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

An 18-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.

To determine the tumorigenicity of the UTOS-1 cell line in vivo, 1 × 10⁸ cells were washed, suspended in phosphate-buffered 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.

Tumor cells were seeded in a 25 cm² 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% CO₂ and 95% air. The medium was replaced once per week. When semiconfluent layers were obtained, the cells were dispersed with Ca²⁺- and Mg²⁺-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.

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% CO₂ 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.

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 anti-vimentin. 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.

Expression of osteoblastic differentiation markers was assessed using RT-PCR. UTOS-1 cells were grown to confluence, 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.

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].

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.

Approximately 5 weeks after implantation, all SCID mice had palpable elastic hard nodules with a volume of about 1000 mm³ (Figure 2). The tumor volume was about 4000 mm³ at 6 weeks after implantation, and was > 10,000 mm³ 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).

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).

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).

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

A representative karyotype is shown in Figure 8. 50 UTOS-1 cells exhibited a complex karyotype. The karyotypes of UTOS-1 cells at passage 15 were similar to those of the original tumor. The composite karyotypes were as follows: 73~85, Y, -X [7],+Y[10],add(X)(q11)[9],add(X)(q11)[2],+1[8],del(1)(q11)[9], der(1)add(1)(p11)add(1)(q42)[9],der(1)add(1)(p22)add(1)(q32)[2],der(1)add(1)(p32)add(1)(q42)[6],-3[10],-4[3],add(4)(q11)[9],-5 [4],del(5)(p13)[9], +add(6)(q11)[3],der(6)del(6)(p21)add(6)(q2?)[10],der(6)del(6)(p24)add(6)(q13) ×2,[10]-7[10],add(7)(p22)[6],der(7)t(7;7)(p22;q22)[10],+8[3],-9[10],-9[8],add(9)(q22)[9],-10[10],add(10)(p11)[4],add(10)(q26)[7], der(10)add(10)(p11)add(10)(q26)[3],add(11)(p11)[9],add(11)(p11)[4], del(11)(p11)[6],-12[5],der(12)(q21)[6],der(12)add(12)(p11)add(12)(q24)[10], der(12)add(12)add(12)[7],-13[10],+14[2], add(14)(p11)[10],add(14)[2], add(14)(p11)[8],-15[7],add(15)(p11)[5],add(15)(p11)[4],+16[5], add(16)(p11)[3],add(16)(q24)[2],add(16)(p11)[10],add(16)[4],-17[10],-17[8],add(17)(q24)[3],?del(17)(p11)[3],-18[5],add(18)(p11)[9], add(18)(q21)[5],+19[5],add(19)(p11)[9],add(19)(q13)[8],del(19)(p13)[9],+20[7],add(20)(p11)[7],add(20)(p13)[8],add(20)(q11)[4],+21[8],+21[4],add(21)(p11)[10],add(21)(p11)[4],add(21)(q22)[7],+22[10],+22[7],+22[3],del(22)(q13)[10],del(22)[9],+10~18 mar.

Significant gains of DNA sequences were observed for locus DAB2 at chromosome 5q13, CCND2 at 12p13, MDM2 at 12q14.3-q15, FLI, TOP3A at 17p11.2-p12, and OCRL1 at Xq25. Significant losses of DNA sequences were observed for HTR1B at 6q13, D6S268 at 6q16.3-q21, SHGC17327 at 18ptel, and STK6 at 20q13.2-q13.3. The representative aCGH profile is shown in Figure 9.