Equipment used in the experiments.

Traditional two-dimensional cell cultures are limited in their ability to accurately model cancer progression and treatment response, which has prompted researchers to develop three-dimensional in vitro models, such as spheroids. These models better mimic the cellular interactions and microenvironment found in solid tumors, including features like hypoxic cores and acidic pH, which activate survival pathways and closely mirror in vivo tumor cell behavior. To fully harness the potential of spheroids in cancer research, comprehensive characterization at the morphological, molecular, and ultrastructural levels using various microscopy workflows is essential. Given the challenges associated with traditional handling and processing methods due to the small size of spheroids, we have developed and present here detailed protocols for light and electron microscopy analysis of spheroids formed from the human noncancerous urothelial cell line SV-HUC-1 and the malignant urothelial cancer cell line T24. Spheroids were analyzed by light microscopy using histological staining and immunofluorescence labelling, following either cryosectioning or paraffin embedding. Whole-mount immunofluorescence combined with optical clearing and confocal microscopy enabled visualization of protein expression and localization throughout the entire spheroid. Scanning and transmission electron microscopy provided high-resolution insights into surface morphology and internal ultrastructure, respectively. Each imaging modality, paired with optimized sample preparation, contributed to a comprehensive workflow offering distinct and complementary views of spheroid morphology, protein distribution, and cellular organization. This integrated approach, combining various light and electron microscopy workflows, enables accurate and thorough characterization of spheroids and establishes a comprehensive baseline for downstream functional investigations. The described protocols are adaptable to spheroids derived from various cell types and tissue origins, making them a versatile tool for a broad range of applications.