3D granular mechanical milieus alter the self-organisation dynamics of cancer cells

Self-organized spheroidal aggregates of cancer cells are a common pathological feature, with major implications for disease progression. The widespread adoption of such cancer spheroids as model systems has yielded important insights into the mechanisms underlying malignancy. Most in vitro culturing approaches typically employ suspension cultures, where the self-aggregating behavior of cancer cells drives spheroid formation. However, in physiological regimes, tumors largely form either within solid tissues, which constitute a disordered 3D granular regime. Hence, the fundamental principles underlying spheroidogenesis within such complex microenvironments remain incompletely understood. Here, we introduce a chemically inert, optically transparent, and mechanically tunable 3D growth medium which mimics the disordered pore space geometry and granular nature of tissues. We show that in sharp contrast to free-floating liquid suspension cultures, spheroid formation via self-assembly achieves dramatically different outcomes in the 3D granular milieu. By systematically altering the mechanical stiffness, matrix porosity, cell densities, and cellular activity, we characterize diverse outcomes in spheroid formation - highlighting the crucial influence of spatial architecture and environmental mechanics on collective behavior.