Mechanical cues guide the formation and patterning of 3D spheroids in fibrous environments

Multicellular spheroids have shown great promise in 3D biology. Many techniques exist to form spheroids, but how cells take mechanical advantage of native fibrous extracellular matrix (ECM) to form spheroids remains unknown. Here, we identify the role of fiber diameter, architecture, and cell contractility on spheroids' spontaneous formation and growth in ECM-mimicking fiber networks. We show that matrix deformability revealed through force measurements on aligned fiber networks promotes spheroid formation independent of fiber diameter. At the same time, larger-diameter crosshatched networks of low deformability abrogate spheroid formation. Thus, designing fiber networks of varying diameters and architectures allows spatial patterning of spheroids and monolayers simultaneously. Forces quantified during spheroid formation revealed the contractile role of Rho-associated protein kinase in spheroid formation and maintenance. Interestingly, we observed spheroid-spheroid and multiple spheroid mergers initiated by cell exchanges to form cellular bridges connecting the two spheroids. Unexpectedly, we found large pericyte spheroids contract rhythmically. Transcriptomic analysis revealed striking changes in cell-cell, cell-matrix, and mechanosensing gene expression profiles concordant with spheroid assembly on fiber networks. Overall, we ascertained that contractility and network deformability work together to spontaneously form and pattern 3D spheroids, potentially connecting in vivo matrix biology with developmental, disease, and regenerative biology.