A 69-year-old Japanese man consulted a local doctor because of a right flank pain. He was referred to our hospital in 2009 for further examination and treatment. Laboratory values upon admission indicated liver dysfunction and obstructed jaundice. Endoscopic retrograde cholangiopancreatography and abdominal computed tomography suggested adenocarcinoma of the ampulla of Vater with lymph node metastasis. No metastasis to the liver and the other organs was observed. On the basis of these findings, pylorus-preserving pancreatoduodenectomy with lymph node dissection was performed (Fig. 1A). Pathological diagnosis was hepatoid adenocarcinoma of the ampulla of Vater with lymph node metastasis (Fig. 1B) considering the positive immunoreactivity for AFP (Fig. 1C). Small part of the viable lesion of the lymph node metastasis was resected surgically during an operation, rinsed with phosphate-buffered saline (PBS), and minced with scissors, followed by digestion in 1 mg/ml collagenase (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 1000 PU/ml dispase (Godo Shusei, Tokyo, Japan) in the mixture of RPMI-1640 (Nissui, Tokyo, Japan) and Ham’s F12 (Nissui) at the rate of 1:1 at 37°C for 4 h. The remaining part was used for pathological diagnosis. Then the solution was centrifuged at 700 rpm for 3 min and the pellet was resuspended in the same medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (JRH Biosciences, KS, USA), 100 IU/ml penicillin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), 100 μg/ml streptomycin (Meiji Seika Pharma Co., Ltd.), 20 mM N-2-hydroxyethyl piperazine-N′-2-ethane sulfonic acid (HEPES) (Nakalai, Kyoto, Japan), pH 7.4. The flasks were cultured in a fully humidified atmosphere of 5% CO₂ in air at 37°C. Informed consent was obtained from this patient and the protocol was approved by the ethics board of the Faculty of Medicine, University of Miyazaki. Mycoplasma infection was examined by a PCR Mycoplasma Detection kit (Takara Bio Inc., Shiga, Japan).

Growth curves were established by seeding 1×10⁵ cells/5 ml in growth medium onto 60-mm culture dishes. Triplicate dishes were harvested and counted daily. Throughout the entire procedure, cell viability was determined by means of the trypan blue exclusion method. Doubling time was determined during the exponential phase of growth.

Exponentially growing cultures of the cell line, passage number 50, was harvested for chromosome preparations with standard procedures by SRL (Tokyo, Japan). Fifty cells were analyzed for chromosome number and the Giemsa banding technique for karyotype analysis. STR profiling at the passage number 1 and 50 were performed by Takara Bio Inc. to identify the cross-contamination of another cell line.

For light microscopy, VAT-39 cells were cultivated on Chamber Slide (Nunc Inc., IL, USA) and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 30 min at room temperature (RT). After washing with PB, the cells were observed with hematoxylin-eosin (H&E) or Periodic acid-Schiff (PAS) staining to examine their morphology and the existence of glycogen, respectively.

For electron microscopy, high-pressure freezing (HPF) technique was applied for fine structural observation (20,21). In brief, VAT-39 cells were cultivated on sterilized 10 μm-thin stainless foil (YUS205-M1, Nippon Steel, Tokyo, Japan). At semi-confluency, the attached VAT-39 cells on the stainless foil were immediately cryofixed in the HPF machine (HPM010, BAL-TEC, Liechtenstein). The assembly with cryofixed cells was transferred to 1 ml of freeze substitution medium (1% osmium tetroxide in acetone), and then freeze substituted in a Leica AFS machine (Leica, Vienna, Austria). After embedding into epoxy resin, ultrathin sections (60–80 nm) were cut and stained with uranyl acetate and lead citrate to be observed in a transmission electron microscope (HT7700, Hitachi, Tokyo, Japan).

Electron microscopic localization of cytoplasmic glycogen was performed by means of periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method. Briefly, the VAT39 cells cultivated on the micro-cover glass (thickness no. 5, Matsunami, Osaka, Japan) (22) were fixed with a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M PB for 2 h at RT. After washing with in PBS, without post-fixtaion by 1% osmium tetroxide, the cells were dehydrated and embedded in epoxy resin. Ultrathin sections mounted on gold meshes were treated in 1% sodium periodate (Wako Pure Chemical Industries, Ltd.) for 30 min followed by 1% thiocarbohydrazide (Sigma-Aldrich, MO, USA) for 1 h. After washing, the sections were incubated in 1% silver protein (Merck, Germany) for 1 h and observed as described above.

Conventionally prepared paraffin sections were deparaffinized, and incubated in 0.3% H₂O₂ in methanol to block endogenous peroxidase for 30 min. After rinsing in PBS, the sections were treated with 1% bovine serum albumin (BSA) in PBS for 20 min, and then incubated with a polyclonal rabbit anti-human AFP (Dako, Tokyo, Japan; 1:10 diluted with 1% BSA in PBS) for 1 h at RT. After washing with PBS, the sections were incubated with biotinylated horse anti-rabbit IgG (Vector Laboratories, CA, USA; diluted 1:300 with 1% BSA in PBS) for 40 min at RT. After washing with PBS, the sections were incubated in a freshly prepared solution of avidin-biotinylated horse-radish peroxidase complex (ABC) kit (Vector Laboratories) for 30 min (23), and then peroxidase reaction was developed by incubating in 0.05% 3,3-diaminobenzidine tetrahydrochloride in 0.05 M Tris buffer, pH 7.6, containing 0.001% H₂O₂ for 5–10 min. After washing, the sections were briefly counterstained with hematoxylin.

The sera, preoperative and postoperative conditions, were collected as a routine follow up of operation. The supernatants of VAT-39 at passage 50 were harvested after 24, 48 and 72-h culture in 5 ml of serum-free media in a 25 cm² flask. The AFP concentrations in these samples were measured by Architect AFP using Architecto i200SR (Abbott, Chiba, Japan).

Human biliary tract cancer cell lines (HuCCt1, HuH-28, TFK-1, TKKK, TGBC2TKB, TGBC14TKB and TGBC18TKB) were obtained from Riken Cell Bank (Tsukuba, Japan). The two other human biliary tract cancer cell lines (IHGGK and G415) were obtained from Tohoku University (Miyagi, Japan). Total RNAs were extracted with TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA), followed by Cloned DNase I (Takara Bio. Inc.) treatment and phenol/chloroform/isoamylalcohol extraction. For RT-PCR, 6 μg of total RNA was reverse transcribed with a mixture of oligo (dT) and random primer using 200 units of ReverTra Ace^® (Toyobo, Osaka, Japan), and 1/90 of the resultant cDNA was processed for each PCR with 0.25 μM of both reverse and forward primers and 1.25 units of HotStar^® Taq DNA polymerase (Qiagen, Tokyo, Japan). For detection of hepatoid adenocarcinoma-related gene mRNA, the following primers were designed: AFP, sense 5′-GACATCCTCAGCTTGCTGTC-3′ and antisense 5′-GAGGCCGCAGCTTCGCTTTG-3′ (173 bp); GPC3, sense 5′-GCAGGAAAGCTGACCACCAC-3′ and antisense 5′-GACATGGTTCTCAGGAGCTGG-3′ (419 bp); glyceraldehydes-3-phosphate dehydrogenase (GAPDH), sense 5′-GTGAAGGTCGGAGTCAACG-3′, and antisense 5′-GGTGAAGACGCCAGTGGACTC-3′ (300 bp). The PCR products were analyzed by 1.5% agarose gel electrophoresis.

We evaluated the effect of several chemotherapeutic reagents, generally used to the patient of biliary tract cancer, on the growth of VAT-39 at the passage number 50. Gemcitabine, 5-fluorouracil, epirubicin and mitomycin C were purchased from Wako Pure Chemical Industries, Ltd.; paclitaxel, oxaliplatin and doxorubicin were from Nakalai; cisplatin was from Sigma-Aldrich. The VAT-39 cells (1×10⁴) were seeded in 96-well plates and cultivated overnight in 100 μl of the same medium containing 10% FBS. The medium was removed and replaced with the same medium containing various concentrations of the chemical agents. After 24 h incubation, 10 μl of Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) was added to each well. Following further 3 h incubation, the absorbance at 450 nm was monitored using ImmunoMini NJ-2300 (Biotec Co. Ltd., Tokyo, Japan).

Tumorigenicity and metastatic potential of VAT-39 cells were analyzed using 6 weeks old BALB/c athymic mice (Kyudo, Kumamoto, Japan). After trypsinization of cultured cells by 0.25% trypsin and 0.02% ethylenediamine tetraacetic acid (TE, Sigma-Aldrich), the cells were centrifuged immediately at 1000 × g for 5 min, and the resuspended cells (1×10⁶ in 0.2 ml PBS) were injected subcutaneously at the abdominal flank. At 8 weeks or when the mice became moribund, they were sacrificed or necropsied. Under general anesthesia using diethyl ether, the xenograft, brain, lung, liver, kidneys and gastrointestinal tract were harvested and examined under a dissecting microscope. Tumor volume was calculated weekly using the formula, length × (width)² × 0.5. For light microscopy, xenografted tumors were fixed with 4% paraformaldehyde in 0.1 M PB at 4°C overnight, and embedded in paraffin. The sections were stained with H&E.

All animal procedures were carried out under protocols approved by the University of Miyazaki Animal Research Committee, in accordance with international guiding principles for biomedical research involving animals.

The tumor cells formed colonies surrounded by fibroblasts in two months of primary culture. The fibroblasts were eliminated by a weak trypsin and TE treatment for one month. The first passage was done at a split ratio of 1:4. The cell line was designated as VAT-39, and it has already undergone more than 100 passages.

The VAT-39 cells proliferated well in culture media containing 10% FBS. The polygonal cells with prominent nucleoli formed adhesive sheet-structures as shown in phase-contrast microscopy (Fig. 2A). Morphological findings of VAT-39 (Fig. 2B) were mostly in accordance with those of the lymph node metastasis (Fig. 1B) in which mitotic figures were frequently seen. Growth characteristics were determined at passages 1 and 50. The doubling times at passage 50 were 34 h (data not shown). The cell line was free from mycoplasma infection.

The STR analysis was performed at passages 1 and 55 of the VAT-39 cells, and the obtained STR profile ∼10 loci and detected peaks were: AMEL (X), CSF1PO (12), D13S317 (11), D16S539 (9,12), D21S11 (30), D5S818 (12), D7S820 (12,13), TH01 (9), TPOX (8,11), vWA (14,17). The STR analysis at passage 1 was identical to that of 55, which confirmed that VAT-39 cell lines maintained the same genetic feature without possible contamination.

The number of chromosomes was between 50–54 at passage 50 with a modal chromosome number 53. The result of karyotype analysis of 10 cells were as follows; X, -Y, +add(1)(p11), add(1)(q21), +del(3)(p11), der(3)add(3)(q21)del(3)(q?), add(7) (p11.2), add(9)(p13), der(11)add(11)(p11.2)add(11)(q23), der(11) add(11)(p15)add(11)(q23), add(13)(p11.2)×2, -14, +del(16)(p?), del(16)(q?), add(19)(p13), -20, add(21)(q22), +mar1, +mar2, +mar3, +mar4×2. No change was observed in chromosome number 4 and X where the AFP and GPC3 genes are located, respectively.

Transmission electron microscopic study by HPF revealed electron lucent granules in the cytoplasm of VAT-39 cells (Fig. 3A), suggesting accumulations of glycogen granules. Light microscopy by PAS staining demonstrated strongly stained numerous granules in the cytoplasm (Fig. 3B). At the electron microscopic level, PA-TCH-SP method clarified the accumulation of glycogen in the electron lucent granules (Fig. 3C).

Immunohistochemistry of AFP demonstrated a distinct immunoreactivity around the perinuclear region of VAT-39 cells (Fig. 4A). The expression of AFP mRNA in VAT-39 cells was evaluated by RT-PCR (32 cycles of amplification), in comparison with various cell lines derived from biliary tract cancer. As a result, the VAT-39 cell line exceptionally expressed AFP mRNA (Fig. 4B). The expression was confirmed in the lymph node metastasis, from which the VAT-39 cell line was established, as well as in the VAT-39 cells of varying passage numbers (Fig. 4C). Moreover, the VAT-39 cell line and the lymph node metastasis also expressed GPC3 mRNA (Fig. 4B and C) that is highly expressed in HCC. The AFP protein concentrations were highly elevated in the culture media depending on the culture period (Table IB).

Although the APF protein concentration in the preoperative serum indicated a remarkably high level, the concentration was drastically decreased in the postoperative serum (Table IA). Taken together, the elevated serum AFP was considered to be secreted from the tumor, and this phenotype was well-preserved in this established cell line.

The VAT-39 cells were subjected to various chemotherapeutic reagents to evaluate their sensitivities to each reagent concerned with 50% inhibition concentration (Table II). As a result, the VAT-39 cell line exhibited no sensitivity to 5-FU and gemcitabine.

Tumorigenicity was tested by subcutaneous injection of VAT-39 cells at the abdominal flank of nude mice. The cells grew rapidly to form solid tumor masses, which were detected within 4 weeks (Fig. 5A, inset). The tumor doubling time was calculated as 2.5 days (data not shown). The VAT-39 cell pellets showed 100% tumorigenicity in all the transplanted mice (n=5; data not shown). It should be noted that obvious tumor mass necrosis was seen on a cut surface of the xenografted tumor. Histopathology demonstrated central necrosis, surrounded by viable polygonal tumor cells in the peripheral region. The viable cells formed sheet-structures, showing hyperchromatic nuclei, prominent nucleoli, foamy and clear cytoplasm, and numerous mitotic figures (Fig. 5A). The expression of AFP was also noted in the explanted tumor of the nude mouse (Fig. 5B), preserving the characteristics of AFP expression. These findings were consistent with the original tumor and lymph node metastasis (Fig. 1B). In the explanted mice, no metastatic lesion was identified in the other organs.