Neural Crest-Derived Stem Cell Secretomes and Extracellular Vesicles Disrupt Glioblastoma through Dual-Pathway Inflammatory Rebalancing.

Glioblastoma multiforme is characterised by resistance to conventional treatments via sustained pro-inflammatory signalling. This study investigated whether secretomes and small extracellular vesicles (sEVs) from human oral mucosa neural crest-derived stem cells (NCSCs) could disrupt multiple glioblastoma features by rebalancing pro- and anti-inflammatory pathways. NCSCs were characterised, and their secretomes/sEVs isolated, and analysed using a range of state-of-the-art methods. Anti-tumour effects were evaluated using multiple glioblastoma cell lines (U251, U373, U87) through viability, proliferation, migration, and tumourigenicity assays. Mechanistic studies employed dual nuclear factor-κB (NF-κB) and quadruple NF-κB/interferon regulatory factor 3 (IRF3) reporter systems, cytokine arrays, and immunocytochemistry. Therapeutic potential was assessed using temozolomide (TMZ) chemosensitivity assays and organotypic mouse brain slice models. NCSC secretomes and sEVs demonstrated consistent anti-glioblastoma activity across all functional assays. Both products potently suppressed NF-κB activation across multiple pro-inflammatory stimuli whilst simultaneously enhancing IRF3 nuclear translocation and transcriptional activity. This dual pathway modulation reprogrammed glioblastoma cytokine secretion towards anti-inflammatory profiles, and inhibited tumourigenicity in both 3D culture and ex vivo brain slice models. Notably, secretomes and sEVs enhanced the efficacy of TMZ, reducing colony size compared to monotherapy, without compromising tissue viability. These findings demonstrate the first evidence of dual NF-κB/IRF3 pathway rebalancing by NCSC-derived products in glioblastoma. The simultaneous suppression of tumour-promoting inflammation whilst enhancing anti-tumour immune signalling represents a novel therapeutic paradigm that addresses multiple resistance mechanisms simultaneously. This strategy redefines stem cell-based therapy for glioblastoma by integrating immunomodulation with chemosensitisation in a single, Good Manufacturing Practice (GMP)-compatible product.