video densitometry analysis (Biorad 620; New York, NY) as previously reported (Friess et al. 1998a , Friess et al. 1998b ). The ratio between the galectin-1 and galectin-3 signals and the corresponding 7S signal was calculated for each sample.

In Situ Hybridization
ISH was performed as reported in detail previously using DIG-labeled cRNA probes (Lu et al. 1997 ; Guo et al. 1998 ). Tissue sections of normal and cancerous tissue samples were always processed simultaneously. In addition, tissue slides were processed consecutively, one slide being incubated with the sense probe and the other simultaneously with the antisense probe. Four-µm tissue sections were deparaffinized, rehydrated, and incubated in 0.2 mol/liter HCl for 20 min. After washing with 2 x SSC, the tissues were permeabilized with proteinase K at a concentration of 35 µg/ml for 15 min at 37C. After a postfixation with 4% paraformaldehyde in saline phosphate buffer (5 min) and washing in 2 x SSC, the sections were prehybridized for 1 hr at 60C in a buffer containing 50% formamide, 4 x SSC, 2 x Denhardt's reagent, and 250 µg RNA/ml. Hybridization was performed overnight at the same temperature in 50% formamide, 4 x SSC, 2 x Denhardt's reagent, 500 µg RNA/ml, and 10% dextran sulfate. The final concentration of the DIG-labeled probes (antisense or sense) was approximately 0.5 ng/µl. After hybridization, excess probe was removed by washing in 2 x SSC and by RNase treatment: 100 U/ml RNase T1 and 0.2 µg/ml RNase DNase-free (Roche Diagnostics; Rotkreuz, Switzerland) at 37C for 30 min. Washings were performed at 60C for galectin-1 and 63C for galectin-3 in 2 x SSC (10 min) and twice in 0.2 x SSC (10 min each). Then the sections were incubated with an anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche Diagnostics). For the color reaction, 5-bromo-4-chloro-3-indolyl phosphatase and nitroblue tetrazolium (Sigma; Buchs, Switzerland) were used.

Pretreatment of the slides with RNAse abolished the hybridization signals and hybridization with the appropriate sense probes failed to produce an ISH signal.

The ISH signals were semiquantitatively evaluated by two independent observers unaware of patient status. This was followed by resolution of any differences by joint review and consultation with a third observer. The ISH results were scored as previously described (Guo et al. 1998 ): -, no detectable signal; +, weak detectable signal; ++, moderate detectable signal; and +++, strong detectable signal.

Preparation of the Galectin cRNA Probes
A 245-bp fragment of the human galectin-1 cDNA (corresponding to nucleotides 165–410) and a 352-bp fragment of the human galectin-3 cDNA (corresponding to nucleotides 382–734) were generated by RT-PCR and subcloned into the pGEM-T Easy vector (Promega Biotechnology; Madison, WI), which contains promoters for DNA-dependent SP6 and T7 RNA polymerases. The identity of the cDNA fragments was confirmed by sequence analysis using the dye terminator method (ABI 373A; Perkin Elmer, Rotkreuz, Switzerland). After linearization of the plasmid, the antisense galectin-1 and -3 probes were transcribed using SP6 polymerase and the Ribomax System (Promega Biotechnology). 32P-labeled galectin-1 and -3 cRNA probes were used for Northern blotting analysis, whereas ISH was performed with DIG-labeled cRNA probes, generated by using the Ribomax System (Promega Biotechnology) (Lu et al. 1997 ). Specificity of the ISH reaction was tested by comparing sense and antisense DIG-labeled probes.

Preparation of 7S cDNA Probe
To verify equivalent RNA loading and transfer on Northern blotting membranes, all filters were rehybridized with a 212-bp fragment of 7S cDNA. The 7S cDNA probe was radiolabeled with [{alpha}-32P]-dCTP using a random primer labeling system (Pharmacia Biotech; Dubendorf, Switzerland) (Korc et al. 1992 ).

Immunohistochemistry
After deparaffinizing and rehydrating, 3-µm tissue sections were submerged for 15 min in Tris-buffered saline (10 mmol/liter Tris-HCl, 0.85% NaCl, pH 7.4) containing 0.1% Triton X-100 and briefly rinsed three times for 1–2 min in TBS solution. After incubation in methanol containing 0.6% hydrogen peroxide for 30 min to block endogenous peroxidase activity, the slides were covered with 10% normal goat serum at 23C for 30 min before overnight incubation at 4C with a polyclonal rabbit anti-galectin-1 antibody (kindly supplied by Dr. Douglas N.W. Cooper (Langley Porter Psychiatric Institute; University of California, San Francisco) or anti-galectin-3 antibody. The specificity of the galectin-1 and galectin-3 antibodies has already been shown in previous studies (Cooper and Barondes 1990 ; Frigeri and Liu 1992 ). The galectin-1 antibody was generated by immunization of rabbits with the whole rat galectin-1 antibody. The specificity of the antibodies has been shown previously (Cerra et al. 1985 ; Cooper and Barondes 1990 ). The galactin-3 antibody was raised in rabbits using recombinant purified galectin-3. Because of the lectin activity it was not necessary to produce a fusion or His-tagged protein. The antibody was affinity-purified from the immune serum with ammonium sulfate, followed by affinity-purification using purified galectin-3 bound to CNBr-activated Sepharose, as reported previously (Frigeri and Liu 1992 ).

After washing with TBS buffer, biotinylated goat anti-mouse immunoglobulin and streptavidin–peroxidase complex (Kirkegaard & Perry Laboratories; Gaithersburg, MD) were added at 23C for 45 min, followed by incubation with a 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide mixture (Lu et al. 1997 ). The slides were counterstained with Mayer's hematoxylin. To ensure specificity of the primary antibodies, consecutive sections were incubated either in the absence of the primary antibody or with a non-immunized rabbit IgG antibody. In these cases no immunostaining was detected.

The IHC results from all pancreatic cancer tissues were analyzed semiquantitatively by two independent observers, followed by resolution of any differences by joint review and consultation with a third observer. The intensity of immunostaining and the percentage of immunoreactive cancer cells were evaluated. The staining intensity was recorded as 0, no immunostaining; 1, weak immunostaining; 2, moderate immunostaining; and 3, intense immunostaining. An IHC staining score was calculated for each sample as intensity times percentage of immunopositive cancer cells, as reported previously (Lu et al. 1997 ).

Western Blotting Analysis
Approximately 200 mg of tissue samples was powdered in liquid nitrogen and then homogenized in lysis buffer (150 mmol/liter NaCl, 10 mmol/liter Tris-HCl, pH 7.5) supplemented with protease inhibitor cocktail (Roche Diagnostics). The lysate was collected and centrifuged at 4C for 30 min at 14,000 rpm to remove the insoluble material. The protein concentration of the supernatant was measured by spectrophotometry using the BCA protein assay (Pierce; Rockford, IL). Protein extracts were subjected to 12% (galectin-1) and 7% (galectin-3) SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose filters. For immunoblotting, unspecific binding was blocked with 5–10% non-fat milk and the membranes were incubated with the primary antibodies (0.01 µg/ml for anti-galectin-1 and 0.5 µg/ml for anti-galectin-3) at room temperature (RT) for 1 hr. Then the filters were washed in TBST buffer (5% non-fat milk in 20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) and incubated with a horseradish peroxidase-conjugated donkey anti rabbit IgG at RT for 30 min. After washing in TBST buffer, the signal was detected using the enhanced chemiluminescence (ECL) method. The intensity of the radiographic bands for each normal and pancreatic cancer sample was quantified by video densitometry.

Statistical Analysis
Results are expressed as median and range or as mean ± SD. For statistical analysis, the Student's t-test, the Pearson correlation analysis, and a multivariate Cox analysis were used. Significance was defined as p<0.05.


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Northern Blotting Analysis
Northern blotting analysis revealed high expression levels of galectin-1 and galectin-3 mRNA in pancreatic cancer samples compared with normal controls. As shown in Fig 1, galectin-1 and galectin-3 mRNA signals in pancreatic organ donor tissues were weak or too low to be detected by Northern blotting analysis (Fig 1, Lanes 1–10). In contrast, galectin-1 and galectin-3 mRNA levels were markedly increased in most of the pancreatic cancer tissues (Fig 1, Lanes 11–22). For galectin-1, 88% (30/34) of the pancreatic cancer tissues exhibited elevated mRNA levels in comparison with normal controls. By densitometry, galectin-1 mRNA levels were 11.9-fold higher in the cancer samples than in normal controls (p<0.001). Galectin-3 mRNA expression was increased in 77% (26/34) of the cancer samples. Densitometric analysis revealed that galectin-3 mRNA expression levels were 4.7-fold higher in the cancer samples compared with the normal controls (p<0.005). In the remaining pancreatic cancers, galectin-1 and galectin-3 mRNA expression was similar to that of the normal controls.



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Figure 1. Northern blotting analysis of galectin-1 and galectin-3 mRNA in normal pancreas (Lanes 1–10) and pancreatic cancer (Lanes 11–22). Galectin-1 and galectin-3 mRNA levels were increased in most pancreatic cancer samples.

When galectin-1 and galectin-3 mRNA expression was analyzed simultaneously in pancreatic cancer samples, 74% (25/34) of the pancreatic cancers exhibited concomitant overexpression of galectin-1 and -3. In 9% (3/34) of the pancreatic cancers, galectin-1 and -3 were simultaneously not increased; in 14% (5/34) galectin-1 was increased but not galectin-3, and in 3% (1/34) of the cancers galectin-3 was increased but not galectin-1.

In Situ Hybridization
ISH was performed to determine the exact site of galectin-1 and galectin-3 mRNA expression in the normal and cancerous tissue. Consecutive slides were hybridized with the galectin-1 and galectin-3 antisense and sense probes. In the normal pancreatic tissue samples, only faint or no mRNA signals of galectin-1 (Fig 2A) were present in some fibroblasts but not in acinar or ductal cells. Weak galectin-3 mRNA signals (Fig 2B) were found in some ductal cells in the normal pancreas. In contrast, there were moderate to strong galectin-1 mRNA signals in most of the fibroblasts of the extracellular matrix in and around the pancreatic cancer mass (Fig 2C and Fig 2E), but no signals were seen in the pancreatic cancer cells. In contrast, strong galectin-3 mRNA staining was present in most pancreatic cancer cells (Fig 2D) and was absent in the extracellular matrix.



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Figure 2. In situ hybridization. In the normal tissue, faint galectin-1 mRNA signals (A) were present in fibroblasts and faint galectin-3 mRNA signals (B) in ductal cells. In pancreatic cancer, galectin-1 mRNA signals (C,E) were strongly present in most fibroblasts of the extracellular matrix around the cancer mass, whereas the cancer cells were negative. In contrast, intense galectin-3 mRNA signals (D) were present in pancreatic cancer cells. Galectin-1 (C, inset) and galectin-3 (D, inset) mRNA signals were also present in the pancreatic nerves. (F) A section with pancreatic cancer cells hybridized with the galectin-3 sense probe. Original magnifications: A–C x 200; D–F x 400.

In addition, moderate to strong galectin-1 (Fig 2C, inset) and galectin-3 (Fig 2D, inset) mRNA signals were present in pancreatic nerves in normal and in cancerous tissue samples.

Immunohistochemistry
The IHC staining pattern in the normal and cancerous pancreatic tissues was similar to that of ISH. In the normal controls, no galectin-1 (Fig 3A) immunostaining was found in the acinar, ductal, and islet cells. Faint galectin-1 immunostaining was present in some fibroblasts of the intra- and interlobular stroma (Fig 3A) of the normal pancreas. Faint galectin-3 immunoreactivity was present in some ductal cells, whereas no signals were found in acinar cells (Fig 3C). Pancreatic cancer cells showed moderate to strong immunoreactivity for galectin-3 (Fig 3D) but not for galectin-1 (Fig 3B). In contrast, strong galectin-1 immunoreactivity was present in the fibroblasts of fibrotic tissue strains in and around the tumor mass (Fig 3B). Furthermore, strong galectin-3 immunoreactivity was present in the cancer cells of metastatic lesions in lymph nodes (Fig 3E, arrows) and in the liver (Fig 3F), whereas no immunostaining was found for galectin-1 in metastatic pancreatic cells (data not shown).



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Figure 3. Immunohistochemical staining. Some fibroblasts in the normal pancreas exhibit faint galectin-1 immunoreactivity (A, arrows). Strong galectin-1 immunostaining was observed in fibroblasts of the extracellular matrix surrounding the cancer mass (B). Galectin-3 immunostaining was present in some ductal cells in the normal pancreas (C). In contrast, most of the pancreatic cancer cells exhibited intense galectin-3 immunoreactivity (D). Galectin-1 (B, inset) and galectin-3 (D, inset) immunoreactivity was also found in pancreatic nerves. Pancreatic cancer cells (arrows) that had metastasized to lymph nodes (E) or to the liver (F) exhibited strong galectin-3 immunoreactivity but were negative for galectin-1 (data not shown). (F) Arrowheads mark border of normal liver. Original magnification x 200.

Pancreatic nerves in the normal and in the cancerous tissues exhibited strong galectin-1 (Fig 3B, inset) and galectin-3 (Fig 3D, inset) immunoreactivity.

Western Blotting Analysis
To semiquantitatively compare the presence of galectin-1 and galectin-3 proteins in the normal and cancerous tissue samples and to confirm the stronger immunoreactivity in pancreatic cancer than in the normal pancreas, Western blotting analysis was performed. Fig 4 shows markedly higher galectin-1 and galectin-3 protein levels in pancreatic cancer tissue samples (Fig 4, Lanes 3–8) in comparison with normal controls. Densitometry analysis revealed that galectin-1 protein levels were 8.6-fold higher and those of galectin-3 were 2.5-fold higher in pancreatic cancer than in the normal pancreatic tissue samples.



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Figure 4. Western blotting analysis. Galectin-1 and galectin-3 were increased in pancreatic cancer samples (Lanes 3–8) compared with normal controls (Lanes 1 and 2).

Correlation Between the Molecular Findings and the Histopathological Parameters
The fold increase of galectin-1 and galectin-3 mRNA levels above the normal controls obtained by Northern blotting analysis was compared in tumor samples without metastases (Stages I + II) and tumors with lymph node metastases (Stage III) and for well/moderately differentiated (G1 + 2) and poorly differentiated (G3) tumors. Statistical analysis revealed no difference in galectin-1 and galectin-3 mRNA levels between tumors with and without metastases. Galectin-3 mRNA levels were also not different between G1 + 2 and G3 tumors; however, galectin-1 mRNA levels were significantly higher (p=0.01) in G3 tumors compared with G1 + 2 tumors. In awareness of the limitation of such whole tissue examinations, we performed similar analysis using the IHC staining score in the 33 pancreatic cancer samples (Table 1). There was no difference in the mean immunohistochemical staining score of galectin-1 and galectin-3 between metastatic (Stage III) and non-metastatic (Stage I/II) tumors. The mean galectin-3 immunohistochemical staining score was also not different in G1 + 2 tumors vs G3 tumors. However, in G3 tumors the galectin-1 IHC staining score was significantly higher than in G1 + 2 tumors (p=0.02).


 
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Table 1. Galectin-1 and galectin-3 immunohistochemical staining scores depending on the tumor stage and tumor differentiationa

Pearson correlation analysis revealed no relationship between the galectin-1/galectin-3 IHC staining score and the tumor stage or between the galectin-3 IHC staining score and the tumor grading. However, a significant relationship (r=0.38, p=0.03) was present between the galectin-1 IHC staining score and the tumor grading. Furthermore, multivariate Cox analysis, including age, presence of metastasis, tumor stage, tumor grading, the galectin-1 or galectin-3 IHC staining score, and the galectin-1 or galectin-3 mRNA expression levels, showed no significant relationship. No relationship was found either between the galectin-1 and galectin-3 mRNA levels and postoperative survival or between the IHC staining score of galectin-1 and galectin-3 and postoperative survival.


  Discussion
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Pancreatic cancer is characterized by extremely aggressive growth, with early development of metastases in lymph nodes and distant organs (Ellenrieder et al. 1999 ). The early invasive properties lead to growth of the tumor around and in major abdominal vessels, the neighboring organs, and the retroperitoneal bed, which makes curative resection often impossible (Gudjonsson 1987 ; Warshaw and Fernandez-del Castillo 1992 ; Parker et al. 1997 ).

In the processes of local invasion and metastasis, tumor cells leave their primary tumor, protrude through the surrounding extracellular matrix and the endothelium, invade blood and lymph vessels, and finally attach and proliferate at a distant site (Ellenrieder et al. 1999 ). At present, little is known about the molecular and cellular mechanisms that contribute to this cascade of events that leads to local tumor invasion and formation of distant metastases. However, we know that every normal cell expresses at its membrane surface a large variety of receptor molecules (cell adhesion molecules). These receptor molecules are involved in cell-to-cell communication and characterize the cell's position and function in the community with other cells and the extracellular matrix (Zetter 1993 ; Ellenrieder et al. 1999 ).

Through malignant cell transformation, the pattern of surface molecules can be dynamically changed. Thus, the cancer cells develop the ability to disrupt and invade normal tissue structures and finally form metastases in distant organs (Woodhouse et al. 1997 ; Meyer and Hart 1998 ).

Galectins have been implicated as key factors in the processes of malignant transformation and metastasis in a variety of gastrointestinal tumors, including stomach, hepatocellular, and colon cancer (Lotz et al. 1993 ; Lotan et al. 1994 ; Schoeppner et al. 1995 ; Bresalier et al. 1997 ; Sanjuan et al. 1997 ; Hsu et al. 1999 ). In gastric and hepatocellular cancer, significantly higher levels of galectin-3 were demonstrated in the cancer cells in comparison to the normal mucosa cells or normal hepatocytes (Lotan et al. 1994 ; Hsu et al. 1999 ), and overexpression of galectin-3 in gastric cancer is correlated with metastasis formation (Lotan et al. 1994 ). However, controversial results were reported in colon cancer. Several recent studies showed significantly higher levels of galectin-1 and galectin-3 in colon cancer in comparison to the normal mucosa, but also that their overexpression is associated with advanced tumor stages and shorter survival and therefore worsens the prognosis (Schoeppner et al. 1995 ; Bresalier et al. 1997 ; Sanjuan et al. 1997 ). Furthermore, transfection of galectin-3 in colon cancer cells enhances their potential to form liver metastases in athymic mice (Bresalier et al. 1998 ). In contrast, earlier studies reported decreasing galectin-3 levels in colon cancer progression and in other malignancies such as breast and thyroid cancer (Lotz et al. 1993 ; Xu et al. 1995 ; Castronovo et al. 1996 ). The meaning of these contradictory results cannot be completely explained at present. However, in addition to different technical approaches in galectin detection, bi-modal functions of galectins, including positive and negative growth-regulatory properties, depending on various tumor-specific factors, might be of importance, because they have already been described for galectin-1 and other tumor factors, like TGF-ß.

In a first immunohistochemical analysis in a small number of pancreatic cancer samples and cell lines, increased immunoreactivity of galectin-3 was reported (Schaffert et al. 1998 ). These findings suggested that galectins may have some implications in tumor progression and metastasis formation in pancreatic cancer as well.

It was the goal of this study to further analyze galectin-3 in a larger series of human pancreatic cancer samples and corresponding metastatic lesions. Furthermore, we also investigated galectin-1, which had not been previously studied in pancreatic cancer. Our present data show that galectin-3 and galectin-1 are strongly overexpressed at the mRNA and protein level in human pancreatic cancer in comparison to normal human pancreas. ISH and IHC confirmed the results of the Northern and Western blotting analyses. Galectin-3 mRNA and protein signals were present in most cancer cells in more than two thirds of the pancreatic cancer samples. In comparing the immunohistochemical staining pattern of metastatic and non-metastatic primary pancreatic cancer samples, we found no difference. The presence of galectin-3 in pancreatic cancer cells was not associated with advanced disease, as has been proposed in other malignancies (Lotz et al. 1993 ; Lotan et al. 1994 ; Schoeppner et al. 1995 ; Bresalier et al. 1997 ; Sanjuan et al. 1997 ; Hsu et al. 1999 ). Similar observations of increased galectin-3 levels were made in pre-neoplastic hamster pancreatic ducts, suggesting a role of galectin-3 even in the early course of pancreatic cancer (Schaffert et al. 1998 ). On the other hand, metastatic pancreatic cancer cells in lymph nodes and in the liver showed strong galectin-3 immunoreactivity in this study, indicating that galectin-3 may affect tumor cell spread. In summary, galectin-3 appears to have a role in pancreatic cancer lesions from an early stage onward, and to maintain this function also after development of metastatic disease. Furthermore, galectin-3 is able to promote longer survival of cells through its anti-apoptotic properties (Yang et al. 1996 ). It is believed that this effect is functional based on the NWGR motif, which is found in the COOH end of galectin-3. This motif is a highly conserved domain in the well-known anti-apoptotic Bcl-2 protein family (Akahani et al. 1997 ). Recently, it was also shown that breast carcinoma cell lines, which are stable when transfected with galectin-3, have improved adhesive properties with regard to the extracellular matrix and are consequently much more resistant to apoptosis (Matarrese et al. 2000 ). This anti-apoptotic effect of galectin-3 could explain the role of the overexpression of this protein in tumor cells in the course of pancreatic cancer.

Thus far, no information has been available on galectin-1 in pancreatic cancer. mRNA and protein analysis of galectin-1 revealed its low abundance in the normal pancreas. However, strong immunostaining was observed in most fibroblasts in the stromal strains of desmoplastic tissue in and around the pancreatic cancer mass. These galectin-1 signals in the extracellular matrix were more intense and were uniformly present in the fibroblasts in and around the cancer lesions in comparison to the normal pancreatic stroma. However, no galectin-1 expression was detectable in pancreatic cancer cells, which is similar to the findings in colon cancer, in which galectin-1 overexpression in the stromal component has already been observed (Schoeppner et al. 1995 ). Moreover, galectin-1 has also been shown to be involved in apoptosis regulation. In contrast to galectin-3, it appears to induce apoptosis, especially in activated T-cells (Perillo et al. 1995 , Perillo et al. 1997 ). This could affect cancer growth in two ways. First, through its immunomodulatory properties, galectin-1 could give pancreatic cancer the possibility of escaping the immune response. More probably, because the galectin-1 is overexpressed in fibroblasts, it plays a role in remodeling the extracellular matrix in the formation of the desmoplastic reaction.

Overexpression of galectin-1 and -3 in pancreatic cancer did not show a relationship with patient or histopathological tumor parameters, nor was a relationship found with the tumor stage or postoperative survival. However, there was a tendency toward higher galectin-1 and galectin-3 expression in more dedifferentiated cancer samples (G3).

In conclusion, the expression pattern of galectin-1 and galectin-3 in pancreatic cancer tissues implies a role of galectin-1 in the desmoplastic reaction occurring around the cancer cells, whereas galectin-3 appears to be involved in cancer cell development and growth.


  Acknowledgments

Supported by the Swiss National Foundation (SNF Grant 32-049494) and by a Research Grant of the Department of Clinical Research of the University of Bern.

We would like to thank Dr Cooper Douglas for giving us his anti-galectin-1 antibody and Dr Hans Graber for his technical support in preparing the galectin-1 and galectin-3 cDNA probes.

Received for publication June 1, 2000; accepted November 22, 2000.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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