ABSTRACT: Neurological toxicity, including brain atrophy, is a commonly observed side effect of chemotherapy, often referred to as "chemotherapy brain," particularly prevalent among young cancer patients with developmental disorders. Methotrexate (MTX), a widely used chemotherapeutic reagent and immunosuppressive medication, is associated with a spectrum of adverse effects. While many researchers have highlighted neurotoxic-related issues in both cancer patients and animals treated with MTX, the mechanism of chemotherapy's impact on brain development, including in children or fetuses, remains poorly understood. In this study, we investigate the effects of MTX exposure on brain cortical organoids (COs), three-dimensional in vitro models designed to mimic human cortex development. Our primary objective is to assess their potential for exhibiting developmental toxicity when exposed to MTX. We observed a significant decrease in both the size and viability of COs morphologically and statistically after MTX exposure (MTX COs) on day 45 of differentiation. Additionally, compared with control COs, the MTX COs showed increased expression of immature neuron and GABAergic neuron-related genes. Furthermore, we detected a significant increase in intracellular ROS levels and the expression of ER-stress, cell cycle, and DNA damage-related genes in MTX COs. The expression of the apoptosis-related protein, cleaved caspase-3, was also significantly elevated in the MTX COs. Moreover, through RNA sequencing analysis, we found a significant decrease in SGK3 (serum/glucocorticoid-regulated kinase family member 3), a regulator gene of proliferation, in MTX COs. Consequently, our findings suggest that MTX could impact cortex development in the brain by increasing intracellular ROS and ER stress, ultimately inducing apoptosis, possibly through the inhibition of the SGK3 pathway. Understanding the developmental neurotoxicity of MTX in brain COs is crucial, as it provides valuable insights into the risk factors associated with prenatal exposure to the drug.
