A microbial therapy-mimicry assay shows how spatial resource dynamics control resistance escape

Abstract The evolution of therapy resistance in structured populations such as biofilms and solid tumours is shaped by emergent spatial organization, with profound consequences for evolution-based therapies. However, how treatment reshapes these patterns remains poorly understood. Here we show that intermittent treatment pulses transiently reconfigure the resource landscape, reorganize spatial growth zones and can enable resistant mutants to escape spatial confinement and drive therapy failure. We introduce a spatial evolution assay in which populations expand from single, genetically tailored yeast cells, enabling quantitative tracking of the full spatiotemporal trajectories of continually emerging resistant mutants under intermittent treatment. By integrating these observations with a mechanistically interpretable computational model in a real-to-sim-to-real loop, we identify a dynamic phase-transition-like boundary in schedule space that defines a candidate optimal balance between population control and sustained resistance confinement, which we test experimentally. Together, our results establish resource-mediated spatial confinement as a central organizing principle of resistance evolution and provide a mechanistic foundation for spatially informed, evolution-based therapies.