Protocol to generate scaffold-free, multicomponent 3D melanoma spheroid models for preclinical drug testing

Three-dimensional (3D) models play an increasingly important role in preclinical drug testing as they faithfully mimic interactions between cancer cells and the tumor microenvironment (TME). Here, we present a protocol for generating scaffold-free 3D multicomponent human melanoma spheroids. We describe steps for characterizing models using live-cell imaging and histology, followed by drug testing and assessment of cell death through various techniques such as imaging, luminescence-based assays, and flow cytometry. Finally, we demonstrate the models' adaptability for co-cultures with immune cells.