Fig. 1

Schematic representation of monolayers (2D) and spheroid (3D) cell culture, where cell type 1 depicts a tumor cell and cell type 2 exemplifies either another tumor cell type or non-tumor cell. A Monolayer tumor cell culture—where cells are grown in a two-dimensional environment, providing simplicity and ease of use but lacking the 3D architecture and microenvironmental complexity of in vivo tissues; B Multicellular tumor spheroid—single-cell line spheroids are 3D cell aggregates that better mimic tumor architecture and gradients. From outer to inner layers, spheroids have a proliferative zone where cell cycle is progressing, a quiescent zone where cells are not dividing, and a necrotic zone, where cells are either dead or in the process of cell death; C Heterotypic multicellular tumor spheroid—Co-culture spheroids can incorporate multiple cell types, such as stromal or immune cells, to simulate intercellular interactions within the tumor microenvironment