Dermokine Expression in Intraductal Papillary-Mucinous Neoplasm and Invasive Pancreatic Carcinoma

Materials and Methods

Cell culture. A total of six pancreatic cancer cell lines were purchased from RIKEN BioResource Center (Tsukuba, Japan). All cells were maintained in Dulbecco's modified Eagle's medium (Sigma–Aldrich Japan, Tokyo, Japan), supplemented with 10% fetal calf serum.

Patients and samples. Serum samples and specimens of pancreatic neoplasms were obtained from patients (n=26) under a protocol approved by the Institutional Review Board of Kyoto Prefectural University of Medicine (KPUM) at the KPUM Hospital from 2006 to 2009. The eligibility criteria of patients were: (i) histologically-proven primary pancreatic tumor; (ii) no active double-cancer (synchronous or metachronous double-cancer); and (iii) no prior chemotherapy or radiotherapy for any other malignancy. Serum samples from randomly selected healthy volunteers were also collected from KPUM (n=25). All patients gave written informed consent, and all aspects of these studies were approved by the Ethics Committees of KPUM.

Blood was collected with the Vacutainer blood collection system (Kyokuto Pharmaceutical Industrial Co. Ltd. Tokyo, Japan). All serum samples were centrifuged (3,000 rpm, 5 min), aliquoted and stored at −80°C. Pancreatic tissues were fixed in 10% paraformaldehyde, and whole-tissue was prepared to make continuous sections along the pancreatic duct. At the Department of Pathology of KPUM, paraffin-embedded sections were subjected to immunohistochemical and hematoxylin and eosin (HE) staining to map for tumor distribution in the whole resected specimen. We analyzed samples of intraductal papillary mucin-producing adenoma (IPMA)/ intraductal papillary mucin-producing carcinoma (IPMC)/ordinary invasive ductal carcinoma derived from IPMN (n=12, cases1-10, 16, and 17) and IPMN with ordinary invasive carcinoma (n=5, cases11-15), as shown in Table I. Subtypes were of gastric type (n=12), intestinal type (n=2) and pancreatobiliary type (n=3), but there was no oncocytic type. One case of very mild dilatation of the pancreatic duct was included as IPMN because of the microscopically aggregated atypical and papillary lesion in the head of the pancreas.

Reverse-transcriptase polymerase chain reaction. For the reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of pancreatic cancer cell lines, total RNA was extracted from the pancreatic cancer cell lines using TRIsure (Nippon Genetics Tokyo, Japan). For detection of DK-γ mRNA and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), first-strand cDNA synthesis and RT-PCR were performed in duplicate with PrimeScript™ II 1st strand cDNA Synthesis kit (Takara Bio, Otsu, Japan), with oligo-dT primers. PCR was performed with AmpliTaq Gold®PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and PCR Thermal Cycler Dice (Takara Bio), according to the manufacturer's protocol. Primers for the experiment were as follows: DK-γ forward 5’-ATGCCATAAACAAGGACCAGAGAA-3’, reverse 5’-ACACCACC CTCTCATCACTAATCTC-3’; GAPDH forward 5’-ACCTGCC GTCTAGAAAAACCTGC-3’, reverse 5’-CTCCTCACAGTTGC CATGTAGACC-3’.

Antibody preparation. Monoclonal antibody (mAb) generation was performed by Kohjin Bio Co. Ltd. (Sakado, Japan) using the antigen hDK-βΔC-SEAP (His)6, as previously described (4). To detect serum DK-β/γ in patients with colorectal cancer, we established a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) with anti-DK-β/γ mAbs, as previously described (4). We generated anti-DK-β/γ mAb, not anti-DK-γ mAb, because the DK-γ-specific mAb was difficult to generate.

Immunohistochemistry. Paraffin sections (5-μm-thick) of tumor tissues were subjected to immunohistochemical staining for the DK protein with the tyramide amplification method, which uses fluorescyl-tyramide. Antigen retrieval was performed by heating the samples in Dako REAL Target Retrieval Solution (DAKO Japan. Tokyo, Japan), for 40 min at 98°C. Endogenous peroxidases were quenched by incubating the sections for 30 min in 3% H₂O₂. After a brief wash with phosphate-buffered saline (PBS) (pH 7.2) and 0.3% polyoxyethylene sorbitan monolaurate (Sigma–Aldrich), the sections were incubated for 45 min at room temperature with blocking reagent (Block Ace®; DS Pharma Biomedical Co. Osaka, Japan), to reduce the background signals. Each section was incubated at 4°C overnight with the anti-DK mAb. CSAII (Dako) was used for color development according to the manufacturer's protocols. The sections were counterstained with hematoxylin.

Detection of serum DK by ELISA. We developed a DK-specific ELISA that used an mAb against DK-β/γ. DK-β/γ was captured in 96-well plates for ELISA as follows. First, the captured mAb anti-DK-β/γ (IgG1) was added to each well of a 96-well plate and plates were incubated overnight at 4°C. Wells were then washed with PBS and incubated with PBS, containing 1% Block Ace (DS Pharmaceutical) to block non-specific antibody binding. The serum samples (prepared as described above) were added to each well, and the plates were incubated overnight at 4°C. Horseradish peroxidase-conjugated anti-DK-β/γ mAb (IgG2a) was added and the plates were incubated at room temperature for 1 h. DK was then detected with the tetramethylbenzidine Liquid Substrate System for ELISA (Sigma–Aldrich). The standard used in these assays was recombinant DK-β, expressed in 293/EBNA-1 cells. Standard curves were prepared for each assay. Limit of detection was estimated as 26.3 U/ml as mean of 20-control sample (from 10 separate sources) assay results plus 3-times the standard deviation of the mean. Limit of quantitation was estimated to be 36.7 U/ml as the mean of the same results plus 10-times of the standard deviation. Serum CEA, CA19-9 (Abbott Japan Co., Ltd., Tokyo, Japan) SPAN-1, DUPAN-2, elastase and NCC-ST-439 were quantified at a commercial laboratory (Falco Biosystems Ltd., Kyoto, Japan).