Sonodynamic therapy is recognized as a minimally invasive cancer treatment method that generates reactive oxygen species (ROS) with high spatial and temporal precision, offering significant potential for treating glioblastoma (GBM) beneath the skull. However, there is an urgent need for highly efficient sonosensitizers capable of penetrating the blood-brain barrier (BBB) and reshaping GBM's suppressive immune microenvironment. To address this, a biomimetic hybrid vesicle-coated sonodynamic nanoplatform (CpG@E-OMV@BTO@Pt) is proposed for enhanced glioblastoma therapy. This innovative platform generates ROS via piezo-catalysis, causing mitochondrial damage and the release of mitochondrial DNA (mtDNA), which in turn triggers immunogenic cell death (ICD) in GBM cells. Furthermore, the hybrid vesicles, composed of bacterial outer membrane vesicles (OMVs) and Angiopep-2 peptide-modified engineered exosomes, facilitate effective BBB penetration and targeted delivery to the tumor site. In vitro, in vivo experiments, and RNA sequencing (RNA-seq) demonstrate that CpG oligodeoxynucleotide-modified OMVs interact with TLR receptors, activating the NF-kappa B pathway, promoting dendritic cell (DC) maturation, and stimulating the adaptive immune response, thereby achieving a comprehensive therapeutic effect against GBM. This study presents a novel therapeutic system for the multimodal treatment of GBM.

Figure S5. Blocking GSC-induced neuronal activity reverses microglial M2 polarization in vivo.

Abstract Neuronal activity can drive progression of high-grade glioma by mediating mitogen production and neuron-glioma synaptic communications. Glioma stem cells (GSC) also play a significant role in progression, therapy resistance, and recurrence in glioma, which implicates potential cross-talk between neuronal activity and GSC biology. Here, we manipulated neuronal activity using chemogenetics in vitro and in vivo to study how it influences GSCs. Neuronal activity supported glioblastoma (GBM) progression and radioresistance through exosome-induced proneural-to-mesenchymal transition (PMT) of GSCs. Molecularly, neuronal activation led to elevated miR-184–3p in neuron-derived exosomes that were taken up by GSCs and reduced the mRNA N6-methyladenosine (m6A) levels by inhibiting RBM15 expression. RBM15 deficiency decreased m6A modification of DLG3 mRNA and subsequently induced GSC PMT by activating the STAT3 pathway. Loss of miR-184–3p in cortical neurons reduced GSC xenograft growth, even when neurons were activated. Levetiracetam, an antiepileptic drug, reduced the neuronal production of miR-184–3p-enriched exosomes, inhibited GSC PMT, and increased radiosensitivity of tumors to prolong survival in xenograft mouse models. Together, these findings indicate that exosomes derived from active neurons promote GBM progression and radioresistance by inducing PMT of GSCs. Significance: Active neurons secrete exosomes enriched with miR-184–3p that promote glioblastoma progression and radioresistance by driving the proneural-to-mesenchymal transition in glioma stem cells, which can be reversed by antiseizure medication levetiracetam.

Figure S10. miR-200c-3p promotes the microglial M2 polarization by targeting ZC3H13.

Figure S9. miR-200c-3p promotes the M2 polarization of microglia in vivo.

Figure S4. hM3Dq activated neurons promote microglial M2 polarization through exosomes.

Figure S6. A-NDEs promote mouse microglial M2 polarization.

Figure S14. Levetiracetam inhibited the activated-neuron induced microglial M2 polarization in vivo.

Figure S12. DUSP9 knockdown promotes the microglia M2 polarization.

Figure S1. Hypoxia drives glioma stem cells to activate neurons via glutamate.

Figure S13. A-NDEs promote microglial M2 polarization via the miR-200c-3p/ZC3H13/DUSP9/ERK pathway.

Figure S7. A-NDEs reduce the m6A level in microglia.

AbstractPurpose:Neuronal activity in the brain has been reported to promote the malignant progression of glioma cells via nonsynaptic paracrine and electrical synaptic integration mechanisms. However, the interaction between neuronal activity and the immune microenvironment in glioblastoma (GBM) remains largely unclear.Experimental Design:By applying chemogenetic techniques, we enhanced and inhibited neuronal activity in vitro and in a mouse model to study how neuronal activity regulates microglial polarization and affects GBM progression.Results:We demonstrate that hypoxia drove glioma stem cells (GSC) to produce higher levels of glutamate, which activated local neurons. Neuronal activity promoted GBM progression by facilitating microglial M2 polarization through enriching miR-200c-3p in neuron-derived exosomes, which decreased the expression of the m6A writer zinc finger CCCH-type containing 13 (ZC3H13) in microglia, impairing methylation of dual specificity phosphatase 9 (DUSP9) mRNA. Downregulation of DUSP9 promoted ERK pathway activation, which subsequently induced microglial M2 polarization. In the mouse model, cortical neuronal activation promoted microglial M2 polarization whereas cortical neuronal inhibition decreased microglial M2 polarization in GBM xenografts. miR-200c-3p knockdown in cortical neurons impaired microglial M2 polarization and GBM xenograft growth, even when cortical neurons were activated. Treatment with the anti-seizure medication levetiracetam impaired neuronal activation and subsequently reduced neuron-mediated microglial M2 polarization.Conclusions:These findings indicated that hypoxic GSC-induced neuron activation promotes GBM progression by polarizing microglia via the exosomal miR-200c-3p/ZC3H13/DUSP9/p-ERK pathway. Levetiracetam, an antiepileptic drug, blocks the abnormal activation of neurons in GBM and impairs activity-dependent GBM progression.See related commentary by Cui et al., p. 1073

Figure S3. Activated neuron promote microglial M2 polarization.

Figure S11. ZC3H13 regulates the expression of DUSP9.

Figure S2. Characterization of neuron-derived exosomes.

Table S2. siRNA, miRNA mimics and antagomir used.

Figure S8. miR-200c-3p promotes the M2 polarization of microglia in vitro.