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.传统二维细胞培养在精准模拟癌症进展与治疗应答方面存在显著局限，这促使研究者开发出细胞球（spheroids）等三维体外模型。此类模型可更好地模拟实体瘤中的细胞互作与微环境，涵盖缺氧核心、酸性pH等特征——这些特征能够激活肿瘤细胞的生存通路，且可高度复现体内肿瘤细胞的行为模式。为充分发挥细胞球在癌症研究中的应用潜力，借助多种显微成像流程对其开展形态学、分子层面及超微结构水平的全面表征至关重要。鉴于细胞球体积微小，传统处理与制备方法存在诸多技术瓶颈，本文研发并详述了针对两类细胞来源细胞球的光镜与电镜分析方案：一类源自人非癌性尿道上皮细胞系SV-HUC-1，另一类源自人恶性尿道上皮癌细胞系T24。针对细胞球的光镜分析可采用组织化学染色与免疫荧光标记技术，样本制备可选择冷冻切片或石蜡包埋两种路径。结合光学透明化技术与共聚焦显微镜（confocal microscopy）的整体免疫荧光染色方案，可实现整个细胞球内蛋白质表达与定位的全范围可视化观测。扫描电子显微镜（scanning electron microscopy）与透射电子显微镜（transmission electron microscopy）则可分别获取细胞球表面形貌与内部超微结构的高分辨率观测结果。每一种成像技术搭配优化后的样本制备流程，共同构成一套完整的成像工作体系，可从独特且互补的视角呈现细胞球的形态特征、蛋白质分布及细胞组织结构。这套整合了多种光镜与电镜成像流程的一体化方案，可实现细胞球的精准且全面的表征，并为后续功能研究建立完整的基准参照。本文所述的实验方案可适配源自不同细胞类型与组织来源的细胞球，因此可作为通用工具应用于诸多研究场景。