Ultrastructural Evidence of Lithium-Induced Mitophagy in 3D Endometrial Cancer Spheroids

Autophagy is a tightly regulated catabolic process responsible for the degradation of dysfunctional organelles and protein aggregates via lysosomes. When this process targets damaged mitochondria, it is referred to as mitophagy, a mechanism crucial for maintaining mitochondrial quality control, especially under stress conditions. While lithium chloride (LiCl) is widely used as a mood stabilizer, emerging studies suggest it also modulates autophagy and mitophagy, potentially through inositol monophosphatase inhibition. However, its ultrastructural effects in three-dimensional (3D) cancer models have not been clearly elucidated.In this study, we investigated the mitophagic effects of LiCl in 3D spheroids derived from Ishikawa endometrial cancer cells and SH-SY5Y neuroblastoma cells. Spheroids were treated with increasing concentrations of LiCl (1, 10, and 50 mM), and analyzed by transmission electron microscopy (TEM) and flow cytometry. TEM revealed hallmark mitophagic structures, including double-membrane-bound autophagosomes encapsulating defective mitochondria. Flow cytometry demonstrated a dose-dependent reduction in mitochondrial content and cell viability, supporting the occurrence of mitophagy-related cell death.Although no molecular markers were directly assessed, the morphological and functional data strongly suggest the activation of selective mitophagy pathways. Based on previous reports, we hypothesize that LiCl may promote mitophagy via LC3 recruitment and involvement of mitochondrial outer membrane receptors such as NIX, BNIP3, and FUNDC1. These findings underscore the utility of 3D tumor models for visualizing mitophagic dynamics and provide a structural foundation for future mechanistic studies of lithium-induced mitochondrial clearance.