Frozen normal human tissues were received from the Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan. PBMCs were obtained from healthy Japanese adult volunteers. HEK293 and 293FT cells were purchased from Invitrogen Corporation (Carlsbad, CA, USA). The human pancreatic cancer cell line PANC-1 was provided by RIKEN (Tsukuba, Japan), PK-9 and PK-45P were from Tohoku University (Sendai, Japan), and SUIT-2 was from Health Science Research Resources Bank (Osaka, Japan). Human pancreatic cancer xenografts were established by subcutaneous injection of these cell lines into female BALB/c-SCID mice.

Total RNA from normal human adrenal gland was extracted by the RNeasy Mini kit (Qiagen, Tokyo, Japan). The cDNA synthesis reaction was performed as previously described (19). ETBR-cDNA was amplified by PCR. Briefly, each 50-μl reaction mixture contained 1 μl of reverse transcription reaction products, 1 unit of KOD-Plus-DNA polymerase, 5 μl PCR buffer, 5 μl of 2 mM deoxynucleotide triphosphate, 2 μl of 25-mM MgSO₄ (all from Toyobo, Osaka, Japan), and 1.5 μl of each 10-μM 3′ and 5′ primer specific for ETBR (sense, 5′-CGGCTAGCCCTTCTGGAGCAGGTA-3′ and antisense, 5′-CGCGGATCCTCAAGATGAGCTGTA-3′). ETBR cDNA was amplified for 30 cycles. Conditions for ETBR PCR were: 94°C for 15 sec, 40°C for 30 sec and then 68°C for 3 min. All PCR products were electrophoresed in a 2.0% agarose gel and visualized by ethidium bromide staining. Plasmids expressing ETBR were generated by the PCR amplification of ETBR cDNA and cloning into the NheI and BamHI sites of pcDNA3.1(+) (Invitrogen).

Subsequently, pcDNA-3.1(+)-ETBR-IRES-GFP (pETBR) was transfected into 293FT or HEK293 cells using Lipofectamine^® 2000 (Invitrogen). Transfected cells were incubated at 37°C in a 5% CO₂ incubator for 20 h prior to harvest.

Total RNA extraction and cDNA synthesis of normal human tissues were performed as described above. Multiplex PCR was performed using primers specific for ETBR or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Briefly, each 20-μl reaction mixture contained 1 μl reverse transcription reaction products, 0.2 μl Taq DNA polymerase, 4 μl reaction buffer (both from Promega, Madison, WI, USA), 0.5 μl of 10-mM deoxynucleotide triphosphate, and 0.5 μl of each 10-μM 3′ and 5′ primer specific for ETBR (as described above) and GAPDH (sense, 5′-ACCCCTTCATTGACCTCAACT-3′ and antisense, 5′-TGAGTCCTTCCACGATACCAA-3′). ETBR and GAPDH cDNA was amplified for 25, 30, 35 and 40 cycles. Conditions for ETBR and GAPDH PCR were: 95°C for 30 sec, 50°C for 80 sec, and then 72°C for 5 min. All PCR products were electrophoresed in a 2.0% agarose gel and visualized by ethidium bromide staining.

Western blot analysis was performed to analyze ETBR and GFP protein expression in transfected 293FT and HEK293 cells, normal human tissues, human pancreatic cancer cell lines and xenografts. Briefly, lysates from cells, tissues and xenografts were resolved using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride microporous membranes (Millipore, Billerica, MA). Anti-ETBR rabbit polyclonal antibodies (Ab1, 3, 4), mouse monoclonal antibody (Ab2), and anti-GFP and anti-β-actin mouse monoclonal antibodies were used as the primary antibodies. Peroxidase-conjugated goat anti-rabbit or mouse IgG was used as the secondary antibody (1:10,000 dilution). The detection of bound antibodies was performed using the electrogenerated chemiluminescence (ECL) system (Amersham, Aylesbury, UK).

Flow cytometry was performed by FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, pETBR-transfected 293FT cells [293FT (pETBR)] were harvested by 0.25% trypsin, and then anti-ETBR antibodies (Ab1–4) were used as the primary antibody. PE-conjugated goat anti-rabbit or mouse IgG (Beckman Coulter, Fullerton, CA, USA) was used as the secondary antibody.

Tumor specimens were obtained from the same 80 patients reported previously (3). This examination was carried out with the approval of the Hokkaido University Ethics Committee.

Immunohistochemical reactions were carried out using the universal immunoenzyme polymer method. Sections were deparaffinized with xylene, rehydrated through a graded series of ethanol/water and treated in a pressure cooker for 20 min. Endogenous peroxidase activity was blocked by 30-min incubation with 0.3% hydrogen peroxide in methanol. After washing in TBS-T, specimens were saturated with 10% normal goat serum [Histofine MAX-PO (Multi) kit; Nichirei Corp., Tokyo, Japan] for 30 min. Sections were then incubated overnight with anti-ETBR antibodies (Ab1–4) at 4°C. After three additional washes, the sections were incubated with Histofine Simple Stain MAX-PO (Multi) (Nichirei Corp.) for 20 min at room temperature. Reaction products were observed by incubation for ~5 min with 3,30-diaminobenzidine tetrahydrochloride (Nichirei Corp.). Sections were counterstained in hematoxylin for 1 min and then mounted in Marinol (micro slides; Muto-Glass, Tokyo, Japan).

Three vectors were constructed as follows (Fig. 1A): pcDNA3.1(+) (pEmpty Vector), pcDNA3.1(+)-IRES-GFP (pGFP), and pcDNA3.1(+)-ETBR-IRES-GFP (pETBR). Four sense and antisense primers were synthesized (Fig. 1B), and the sequence after subcloning was confirmed (data not shown). The amino acid sequence corresponding to the ETBR base sequence was equal to the ETBR sequence described in The National Center for Biotechnology Information (NCBI) (Fig. 1C). Transfection was then performed, and the detectability of ETBR expression by western blot analysis (WB), flow cytometry and IHC was evaluated. On WB, the expression of GFP protein was confirmed in both 293FT (pGFP) and 293FT (pETBR) lysates. For the ETBR protein, bands (100, 37, 25 kDa) were shown only in the 293FT (pETBR) lysate by all four ETBR antibodies. The density of the 25-kDa band appeared different with each antibody (Fig. 2). HEK293 cell lysates showed lower density bands of the same size as compared with 293FT. In the normal human tissue sample, specific bands of the same size as the positive control [PC; 293FT (pETBR)] were not observed with all tissue samples. On the other hand, different size bands from PC were found. This tendency was different with each antibody (Fig. 2). On flow cytometry, ETBR expression of 293FT (pETBR) was observed with all antibodies. However, the ratio of ETBR positivity varied by antibody (Fig. 3). On IHC, no immunostaining was observed with the negative control [NC; 293FT (pGFP)] by all antibodies, and immunostaining was found on the membrane surface with the PC by all antibodies. No immunostaining was seen with isotype IgG (Fig. 4). This result remained the same under different pH conditions (data not shown). In normal human tissues, excluding the adrenal gland, obvious immunostaining such as 293FT (pETBR) was not shown. In the adrenal gland, the staining region (cortex or medulla) and intensity were different with each antibody (Fig. 4). These staining results changed under different pH conditions (data not shown).

The mRNA of normal human tissues was extracted, and semi-quantitative RT-PCR was performed with the ETBR primer. ETBR mRNA expression was weak only in the adrenal gland sample (Fig. 5).

On WB, no specific band of ETBR was shown by two antibodies (Fig. 6A). On IHC, immunostaining was shown only in the adrenal medulla section by Ab1, maintaining consistency between cell cultures and xenografts (Fig. 6B). Thus, it was considered that Ab1 was more suitable for IHC than Ab2 as almost all immunostaining by Ab2 was shown to be non-specific. Ab1 was then used for IHC of PDAC tumor specimens. No biologically significant staining was shown in the vascular endothelial cells and the pancreatic cancer cells. Some staining was shown in the pancreatic acinar cells and pancreatic islets (Fig. 7).