Cell migration through the 3D extracellular matrix (ECM) is a fundamental behavior that has profound importance for both normal and pathological physiology. Despite the ubiquity and necessity of cell migration, it is not well understood how complex cell-environment interactions integrate to drive emergent migration behaviors. This dissertation helps resolve important questions related to how cells move within 3D environments, leading to novel insights relevant to both basic and applied biological questions. Chapter 1 describes a novel biophysical imaging platform capable of measuring multiple cell-ECM interactions simultaneously within 3D environments. This approach reveals how cytoskeletal, adhesion, contractility, and matrix remodeling machinery are coordinated by cells to achieve different modes of movement and how cell-matrix interactions can be used to predict cell migration. This in turn provides critical insights into the sources of migration heterogeneity and reveals a breakdown in the universal coupling of speed and persistence law regulated by adhesion through integrin beta 1. In Chapter 2, migration heterogeneity is leveraged to identify the differential regulation of gene expression that results from distinct modes of cell-matrix interactions, revealing a central role for iron in regulating 3D migration through both biophysical and metabolic pathways. Early upregulation of the enzyme heme oxygenase-1 (Hmox1) promotes invasive migration through endogenous free-iron generation, and the iron-sensitive enzyme aconitase-1 (Aco1) is identified as a sustained marker of invasive migration. The findings and discussion presented in this dissertation represent key advances in methodology, analysis, and biological insight related to 3D cell migration.