Integrated light and electron microscopy workflow for morphological, molecular and ultrastructural analysis of spheroids

Tratnjek, Larisa (author), Janev, Aleksandar (author), Resnik, Nataša (author), Cerkvenik, Uroš (author), Erdani-Kreft, Mateja (author)

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.

• Language: English
• Keywords: spheroids, morphology, ultrastructure, microscopy, cell biology
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: 21 str.
• Numbering: Vol. 21, iss. 3, art. e0342659
• PID: 20.500.12556/RUL-185266
• UDC: 576
• ISSN on article: 1932-6203
• DOI: 10.1371/journal.pone.0342659
• COBISS.SI-ID: 270473475
• Publication date in RUL: 30.07.2026

Record is a part of a journal

• Title: PloS one
• Publisher: Public Library of Science
• ISSN: 1932-6203
• COBISS.SI-ID: 2005896

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: sferoidi, morfologija, ultrastruktura, mikroskopija, celična biologija

Projects

• Funder: ARIS - Slovenian Research and Innovation Agency
• Project number: J7-2594
• Name: Uporaba amnijske membrane za inovativno multimodalno zdravljenje bakterijskega cistitisa in raka sečnega mehurja: učinek njene protimikrobne, imunomodulatorne in protirakave aktivnosti

• Funder: ARIS - Slovenian Research and Innovation Agency
• Project number: P3-0108
• Name: Celična biologija in molekularna genetika v biomedicini