A 3D Tumor Spheroid Platform to Model Fusobacterium nucleatum-Induced Tumorigenesis

Cancer cell line spheroids provide a three-dimensional (3D) culture platform that enables the evaluation of cancer cell aggregation, tumor initiation, cell proliferation, and overall tumor growth. Compared with conventional two-dimensional monolayer cultures, spheroid models more faithfully recapitulate the architectural, physiological, and microenvironmental features of solid tumors, making them particularly well suited for studying tumor biology and microbe-host interactions. Previous work has leveraged this 3D system to investigate the role of the oral oncobacterium Fusobacterium nucleatum in promoting tumorigenesis. Here, we present a detailed protocol in which colorectal cancer cells are seeded into ultralow attachment plates and co-cultured with F. nucleatum either prior to spheroid formation, to model early tumor development, or after spheroids have been established, to model tumor progression. This assay provides a tractable platform for dissecting cancer cell-microbe interactions and supports a contributory role for F. nucleatum in colorectal tumor development.