Preparation and analysis of monotypic and organotypic tumor spheroids

• Published in: Methods in cell biology Vol. 196; pp. 139 - 159
• Main Authors: Domingues, Ana Carolina M., Palin, Claire, Sun, Yi, Xie, Hongyan, Woods, Elliot C., Jenkins, Russell W., Revach, Or-Yam
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 2025
• Subjects: 3D tumor models; Animals; Cancer immunotherapy; Cell Culture Techniques - methods; Cell Culture Techniques, Three Dimensional - methods; Cell Line, Tumor; Humans; Immunotherapy - methods; Lab-On-A-Chip Devices; Mice; Neoplasms - immunology; Neoplasms - pathology; Organotypic tumor spheroids; Patient-derived tumor models; Spheroids, Cellular - immunology; Spheroids, Cellular - pathology; Tumor Cells, Cultured; Tumor Microenvironment; Patient-derived tumor models; Organotypic tumor spheroids; Cancer immunotherapy; 3D tumor models
• ISSN: 0091-679X
• DOI: 10.1016/bs.mcb.2024.11.003

Patient-derived tumor models are emerging as promising tools to explore patient-specific tumor behavior and to address a central gap in tumor immunology and immunotherapy drug development – the need for clinically relevant tumor models that recapitulate the complexity of the human tumor ecosystem, in which cancer cells are interacting with various immune and stromal elements. Patient-derived organotypic tumor spheroids (PDOTS), a biomimetic 3D-patient tumor avatar (3D-PTA), are comprised of cancer cells and autologous tumor-infiltrating immune and stromal cells that are grown within collagen hydrogels embedded in a 3D microfluidic culture device to model physiologic conditions and enable the study of tumor-immune dynamics. PDOTS and their murine counterparts (MDOTS, murine-derived organotypic tumor spheroids) are responsive to immune checkpoint blockade (ICB) and mirror in vivo response dynamics. We have also confirmed the utility of MDOTS/PDOTS in examining novel therapeutic strategies to overcome ICB resistance and testing the efficiency of T cell-based immunotherapies, demonstrating the utility of PDOTS profiling in examining the tumor-immune dynamics of immunotherapy response and resistance. Here, we provide a detailed protocol for processing, ex vivo culture, and analysis of patient-derived tumor organotypic tumor spheroids (PDOTS) in 3D microfluidic culture for immuno-oncology applications. The protocol can be readily adapted for ex vivo profiling of murine-derived organotypic tumor spheroids (MDOTS) and cancer cell line-derived monotypic tumor spheroids (MTS) for robust and iterative testing of immuno-oncology targets.