Abstract
Paired isolation and characterization in 3D hypoxic microenvironments (HyPIC-3D) is a platform for delineating migratory heterogeneity among cancer cell subpopulations within 3D hypoxic tumor microenvironments. Here, we present the protocol for implementing HyPIC-3D using standard laboratory and computational resources. We describe the steps to perform migration assays with cancer spheroids, collect paired non-migrated and migrated subpopulations, and characterize their heterogeneous phenotypes through quantitative imaging, molecular, and cellular analyses. This protocol has potential for applications in diverse 3D migration contexts. For complete details on the use and execution of this protocol, please refer to Schito and Rey-Keim1.