HyPIC-3D enables characterization of migratory cancer cell subpopulations in 3D hypoxic microenvironments

Malignant tumors harbor cancer cell subpopulations with heterogeneous migratory behaviors. Identifying the molecular programs specifying whether these subpopulations migrate or remain stationary is essential for preventing metastasis and requires experimental approaches that directly separate and analyze these subpopulations as they move throughout 3D microenvironments. Here, we present "paired isolation and characterization in 3D hypoxic microenvironments" (HyPIC-3D), a method to isolate and characterize migratory and non-migratory cancer cell subpopulations from the same pool of spheroids. By adapting tissue culture inserts used for migration assays, we established a migration platform that preserves oxygen gradients and the spheroid architecture. HyPIC-3D shows that the pattern and extent of migration differ among cancer cell types while identifying distinct molecular switches and oxygen requirements underlying this heterogeneity. Integrated with image analysis tools, HyPIC-3D is implemented with standard computational and laboratory equipment and is amenable to diverse downstream applications, enabling mechanistic dissection of migration and discovery of metastatic regulators.