GoKawiilCell lines and patient-derived GSC cultures
Human GBM samples were obtained from patients who provided written informed consent for tissue collection and use in research and publication, under the approval of the Hamilton Health Sciences and McMaster Health Sciences Research Ethics Board (no. 16078). Tumour samples were processed using established protocols60. GBM cells were cultured in Neurocult Complete (NCC) medium, a commercially available serum-free NSC medium (STEMCELL Technologies, 05751), supplemented with human recombinant epidermal growth factor (20 ng ml−1; STEMCELL Technologies, 78006), basic fibroblast growth factor (20 ng ml−1; STEMCELL Technologies, 78006), heparin (2 μg ml−1; STEMCELL Technologies, 07980) and antibiotic–antimycotic solution (1×; Wisent, 450-115-EL). NSCs were cultured and maintained using similar protocols as described previously61. SK-MEL-2 cells, MDA-MB-231 and HEK293T cells were purchased from the American Type Culture Collection (ATCC) and grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% non-essential amino acids (NEAA; Thermo Fisher, 11140050). NHAs were purchased from ATCC and grown in DMEM/F12 medium (Gibco, 11320033) supplemented with 10% FBS, and epidermal growth factor and fibroblast growth factor as above.
Animal studies
All animal experiments were conducted in compliance with the ethical guidelines approved by the Animal Use Protocols (22-12-38) of the McMaster University Central Animal Facility. Mice were housed in pathogen-free, temperature-controlled, 12 h light and dark cycle environment and were fed ad libitum. Intracranial injections were performed on 6–12-week-old NOD/SCID gamma (NSG) or C57BL/6 mice, following previously described methods60. GBM4, GBM8 and MBT06 cell lines (106 cells per mouse) or GL261 cells (105 cells per mouse) were suspended in 10 μl PBS and injected into the right frontal lobe using a Hamilton syringe (Hamilton, 7635-01). A burr hole, 2 mm posterior to the coronal suture and 3 mm lateral to the sagittal suture, was drilled for intracranial access60. At the humane end-point, mice were euthanized, perfused with 10% formalin, and the brains were sectioned into 2 mm slices using a brain matrix for paraffin embedding and haematoxylin and eosin (H&E) staining. Digital images were captured using an Aperio Slide Scanner (Leica Biosystems) and analysed with ImageScope v11.1.2.760 software. Kaplan–Meier survival analyses were based on the time from surgery to end-point. For humanized studies, NOG-EXL mice (Taconic, NOD.Cg-PrkdcscidIl2rgtm1SugTg(SV40/HTLV-IL3,CSF2)10-7Jic/JicTac, 13395-F) were purchased and utilized as outlined above. Intracranial injections followed the previously described protocol for the BT972 cell line (106 cells per mouse). Mice were monitored post-injections, and survival data were analysed using the Kaplan–Meier method.
RNA-seq and differential gene expression ranking
Cells were subjected to RNA extraction, followed by RNA sequencing on the Illumina HiSeq 2500 platform. Four comparative analyses were performed: (1) GBM CD133+ versus CD133−; (2) NSC CD133+ versus CD133−; (3) CD133+ NSC versus CD133+ GBM; and (4) CD133− NSC versus CD133− GBM. Data filtering was performed using a CPM threshold of 3.5. Multidimensional scaling plots indicated clear sample separation across all comparisons. A smear plot analysis confirmed consistent gene expression patterns without significant artifacts at this CPM threshold. DEGs were identified using the quasi-likelihood F-test in edgeR, chosen due to its stringency and appropriateness for datasets with minimal sample sizes (n = 4, with ≥2 per group). Genes were ranked on the basis of P values and fold changes using the formula: ranking score = sign(log(fold change)) × –log 10 (P value), where the sign(log(fold change)) indicates the direction of expression change (positive for upregulation, negative for downregulation), and –log 10 (P value) reflects the significance level. Genes were ranked from highest upregulation to highest downregulation, with rankings exported as.rnk files for GSEA.
GSEA and enrichment mapping
GSEA was conducted for all four comparisons using the.rnk files and the Human_GOBP_AllPathways_with_GO_iea_December_24_2015_symbol.gmt gene set. A total of 1,000 permutations were performed using a random seed of 349. Comparative GSEA results for the four analyses were compiled in Pathway.xlsx, including normalized enrichment scores and false discovery rate (FDR) q-values. Differences in gene filtering between the comparisons necessitated reanalysis of the RNA-seq data using only protein-coding genes, allowing consistent gene sets across all analyses. Owing to high similarity between: (1) CD133+ versus CD133− in NSC (GSEA2) and GBM (GSEA1); and (2) NSC versus GBM in CD133+ (GSEA3) and CD133− (GSEA4), combined enrichment maps were generated (map A: GSEA1 and GSEA2; map B: GSEA3 and GSEA4). These maps were constructed using a Jaccard coefficient of 0.25 (for edges) and an FDR q-value cutoff of 0.0001 (for nodes). Cytoscape files (.cys) containing both stringent and relaxed conditions (FDR q-value < 0.1, P value < 0.05) are available. To ensure direct comparability across the GSEA analyses,.rnk files were regenerated using all protein-coding genes without CPM filtering. z-scores were calculated for each pathway on the basis of the direction of enrichment and nominal P values. Pathways with significant differences (P < 0.01) were identified, and corresponding enrichment maps were generated.
Interrogation of public databases
Publicly available datasets were incorporated using the GEPIA2 interface to compare gene expression correlations between GBM samples from TCGA and normal brain tissue from the GTEx portal, following established methods62.
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