Tumor tissues and cell lines
All studies reported here were performed with approval of the Institutional Review Board of the Seoul National University Hospital. Snap-frozen medulloblastoma tissue from 39 patients was retrieved from the Brain Bank of the Division of Pediatric Neurosurgery, Seoul National University Children’s Hospital. Normal cerebellar tissue was retrieved from the same tissue bank for use as a control. Patient selection was based on the availability of snap-frozen tissues. The person who selected the patients was blind to patient’s clinical information except diagnosis. Human medulloblastoma cell lines (D283 and Daoy) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). D283 cells were cultured in Minimum Essential Medium Eagle (EMEM; ATCC), and Daoy cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen) and penicillin-streptomycin (1× final concentration; Invitrogen). All cells were incubated at 37°C in a 5% CO2/ 95% air atmosphere.
Real-time quantitative polymerase chain reaction (PCR)
The levels of mRNA transcription were assessed by real-time quantitative PCR (RT-qPCR) using TaqMan probes (Applied Biosystems, Carlsbad, CA) in an ABI 7000 system. TaqMan probes for ID1 (ABI Catalog number Hs00357821), ID2 (Hs00747379), ID3 (Hs00171409), ID4 (Hs00155465), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Hs99999905) were used. The reactions were performed under the conditions specified in the ABI TaqMan Gene Quantitation assay protocol, and all reactions were repeated in triplets. The comparative threshold cycle (ΔCt) method calculated the relative gene expression, normalized to GAPDH and relative to normal brain expression .
siRNA and shRNA knockdown of ID3
siRNA and shRNA were used to knock down ID3 expression in the D283 cell line. Control siRNA (#SN1002) and ID3-siRNA (#1072358) were designed and synthesized by Bioneer (Daejeon, Korea) for the in vitro studies. Transfection of the control-siRNA and ID3-siRNA was performed using Lipofectamine RNAiMax (Invitrogen) following the manufacturer’s instructions. Lentiviral particles containing shRNA targeting the human ID3 (SHCHLNG-NM_002167), nontargeting shRNA (SHC002V), and GFP (SHC003V)-containing control transduction particles were obtained from Sigma-Aldrich (St. Louis, MO) for in vivo studies. D283 cells were seeded in 96-well plates and transduced in 110 μl of EMEM containing 10% FBS and 8 μg/ml hexadimethrine bromide. The cells were reseeded in 6-well plates 24 hrs after incubation and selected using 1 μg/ml puromycin for 7 days. Knockdown efficiency and specificity with siRNA and shRNA was confirmed using RT-qPCR with the gene expression normalized to GAPDH. Knockdown of ID gene expression was further confirmed by western blot.
ID3 rescue experiment
To prove the specificity of the ID3-shRNA knockdown, full-length ID3 cDNA (360 bp) was synthesized using the RT–PCR Kit (Clontech, Mountain View, CA) from RNA extraction of D283 cells. Constructs were inserted into the BamH1/Xho1cloning site of pEGFP.C2 (BD Biosciences, San Jose, CA) and then transfected into the ID3 knockdown D283 cell line using the Neon® Transfection (Invitrogen) according to the manufacturer’s instructions with some modifications. ID3-shRNA knockdown cells (1 × 107) were resuspended in 120 μl of Neon® Resuspension Buffer R with 12 μg of plasmid DNA pulsed once according to the manufacturer’s instructions. After the pulse, cells were quickly transferred into EMEM media containing 10% FBS. Cells transfected with a pEGFP.C2 vector were used as a control. Expression of green fluorescent protein (GFP) was observed by fluorescence microscopy 24 hrs after nucleofection. The cells were then incubated for 48 hrs before RNA and protein collection for further experiments.
After Transfection with siRNA negative control or ID3-siRNA, cells were resuspended in protein extraction solution (Intron Biotech, Seongnam, Korea), according to the manufacturer’s protocol. Protein concentrations were determined using BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). For immunoblottg, 40 μg of protein was prepared in SDS sample buffer (Invitrogen), boiled for 10 min at 70°C and electrophoresed on a 4–12% gradient bis-Tris gel (Invitrogen). The proteins were then electotransfered to a polyvinylidene fluoride membrane (Invitrogen) using iblot transfer system (Invitrogen). After the membrane had been blocked with Tris-buffered saline (TBS) containing 5% nonfat dry milk, it was incubated overnight at 4°C with the following anti-ID1 antibody (1:500; Abnova, Taipei, Taiwan), anti-ID2 antibody (1:500; Cell Signaling Technology, Danvers, MA), anti-ID3 antibody (1:500; Cell Signaling Technology), anti-ID4 antibody (1:250; Abcam, Cambridge, UK) and monoclonal anti-β-actin antibody (1:10,000; Sigma-Aldrich) in TBS containing 0.1% Tween-20. After the blot was washed, it was incubated with horseradish-peroxidase–conjugated species-specific secondary antibody (1:5,000; Jackson Laboratory, West Grove, PA) for 1 hr at room temperature. After the blots had been washed several times in TBS with 0.1% Tween-20, they were developed with enhanced chemiluminescence reagent (Invitrogen) and exposed to Kodak BioMax autoradiography film, and developed.
D283 cells were transfected with control-siRNA or ID3-siRNA, seeded in 96-well plates (5 × 103), and incubated for 48 hrs. CCK (cell counting kit; Dojindo, Kumamoto, Japan) was added and incubated for 2 hrs. Then, absorbance of each well was measured at 540 nm using a micro-ELISA reader (Molecular Devices, Sunnyvale, CA). The percentage of cellular survival was determined using the relative absorbance of ID3-siRNA-transfected cells versus control-siRNA-transfected cells. All in vitro assays were performed in triplicate.
The proliferation rates of D283 cells were measured using a BrdU ELISA kit (Roche Diagnostics, Basel, Switzerland) 48 hrs after transfection with control- or ID3-siRNA. The cells were plated in 96-well plates at an equal density (5 × 103). BrdU was added to the cells for 4 hrs, and the cells were treated according to the manufacture’s protocol. The optical density at 450 nm was measured using an ELISA plate reader.
TUNEL assay was performed for the detection of apoptotic cells using an ApopTag Peroxidase In situ Apoptosis Detection Kit (Chemicon, Temecula, CA). D283 cells were transfected with control- or ID3-siRNA (1 × 104) and cultured in 2-well chamber slides for 24-48 hrs. The cells were fixed and stained according to the manufacturer’s instructions. Apoptotic cells were observed and quantified in 5 randomly chosen high-power fields under a light microscope. The apoptosis index was defined as a percentage of the observed apoptotic cells in 1,000 cells.
Cellular senescence assay
Senescence-associated ß-galactosidase (SA-ß-gal) activity was detected using the Cellular Senescence Assay Kit (Chemicon), according to the manufacturer’s instructions. D283 cells were transfected with control- or ID3-siRNA (1 × 104), seeded in 6-well plates, and incubated for 16 hrs at 37°C. Representative microscopic fields were photographed under a 10× objective lens.
Cell cycle analysis
D283 cells were transfected with control- or ID3-siRNA (1 × 106) and detached by scraping. The cells were fixed in 70% iced-cold ethanol with vortexing and incubation for 1 hr at 20°C. The cells were washed with cold PBS and resuspended with 0.5 mg/ml Rnase A (Sigma-Aldrich). After 1 hr at 37°C, 10 μg/ml propidium iodine solution (Sigma-Aldrich) was added in the dark at 4°C and the cells were observed with fluorescent microscopy. The cells were analyzed using fluorescence-activated cell sorting (FACS).
D283 cells were transfected with control- or ID3-siRNA prior to seeding onto the upper chamber of a Transwell (8 μm pore size; Corning Costar, Corning, NY). The cells (5 × 104/ well) were harvested after transfection and introduced into the upper chamber. The cells in the upper chamber were maintained in serum-free medium that included mitomycin-C (10 μg/ml; Sigma-Aldrich), and the lower chamber was filled with culture medium supplemented with 10% fetal bovine serum as the chemoattractant. The cells without siRNA treatment were included as reagent control. The remaining cells at the upper surface were completely removed using a cotton swab after 16 hrs. The migrated cells on the lower surfaces of the membranes were fixed and stained in a solution of 1% (w/v) crystal violet in 2% ethanol for 30 seconds and rinsed in distilled water. The migrated cells were quantified in 5 randomly chosen fields. The assays were performed in triplicate.
mRNA miniarray for 94 genes related to cellular invasion and migration
The mRNA expression of 94 cellular invasion and migration gene was analyzed using a ready-to-use Array Human Extracellular Matrix & Adhesion Molecules 96-well Plate (Applied Biosystems; Catalog number, 4414133) and the ABI 7500 Real-Time qPCR system. Selected genes that demonstrated large discrepancies were confirmed using RT-PCR. The primer sequences and PCR parameters are summarized in Additional file 1: Table S1.
Reverse transcriptase polymerase chain reaction
Total RNA was isolated from human tissues and tumor cell lines using a PureLink RNA mini-kit (Invitrogen). cDNA synthesis was performed using EcoDry Premix-Random hexamers (Clontech, Madison, WI), following the manufacturer’s instructions. PCR amplification was performed using AccuPower PCR premix (Bioneer). The primer sequences and PCR parameters are summarized in Additional file 1: Table S1. The PCR products were resolved on a 1% agarose gel stained with ethidium bromide and visualized using a UV transilluminator.
Four paraffin-embedded medulloblastoma tissues (2 tissues from patients with seeding and 2 tissues from patients without seeding) were sectioned at 4 μm using a microtome and transferred to silane-coated slides. Immunohistochemistry was performed as described previously . Primary antibodies and their concentrations were applied as follows: ID3 (1:50; Spring Bioscience, Pleasanton, CA), tissue inhibitor of metalloproteinase 3 (TIMP3; 1:100; Novus Biologicals, Littleton, CO), integrin beta 4 (ITGB4; 1:50; Abcam), collagen type XII alpha1 (COL12A1; 1:1; Abnova), ADAM metallopeptidase with thrombospondin type 1 motif 8 (ADAMTS8; 1:100; Abbiotec, San Diego, CA), tenascin C (TNC; 1:100; Abcam), connective tissue growth factor (CTGF; 1:600; Abcam), and intercellular adhesion molecule 1 (ICAM1; 1:25; Cell Signaling Technology).
Animal model and inhibition of tumor seeding in vivo
The Institutional Animal Care and Use Committee of Seoul National University College of Medicine approved all animal experiment protocols. Transplantation of cells into female BALB/cnude mice was performed under aseptic conditions. D283 cells were labeled using fluorescent magnetic nanoparticle (LEO-LiveTM-675; Biterials, Seoul, Korea) for live in vivo imaging or chloromethylbenzamido-DiI (Molecular Probes, Eugene, OR) for Immunofluorescence staining. The cells were washed three times after a 24-hour incubation and suspended in PBS at a concentration of 1.5 × 106 cells per 30 μl. Mice were anesthetized using an intraperitoneal injection of 100 mg/kg ketamine (Yuhan, Seoul, Korea) and 10 mg/kg xylazine (Bayer Korea, Seoul, Korea). The mouse heads were fixed in a stereotactic guiding device (David Kopf Instruments, Tujunga, CA), and the cisterna magna was exposed under a microscopic view. Labeled cells were slowly injected into the subarachnoid space of the cisterna magna using a 30-gauge needle .
Live in vivo image acquisition and analysis were performed using an in vivo multispectral imaging system (CRi Maestro™ In-Vivo Imaging System; Cri, Hopkinton, MA). The injected cells were observed using an in vivo multispectral imaging system every 3–4 days. The regions of interest (ROI) were drawn over the tumor and normal tissue, and the average signal (×106 photons/cm2 × s) for each area was measured.
The longitudinal length from the cranial to caudal ends of the signal was measured to evaluate the extent of seeding. The mice were perfused with 4% paraformaldehyde under deep anesthesia and sacrificed 30 days after cellular implantation. Whole brains and spinal cords were fixed and dehydrated in graded sucrose concentrations. The tissues were embedded in OCT compound (Tissue-Tek®; Sakura, Tokyo, Japan) and stored at −80°C. The brains were sectioned sagittally into 10 μm-thick slices using a cryostat. Spinal cords were sectioned in 5-μm intervals beginning at the cervicomedullary junction. The sections were stained with hematoxylin and eosin.
Immunofluorescence staining was further performed on the sections to confirm the presence of proliferating and apoptotic cells. Sectioned tissues were washed and the primary antibodies were applied. Primary antibodies used were anti-ID2 antibody (1:100; Cell Signaling Technology), anti-ID3 antibody (1:200; Cell Signaling Technology), anti-ID4 antibody (1:50; Abcam), and anti-Ki-67 nuclear antigen (1:400; Abcam) for proliferating cell staining, anti-caspase-3 (1:200; Cell Signaling Technology) for apoptotic cell staining, and anti-human nuclei (1:250; Millipore, Billerica, MA). Alexa Fluor 488 or 594-conjugated goat anti-rabbit or mouse immunoglobulin-G (IgG) (1:200; Invitrogen) were used for secondary antibodies. After washing, tissue sections were mounted with an anti-fading solution containing 4′-6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) and negative control staining were established by omitting the primary antibodies. The apoptotic and proliferating indices were defined as the percentage of positive nuclei within the total number of nuclei in three random fields. The sections were observed using a confocal microscope (Carl Zeiss, Oberkochen, Germany).
Review of patient clinical data
The electronic medical records (EMR) of selected patients were reviewed retrospectively. All patients received initial surgical resection at the Seoul National University Children’s Hospital from Nov. 1998 to Apr. 2009. Clinical data included the patient’s age at diagnosis, sex, tumor histology, and tumor seeding at presentation. Patient survival was confirmed from follow-up sheets in the EMR. Tumor progression was defined as radiological documentation of a new tumor or the growth of preexisting masses (> 25% in 2-D analyses). All patients were followed up from the time of initial surgery (initial diagnosis) until the date of death or until Jan 29, 2013, whichever occurred first. Death certificate information was retrieved from the National Statistical Office and the Ministry of Public Administration and Security. A structured data extraction form was developed in which clinical data from the EMR was merged with the death certificate information and experimental data (i.e., the expression levels of specific genes).
Identification of molecular subgroup of tumors
Medulloblastomas are heterogeneous tumors consisting of at least 4 distinct molecular subgroups . Tumor tissues from 31 patients out of the 39 patients comprising this study were also included in a large-scale genomic study of Dr. Taylor MD, in which subgroup affiliation was performed using a nanoString-based RNA assay . The information of subgroup allocations were obtained for 30 patients and the tumors were divided into WNT, SHH, Group 3, and Group 4 medulloblastoma (information courtesy of Dr. Taylor MD).
A Mann–Whitney U test or Student t test were applied to compare continuous variables between 2 groups. Analysis of variance (ANOVA) was used for comparison of data between 3 groups. Progression-free survival (PFS) was defined as the time interval from the day of initial surgery to the date that tumor progression was documented radiologically or the date of the last follow-up. Overall survival (OS) was defined as the time interval from the day of initial surgery to the date the patient died or the date of the last follow-up. We explored the best cutoff point of ID3 for predicting occurrence of death or progression of MB using receiver operator characteristic (ROC) analyses without considering the length of follow-up. Since the value of 6.007 was optimal for both outcomes, a high expression level of ID3 was defined as a greater than 6.007-fold increase in mRNA expression in RT-qPCR normalized to controls. Survival in each group was analyzed using a Kaplan-Meier method. A log-rank test was used for comparisons of survival data between groups. Multivariate analyses of PFS and OS were conducted using the Cox proportional hazard model. Clinical variables with a P value less than 0.1 in univariate analyses were included in the multivariate models. Basic clinical variables such as sex and age at diagnosis were included in multivariate analyses regardless of their P values. All tests were two-sided, and a P value less than 0.05 was considered significant. MedCalc version 12.4.0 (MedCalc, Ostend, Belgium; a free-trial version) was used for ROC analysis and IBM-SPSS version 19.0 software (IBM, Armonk, NY) was used for all the other statistical analyses.