Establishment of a new human osteosarcoma cell line, UTOS-1: cytogenetic characterization by array comparative genomic hybridization

The cytogenetic characteristics of osteosarcoma (OS) remain controversial. The establishment of a new human OS cell line may improve the characterization. We report the establishment of a new human osteosarcoma cell line, UTOS-1, from a typical osteoblastic OS of an 18-year-old man. Cultured UTOS-1 cells are spindle-shaped, and have been maintained in vitro for over 50 passages in more than 2 years. Xenografted UTOS-1 cells exhibit features typical of OS, such as production of osteoid or immature bone matrix, and proliferation potency in vivo. UTOS-1 also exhibit morphological and immunohistochemical characteristics typical of osteoblastic OS. Chromosomal analysis by G-band show 73~85 chromosomes with complicated translocations. Array CGH show frequent gains at locus DAB2 at chromosome 5q13, CCND2 at 12p13, MDM2 at 12q14.3-q15, FLI and TOP3A at 17p11.2-p12 and OCRL1 at Xq25, and show frequent losses at HTR1B at 6q13, D6S268 at 6q16.3-q21, SHGC17327 at 18ptel, and STK6 at 20q13.2-q13.3. The UTOS-1 cell line may prove useful for biologic and molecular pathogenetic investigations of human OS.


Introduction
Osteosarcoma (OS) is the most common malignant bone tumor in adolescents and young adults, and is characterized by proliferation of tumor cells which produce osteoid or immature bone matrix. Despite recent advances in multimodality treatment consisting of aggressive adjuvant chemotherapy and wide local excision, pulmonary metastasis occurs in approximately 40 to 50% of patients with OS and remains a major cause of fatal outcome [1][2][3].
There have been several reports describing xenotransplantation models of human OS [4][5][6][7], but characterization of human OS at the molecular cytogenetic level has been limited [8,9]. We describe the establishment and characterization of a new human OS cell line, designated as UTOS-1, derived from a conventional osteoblastic OS. In addition, we analyze chromosomal aberrations and DNA copy number changes in UTOS-1 by array comparative genomic hybridization (aCGH). year-old Japanese man noticed swelling and pain of the left shoulder for 2 months. Radiographs showed a periosteal reaction and an osteosclerotic change in the proximal metaphysis of the left humerus. An open biopsy of this humeral tumor confirmed that it was conventional osteoblastic OS ( Figure 1). Immunohistochemically, most of the tumor cells were strongly positive for vimentin, alkaline phosphatase (ALP), osteopontin (OP), and osteocalcin (OC). Despite intensive chemotherapy, the patient died of lung metastasis 2 months after open biopsy. The present study was conducted after a human experimentation review by our ethics committee.

Tumorigenicity in severe combined immunodeficiency (SCID) mice
To determine the tumorigenicity of the UTOS-1 cell line in vivo, 1 × 10 8 cells were washed, suspended in phosphatebuffered saline (PBS), and injected subcutaneously into the leg of 4-week-old male SCID mice (CB-17/Icrscid; Clea Japan Incorporation, Osaka, Japan). Also, tumor growth in vivo was measured by calculating tumor volume based on the measurement of 2 perpendicular diameters using a caliper [10]. The volume was estimated using the following formula: 0.5 × L × (S) 2 , where L and S are the largest and smallest perpendicular tumor diameters, respectively.

Establishment of the tumor cell line
Tumor cells were seeded in a 25 cm 2 plastic flask (35-3109; Falcon, Franklin Lakes, NJ, USA) [11]. These cells were cultured in RPMI 1640 (MP Biomedicals, Solon, OH, USA), supplemented with 100 mg/ml streptomycin (Meiji Seika, Tokyo, Japan), 100 U/ml penicillin (Meiji Seika) and 10% fetal bovine serum (FBS; Funakoshi, Tokyo, Japan), at 37°C in a humidified atmosphere of 5% CO 2 and 95% air. The medium was replaced once per week. When semiconfluent layers were obtained, the cells were dispersed with Ca 2+ -and Mg 2+ -free PBS containing 0.1% trypsin and 0.02% EDTA solution, and were then seeded in new flasks for passage. The configuration of tumor cells was almost equalized after the 3rd generation. These procedures were serially performed until the UTOS-1 cell line was established.

Cell growth in vitro
To determine the doubling time, UTOS-1 cells were seeded in each well of 96-well dishes (Corning Costar, Tokyo, Japan) with fresh medium containing 100 μl of RPMI 1640 with 10% FBS. Cell growth was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Cell Counting Kit-8, Dojindo, Tokyo, Japan) [12]. A volume of 10 μl of MTT was added to each well, followed by mixing. Plates were incubated for 3 hours at 37°C in a humidified atmosphere of 5% CO 2 and 95% air. Formazan levels, which correspond to the number of viable cells, were quantified using a microplate reader (model 450; Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 450 nm. The absorbance of each well was evaluated at 6, 12, 24, 48, 72, 96 and 120 hours after seeding. Triplicate wells were used for each observation.

Immunohistochemistry
Cells were cultured in chamber slides (Lab-Tek; Nalge Nunc International, Naperville, IL, USA). For the detection of mesenchymal phenotype, we used 3 monoclonal antibodies: anti-AE1/AE3, anti-keratin mix, and antivimentin. Also, to assess osteoblastic differentiation, we used 2 monoclonal antibodies: anti-OP and anti-OC. ALP activity of UTOS-1 cells was estimated using a modified version of a cytochemical method described elsewhere [13], with naphthol AS-MX phosphate-fast blue RR staining (ALP staining kit; Muto Pure Chemicals Corporation, Tokyo, Japan).
Cells grown in chamber slides were washed in PBS, fixed in 4% paraformaldehyde for 15 minutes at room temperature, and then fixed in methanol for 20 minutes at -20°C. The cells were incubated with each of the primary antibodies for 24 hours at 4°C. Immunoreaction products were detected using DAKO envision (DAKO Sytomation, Carpinteria, CA, USA), and were visualized after adding diaminobenzidine (DAB; DAKO) as the chromogen.

RNA extraction and reverse-transcription polymerase chain reaction (RT-PCR)
Expression of osteoblastic differentiation markers was assessed using RT-PCR. UTOS-1 cells were grown to con-Histologic appearance of the original tumor fluence, and total cellular RNA was isolated using a TRIzol ® Reagent (Invitrogen, San Diego, CA, USA). Total RNA was used as a template for cDNA synthesis using the SuperScript First-strand Synthesis System (Invitrogen). PCR was performed to assess expression of ALP, OP and OC. The oligonucleotide primer sequences and PCR conditions for ALP, OP and OC are shown in Table 1. Amplified products were analyzed by 2% agarose gel (Cambrex Bio Science Rockland Incorporation, Rockland, ME, USA) electrophoresis and ethidium bromide staining (Invitrogen). For comparison, Saos-2 [7], which is one of the most popular OS cell lines, was used as a positive control.

Cytogenetic analysis
For cytogenetic analysis, preparations of metaphase chromosomes from UTOS-1 cells at passage 15 were obtained, and were banded with Giemsa-trypsin [14]. Karyotypes were described using the short version of the International System for Human Cytogenetic Nomenclature [15].

DNA extraction and array CGH
Genomic DNA was extracted from UTOS-1 cells at passage 15. The CGH procedure used was similar to published standard protocols [16]. Genomic DNA was isolated from tumor samples using standard procedures including proteinase K digestion and phenol-chloroform extraction.
Array CGH was performed using the GenoSensor Array 300 system, following the manufacturer's instructions (Vysis, Downers Grove, IL, USA). This array contains the 287 chromosomal regions that are commonly altered in human cancer, such as telomeres, regions involved in microdeletions, oncogenes, and tumor suppressor genes. Tumor DNA (100 ng) was labeled by random priming with fluorolink cy3-dUTP, and normal reference (control) DNA was labeled using the same method with cy5-dUTP. The tumor and control DNAs were then mixed with Cot-1 DNA (GIBCO-BRL, Gaithersburg, MD, USA), precipitated, and resuspended in microarray hybridization buffer containing 50% formamide. The hybridization solution was heated to 80°C for 10 minutes to denature the DNA, and was then incubated for 1 hour at 37°C. Hybridization was performed for 72 hours in a moist chamber, followed by a post-hybridization wash in 50% formamide/2 × SCC at 45°C. Slides were mounted in phosphate buffer containing 4',6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO, USA). Fluorescence intensity images were obtained using the GenoSensor Reader System (Vysis) according to the manufacturer's instructions. For each spot, the total intensity of each of the 2 dyes and the ratio of their intensities were automatically calculated. The diagnostic cut-off levels representing gains and losses were set at 1.2 (upper threshold) and 0.8 (lower threshold). This assay was performed in triplicate, and common aberrations were considered to be meaningful aberrations.

Tumor growth in vivo
Approximately 5 weeks after implantation, all SCID mice had palpable elastic hard nodules with a volume of about 1000 mm 3 ( Figure 2). The tumor volume was about 4000 mm 3 at 6 weeks after implantation, and was > 10,000 mm 3 at 8 weeks after implantation. The cut surfaces of these tumors were solid and white-gray with small necrotic foci. Histopathologically, the tumors contained primarily atypical tumor cells, and exhibited formation of osteoid or immature bone matrix, which is similar in characteristics to the original tumor ( Figure 3).

Cell growth and morphological findings in vitro
UTOS-1 cells were spindle-shaped, contained several nucleoli, and formed clumps. Two weeks after initial cultivation in primary culture, the tumor cells reached subconfluence with some piled-up foci of cells ( Figure 4A). After the cells were serially subcultured for about 3 months, they began to grow rapidly at passage 6 ( Figure  4B).
This new cell line has been maintained in vitro for more than 50 passages over more than 2 years. In the exponential phase of cell growth, the population-doubling time was 40 hours ( Figure 5).

Immunohistochemical and cytochemical findings
All UTOS-1 cells were negative for AE1/AE3 and keratin mix. Most UTOS-1 cells were positive for vimentin. All UTOS-1 cells were positive for OP, OC and ALP ( Figure  6).

RT-PCR
UTOS-1 cells expressed ALP, OP and OC, which is similar to the results for Saos-2 ( Figure 7).

Discussion
There have been several reports describing xenotransplantation models of human OS [4][5][6][7]. In the present study, the parent tumor, the cultured tumor cells, and the xenografted tumor exhibited features typical of OS, as Immunohistochemical findings Osteoblast marker expression in UTOS-1 cells Figure 7 Osteoblast marker expression in UTOS-1 cells. The expression of several osteoblast markers, including ALP, OP and OC, is shown. Saos-2, which is one of the most popular OS cell lines, is used as a positive control for osteoblastic markers in UTOS-1 cells. These cells express ALP, OP and OC, which is similar to Saoa-2.
A representative G-banded karyotype of a UTOS-1 cell reported previously [15,17]. Cultured UTOS-1 cells have a spindle shape with several nucleoli, which is similar to the original tumor cells. Biochemical characteristics of UTOS-1, such as cell growth rate and osteoblastic activity, have not changed during the 2 years that they have been maintained. Immunohistochemically, the UTOS-1 cells remain positive for ALP, OP and OC. After implantation from cell culture into SCID mice, UTOS-1 cells grew in vivo, producing osteoid resembling that of the original tumor. Abundant osteoid tissue formed in the xenografted tumors and reimplanted tumors. These findings suggest that UTOS-1 cells have an osteoblastic phenotype and retain the characteristics of the original tumor. The population-doubling time of UTOS-1 cells in vitro is 40 hours, which is similar to that of other OS cell lines [4,6,18].
In the present study, chromosome 12 showed several distinct regions of focal amplification, occurring at gains of CCND2 at 12p13 12q13 and MDM2 at 12q14.3-q15. Previous CGH studies of OS have revealed abnormalities of chromosome 12, including gains at bands 12p12-p13 [24], 12q12-q13 [27], and 12q13-q14 [28]. Expression of the CCND2 gene, which is located at chromosome 12p13, has been observed in various malignancies, including prostate cancer and breast cancer [29][30][31]. CCND2 encodes a protein belonging to the cyclin family of proteins that regulate cyclin-dependent kinase (CDK) kinases [32]. CDK activity controls the cell cycle G1/S transition by regulating phosphorylation of the tumor suppressor protein Rb [33]. These facts suggest that CCND2 controls proliferation of UTOS-1 tumor cells.
Some studies indicate that 14 to 27% of OS tumors have abnormal MDM2 expression [34,35]. MDM2 is a target gene of the transcription factor tumor protein p53 [36]. The encoded protein is a nuclear phosphoprotein that binds and inhibits transactivation by tumor protein p53, as part of an autoregulatory negative feedback loop [37,38]. Overexpression of MDM2 gene can result in excessive inactivation of tumor protein p53, diminishing its tumor suppressor function. These findings suggest the possible involvement of the p53 tumor suppressor gene, which is associated with development of OS in UTOS-1 cells.
In a study of OS using a cDNA array, Squire et al. observed amplification of the genes MYC, GAS7, and PM1 in OS cells [45]. Other reports indicate that losses of chromosome bands 6q16 and 6q21-q22 occur in high-grade OS [46]. These findings and those of the present study suggest that gene losses on chromosome 6q, including HTR1B and D6S268, contribute to OS tumorigenesis.
One of the most remarkable breakpoint clusters that have been found in OS tumors was detected on chromosome Genetic instability analyzed by aCGH Figure 9 Genetic instability analyzed by aCGH. The line in the middle (gray) is the baseline ratio (1.0); The upper (red) and lower (green) bars in each frame indicate losses and gains, respectively. The arrow shows the axes of X and Y chromosomes. 20 by spectral karyotyping (SKY) analysis [47]. Chromosome 20 is one of the smaller chromosomes, suggesting that it is particularly vulnerable to structural rearrangement. However, there is little evidence that chromosome 20 is frequently involved in chromosomal imbalances [26,28]. In the present study, the only loss that involved chromosome 20 occurred at band 20q13.2-q13.3. Many chromosomal changes have been observed in CGH studies of high-grade OS [46]. Reports indicate that the genes involved in OS tumorigenesis include DAB2 (at chromosome 5q13), OCRL1 (at Xq25), and SHGC17327 (at 18ptel). However, many of these genes were not previously known to be associated with OS tumorigenesis.
In conclusion, we have isolated and characterized a new permanent human cell line, UTOS-1, established from an osteoblastic OS. This cell line retains the morphology, osteoblastic activities and cytogenetic characteristics of the original tumor in vitro. The UTOS-1 cell line is useful for biologic and molecular pathogenetic studies of human OS.