Abstract
<jats:p>Background: Glioblastoma (GBM) is characterized by neurological dysfunction caused by tumor cells that interact with and alter neuronal circuits. However, the specific neuronal populations and molecular mechanisms most susceptible to GBM invasion remain poorly understood. Methods: We created a human tumor brain organoid model by combining U87 glioblastoma cells with iPSC derived neural organoids. This system enabled us to study tumor neural interactions over an extended period under standard temozolomide (TMZ) treatment. We used single-cell transcriptomics to monitor cell type specific responses. Results: Our model recapitulated the diffuse infiltration observed in patients, leading to extensive structural remodeling and a profound loss of neuronal and glial populations. Single cell analysis revealed that TMZ suppressed proliferative and biosynthetic programs but enriched for stress-responsive, mesenchymal like, and therapy-adapted tumor states. Notably, GABAergic neurons exhibited the greatest transcriptional vulnerability, with ~36% (7,499 of 20,659) of genes differentially expressed. Invasion triggered endoplasmic reticulum stress and shut down metabolic, respiratory, synaptic, and ion-homeostatic pathways. Crucially, SLC12A5 expressing GABAergic neurons plummeted from 31% to 12%, accompanied by a sharp decline in KCC2 protein expression. While TMZ partially rescued neuronal metabolic and electron transport chain function, it failed to restore SLC12A5/KCC2 expression or inhibitory signaling. Conclusions: GBM invasion leads to a continued imbalance of chloride in GABAergic networks, and this disruption remains even after undergoing tumor targeted chemotherapy. This human iPSC derived tumor brain organoid platform provides a reliable and scalable system for studying complex tumor-neural interactions and exploring therapeutic approaches that aim to eliminate the tumor while preserving neural function.</jats:p>