Matrix stiffness modulates 3D spheroid sorting and burst-like collective migration.

While it is known that cells with differential adhesion tend to segregate and preferentially sort, the physical forces governing sorting and invasion in heterogeneous tumors remain poorly understood. To investigate this, we develop a composite hydrogel that uncouples matrix stiffness and collagen fiber density, mimicking changes in the stiffness of the tumor microenvironment, to explore how physical confinement influences individual and collective cell migration in 3D spheroids. The mechanical properties of the hydrogel can be tuned through crosslinking and crosslink reversal. Using this hydrogel system and computational Self-Propelled Voronoi modeling, we show that spheroid sorting and invasion into the matrix depend on the balance between cell-generated forces and matrix resistance. Sorting is driven by high confinement and reducing matrix stiffness triggers a collective fluidization of cell motion. Cell sorting, which depends on cell-cell adhesion, is crucial to this phenomenon, and burst-like migration does not occur for unsorted spheroids irrespective of matrix stiffness. The findings support a model where matrix stiffness modulates 3D spheroid sorting and unjamming in an adhesion-dependent manner, providing insights into the mechanisms of cell sorting and migration in the primary tumor and toward distant metastatic sites.