Male C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME), maintained in our laboratory and used at ages between 6–9 weeks. All mice were provided autoclaved food pellets and water ad libitum. All surgical procedures were completed in accordance with an approved protocol by the University of Chicago IACUC.
Mouse glioma cell line
GL261 cells carry a point mutation in the K-ras and p53 genes, do not express major histocompatibility complex II and have been extensively characterized (Szatmári et al., 2006
). GL261 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10 % fetal calf serum as well as streptomycin (100 mg/ml) and penicillin (100 U/ml) at 37 °C in a humidified atmosphere of 95 % air / 5 % CO2
. All cell culture products were purchased from Gibco Invitrogen (Carlsbad, CA).
Mouse intracranial-injection model
Mice were anesthetized with an intraperitoneal injection of 0.1 ml of a stock solution containing ketamine HCl (25 mg/ml), xylazine (2.5 mg/ml), and 14.25 % ethyl alcohol (diluted 1 : 3 in 0.9 % NaCl). For the stereotactic intracranial injection, the surgical site was shaved and prepared with 70 % ethyl alcohol. A midline incision was made, and a 1 mm diameter right parietal burr hole, centered 2 mm posterior to the coronal suture and 2 mm lateral to the sagittal suture, was drilled. Mice were placed in a stereotactic frame and 2.5 µL saline or 4 × 105 GL261 cells in 2.5 µL saline were injected intracranially with a 26-gauge needle at a depth of 3 mm. The needle was removed and the skin was sutured with 4-0 nylon thread.
Brains were taken from mice with GL261-cell based brain tumors at 3 weeks post-operative (WPO) and were flash frozen [in 62.5 % n-Butyl Bromide (Fisher Scientific) + 37.5 % 2-methylbutane (Fisher Scientific) surrounded by crushed dry ice] as previously described by Wainwright et al. (2010)
. Briefly, frozen tissue was immersed into Tissue Teck O.C.T. Compound (Sakura Finetek) and sectioned at a temperature of −24 °C at 8 µm intervals and thaw-mounted onto pre-cleaned SuperFrost slides (Fisher Scientific). Sections were post-fixed with 4 % paraformaldehyde, blocked for endogenous biotin for 5 min (1 % H2
in PBS), and blocked for non-specific staining with 10 % bovine serum albumin (Sigma-Aldrich) in PBS for 1 hr. Sections of mouse tissue were incubated with anti-BST2-alexa fluor 647 (eBio927, 1:500; Ebioscience; San Diego, CA) and anti-CD11c-alexa fluor 488 (N418, 1:50; Ebioscience) in PBS at 4 °C overnight. Following extensive washing in PBS, sections were covered with Ultra Cruz mounting media with DAPI (Santa Cruz). Images of antibody-stained sections were captured using either the Sp5 Tandem Scanner 2-photon confocal microscope (Leica Microsystems; Bannockburn, IL) running LAS AF software (Leica Microsystems) using the argon, and red NeHe laser lines or the Macroscope (MVX10; Olympus; Center Valley, PA) using cellSens digital imaging software (Olympus). Confocal fluorescent images were captured using the 20× or 63× objectives.
Resected specimens from patients who underwent operations in the Section of Neurosurgery at the University of Chicago Medical Center between 2009 and 2010 were evaluated in this study. According to the WHO classification, samples included normal brain, grade II, III or IV astrocytoma. Normal brain control tissue consisted of nonmalignant tissue obtained during resection from a patient with a brain tumor. Histological confirmation of the diagnosis for tumors was obtained in all cases by an attending neuropathologist. The tissue was collected in accordance with a protocol approved by the Institutional Review Board (IRB) at the University of Chicago.
RNA isolation, semi-quantitative PCR and real-time PCR
Total cellular RNA from normal WT mouse brain, mouse brain IC-injected saline at 3 WPO, mouse brain IC-injected with GL261 cells at 3 WPO or normal (non-malignant) human brain and human astrocytoma grades 2 – 4 were isolated using the RNeasy Mini kit (Qiagen; Valencia, CA) according to the manufacturer’s protocol. Equivalent amounts of RNA were reverse-transcribed with the iScript cDNA Synthesis Kit (Bio-rad Laboratories; Hercules, CA). Semi-quantitative PCR was performed using 5 uL Taq PCR Master Mix, 2.5 µM forward primer, 2.5 µM reverse primer, 2 uL RNase-free H2O and 1 ng input cDNA per reaction. Amplification was performed using the iCycler Thermal Cycler (Bio-Rad Laboratories) under the following conditions: 3 min at 95 °C, followed by 30 cycles of 30 sec at 95 °C, 30 sec at 60 °C and 30 sec at 72 °C, followed by 5 min at 72 °C. PCR products combined with 2 uL 6X DNA Loading Dye (Fermentas Life Sciences; Glen Burnie, MD) were ran on a 2 % agarose gel with the GeneRuler 100 bp DNA ladder. The gel was visualized in the ChemiDoc XRS Universal Hood II (Bio-rad Laboratories). Real-time PCR was performed using 5 uL SYBR GreenER qPCR Supermix Universal (Invitrogen; Carlsbad, CA), 0.5 µM forward primer, 0.5 µM reverse primer, 3 uL RNase-free H2O and 1 ng input cDNA per reaction. Amplification and detection were performed using the Opticon 2 Real-Time PCR Detector (Bio-rad Laboratories) using Opticon Monitor software (version 3.1.32; Bio-rad Laboratories) under the following conditions: 15 min hot start at 95 °C, 15 sec denaturation at 95 °C, 20 sec annealing of primers at 60 °C, and 15 sec elongation at 72 °C, for 35 cycles. Triplicate reactions were performed for all cDNA samples. For mouse and human samples, percent change in mRNA levels was calculated using the formula ((Experimental brain group / normal brain group) × 100)) – 100 %. Mouse and human primer sequences used for PCR reactions are found in .
Mouse (m) and human (h) primer sequences. The gene symbols used above indicate: BST2 = bone marrow stromal cell antigen 2; GAPDH = glyceraldehyde 3-phosphate dehydrogenase.
Human brain tumor array
The multiple brain cancer and normal adjacent tissue array (US Biomax; GL1001t) was deparaffinized in xylene and rehydrated through a descending strength ethanol series before being rinsed in tap water. The slide was heated in a sodium citrate buffer (10 mM sodium citrate, 0.05 % Tween-20, pH 6.0) at 97 °C for 40 min and cooled at room temp for 20 min. Sections were rinsed in PBS, serum- and biotin-blocked as described above and incubated with anti-BST2-alexa fluor 488 (26F8, 1:250; Ebioscience) overnight. Following extensive washing in PBS, sections were covered with Ultra Cruz mounting media (Santa Cruz). Photomicrographs of all antibody-stained tissue specimens were taken using the Macroscope (MVX10; Olympus; Center Valley, PA) using cellSens digital imaging software (Olympus). For each representative antibody-stained section, the mean fluorescent intensity was calculated using image J based on standardized arbitrary units. The fluorescent intensities represent averages of individual astrocytoma patient samples that were provided on the array in triplicate.
GL261 cells were adjusted to 1 × 106 and stained in PBS + 1 % bovine serum albumin (Sigma-Aldrich) for 30 min on ice and stained with: anti-BST2-PerCP-eFluor 710 (eBio927; Ebioscience) or IgG2B-PerCP-eFluor 710 (Ebioscience). Cells were washed extensively in PBS and the cellular frequency and geometric mean fluorescence intensity were determined by the LSR II flow cytometer (BD; Franklin Lakes, NJ) and Flowjo analysis software (TreeStar; Cupertino, CA). At least ten-thousand events were collected for each sample.
BST2 knockdown and anti-BST2 pre-incubation with GL261 cells
BST2 shRNA lentiviral particles containing 3 target-specific constructs or shRNA constructs encoding a scrambled sequence were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). To knock down BST2 gene expression, GL261 mouse glioma cells were transduced with control or BST2 shRNA lentiviral particles at a ratio of 5 infectious units of virus per cell in the presence of 8 µg/mL polybrene for 6 hours. The next day, fresh media containing puromycin at 2 µg/mL was added to the media for the selection of cells that had stably incorporated shRNA. Selected cells were expanded and transduced with control or BST2 shRNA lentiviral particles and the protocol described above was repeated. BST2 gene silencing in these cells was confirmed at the protein level by flow cytometry. Finally, cells were stained with anti-BST2-PerCP-eFluor 710 (eBio927; Ebioscience) and sorted on the FACSAria II (BD) isolating the BST2-expressing and BST2-deficient cells from GL261 cells transduced with scrambled and BST2 shRNA, respectively. The cells were then maintained as previously described. For BST2 pre-incubation, 5 × 106 GL261 cells were stained with 50 ug/mL purified anti-BST2 (eBio927; Ebioscience) in PBS + 1 % bovine serum albumin (Sigma-Aldrich) for 30 min on ice and washed in PBS extensively. The cells were then intracranially-injected 1 – 2 hours after pre-incubation.
Survival curves were calculated according to the method of Kaplan–Meier. Overall survival is defined as the time from injection of GL261 cells to the last day of the time course. The p-value was obtained by logrank statistical analysis and was considered significant when p < 0.05. For non-survival curves, data are presented as ± SEM and were analyzed by one-way ANOVA or the Student’s t test and a p < 0.05 was considered significant. All analyses were performed using GraphPad Prism version 4.00 (GraphPad Software, Inc.).