In Vitro Tumorigenic Assay: A Tumor Sphere Assay for Cancer Stem Cells

Cancer stem cells (CSCs) represent a subpopulation of tumor cells that are thought to be responsible for therapy resistance, recurrence, and metastasis through their capacity to self-renew and differentiate into heterogeneous downstream lineages of cancer cells. Understanding the features of CSCs is crucial for managing cancer disease and establishing potential targeted therapeutics. Tumor sphere formation assay is a widely used in vitro method that selects and enriches the CSC subpopulation from the total population of cancer cells, based on their inherent ability to grow and clonally expand in serum-free and nonadherent culture conditions. Here we provide a detailed methodology to generate and propagate spheres from isolated cell suspensions of tumor tissues and cell lines using a semisolid MatrigelTM-based three-dimensional (3D) culture system.