Human glioma cell lines U87, T98, U138, and D54 were obtained from the American Type Culture Collection (Manassas, VA, USA). LN-229 was a kind gift from Dr. Gilbert Cote (MD Anderson Cancer Center, Houston, TX, USA). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen Corp., Carlsbad, CA, USA) in a humidified incubator with 37°C and 5% CO2. For neurosphere induction, cells were cultured in serum-free medium (SFM), which consisted of neurobasal-A medium supplemented with B27, GlutaMAX-I supplement, 1% penicillin-streptomycin (Invitrogen Corp.), 50 ng/ml heparin (Sigma-Aldrich, St. Louis, MO, USA), 20 ng/ml of EGF, and 20 ng/ml bFGF (R&D Systems, Minneapolis, MN, USA). To induce neurosphere formation of LN-229 cells, 200 pM IL-1β and 200 pM TGF-β1 (both from R&D Systems) were added every other day to the serum-free medium. IL-6 and IL-8 (both from R&D Systems) were also used to evaluate their effects on induction of neurospheres.
Self-renewal assay and cell proliferation assay
LN-229 cells at a clonal density of 1 cell/μl in serum-free medium were seeded at 100 μl/well in 96-well plates and treated with or without 200 pM IL-1β and/or 200 pM TGF-β1 for seven days. IL-1β and TGF-β1 were added every other day. The total number of neurospheres in each well was counted under a microscope. Cell proliferation was measured using the cell proliferation kit I (MTT, Roche Applied Science, Indianapolis, IN, USA) as described by the manufacturer.
RNA extraction and RT-PCR
Total RNA was extracted using TRI reagent (Sigma-Aldrich), followed by digestion with a DNase kit (Applied Biosystems, Carlsbad, CA, USA) to remove DNA residues. Reverse transcription was carried out using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) and quantitative real-time PCR was performed using SsoFast Eva Green Supermix kit (Bio-Rad).
Immunostaining and immunoblotting
LN-229 cells were seeded in slide chambers (Fisher Scientific, Hanover Park, IL, USA) and cultured for seven days in serum-free medium in the absence or presence of IL-1β/TGF-β. Then, cells were fixed with 4% paraformaldehyde, permeablized with PBS containing 0.2% Triton X-100, and incubated with mouse anti-nestin (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and followed by secondary chicken anti-mouse IgG (H+L) antibody conjugated with Alexa 488 (1:200, Invitrogen). Cells were then mounted with VECTASHIELD Mounting Medium with DAPI (Vector laboratories, Burlingame, CA, USA) and observed with a confocal microscope.
LN-229 cells were cultured in SFM in the absence or presence of IL-1β/TGF-β for seven days. Cells were then washed with cold PBS, lysed in RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and pelleted by centrifugation. Protein concentrations were determined using a NanoDrop instrument (Thermo Scientific, Wilmington, DE, USA). Cell lysates (30 μg protein for each sample) were incubated for five minutes at 100°C in 2x loading buffer, separated by electrophoresis in 10% polyacrylamide gels, and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% milk in PBS and then incubated with primary antibody anti-Bmi-1 clone F6 (1:1,000 dilution, Millipore), or anti-β-actin (1:1,000 dilution, Sigma), and secondary antibody HRP-conjugated goat anti-mouse IgG-HRP (1:1,000 dilution, Millipore) or anti-rabbit IgG HRP-linked antibody (1:1,000 dilution, Cell Signaling, Danvers, MA, USA), respectively. Detection was performed using HyGLO substrate (Denville Scientific, Metuchen, NJ, USA) and images were taken using the AlphaEaseFC imaging system (Cell Biosciences, Santa Clara, CA, USA).
The invasion assay was performed using 24-well Matrigel invasion chambers (BD Biosciences, San Jose, CA, USA). Single control monolayer cells and IL-1β/TGF-β-induced neurosphere cells were resuspended in serum-free medium and loaded into the top inserts (2.5 × 104/500 μl). DMEM with 20% FBS was added to the lower chamber (750 μl) as a chemoattractant. After incubation for 48 h at 37°C, non-invasive cells were removed from the top of the matrigel with a cotton tipped swab. Invasive cells that migrated through 8-μm pores to the underside of the membrane were fixed with 100% methanol and stained with 0.005% crystal violet before counting under a microscope.
Control monolayer and IL-1β/TGF-β-induced neurosphere cells in serum-free medium were treated with temozolomide (Sigma-Aldrich) at a concentration of 250, 500, 1,000 μM for two days. Then cells were stained with Trypan blue (Amresco Inc., Solon, OH, USA) and counted under a microscope. The viability of the cells was measured by the percentage of live cells over the total of live and dead cells.
Soft-agar colony-forming assay
The soft agar assay was performed in six-well plates containing two layers of Sea Plague Agar (Invitrogen). The bottom layer consisted of 0.8% agar in 1 ml of DMEM with 10% FBS. Control monolayer and IL-1β/TGF-β-induced neurosphere cells were dissociated and placed (1 × 104/well) in the top layer containing 0.4% agar in the same medium as the bottom. Cells were cultured for two to three weeks and colonies were photographed under a microscope and measured using the ImageJ program (http://imagej.nih.gov). Colonies with diameters larger than 80 μm were counted.
Transfection of LN-229 cells with Gaussia luciferase
LN-229 cells were transfected with a pCMV-Gluc-1 vector encoding the Gaussia luciferase gene (Nanolight Technology, Pinetop, AZ, USA) using Lipofectamine 2000 (Invitrogen). The stable transfectants were selected with gentamicin-418 at 1,000 μg/ml. Expression of Gluc was confirmed by a luciferase assay with the IVIS Lumina II imaging system (Caliper Life Sciences, Hopkinton, MA, USA).
Implantation of LN-229 cells into immunocompromised mice
The LN-229 cells stably expressing Gluc were used for an in vivo study. Athymic nude mice (nu/nu, six to seven weeks old; Charles River Laboratories, Wilmington, MA, USA) were stereotactically implanted with the control monolayer cells (3 × 105 cells/mouse) or IL-1β/TGF-β-induced neurosphere cells (3 × 105 cells/mouse) in 3 μl of PBS into the right striatum (1 mm anterior and 2 mm lateral to bregma; 3.5 mm intraparenchymal). Five weeks after implantation, animals were subjected to bioluminescence imaging, followed by euthanization. Brains were removed, placed in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA, USA), and quickly frozen in liquid nitrogen. The frozen tissues were then stored at -80°C until sectioning as described below. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Kansas State University and the IACUC number is 2813.
Bioluminescence imaging was performed using the IVIS Lumina II imaging system (Caliper Life Sciences). To determine the correlation between the number of Gluc-expressing cells and the bioluminescence signal in vitro, various numbers of the monolayer or IL-1β/TGF-β-induced neurosphere cells were used, and images were taken one minute after a substrate for Gluc (60 μg/ml coelenterazine) was added to the serum-free medium. For in vivo bioluminescence imaging, coelenterazine (150 μg/animal in 150 μl of PBS) was given i.v.; animals were anesthetized with 2.5 to 3% isoflurane for approximately two minutes and then placed inside the camera box (Caliper Life Sciences) that maintained 2% isoflurane for the duration of imaging. The imaging scan time for each animal was one minute.
Coelenterazine synthesis and solution preparation
Coelenterazine was synthesized by an extensive modification of the procedure reported by Adamczyk et al. . Aminopyrazine was used as a starting material to generate 3,5-diboromoaminopyrizine as a building block for introducing the pyrazine moiety of coelenterazine. A series of Negishi coupling reactions was used to cross-couple the benzyl and 4-hydroxyphenyl groups to 3,5-diboromoaminopyrizine by using Huo's and Buchwald's approach to zincation [36, 37]. The complete synthesis and characterization of coelentearzine will be published separately.
Coelenterazine and hydroxypropyl-beta-cyclodextrin (Acros Organics, Pittsburgh, PA, USA) (1:50, w:w) was dissolved in methanol (ACS grade). Methanol was removed by high vacuum distillation to obtain a dry yellow powder of the mixture of coelenterazine and cyclodextrin. This mixture was dissolved in autoclaved distilled water prior to imaging. A total of 150 μL of coelentrazine (1 mg/mL coelenterazine), which is the substrate for Gaussia luciferase, was injected i.v. prior to tumor imaging.
Brain sectioning, H&E staining and tumor volume calculation
Mouse brains were cryostat-cut at 10 μm thickness in coronal sections, mounted on Probe One Plus slides (Fisher Scientific) and stained with an H&E stain kit (BBC Biochemical, Stanwood, WA, USA).
Enzyme-linked immunosorbent assay (ELISA)
Cells were treated with or without IL-1β and TGF-β in serum-free medium for six days. Cells were then washed with PBS and cultured in serum-free medium without cytokines for 24 h at 37°C. Cell supernatants were harvested and assayed for IL-1β and IL-8 using Quantikine ELISA Kits (R&D Systems) as described by the manufacturer.
Student's t-test was used to determine statistical significance for all analyzed data except the in vivo bioluminescence signal experiment. A Fisher's exact test was used to compare the percentage of mice with positive bioluminescence signals. In all cases, we consider a two-sided P < 0.05 significant.