Establishment and characterization of pleomorphic adenoma cell systems: an in-vitro demonstration of carcinomas arising secondarily from adenomas in the salivary gland

A fresh tissue sample was obtained from a parotid gland tumor of a 61-year-old woman. The tumor, measuring 23 × 20 × 15 mm in size, was surgically removed with tumor-free margins. The surgical material was fixed in 10% formalin, cut into about 3–5 mm thick slices using whole-organ sectioning, and embedded in paraffin. Sections 5 μm thick were cut from the cut surfaces of the tumor specimens and stained with hematoxylin and eosin (HE). Macroscopically, the tumor was circumscribed with a fibrous capsule, and was grayish white in color, solid, firm and mucinous in its cut surface. There were no signs of local recurrence or metastasis during her three-year postoperative course. The experimental protocol for isolation and analyses of tumor cells was reviewed and approved by the Niigata University Graduate School of Medical and Dental Sciences Ethical Board. Prior to obtaining the tissue samples, our purpose and plan of the experiment were explained to the patient followed by her consent.

A tissue slice obtained from the central and maximum cut surface of the surgical material was minced into small pieces. The tissue pieces were treated with 0.1% (v/w) collagenase (F. Hoffmann-La Roche Ltd., Basel, Switzerland) in Dulbecco's minimal essential medium (DMEM, Gibco, Invitrogen. Co., Carlsbad, CA, USA) containing 10% fetal calf serum (Gibco), 50 IU/ml penicillin and 50 μg/ml streptomycin (Gibco) in a 2 ml tube for 8 hrs at 37°C. Pass-through fractions from a nylon mesh filter were washed and plated in 2 ml of DMEM in 35 mm dishes and incubated at 37°C in humidified 5% carbon dioxide/95% air atmosphere. After two weeks, culture media were replaced with fresh ones and thereafter changed every 7 days. After one month, the cells, which had grown to confluence, were split into 25 cm² flasks. When aggregates of polygonal and bizarre epithelioid cells appeared in the background of spindle-shaped cells in the fourth passage, the cells were split and thereafter passaged every week. After four passages, the cells were served for cloning. The cells prepared as above were plated in 96-well microplates using a conventional method of dilution [28]. Wells with a single cell were observed every 24 hrs by microscope. The culture media were changed every 2 days. After the cells reached subconfluency in the wells, they were transferred into 24 well plates and maintained for up to 1 month until they reached subconfluent conditions, and then the cells were moved to 25 cm² flasks.

All of the established cell systems were used for immunofluorescence studies. The cells were seeded at a cell concentration of 1.2 × 10⁴ onto each well of chamber slides (Lab-Tek II, 4-well type, Nalge Nunc International, Naperville, IL, USA) and cultivated for 6 days. At day 6 after plating, the chamber slides were fixed and served for immunofluorescence experiments [29]. The primary antibodies consisted of rabbit polyclonal antibodies against human keratin (wide spectrum, Dako, Glostrup, Denmark, diluted at 1:25), and human S-100 protein (Dako, 1:100) and mouse monoclonal antibodies against human cytokeratin (CK) 14 (clone CKB1, IgM, 1: 100, Sigma Chemical Co., St Louis, MO, USA), calpoinin (CALP¹, IgG₁, 1:50, Dako), and P53 protein (IgG_2a, specific to the transcription domain in the NH₂-terminal region, clone Bp53-11, Progen Bioteknik GmbH, Heidelberg, Germany). The secondary antibodies were rhodamine-conjugated goat anti-rabbit or mouse IgGs or IgM (ICN Pharmaceuticals, Aurora, OH, USA, 1:50). For control studies, purified non-immune rabbit IgG or mouse IgG_2a (Dako) were used instead of the specific primary antibodies. Xenografts of pleomorphic adenoma cell systems in nude mice were also examined immunohistochemically for perlecan, a basement membrane type heparan sulfate proteoglycan, and fibronectin by using rabbit polyclonal antibodies (diluted at 50 μg/ml, respectively) [29] and the rabbit Envision+/HRP system (Dako). For control studies on the antibodies, the primary antibodies were replaced with preimmune rabbit IgG.

Cellular DNAs were extracted from cells in primary culture and from all of the five established cell systems by using a TRIzol system (Invitrogen). The cells were cultivated for 7 days up to their subconfluency in 25 cm² flasks, and 1 ml of TRIzol reagent was added to each flask. Total RNAs were then extracted from the cell lysate according to the manufacturer's instructions. After complete removal of the aqueous RNA phase, DNA was isolated from the interphase and phenol-phase. Following precipitation with 0.1 M sodium citrate in 10% ethanol, the precipitants were washed with 70% ethanol, and the pellets were air-dried briefly. The DNA samples dissolved in autoclaved water were stored at -20°C.

Primary culture cells in the fourth passages and all of the established cell systems in their subconfluent conditions were arrested for 3.5 hrs with 0.06 μg/ml colcemid in DMEM. They were washed two times with PBS and then removed with 0.05% trypsin at 37°C for 5 min. The cells were recovered from culture dishes with DMEM, suspended in 0.075 M KCl at 37°C for 30 min for hypotonic treatment, and then fixed in 1:3 acetic acid/methanol. The cell suspension was dropped onto glass slides to spread chromosomes from cells in the metaphase at room temperature under 50–55% ambient humidity. Slides were stained with 6% Giemsa solution in 0.067 M phosphate buffer (pH 6.8) for visualization of chromosomes. Metaphase spreads from 20 to 50 cells of the primary culture and each cell system were counted for chromosome numbers. Other slides were treated with 0.005% trypsin at 2°C for 8 min. They were washed and then stained with Giemsa solution for visualization of Giemsa (G)-banding. Ten to twenty cells each were analyzed for G-banded karyotypes in all of the cell systems. For control studies, peripheral blood lymphocytes from the patient were cultured in RPMI 1640 (Gibco) containing 10% fetal calf serum (Gibco), 50 IU/ml penicillin and 50 μg/ml streptomycin (Gibco) for 3 days, and then analyzed for chromosome numbers and G-banded karyotypes in the same way as described above.

The genomic clones encoding the break point regions around t(9;13)(p13;q12) were isolated from the BAC library [30] by the PCR screening method with primer sets flanking the regions of the two chromosomes by using the GenBank database http://​www.​ncbi.​nlm.​nih.​gov/​ as shown in Table 1. About 10 μl of the DIG-labeled BAC DNA was mixed in 2 μl of 10 mg/ml salmon sperm DNA (Roche), 2 μl of 10 mg/ml E. coli tRNA (Roche), 4 μl of 4 M CH₃COONH₄, and 50 μl of 100% ethanol. After storage for 1 hr at -80°C, the mixture was melted and centrifuged in 8 μl of the hybridization mixture consisting of 50% formamide, 10% dextran sulfate, 2 × SSC solution for each slide. After 2 μl of 10.5 mg/ml human placenta DNA (Sigma) was added, the mixtures were denatured for 10 min at 75°C, cooled down, and supplemented with 1 μl BSA. The mixture was pre-annealed for 30 min at 37°C before applying it to the slides. Slide-bound chromosomal DNA was denatured in a solution of 70% formamide in 2 × SSC for 6 min at 75°C on preheated slides for 2 hrs at 60°C and dehydrated in a 4°C ethanol series (70% and 100% ethanol for 5 min each). Ten microliters of the hybridization mixture were applied to each slide, and the slides were sealed with cover glasses. After overnight incubation at 37°C, the cover glasses were removed and the slides were washed once in 50% formamide/2 × SSC for 20 min at 37°C and twice in 2 × SSC for 10 min at 37°C, and then in TNT solution (0.1 M Tris-HCl/0.15 M NaCl/0.05% Tween 20) for 5 min at room temperature. The slides were further incubated with 100 μl of TNB solution (0.1 M Tris-HCl/0.15 M NaCl/0.5% blocking solution (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) to block non-specific reactions. The DIG-labeled probes were reacted with the anti-DIG mouse monoclonal antibody (Roche, 50 μl of 1 μg/ml IgG/4 × SSC) for 30 min at room temperature, rinsed three times in TNT solution for 5 min, and then treated with the secondary antibody (40 μl of 10 μg/ml Alexa flour 488-conjugated goat anti-mouse IgG (Molecular Probes, Inc., Eugene, OR, USA)). Finally, the FISH slides were counterstained with 50 μg/ml propidium iodide (PI, Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. FISH images were obtained by an Olympus confocal laser scanning microscope Fluoroview 1000 (Olympus Co., Tokyo, Japan).

To determine in vivo tumorigenicities of the cloned cell systems, SM-AP cells were transplanted in nude mice. Cells in their 9th to 13th in vitro passages at a concentration of 2 × 10⁶ in 0.3 ml culture media were injected into the lateral back wall of female BALB/c (nu/nu) mice at 4 weeks of age (2 mice each per cell system). The animals were housed in clean boxes in a sanitary and ventilated animal room and maintained under constant conditions (at 22°C and in a 12-hr light/dark cycle) with free access to sterilized solid food and autoclaved water. When tumors reached sizes of around 10–15 mm in diameter, they were surgically removed with the animals under ether anesthesia. The excised tumor tissues were fixed in 10% formalin for 24 hrs at 4°C and embedded in paraffin. Serial sections cut at 5 μm from the paraffin blocks were stained with hematoxylin and eosin, Masson's trichrome and immunoperoxidase for perlecan, fibronectin, keratin, S-100 and P53, and then examined histologically. The experimental research by using animals was reviewed and approved by the Niigata University Graduate School of Medical and Dental Sciences Ethical Board.

The surgical specimen of the parotid gland tumor showed a typical histology of pleomorphic adenoma with a definite capsule (Figure 1A). The tumor contained both myxoid and hyaline areas with varied cellularity but poor vascularity. There were neither apparent foci of carcinoma cells nor pleomorphic adenoma cells with apparent atypical features (Figure 1B).

For the first to third cell passages of the primary culture, it took about 2 months for each of the cells to form a confluent monolayer in a 25 cm² flask ready for splitting. During the period up to the third passage, cells were almost spindle-shaped in isolated colonies but did not show any fascicular modes of packing as seen in fibroblasts (Figure 1C). At the fourth passage, clusters of polygonal cells with ground glass-like cytoplasm started to appear among the spindle-shaped cells (Figure 1D), and the cells reached confluence in 7 days. After four additional passages, during which polygonal cells became enhanced in their atypical features in size and shape of their nuclei and such bizarre cells increased in number in most of the colonies, these cells were served for cloning. From serial dilutions in 96-well microplates, single cells were isolated in individual wells and grew to confluence within one month after plating. They were transferred into 24-well plates and then to a 25 cm² flask. These cloning procedures were repeated twice, and finally, 5 clones were successfully grown from the primary culture. They were designated as SM-AP1 to SM-AP5. After the cloning procedure, the five cloned cell systems were maintained by passages every 7 days. Their doubling times were approximately 31 hrs irrespective of the five cell systems. They were classified into two groups according to their cell shapes: one was polygonal with squamous epithelial characteristics, which was shared by SM-AP1 (Figure 1E), SM-AP2, and SM-AP3, and the other was short spindle shaped, which was characteristic of SM-AP4 and SM-AP5 (Figure 1F).

Clones SM-AP1 to SM-AP5 were stained positive for keratin, a duct-epithelial marker (Figures 2A–2D). SM-AP1, SM-AP2, and SM-AP3 cells were not positive for S-100 protein, a myoepithelial marker (Figures 2C), while SM-AP4 and SM-AP5 cells were strongly positive (Figures 2D) mainly in their nuclei. In addition to S-100 protein, SM-AP4 and SM-AP5 were positive for such myoepithelial markers as CK14 and calponin. All primary cultured cells and five cell systems showed immunopositivities for perlecan but none for P53 protein (not shown).

Chromosome numbers of the five cell systems varied between 64 and 123, which were within tetraploid or pentaploid ranges, with a modal chromosome number of 108 (Figure 3A-a–e). Chromosome numbers of primary cultured cells varied between 107 and 122 with a modal chromosome number of 113 (Figure 3A-f). The G-banded karyotyping revealed a stemline karyotype of 102~114<5n>, XX in SM-AP1; 113~121<5n>, XX in SM-AP2; 98~108<5n>, XXX in SM-AP3; 97~115<5n>, XX, in SM-AP4; 114~121<5n>, XX in SM-AP5. Figure 3B represents the SM-AP5 stemline karyotype. All of the cell systems had numerous chromosomal abnormalities, such as deletion, translocation and marker chromosomes as shown in Table 2. Among them, additional materials of unknown origin such as add(X)(p11), der(3)add(3)(p11)der(3)(q2?), add(4)(q35) and translocations such as der(9)t(9;13)(p13;q12), add(12)(p11), and add(14)(q32) were shared by all of them. Since the der(9)t(9;13)(p13;q12) was the only abnormality in which the origin of translocated chromosomes was evident, this translocation was further analyzed for its break points by the FISH technique as described below. Chromosome 17, where the p53 gene is located at p13, decreased in number in all of the cell systems. In addition, del(17)(p11) was shared by four cell systems except for SM-AP4. SM-AP3 cells had add(17)(p13), which might have affected the p53 gene locus.

Primary cultured cells shared the same translocations as those of the established SM-AP cell systems, such as der(9)t(9;13)(p13;q12) and add(12)(p11) (Table 2). Peripheral blood lymphocytes from the patient had normal chromosome numbers and karyotype (not shown).

To restrict the break points for the translocation t(9;13)(p13;q12), which was shared by all five cell systems, FISH analyses for chromosome 9p13 and 13q12 regions were performed. By using the primer sets listed in Table 1, three clones for chromosome 9 (p13) and four clones for chromosome 13 (q12) were isolated by PCR to map their chromosomal locations. Clear single or paired FISH signals were demonstrated by the BAC clones, 1537E12 (Figure 4A-b) and 1405F1 (Figure 4A-c), which are located in chromosome 9p13.2 and 9p13.1, respectively (Figure 4A-a). BAC clones by 1325C2 in chromosome 13q12.2 (Figure 4B-a) were localized on chromosomes with translocation t(9;13)(p13;q12) (Figure 4B-c). FISH signals for 1213F4, which is located in chromosome 13q13.1, were not found on any chromosomes (Figure 4B-b). Thus, break points for the translocation, t(9;13)(p13;q12), were thought to be located in restricted regions of 9p13.3 and 13q12.3.

PCR amplicons for exons 5, 6 and 7 of the p53 genes from the primary cultured cells as well as from all of the SM-AP cell systems were sequenced, and deletion of the last base G of codon #249 (AGG to AG_) of exon 7 was found in all of them including the primary culture (Figure 5). Owing to this point mutation, a nonsense polypeptide should have been generated on and after codon #249, resulting in non-functional p53 gene products. No other mutations were found in exons 5 and 6.

Five cell systems, SM-AP1 to SM-AP5, successfully formed subcutaneous tumors in nude mice after 5 to 19 weeks of injections (Figure 6A, Table 3). The tumors which had grown to 10 to 15 mm in diameter were harvested for histopathological examinations (Figure 6B). Histologically, transplanted tumors showed rather expansive growths mostly limited to the range of the dermis to the subcutis or to only the surface part of the muscle layer, although they had no definite fibrous capsules. Tumor cell nests varied in size, and some of them contained keratinization centers, irrespective of transplanted tumors. Hence, squamous cell carcinomas were basically diagnosed for all of the tumors formed by the five cell systems. However, their keratinization seemed to occur suddenly without gradual differentiation tendencies in the tumor cell nests, which had no basal cell alignment at the periphery of the tumor cell nests (Figure 6C), which were usually not seen in squamous cell carcinomas of the oral mucosal origin. In the transplanted tumors of SM-AP1 to SM-AP3, which had been regarded as duct-epithelial by immunofluorescence in vitro, there were tendencies to form mimics of ductal structures (Figure 6D, arrow). Those of SM-AP1 and SM-AP2 contained tumor cells with plasmacytoid appearances (Figure 6E). In those of SM-AP4 and SM-AP5, which had rather myoepithelial natures in vitro, tumor cell nests were sheet-like with some tendencies toward keratinization, and they showed ground glass-like cytoplasm (Figure 6F). Mitotic figures were observed in each tumor but more frequently in SM-AP4 and SM-AP5 (Figure 6G, arrows), suggestive of their malignant natures. Hyaline stromal spaces without definite vascular channels and lymphocytic infiltration were formed between tumor cell nests in every tumor, which were also unusual in oral squamous cell carcinomas (Figure 6H). Immunohistochemically, the hyaline stroma was strongly positive for perlecan (Figure 6I) as well as for fibronectin (Figure 6J). Although there was no area with a typical histology of benign pleomorphic adenoma in the transplanted tumors, the presence of ductal mimics, plasmacytoid cells, ground-glass appearance of tumor cell cytoplasm and predominant hyaline stroma mentioned above might indicate their pleomorphic adenoma origin. However, there were no foci of salivary duct carcinoma, which is common in carcinoma ex pleomorphic adenoma. Immunohistochemically, the transplanted tumors of SM-AP1 to SM-AP5 were stained positive for keratin. SM-AP1, SM-AP2, and SM-AP3 cells were not positive for S-100 protein, while SM-AP4 and SM-AP5 cells were strongly positive for other myoepithelial markers, such as CK14 and calponin, in addition to S-100 protein. All five transplanted cell systems showed no P53 protein immunopositivities (not shown).