(A) Cells grow as a blob in a specified volume of medium (0.1–0.2 mL). Cells consume O2 and glucose. O2 is supplied from the upper boundary and glucose is depleted from the medium unless replenished. Model parameters for diffusion and uptake of constituents, rate of cell growth and death, are acquired from experiments with monolayers and multicellular layers (MCL). Parameters were fitted using the monolayer ABM and Matlab MCL program as described in methods. Time-varying concentration fields of the constituents are solved for in the medium and within the spheroid on a course grid and fine grid respectively (see Figure C), the cells acting as sinks. (B) Simulation illustrating cell states as a function of local O2 and glucose concentrations. Once cells double in size they undergo mitosis (pink cells). As the spheroid grows, O2 and glucose levels decrease in medium near the spheroid (indicated by a cyan color gradient), and decrease in the spheroid core to very low levels with cells becoming hypoxic (< 0.15 μM O2, dark green cells). Cells starved of O2 or glucose eventually are tagged to die (blue cells) and then, after a delay, undergo cytolysis, creating a necrotic core (black). (C) Bright field image of a growing, 4-day HCT116 spheroid seeded at 1000 cells per well.