The human glioblastoma cell line U87MG was purchased from ATCC. U87MG is derived from a human glioblastoma and is widely used in biological studies on gliomas, particularly those examining the pathway involved in glioma proliferation. We initially cultured naive U87MG cells in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS) (Fig. 1A) which was, according to a previous study (4), subsequently changed to non-serum DMEM/F12 containing B27 supplement (Gibco-Invitrogen, Grand Island, NY, USA), basic FGF (PeproTech EC Ltd., Rocky Hill, NJ, USA) and EGF (Upstate Biotechnology, Lake Placid, NY, USA). CD133⁺ cells were then selected by using an autoMACS™ Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany) and neurosphere-forming cells were cultured in the same medium (Fig. 1B). Serial passage of the cells was performed by a mechanical dissociation method and passaged cells, i.e., GSLCs, were used for subsequent experiments.

Characterization of U87 GSLCs was carried out by immunocytochemistry using the following antibodies: anti-CD133 (Santa Cruz Biotechnology, San Diego, CA, USA), anti-STAT3 (Cell Signaling Technology, Danvers, MA, USA), anti-JAK2 (Santa Cruz Biotechnology), anti-VHL (Santa Cruz Biotechnology), anti-Elongin A (Abgent, San Diego, CA, USA), anti-PTEN (Santa Cruz Biotechnology), anti-NeuroD (Santa Cruz Biotechnology), anti-MAP2 (Sigma-Aldrich, St. Louis, MO, USA) and anti-GFAP antibodies (Dako, Glostrup, Denmark), as described below. In addition, cell proliferation, tumorigenicity in the subcutaneous tissue of SCID mice (Charles River, Yokohama, Japan), as well as soft agar colony and neurosphere formation were examined as described below.

We hypothesized that STAT3 was inhibited by VHL, since most BC-box family proteins, including VHL, have the ability to inhibit the JAK/STAT pathway. STAT3 plays a role in stem cell maintenance. Therefore, we investigated whether VHL was able to downregulate STAT3 in GSLCs. Initially, we constructed a VHL-expressing adenovirus vector, as previously described (15).

The VHL-expressing adenovirus vector was prepared as previously described (15). Adenovirus vector encoding human VHL (VHL54-213 amino acids) was generated by the use of the cosmid vector pAxCAwt. As a control vector, the vector for green fluorescent protein (GFP), generated by the use of pAxCAwt, was obtained from the Riken Gene Bank (Saitama, Japan). For adenovirus infection, U87 GSCs were seeded into 6-cm dishes (1×10⁶ cells/cm²) 1 day prior to infection. The cells were then incubated for 1 h with 5 μl of the virus solution diluted to 1×10⁷ plaque-forming units per milliliter in DMEM/F12 medium containing 5% FCS at a multiplicity of infection of 10, which is a condition sufficient for nearly 100% cell infection. VHL-expressing adenovirus vector was transferred to U87 GSLCs following dissociation of the neurospheres into single cells. Two days following the transfection in dishes, some of the cultured cells were transferred onto a cover glass and subsequently fixed for immunocytochemical study while the remaining were used to extract protein for western blot analysis.

Naive U87MG cells attached to round cover slips were fixed for 24 h with 10% formalin naturally deodorized buffer solution. After irrigating twice with phosphate-buffered saline (PBS), the fixed cells were blocked with 5% normal donkey serum for 30 min. Subsequently, primary antibodies (anti-NeuroD, anti-MAP2, anti-STAT3, anti-VHL, anti-PTEN, anti-JAK2 and anti-GFAP) were diluted with PBS (1:200) and incubated with the cells for 1 h at room temperature. Following irrigation 3 times with PBS, the cells were incubated in the dark for 45 min at room temperature with FITC-linked anti-mouse IgG antibody (Sigma-Aldrich) or TRIC-linked anti-rabbit IgG antibody (Sigma-Aldrich) as secondary antibodies. Following a further 3 irrigations with PBS, the cells were incubated for 5 min with DAPI (Molecular Probes, Eugene, OR, USA) diluted 1:3,000 with PBS. Finally, the cover glasses bearing the cells were placed on glass slides following application of ProLong Gold Antifade Reagent (Life Technologies, Grand Island, NY, USA) to the slides. The cells were then observed under an FV300 confocal microscope (Olympus, Tokyo). Cell nuclei appeared as red fluorescence and primary antibody-reactive antigens as green fluorescence.

Protein was extracted from the cells with RIPA buffer (Thermo Scientific, Rockford, IL, USA; 0.05 M Tris-HCl, pH 7.4, containing 0.15 M NaCl, 0.25% deoxycholic acid, 1% NP-40, 0.1% SDS, 1 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 μg/ml leupeptin and 1 μg/ml aprotinin). Extracted proteins were electrophoresed on 8–15% SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes by using an iBlot™ Gel Transfer Device (Life Technologies) for 7 min. The blots were probed with primary and secondary antibodies by using a SNAP id Protein Detection system (Merck Millipore, Darmstadt, Germany). Immunoreactive bands were visualized by chemiluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, Japan). Images were analyzed with LAS-1000 (Fujifilm, Tokyo, Japan) and the density of the bands was determined by using Image Gauge software (Fujifilm). The following primary antibodies were used: anti-CD133 (Biorbyt, Cambridge, UK), anti-STAT3, anti-JAK2, anti-VHL and anti-PTEN. Horseradish peroxidase (HRP)-linked anti-rabbit IgG and HRP-linked anti-mouse IgG were used as the secondary antibodies.

The neurosphere formation assay was performed as follows (16): neurosphere-forming U87 GSLCs were dissociated into single cells by continuous pipetting for 10 min. Following confirmation of their single-cell status and dilution up to 5 cells/ml, 200 μl of the cell solution was placed into each well of a 96-well plate (mean, one cell per well). Subsequently, neurospheres ≥50 μm in diameter in a single plate were counted under a phase-contrast microscope 1 week following placement of the cells.

This assay was performed as previously described (17). Briefly, following dissociation of the U87 GSLC neurospheres, a single-cell suspension (1,000 cells/ml) in 0.5 ml of 0.3% agar in a medium consisting of 10% FCS in DMEM/F12 was overlaid onto 0.5 ml of 0.6% agar medium in the wells of 24-well plates. Following 3 weeks of cultivation in a 5% CO₂ incubator, each well was examined under a stereoscopic microscope for colonies consisting of >40 cells.

Cell proliferation was examined as previously described (18). After U87 GSLCs had been dissociated into single cells by 10-min continuous pipetting using a long thin pipette, the cells were transfected with the VHL-expressing adenovirus vector or GFP-expressing adenovirus vector as a control. They were then cultured in the U87 GSLC maintenance medium. Starting 1 day after the cultures had been prepared, the number of cells was counted as follows: control vector-transfected and VHL-expressing vector-transfected U87 GSLCs were washed once with PBS, then dissociated into single cells by 10-min continuous pipetting using a long thin pipette. The number of viable cells was counted following staining with trypan blue.

Subcutaneous implantation of control- or VHL-transfectant U87 GSLCs (1×10⁴ U87 GSLCs) into 4 SCID Hairless Outbred (SHO-Prkdc Hr) mice (Charles River) for each vector was performed. The mice were housed in a clean, temperature-controlled room on a 12-h day/night cycle with free access to food and water. One month following implantation, the mice were examined for subcutaneously-formed tumors. Formed tumors were histologically examined with hematoxylin-eosin staining and immunohistochemically with anti-CD133, anti-GFAP and anti-STAT3 antibodies. The animal experimental procedure was approved by the Institutional Animal Use Committee of Yokohama City University and was in accordance with the National Institutes of Health Guidelines for the care and use of laboratory animals.

Data were analyzed by ANOVA (SPSS II; SPSS, IBM, Tokyo, Japan) and a P-value of <0.05 was considered to indicate a statistically significant difference.

Naive cells of the U87MG cell line were cultured as substrate-attached cells in DMEM containing 10% FCS and the cells were passaged every 4–5 days. The cells displayed 2 or 3 cellular processes and were Elongin A positive, rarely CD133 positive, VHL negative and MAP2 negative. Also, the majority of the cells were STAT3 positive, partially JAK1 positive and PTEN negative.

To obtain GSLCs from the U87MG cell line, we changed the medium to non-serum DMEM containing basic FGF, EGF and B27 supplement. One month later, the cultured cells tended to float. By using the MACS method with anti-CD133 antibody, we collected CD133-positive fractioned cells, according to the manufacturer’s instructions, and then cultured them in the above medium for 2–3 weeks. The cultured cells formed numerous floating neurospheres. The neurospheres were dissociated and some of the single cells were stained by fluorescence immunocytochemistry for characterization, whereas the remaining were used for soft agar colony and neurosphere formation, as well as for cell proliferation assays. In addition, protein was extracted from neurospheres for western blot analysis.

Results of the immunocytochemical analysis of U87 GSLCs are depicted in Figs. 2 and 3A. The percentage of CD133-positive cells as well as STAT3- and JAK2-positive cells was higher in the control-vector than in the VHL-vector transfectants. The majority of the naive U87 cells were Elongin A positive, as were the U87 GCLSs. U87 GSLCs were also VHL and PTEN negative, similar to U87 naive cells. Approximately half of those cells were NeuroD positive, of which some were GFAP positive.

The results of western blot analysis (Fig. 3B) supported the immunocytochemical data. U87 GSLCs expressed CD133, STAT3 and JAK2, but not VHL or PTEN. Expression of CD133, STAT3 and JAK1 was not detected following VHL gene transfer, contrary to VHL and PTEN expression, which was detected. Following VHL gene transfer to U87 GSLCs, the percentage of VHL and PTEN positive cells was increased, whereas that of CD133, STAT3 and JAK2 positive cells was decreased.

The results of the colony-formation assay in soft agar medium demonstrated significantly greater colony formation in the control vector-transfected compared to the VHL-expressing vector-transfected U87 GSLCs (P<0.001) (Fig. 4A). Neurosphere formation, which is a reflection of the self-renewal ability, was also significantly greater in the former than in the latter (P<0.001) (Fig. 4B). Proliferation of control vector-transfected U87 GSLCs was significantly more pronounced compared to the VHL gene-transfected GSLCs 7 days following transfection (P<0.01) (Fig. 4C), although the difference between them was smaller than in the case of the soft agar colony or neurosphere formation assay.

Although subcutaneous transplantation of U87 GSLCs into SCID mice always resulted in tumor formation, transplantation of VHL gene-transfected U87 GSLCs resulted in markedly reduced or no tumor formation. The tumors arising from the U87 GSLCs transplanted into the SCID mice exhibited the pathological features of glioblastoma and most of the cells in the tumor tissue were CD133- and STAT3- positive but showed no immunoreactivity indicative of GFAP (Fig. 5).