Mitochondrial Injury Accompanied by Intermediate Filament Remodeling Following Lithium Chloride Exposure in 3D Endometrial Cancer Spheroids

Background/Objectives: Endometrial cancer frequently develops resistance to thera-py, partly due to the ability of tumor cells to adapt to cellular stress through non-apoptotic mechanisms. Mitochondrial dysfunction and cytoskeletal remodeling are increasingly recognized as key components of stress adaptation; however, their structural relationship under pharmacological stress in three-dimensional (3D) tumor models remains poorly characterized. The present study aimed to investigate the ul-trastructural and phenotypic effects of lithium chloride (LiCl)–induced stress in 3D endometrial cancer spheroids, with a particular focus on mitochondrial alterations and intermediate filament organization. Methods: Three-dimensional spheroids gen-erated from Ishikawa endometrial cancer cells were exposed to lithium chloride at concentrations of 1, 10, or 50 mM for defined time periods. Cell viability, proliferative activity, and clonogenic capacity were assessed using Trypan Blue exclusion, BrdU in-corporation, and soft agar assays. Ultrastructural changes were examined by trans-mission electron microscopy to evaluate mitochondrial morphology, cytoplasmic or-ganization, and intermediate filament distribution. Results: LiCl exposure resulted in a dose- and time-dependent reduction in cell viability, proliferation, and clonogenic potential in 3D spheroids. Ultrastructural analysis revealed pronounced mitochondrial swelling, cristae disorganization, and membrane-associated mitochondrial alterations. These changes were consistently accompanied by conspicuous accumulation and re-organization of intermediate filaments in close spatial proximity to damaged mito-chondria. Across all experimental conditions, classical apoptotic ultrastructural fea-tures, including chromatin condensation and apoptotic body formation, were not ob-served, despite substantial organelle-level alterations. Conclusions: Together, these observations indicate that lithium chloride elicits a stress phenotype in 3D endometrial cancer spheroids that primarily manifests at the organelle and cytoskeletal levels, ra-ther than through classical apoptotic execution. Although descriptive in nature, the present study highlights intermediate filament accumulation as a prominent structural feature of lithium-induced mitochondrial stress and establishes a structural reference point for future studies aimed at dissecting mitochondrial–cytoskeletal interplay dur-ing pharmacological stress in endometrial cancer.