Collective Mechanical Memory Encoded by Long-Lasting Supracellular Cytoskeletal Structures in Multicellular Spheroids

Animal cells can sense and "remember" the stiffness of their extracellular environment, resulting in sustained changes in form and function. Such "mechanical memory" has been previously explored using individual cells and attributed to epigenetic changes and transcriptional activity. However, it is unclear whether such memory is retained across collective cells. Here, we report that collective cells sustain mechanical memory through self-organized actin-CK18 networks spanning multiple cell lengths, even under dramatically changing mechanical environments, such as those encountered during cancer metastasis. As a case study, we modeled ovarian cancer metastasis and found that cells initially cultured on different stiffness retained distinct migratory phenotypes throughout the environmental transitions of the metastasis model. Notably, soft-primed cells, in particular, demonstrated stronger cell-cell adhesions than stiff-primed cells. Upon aggregation into multicellular spheroids, mimicking malignant spheroids found in patient ascites, the soft-primed spheroids exclusively developed a dense cage-like supracellular actin-CK18 structure at their peripheral surfaces. Furthermore, these soft-primed spheroids exhibited impeded collective invasion, instead becoming confined by the long-lasting cytoskeletal cage. Inhibition of gap junctions attenuated the formation of cytoskeletal cages, indicating that dynamic intercellular communication via gap junctions is essential for maintaining collective mechanical memory. This work demonstrates a collective mechanism of mechanical memory that is not solely dependent on epigenetic and transcriptional activation, advancing our understanding of the elevated metastatic potential of tumor cell clusters originating in stiffened matrices.