No abstract

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.

The endoplasmic reticulum (ER) is a dynamic network of interconnected sheets and tubules that orchestrates the distribution of lipids, ions, and proteins throughout the cell. The impact of its complex, dynamic morphology on its function as an intracellular transport hub remains poorly understood. To elucidate the functional consequences of ER network structure and dynamics, we quantify how the heterogeneity of the peripheral ER in COS7 cells affects diffusive protein transport. In vivo imaging of photoactivated ER membrane proteins demonstrates their non-uniform spreading to adjacent regions, in a manner consistent with simulations of diffusing particles on extracted network structures. Using a minimal network model to represent tubule rearrangements, we demonstrate that ER network dynamics are sufficiently slow to have little effect on diffusive protein transport. Furthermore, stochastic simulations reveal a novel consequence of ER network heterogeneity: the existence of 'hot spots' where sparse diffusive reactants are more likely to find one another. Intriguingly, ER exit sites are disproportionately found in these highly accessible regions. Combining in vivo experiments with analytic calculations, quantitative image analysis, and computational modeling, we demonstrate how structure guides diffusive protein transport and reactions in the ER. SIGNIFICANCE The endoplasmic reticulum (ER) is the largest organelle in the eukaryotic cell, forming a web of interconnected hollow tubules and sheets. The ER is central to the transport of many cellular components such as lipids, ions, and proteins. However, the impact of the ER's complex network architecture on these transport processes remains opaque. Using live-cell experiments and simulations, we demonstrate that structural heterogeneity leads to non-uniform transport of proteins to nearby regions of the ER. As a consequence, certain regions of the network function as 'hot spots' where diffusive reactants are more likely to find each other. In live cells, sites of protein export are preferentially localized to these regions.