Dual tumour-myeloid targeting of glioblastoma with GPNMB CAR-T cells

Key Points:

• Human glioblastoma (GBM) and neural stem cells (NSCs) were cultured using standardized serum-free media supplemented with growth factors, while various cell lines were maintained in appropriate media with fetal bovine serum.
• Intracranial injections of GBM cells into immunodeficient mice were performed to study tumor growth and survival, with ethical approval and humane endpoints observed; humanized mouse models were also utilized for specific cell lines.
• RNA sequencing and gene set enrichment analyses were conducted to compare gene expression profiles between GBM and NSC populations, focusing on CD133+ and CD133− subpopulations, with rigorous data filtering and statistical methods applied.
• Functional assays including flow cytometry, immunohistochemistry, immunofluorescence, and Western blotting were used to characterize protein expression and cell phenotypes; gene knockout and CAR-T cell generation protocols were established for experimental interventions.
• CAR-T cell cytotoxicity, activation, and cytokine release were assessed in vitro and in vivo, including murinized CAR-T cells for mouse models; macrophage polarization and triple co-culture systems were employed to study tumor-immune interactions, complemented by advanced single-cell RNA sequencing and multiplexed immunofluorescence imaging techniques.