Extracellular Vesicles Derived from L-MYC Neural Stem Cells Mediate Neuroprotection in 3D Models of Chemotherapy- and Radiation-Induced Neurotoxicity

Abstract

Background/Objectives Cancer survivors frequently experience long-term neurocognitive impairments following chemotherapy and cranial irradiation, yet experimental models that enable mechanistic investigation of therapy-induced neurotoxicity at the transcriptional level remain limited. This study aimed to develop a human three-dimensional (3D) neural tissue model derived from L-Myc immortalized neural stem cells (LMNSCs) and use transcriptomic profiling to identify molecular pathways underlying chemotherapy- and radiation-induced neural injury and extracellular vesicle (EV)-mediated recovery. Methods LMNSCs were differentiated in a 3D, methylcellulose-based culture to generate neural tissue containing neurons, astrocytes, and oligodendrocytes. Cultures were exposed to methotrexate (MTX) or ionizing radiation to induce neural injury and subsequently treated with LMNSC-derived EVs. Neural injury and repair mechanisms were evaluated by immunocytochemistry and bulk transcriptomics. Results MTX and irradiation induced dose-dependent injury, exhibited by loss of neuronal complexity and reduced glial populations. LMNSC-EV treatment promoted recovery of neuronal and glial populations following MTX- and irradiation-induced injury. Transcriptomic analysis of irradiated cultures revealed activation of inflammation, DNA damage, and stress-response pathways, which were attenuated after treatment with LMNSC-EVs. Conclusions LMNSC-based 3D neural tissue provides a human-relevant platform for modeling cancer therapy-induced neurotoxicity. Furthermore, LMNSC-EVs represent a promising cell-free regenerative therapeutic that restores injury-associated inflammatory, stress, and metabolic transcriptional programs after radiation-induced neural injury. Graphical Abstract Simple Summary Many cancer survivors experience persistent problems with memory, attention, and learning after chemotherapy or radiotherapy, yet the biological mechanisms underlying these cognitive side effects remain poorly understood. Progress has been limited by the lack of human laboratory models that accurately reproduce treatment-induced brain injury. To address this need, we developed a three-dimensional human neural tissue model containing multiple brain cell types derived from human neural stem cells. This platform was used to model cancer therapy-induced neural injury and evaluate the regenerative potential of neural stem cell-derived extracellular vesicles (EVs), small bioactive particles released by stem cells. We found that cancer therapies caused significant injury to neuronal and glial cell populations, whereas EV treatment promoted recovery of these cells and activated biological pathways associated with tissue repair. These findings establish a reproducible human neural tissue platform for studying cancer therapy-related neurotoxicity and support neural stem cell-derived EVs as a promising regenerative therapeutic strategy to prevent or reduce treatment-induced cognitive impairment.