Engineering tumor spatial heterogeneity in vitro.

Spatial heterogeneity is a fundamental feature of the tumor microenvironment, characterized by structured variations in cellular composition, phenotypic states, extracellular matrix (ECM) organization, and biochemical and biophysical gradients. These spatial patterns shape tumor evolution, modulate immune infiltration, and underlie resistance to therapy. Advances in spatial transcriptomics and multiplex imaging have revealed dynamic and region-specific niches, such as hypoxic cores, immune-excluded zones, and fibroblast-dense invasive fronts, that correlate with clinical outcomes. However, most in vitro models fail to capture this architectural complexity. Recent engineering technologies, including 3D bioprinting, organoid assembloids, organ-on-a-chip systems, and ECM-mimetic scaffolds, now enable controlled reconstruction of tumor spatial organization and microregional heterogeneity. These technologies allow integration of patient-derived cells, tunable matrix environments, and spatially defined signaling to mimic in vivo pathophysiology. When integrated with spatial transcriptomics and proteomics, these models enable mechanistic exploration of microregional tumor biology, evaluation of therapeutic responses, and investigation of immunotherapy resistance. This review integrates our current understanding of spatial heterogeneity in cancer with enabling engineering strategies to guide future developments in tumor biology and therapeutic innovation.