Human bile duct cancer cell lines (TFK-1, HuH-28, and MEC) were obtained from Cell Resource Center for Biomedical Research, Tohoku University. Bile duct cancer cell line TKKK and gallbladder carcinoma cell lines TGBC1TKB, TGBC2TKB, and TGBC14TKB were purchased from Riken Cell Bank (Tsukuba, Japan). Cells were cultured in RPMI1640 or DMEM supplemented with 10% fetal bovine serum. Formalin-fixed, paraffin-embedded sections of 61 biliary tract carcinomas (30 extrahepatic bile duct carcinomas, 18 gallbladder carcinomas, 13 carcinomas of the ampulla of Vater) were used for immunohistochemically. Sections containing the most invasive part of each tumor were used. Fresh specimens of extrahepatic bile duct carcinoma (n = 10), gallbladder carcinoma (n = 7), and carcinoma of the ampulla of Vater (n = 3) were obtained from patients who had undergone surgical treatment. Specimens were immediately frozen in liquid nitrogen at the time of surgery and stored at -80°C. Each tissue specimen was used for casein zymography. Histopathological features of the specimens were classified according to the pathological tumor-node-metastasis (TNM) classification system of the International Union Against Cancer. Informed consent was obtained from each subject and the institutional review committee approved the experiments.

Semi-quantitative RT-PCR was carried out as described previously[29]. Total RNA was extracted from cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA using SuperScript III reverse transcriptase (Invitrogen) with random hexamers. PCR was performed using primers specific for the LAMA3, LAMB2 and LAMC2 gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. GAPDH served as an internal control of the reaction. Standard curves for semiquantitative RT-PCR were drawn as described previously[30]. All reactions were carried out at least in duplicate and controlled without reverse transcriptase. PCR products were electrophoresed in 2% agarose gels. Real-time RT-PCR was performed using the TaqMan real-time PCR system as described previously[31].

Immunohistochemistry was carried out as described previously[32]. The antibodies used were as follows: anti-LNα3 rabbit polyclonal antibody (1/100 dilution, Santa Cruz, CA, USA), anti-laminin5 (γ2 chain) mouse monoclonal antibody (1/50 dilution, Chemicon, Temecula, CA, USA), anti-LNβ3 rabbit polyclonal antibody (1/100 dilution, Santa Cruz), and anti-MMP7 mouse monoclonal antibody (1/50 dilution, Daiichi Fine Chemical, Takaoka, Japan). Normal mouse or rabbit immunoglobulins were substituted for each primary antibody as negative controls. Cytoplasmic expression was defined as positive when immunoreactivity was observed in more than 10% of carcinoma cells. We defined the cells at the deepest invading part of the tumor as the invasive front.

Casein zymography was performed as previously described with some modifications[28]. Tissue extracts were electrophoresed on 8% polyacrylamide gel containing 1 mg/mL casein. After electrophoresis, gels were washed in 2.5% Triton-X 100 and incubated for 48 h at 37°C in 50 mmol/L Tris-HCl (pH 7.4), 10 mmol/L CaCl₂, 1 mmol/L ZnCl₂, and 0.02% NaN₃, followed by staining with 0.1% Coomassie brilliant blue.

siRNA transfection was performed as described previously[33]. Levels of MMP7 inhibition were analyzed by RT-PCR and Western blotting. siRNA-transfected cells were used for the in vitro invasion assay.

Assays were performed by the modified Boyden Chamber method as described previously[34]. Assays were also performed with 250 ng/mL of TIMP1, an MMP inhibitor. The results were presented as means ± SD for each sample.

Expression was assessed for associations with clinicopathological parameters using the chi-square two-tailed test or Fisher’s exact test. A P value < 0.05 was considered statistically significant. A P value between 0.05 and 0.10 was considered as a trend toward an association.

Expression levels of LNα3, LNβ3 and LNγ2, and MMP7 in cancer cell lines were analyzed using semi-quantitative RT-PCR (Figure 1). LNα3 mRNA was detected in all seven cell lines but at very low levels in TGBC-14TKB. LNβ3 mRNA was detected in six cell lines but at very low levels in MEC and TKKK. LNγ2 mRNA was detected in all seven cell lines but at very low levels in MEC. TFK-1, HuH-28, TGBC-1TKB, and TGBC-2TKB expressed considerable amounts of all 3 chains of LN5. MMP7 mRNA was detected in all seven cell lines. There were no significant correlations between these expression patterns and characteristics of the cell lines. Similar data were observed by real-time RT-PCR (data not shown).

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Figure 1 RT-PCR analysis of the LNα3, LNβ3, LNγ2 genes in biliary tract cancer cell lines. 1: MEC; 2: TFK-1; 3: HuH28; 4: TGBC1TKB; 5: TGBC2TKB; 6: TGBC14TKB; 7: TKKK.

Expression of LNα3 was detected in normal basement membranes but not in carcinoma cells (data not shown). Figure 2 shows representative results of immunohistochemistry for LNγ2 in biliary tract cancer. In carcinoma tissues, the cytoplasm of carcinoma cells was stained for LNγ2 at levels much stronger than those in normal basement membranes. The cytoplasmic immunoreactivity was often more intense at the invasive front. Cancer cells budding or dissociating from the tumor nests showed intense cytoplasmic staining. Sections with immunostaining signals in over 10% of carcinoma cells, which were observed in 35 (57%) of 61 cases, were judged to be positive for LNγ2. LNγ2 positivity was 67% in extrahepatic bile duct cancer, 50% in gallbladder cancer, and 46% in carcinoma of the ampulla of Vater. Figure 3 shows representative results of immunohistochemistry for LNβ3 in biliary tract cancer. In carcinoma tissues, the cytoplasm of carcinoma cells was stained for LNβ3 at levels much stronger than those in normal basement membranes. The expression pattern of LNβ3 was classified into 2 patterns: invasive front dominant pattern and diffuse pattern. The invasive front dominant pattern and diffuse pattern were observed in 23 (38%) and 17 (28%) of 61 cases, respectively. Positivity for invasive front dominant pattern and diffuse pattern was 47% and 17% in extrahepatic bile duct cancer, 28% and 39% in gallbladder cancer, and 31% and 38% in carcinoma of the ampulla of Vater. Figure 4 shows representative results of immunohistochemistry for MMP7 in biliary tract cancer tissues. MMP7 immunoreactivity was intense at the invasive front in several cases. In general, MMP7 immunoreactivity was diffuse rather than invasive front dominant like LNγ2. Sections with immunostaining signals in over 10% of carcinoma cells, which were observed in 42 (69%) of 61 cases, were judged to be positive for MMP7. MMP7 positivity was 80% in extrahepatic bile duct cancer, 50% in gallbladder cancer, and 69% in carcinoma of the ampulla of Vater.

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Figure 2 LNγ2 expression in biliary tract cancer tissues. A, B: Extrahepatic bile duct cancer tissues; A: Moderately differentiated tubular adenocarcinoma positive for staining. Note that LNγ2 is strongly positive in tumor cells at the invasive front; B: Papillary adenocarcinoma negative for staining; C, D: Gallbladder cancer tissues; C: Moderately differentiated tubular adenocarcinoma positive for staining; D: Well differentiated tubular adenocarcinoma negative for staining; E, F: Carcinoma tissues of the ampulla of Vater; E: Moderately differentiated tubular adenocarcinoma positive for staining. Note that LNγ2 is strongly positive in tumor cells at the invasive front; F: Well differentiated tubular adenocarcinoma negative for staining.

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Figure 3 LNβ3 expression in biliary tract cancer tissues. A-C: Extrahepatic bile duct cancer tissues; A: Well differentiated tubular adenocarcinoma positive for invasive front dominant staining; B: Papillary adenocarcinoma positive for diffuse staining; C: Moderately differentiated tubular adenocarcinoma negative for staining; D-F: Gallbladder cancer tissues; A-C: Extrahepatic bile duct cancer tissues; D: Moderately differentiated tubular adenocarcinoma positive for invasive front dominant staining; E: Papillary adenocarcinoma positive for diffuse staining; G-I: Carcinoma tissues of the ampulla of Vater; G: Well differentiated tubular adenocarcinoma positive for invasive front dominant staining; H: Well differentiated tubular adenocarcinoma positive for diffuse staining; I: Well differentiated tubular adenocarcinoma negative for staining.

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Figure 4 MMP7 expression in biliary tract cancer tissues. A, B: Extrahepatic bile duct cancer tissues; A: Moderately differentiated tubular adenocarcinoma positive for staining. Note that MMP7 is strongly positive in tumor cells at the invasive front; B: Papillary adenocarcinoma negative for staining; C, D: Gallbladder cancer tissues; C: Moderately differentiated tubular adenocarcinoma positive for staining; D: Well differentiated tubular adenocarcinoma negative for staining; E, F: Carcinoma tissues of the ampulla of Vater; E: Moderately differentiated tubular adenocarcinoma positive for staining. Note that MMP7 is strongly positive in tumor cells at the invasive front; F: Well differentiated tubular adenocarcinoma negative for staining.

The relationship between LNγ2 positivity and clinicopathological characteristics is summarized in Table 1. LNγ2 positivity was significantly correlated with histologic type (less differentiated type), depth of invasion (invasion into serosa), and advanced stage. The relationship between LNβ3 positivity and clinicopathological characteristics is summarized in Table 2. LNβ3 invasive front dominant pattern was significantly correlated with histologic type (less differentiated type) and advanced stage. There was a tendency that lymph node metastasis was more frequently observed in cases with LNβ3 invasive front dominant pattern than in other cases (P = 0.063). The relationship between MMP7 positivity and clinicopathological characteristics is summarized in Table 3. MMP7 positivity was not significantly correlated with clinicopathological characteristics. The expression of LNγ2, LNβ3 invasive front dominant pattern, and MMP7 were correlated with each other (Tables 456).

Using casein zymography, the levels of secreted matrilysin were analyzed (Figure 5). Nontumorous tissues secreted neither latent (28 kDa) nor activated (19 kDa) MMP7 activity. Latent and activated forms of MMP7 were detected in 16 (80%) and 12 (60%) of 20 carcinoma tissues, respectively. The activity was eliminated by the addition of the metalloproteinase inhibitor EDTA (data not shown). The activated form but not the latent form was correlated with depth of invasion and advanced stage.

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Figure 5 Casein zymography of surgical specimen pairs of biliary tract carcinoma and adjacent nontumor tissue. N and T: Matched samples from nontumor and tumor tissue, respectively.

In vitro invasion assays after treatment with specific siRNA for the MMP7 gene were carried out to assess the direct role of the expression of MMP7 in cancer cell invasiveness. Transfection efficiency determined by fluorescein isothiocyanate-labeled oligonucleotide uptake was 84% ± 6% in TFK-1 cells and 86% ± 5% in TGBC-2TKB cells (data not shown). Transfection with siRNA resulted in over 80% inhibition of mRNA and protein expression (data not shown). Transfection with MMP7-specific siRNA decreased invasiveness of TFK-1 cells compared with control siRNA-transfected counterparts (P < 0.01, Figure 6). This difference was significantly diminished by the addition of TIMP1. Similar results were observed in MMP7-specific siRNA-transfected TGBC-2TKB cells (data not shown).

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Figure 6 In vitro invasion assay with or without TIMP1 (250 ng/mL) in TFK-1 and siRNA transfectants. Each column indicates the means of three experiments; ^bP < 0.01.

Biliary tract cancers are relatively rare human malignancies involving the gallbladder and/or the bile ducts, but the prognosis is poor. Understanding the molecular biological features of biliary tract cancer progression is necessary for improving the prognosis. The potential role of laminin-5 (LN5) and MMP7 in human cancer is receiving increasing attention.

Altered expression patterns of LN5, especially the LNγ2 chain, and MMP7 have been correlated with tumor behavior, such as invasiveness, vascularization, metastatic potential, and patients’ poor prognosis. However, expression of LN5 and MMP7 in biliary tract cancer has not been clearly addressed. In this study, the authors demonstrate that LNγ2, LNβ3, and active MMP7 play a key role in the progression of biliary tract cancer.

This is the first study to report that invasive front dominant expression of LNγ2 and LNβ3, and active MMP7 play a key role in the progression of biliary tract cancer. Furthermore, our in vitro studies suggest that MMP7 plays an important role in biliary tract cancer cell invasion.

Detection of LNγ2, LNβ3, and active MMP7 could be molecular markers for tumor aggressiveness in biliary tract cancer. Understanding how LNγ2, LNβ3, and active MMP7 are induced and how their expression is blocked may represent a future strategy for therapeutic intervention in the treatment of patients with biliary tract cancer.

Laminin: A heterotrimeric glycoprotein composed of three different polypeptide chains (α, β and γ), is a component of the extracellular matrix (ECM) that contributes to the architecture of the basal lamina surrounding the epithelial cells and mediates cell adhesion, growth, migration, proliferation, and differentiation. LN-5/LN-332: LN5, consists of α3, β3 and γ2 chains, and is involved in cell adhesion, migration, and scattering. Altered expression of LN-5 plays an important role in cancer. MMP7: Degradation of ECM components is mostly controlled by proteolytic enzymes called MMP. MMP7, also known as matrilysin, is a minimal domain MMP that exhibits broad proteolytic activity against components of the ECM and non-ECM.

This paper reports the expression of LN-5 chains and MMP-7 in biliary cancer. The authors showed that LNγ2 and LNβ3, in conjunction with MMP7, play a key role in the progression of biliary cancer. The study sounds interesting and confirms the role of LN-5 and MMP7 in human cancer.