Human osteosarcoma cell lines U2-OS and MG-63 were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco), penicillin, and streptomycin (Sigma). All cells were grown in a humidified incubator at 37°C with 5% CO₂.

The retroviral expression vector pQCXIN-ZsGreen-cODC, containing green fluorescence ZsGreen-labeled degron ODC, was kindly provided by Dr Shinji Tanaka. The retroviral vector was transfected into platinum A (Plat-A) to generate a retrovirus. The vector was transfected into Plat-A retroviral packaging cells using FuGENE 6 (Promega); the virus collected from the supernatant of Plat-A was used to infect osteosarcoma cells. The stable transfectants were selected with Geneticin (Invitrogen) and the accumulation of ZsGreen-labeled degron ODC protein was monitored by fluorescence microscopy and flow cytometry. To determine if this was a stable transfection, the cells were exposed to proteasome inhibitor MG-132 (Wako, Japan) for 12 h.

After FACS, ZsGreen⁺ cells were separately plated at a density of 10⁴ cells in 35-mm dishes in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco) and Pen/Strep (Sigma). After incubation in 5% CO₂ at 37°C overnight, cell attachment was confirmed. Image analysis was performed using a FV1200 confocal microscope (Olympus).

We used a FACS Aria II (BD Biosciences, San Jose, CA, USA) for cell sorting. Osteosarcoma cells were washed with phosphate-buffered saline (PBS) and enzymatically dissociated with 0.05% trypsin-EDTA (Invitrogen). Cells were gently triturated and filtered through cell strainer caps (35-μm mesh) to obtain a single cell suspension (~1×10⁷ cells/ml). The presence of ZsGreen allows the selection of cells with low proteasome activity using flow cytometry with standard fluorescein isothiocyanate (FITC) for cell sorting.

Using Proteasome-Glo™ cell-based assays (Promega), the chymotrypsin-like, trypsin-like, and caspase-like activities were measured according to the manufacturer’s protocol. Proteasome-Glo™ buffer was mixed with luciferin detection reagent, and the substrate was added to the mixture and incubated at room temperature for 1 h. An equal volume of proteasome-Glo™ reagent was added to the samples and further incubated for 15 min. The luminogenic substrate containing the Suc-LLVY sequence was recognized by the proteasome. Following the proteasome cleavage, the substrate for luciferase (aminoluciferin) was released, allowing the luciferase reaction to proceed and produce light. The luminescence was measured using a luminometer (Thermo Fisher Scientific, Waltham, MA, USA).

ZsGreen⁺ and ZsGreen⁻ cells were sorted by FACS, plated separately at a density of 10,000 cells on low-attachment 6-well plates (Costar, Corning, NY, USA), and incubated in serum-free Dulbecco’s modified Eagle’s medium/F12 medium (Invitrogen) supplemented with b-FGF, EGF (Sigma), and N2 (Wako). After 14 days, spheres with diameters >100 μm were counted.

Our animal studies were approved by the Animal Experiments Committee of Osaka University (Suita, Japan). Following FACS, the portions containing 1×10³, 1×10⁴ and 1×10⁵ cells were mixed with BD Matrigel (Becton-Dickinson, Franklin Lakes, NJ, USA) at a 1:1 ratio and were subcutaneously injected into 4- to 5-week-old female NOD/SCID mice (Charles River, Japan). The animals were anesthetized with isofluorane and maintained under sterile airflow conditions during the experiments.

Proliferation assays for the U2-OS and MG-63 cells treated with MTX and CDDP (both purchased from Wako, Japan) were performed to test chemosensitivity. Briefly, a total of 2×10³ of both ZsGreen⁺ and ZsGreen⁻ cells were sorted, individually seeded into 96-well plates (Corning), and treated with varying concentrations of the drugs for 72 h. The assay was performed using a commercially available cell counting kit-8 (Dojindo, Japan). The remaining viable cell count was determined by measuring the absorbance at 450 nm using an Enspire plate reader (Perkin-Elmer).

The appropriate number of ZsGreen⁺ and ZsGreen⁻ cells were sorted, individually seeded in 6-cm dishes, and exposed to radiation at 0, 2, 4, 6, and 8 Gy by Cs-137 gamma irradiation generated using a Gammacell 40 Exactor (MDS Nordion, Ottawa, Canada). After incubation for 14 days, the colonies were fixed and stained with crystal violet. Colonies containing >50 cells were counted as survivors. At least three parallel samples were scored in three to five repetitions conducted for each irradiation condition.

Data are expressed as means ± SDs. Statistically significant differences were determined by two-way ANOVA analysis and Student’s t-test, where appropriate, and were defined as p<0.05.

To monitor proteasome activity, two osteosarcoma cell lines, U2-OS and MG-63, were stably transfected with retroviral vector pQCXIN-ZsGreen-cODC. After geneticin selection and single cell cloning, we established two osteosarcoma cell lines transfected with a proteasome sensor vector. We divided these cells into ZsGreen⁺ and ZsGreen⁻ groups according to their fluorescence intensity. We could easily identify ZsGreen⁺ cells (Fig. 1). The FACS analysis showed that the fractions of ZsGreen⁺ cells were 1.7% in U2-OS and 5.1% in MG-63 (Fig. 2A). To validate the proteasome activity monitoring, we performed proteasome inhibition of these established cell lines with MG-132. Fig. 2A shows that proteasome inhibition dramatically increased the fraction of ZsGreen⁺ cells from 1.7 to 93.7% in U2-OS and from 5.1 to 37.8% in MG-63. Furthermore, we directly measured proteasome activity, such as trypsin-like, chymotrypsin-like, and caspase-like activities, using Proteasome Glo; this showed that ZsGreen⁺ cells had lower proteasome activity compared with ZsGreen⁻ cells in MG-63 (Fig. 2B), but not in U2-OS (data not shown).

TICs have self-renewal and multilineage differentiation capacity. To test this, we separately cultured ZsGreen⁺ and ZsGreen⁻ cells after cell sorting for U2-OS. Time-lapse imaging showed asymmetric divisions of the ZsGreen⁺ cells into ZsGreen⁺ and ZsGreen⁻ cells (Fig. 3A), whereas the ZsGreen⁻ cells did not divide into ZsGreen⁺ cells. Similarly, the FACS analysis showed that the ZsGreen⁺ cells of U2-OS regenerated into ZsGreen⁺ and ZsGreen⁻ cells, whereas the ZsGreen⁻ cells divided into only ZsGreen⁻ cells (Fig. 3B). These findings indicate that the ZsGreen⁺ population had the capacity for self-renewal and multilineage differentiation. However, the ZsGreen⁺ and ZsGreen⁻ cells of MG-63 divided into both ZsGreen⁺ and ZsGreen⁻ cells, which indicated that both the populations had the capacity for differentiation.

To evaluate self-renewal, we evaluated the sphere-forming capacity of ZsGreen⁺ and ZsGreen⁻ cells. A total of 10,000 ZsGreen⁺ and ZsGreen⁻ cells were sorted and cultured in serum-free conditions. The frequencies of sphere formation for ZsGreen⁺ and ZsGreen⁻ were 0.16 and 0.013% (p<0.001) in U2-OS and 0.23 and 0.097% (p=0.027) in MG-63 cells (Fig. 4A and B), respectively. The ZsGreen⁺ cells showed a high frequency of sphere formation compared with the ZsGreen⁻ cells.

TICs are primarily characterized by the ability to form tumors in NOD/SCID mice. To determine whether ZsGreen⁺ cells were more tumorigenic than ZsGreen⁻ cells in vivo, we injected both the populations separately into NOD/SCID mice. After 3 months, no tumors were observed (Table I).

To test chemoresistance, ZsGreen⁺ and ZsGreen⁻ cells from two osteosarcoma cell lines were exposed to MTX and CDDP. The difference in chemoresistance between the ZsGreen⁺ and ZsGreen⁻ cells was assessed using the cell proliferation assay. Of the two cell lines, the ZsGreen⁺ cells exhibited significant resistance to MTX, but not to CDDP (Fig. 5A).

Next, to evaluate radioresistance, we performed clonogenic survival assays for ZsGreen⁺ and ZsGreen⁻ cells of the two cell lines. As shown in Fig. 5B, ZsGreen⁺ cells were more radioresistant than ZsGreen⁻ cells in both cell lines. These results indicated that the ZsGreen⁺ cells were resistant to chemotherapy and radiotherapy.