GBM xenograft cell lines used are all established from tumor tissue harvested from patients undergoing surgical resection at the Mayo Clinic, Rochester, Minnesota. The studies were approved by the Mayo Clinic Institutional Review Board and necessary patient consents were obtained. All xenograft lines are from tumors classified as Grade IV gliomas based off of WHO Criteria. Cell lines are tested for mycoplasma infection prior to experiments and are routinely tested as part of maintenance protocols . All cell lines used are mycoplasma free. Cell lines are available upon request from Dr. Sarkaria.
Xenograft cells were labeled with microbeads conjugated to CD133 antibodies (Miltenyi), according to manufacturers’ specifications. Cells were then washed and applied to magnetic columns (Miltenyi). The flow through obtained was designated the CD133-low fraction. The column bound fraction was eluted and then sorted again to improve purity. This double sorted bound fraction was designated CD133-high. Fractions could then be analyzed by flow cytometry to assess and confirm enrichment of CD133.
Cells were fixed on ice for 20 min with 2% paraformaldehyde, blocked with 10% NGS for 1 h, followed by primary antibody incubation with PE-conjugated CD133 (Miltenyi) for 30 min. Labeled cells were analyzed using a BD Calibur, with unlabeled cells serving as negative controls. For cell cycle analysis, cells were pre-treated for 24 h with 100 ng/mL Sema3A, dissociated, and fixed with cold 70% ethanol for 30 min at 4 oC. Cells were washed and treated with RNAse (Qiagen). Propidium Iodide (PI) was added to the cells and analysis was conducted using a BD Calibur, with post-analysis using FlowJo software version 7.6.5.
GBM stem cell culture
All animal studies were approved by the Mayo Clinic Institutional Animal Care and Use Committee. All experiments were performed in compliance with and according to guidelines by the National Institutes of Health (NIH, Bethesda, MD, USA) and the Mayo Clinic (Rochester, MN, USA) Institutional Review Board and Institutional Animal Care and Use Committee guidelines as previously described in Carlson et al. . Established xenograft tumors were harvested from the flanks of athymic nude mice (athymic nude- foxn1nu) (Harlan). Briefly, primary human GBM samples were directly implanted into the flank of 6–8 week old athymic nude-foxn1nu mice in a 1:1 ratio by volume of tumor and Matrigel (Fisher). Tumors were aseptically dissected away from mouse flanks, and dissociated mechanically, then enzymatically with papain. Tumor cells were plated in stem cell media comprised of Neurobasal A (Life Tech), basic fibroblast growth factor (Stem Cell Tech) (20 ng/mL), epidermal growth factor (Sigma) (20 ng/mL), B27 without vitamin A (Life Tech), non-essential amino acids (Life Tech), Glutamax (Life Tech), sodium pyruvate (Life Tech), and penicillin/streptomycin (Life Tech).
Cells were plated on a Matrigel (Fisher) monolayer at a density of 600,000 cells in a 10 cm tissue culture dish, or in the absence of an extracellular matrix to promote tumorsphere formation. Differentiation of BTSCs was induced by culturing xenografts in 10% Fetal Bovine Serum (Atlanta Biologicals) in DMEM (Life Tech) with penicillin/streptomycin for at least 21 days.
Coverglasses were coated with poly-D-lysine (PDL) (10μg/mL, Sigma) followed by fibronectin (40μg/mL, Sigma). Tumor cells were then plated onto the PDL/fibronectin coated coverglasses in stem cell media. After 2 days, cells were immunostained for specific antigens with primary antibodies at 5-10μg/mL, and secondary antibodies at 2μg/mL. CD133 (Miltenyi): Cells were live labeled for 10 min with anti-CD133 at 37 °C. This was followed by fixation with 2% paraformaldehyde, then incubation with an Alexa-488 antibodies secondary antibody (Life Tech). Nestin (Millipore), GFAP (Abcam), β3-tubulin (Abcam), O4 (Abcam): Cells were fixed with 4% paraformaldehyde, followed by blocking and permeabilization with 10% Normal Goat Serum (Jackson ImmunoResearch) and 0.1% Triton-X (Thermo Scientific). Cells were then labeled with primary antibodies for 1 h at room temperature, followed by secondary Alexa-488 antibodies. Nrp1 (Santa Cruz): Cells were fixed with 4% paraformaldehyde, followed by blocking and permeabilization with 10% Normal Donkey Serum (Jackson ImmunoResearch) and 0.1% Triton-X. Cells were incubated with primary anti-Nrp1 overnight, followed by secondary Alexa-488 antibodies. A Zeiss Apotome microscope was used for imaging with a Zeiss AxioCam Mrm and Zeiss AxioVision software. Acquired images were then thresholded to controls to determine background fluorescence using Image J running on Java 6.
All mice were kept in a specific pathogen free (SPF) facility in accordance with Mayo clinic IACUC. Mice were kept in cages with circulating air and water and food ad libitum. Animals were monitored daily for signs of morbidity including but not limited to weight loss, tumor size, and decreased mobility.
Orthotopic tumor growth
BTSCs were dissociated, and stereotactically injected intracranially into athymic nude mice (Harlan), as previously described . Briefly, cells were resuspended in dPBS at a density of 100,000 cells/mL for a total of 300,000 cells per mouse. Athymic nude mice were anesthetized with ketamine/xylazine, and placed into the stereotactic frame. A midline incision was made in the scalp, and a burr hole was made at specific coordinates using bregma as a landmark (1 mm anterior, 2 mm lateral, 3 mm deep). Using a Hamilton syringe, cell suspensions were injected at a rate of 1 μL per minute. The needle was slowly withdrawn, and incisions closed. Mice were given analgesics post-procedure, and monitored daily for signs of neurologic decline, at which point they were euthanized. Moribund mice were anesthetized with ketamine and xylazine, followed by perfusion with 0.1 M PBS and 4% paraformaldehyde (PFA). Brains were carefully removed and post-fixed overnight in 4% PFA, followed by cryoprotection in 30% sucrose. A cryostat was then used to cut 40- μm sections. Floating sections were rinsed in PBS, and permeabilized with 0.1% TritonX-100 and blocked with 10% NGS for 1 h. Overnight incubation of anti-human cytoplasmic antibody, STEM121 (Stem Cell Inc) was used to label tumor cells, followed by secondary Alexa-488 for 1 h. DAPI was used to label total nuclei.
TCGA data analysis
TCGA data analysis was performed using OncoLnc, an online tool that links TCGA mRNA/miRNA/lncRNA data with survival data. Genes of interest were queried and survival correlations were assessed comparing the top quartile of mRNA expression to the bottom quartile of mRNA expression. Statistical analysis was performed using a log-rank test .
RNA was harvested from tumor cells using a Qiagen RNeasy kit and reverse transcribed (Select cDNA Synthesis Kit, BioRad). Amplicons were then transcribed from the cDNA by PCR using specific primer pairs (Platinum PCR, Life Tech). Products were then gel electrophoresed in 2% agarose DNA gels with ethidium bromide (BioRad). Bands were imaged using UV light (BioRad Gel Doc XR). 1 kb ladder (NEB) was used and primer detail with amplicon sizes included in Supp Fig. 8.
BrdU proliferation assay
BTSCs were dissociated from culture using gentle enzymatic dissociation (TrypLE) and plated on PDL/Fibronectin coated coverglasses at 10,000cells per coverglass, and allowed to recover for 2 days. At this point, Time 0, the media was replaced with fresh stem cell media containing bromodeoxyuridine (BrdU)(Roche) with or without recombinant human Sema3A (R&D Systems). After 24 h, cells were fixed with 70% acidic ethanol for 20 min at -20 °C, followed by incubation with the primary anti-BrdU antibody (Roche), then secondary Alexa-488 and DAPI. Coverglasses were imaged using a Zeiss Apotome, imaging at least 13 representative fields per coverglass on an automated stage. BrdU and DAPI positive cells were then counted in ImageJ.
Cell death analysis
GBM Stem cells were plated at 10,000 cells per well onto PDL/Fibronectin coverglasses. Cells were treated as in the BrdU Proliferation Assays for 24 h. Next, plates were placed on ice and incubated with cold PI (Sigma) in stem cell media for 10 min. PI containing media was then removed and cells were fixed with 4% paraformaldehyde, followed by co-staining with DAPI for 1 h. Coverglasses were then imaged shortly thereafter using a Zeiss Apotome. PI and DAPI positive cells were then counted in ImageJ.
Gap migration assay
Gap migration assays were performed as previously described. Briefly, dissociated cells were plated onto PDL/Fibronectin coverglasses around a gap insert (Cell Bio Labs) at a density of 75,000 cells per well. After 48 h, inserts were removed and cell debris was washed away. Cells were then treated with Sema3A for 24 h, at which time coverglasses were fixed and stained with Alexa-488 conjugated phalloidin to label F-actin (Life Tech). Invasion was calculated based on percentage of positive cells in the previously cell free gap.
Tumorsphere invasion assay
Under sterile conditions, individual tumorspheres were carefully removed from culture and embedded into semi-solid Matrigel with or without Sema3A, in a low adhesion 96-well plate at a density of one tumorsphere per well. A layer of stem cell media was gently added with or without Sema3A, corresponding to Matrigel conditions. Initial tumorsphere diameters were measured using light microscopy and subsequent ImageJ analysis. After 24 h, tumorspheres were reimaged and changes in diameter were calculated.
shRNA Lentivirus production
Briefly, shRNA plasmids (Sigma) were obtained and transformed into competent E. coli bacteria by heat shock, and grown in liquid LB media in the presence of ampicillin at 37 °C. Glycerol stocks were made and frozen at -80 °C degrees for future use. Plasmids were then purified by Maxi Prep (Qiagen), and concentrations were determined using a spectrophotometer. 293 T cells were transfected with viral packaging plasmids, VSV-G, Gag, and Pol, in addition to the desired shRNA plasmid, using calcium chloride precipitation. Virions were collected in stem cell media minus growth factors, and stored at -80 °C for single use only. Titers were calculated by limiting dilution infection of 293 T cells, followed by puromycin selection. The number of colonies formed per condition was then calculated to determine the titer.
shRNA Lentivirus knockdown
Viral aliquots were thawed at room temperature, and added to cell cultures for an MOI of approximately 30. Viruses were incubated for 20–24 h. Viral media was then removed, and cells were washed three times with sterile PBS, and replaced with fresh stem cell media. After 4 days, cells were treated with puromycin to select for infected cells at a dose that kills 100% of uninfected cells within 2 days. Knockdown efficiency was determined by comparing mRNA expression between target and control shRNA samples. Briefly, cells were gently dissociated with TrypLE after selection, and mRNA was harvested and reverse transcribed. Specific primers were then used for qRT-PCR to compare gene expression between target and control samples, using the ΔΔCq method, with actin serving as the housekeeping gene as previously described . Constructs with the highest efficiency were selected for use. Virally infected stem cells were only maintained for a single passage to avoid extended culture of the tumor stem cells, and maintain consistent knockdown efficiencies across experiments.
In vivo flank tumor growth assay
Athymic nude- foxn1nu (NU/J) were ordered from Jackson Laboratories. Viral infected tumor cells were harvested and injected into athymic nude mouse flanks at 400,000 cells or 1.2 × 10^6 cells per mouse according to standard protocol. Flank tumors were measured weekly by digital calipers to assess growth, with a final analysis at 7 weeks when tumors approached maximum IACUC approved size. Mice were euthanized using ketamine and xylazine, followed by perfusion with 0.1 M PBS and 4% paraformaldehyde (PFA). Tumors were then harvested for measurement of weight or cultured for re-analysis of expression to ensure maintenance of receptor knockdown.
Statistical analyses were performed using Graphpad Prism software (v7, San Diego, CA, USA). Normally distributed experimental results, as determined by the D’Agostino & Pearson omnibus test, were analyzed using the unpaired 2-tailed student’s t-test for groups of 2, or one-way ANOVA with Bonferroni’s post test for groups of more than 2. Mann Whitney test (groups of 2) or Kruskal-Wallis with Dunn’s post test (> 2) were used for non-parametric results.