Cell lines and patient-derived GSC cultures
Human glioblastoma (GBM) samples were collected from patients who agreed in writing to participate, and this was authorized by the Hamilton Health Sciences and McMaster Health Sciences Research Ethics Board. Tumor samples were processed following established protocols. GBM cells were cultivated in Neurocult Complete (NCC) medium, which is a commercially available serum-free neural stem cell medium, enriched with human recombinant epidermal growth factor, basic fibroblast growth factor, heparin, and an antibiotic solution. Neural stem cells (NSCs) were grown using similar methods to those previously described. Additional cell lines, including SK-MEL-2, MDA-MB-231, and HEK293T, were sourced from the American Type Culture Collection (ATCC) and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with FBS and amino acids. Normal human astrocytes (NHAs) were also obtained from ATCC, cultured in DMEM/F12 medium, and supplemented similarly.
Animal studies
All animal research adhered to ethical standards set forth by the Animal Use Protocols at McMaster University. Mice were kept in a clean and temperature-regulated environment and were given free access to food. Intracranial injections were administered to 6–12-week-old NOD/SCID gamma (NSG) or C57BL/6 mice as per previous methodologies. Various GBM cell lines were injected into the right frontal lobe after making a burr hole. Upon reaching a humane endpoint, the mice were euthanized, and their brains were processed for analysis and imaging. Survival data were tracked from surgery to endpoint using Kaplan–Meier analysis.
RNA-seq and differential gene expression ranking
Cells underwent RNA extraction and sequencing on the Illumina HiSeq 2500 system. Four comparative analyses compared different cell types regarding gene expression. Samples showed clear separation in multidimensional scaling plots, confirming reliable expression patterns. Differentially expressed genes (DEGs) were identified using a quasi-likelihood F-test, which was appropriate for the limited sample sizes. Genes were ranked based on statistical significance and fold changes, with results prepared for subsequent analyses.
GSEA and enrichment mapping
Gene Set Enrichment Analysis (GSEA) was conducted across the four comparisons utilizing ranking files created earlier. After several permutations, results were compiled, showing normalized enrichment scores and false discovery rates. Repetitive gene filtering across comparisons necessitated reanalysis, focusing on protein-coding genes for consistent results. Given the high similarity observed across comparisons, combined enrichment maps were created for better visualization, using specific statistical cutoffs.
Interrogation of public databases
Public datasets were used to analyze gene expression correlations between GBM and normal brain tissue, employing established methods.
Flow cytometry
Cells were dissociated and stained with specific antibodies before analysis using a flow cytometer. Dead cells were excluded from the results.
Immunohistochemistry
Mouse brain tissues were processed and sectioned for immunohistochemical analysis, where antigen retrieval and staining procedures were carried out. Primary antibodies were applied, followed by secondary detection, and the results were imaged and quantified to assess cellular positivity.
Immunofluorescence
Tissue sections underwent a similar protocol as immunohistochemistry but included different staining parameters for fluorescent analysis. Slides were imaged and processed using specialized imaging systems.
Generation of gene-knockout constructs
Guide RNAs targeting specific genes were generated and inserted into lentiviral constructs. These were subsequently packaged into lentiviruses in a laboratory setting, with proper cell lines and conditions applied for optimal results.
Cell proliferation assays
Cells were plated for proliferation assays, with a fluorescent dye added prior to measuring fluorescence to determine growth rates.
Western blotting
Cell lysates were prepared and analyzed for protein content using standard procedures, followed by gel electrophoresis and membrane transfer. Antibodies specific to the proteins of interest were utilized to visualize results.
Generation of CAR-T cells
CAR constructs were produced and plaques prepared as previously outlined. T cells were isolated from blood samples, activated, and subsequently transduced for further research.
CAR-T cytotoxicity assay
Target cells expressing specific markers were treated with effector T cells, and viability was measured to assess cytotoxic effects.
CAR-T activation assays
Effector T cells were co-cultured with target cells to assess activation markers using flow cytometry.
CAR-T cytokine release assays
Cytokine quantification was performed on supernatants collected from co-cultured cells using established ELISA methods.
Generation of murinized anti-GPNMB CAR-T cells
Methods for murine T cell activation and viral production for CAR constructs were adhered to, with progressing stages of cell activation documented.
CAR-T in vivo trials
Different GBM cell lines were implanted into mice; treatment groups received specified doses of CAR-T cells. The mice were monitored for health status periodically post-injection.
Macrophage culturing and polarization
U937 cells were cultured and polarized under specific conditions to replicate various macrophage phenotypes, which were then used for cytotoxicity assays.
GSC–macrophage–T cell triple co-cultures
Triple cultures involving GBM cells, macrophages, and CAR-T cells were set up for further analysis, with cells collected for flow cytometry assessment.
scRNA-seq analysis
Patient and mouse brain samples were processed for single-nucleus RNA sequencing, following established protocols for data alignment and analysis.
Automated multiplexed sequential immunofluorescence imaging
This advanced imaging technique was performed using the COMET platform, which involved a meticulously designed protocol for multi-target analysis of tissue sections.
Reporting summary
Additional research design information is available through associated reporting summaries linked to this article.
