Pre-invasive compaction and spatial organization influence invasion patterns in MDA-MB-231 spheroids

Purpose Cancer metastasis is the primary cause of cancer-related mortality, and this process is initiated by the invasion of tumor cells into surrounding tissues. Three-dimensional (3D) spheroid invasion assays are widely used to study tumor invasion in triple-negative breast cancer (TNBC), yet reported invasion patterns of MDA-MB-231 spheroids in basement membrane extract (BME) systems vary considerably. The physicochemical determinants underlying this variability remain poorly defined. Methods Here, we use time-lapse z-stack imaging combined with quantitative tracking of matrix-associated landmarks to investigate how pre-invasive cellular organization, matrix composition, and spatial heterogeneity influence invasion behavior in MDA-MB-231 multicellular tumor spheroids cultured in the presence and absence of Invasion Matrix (IM). Results Prior to invasion, peripheral cellular aggregates undergo morphological reorganization, while exploratory protrusions exhibit dynamic extension, retraction, merging, and bifurcation events that ultimately give rise to a reduced number of dominant protrusive branches. Matrix composition influences invasion-associated behavior, with reduced collagen density accelerating extracellular matrix (ECM) displacement and altering the spatial organization of spheroid-associated cells. In addition, distinct protrusion morphologies were observed at different focal planes within the same spheroid, highlighting spatial heterogeneity in invasion-associated phenotypes. Conclusion These findings demonstrate that invasion in spheroid models is preceded by dynamic cellular and matrix-associated processes and is influenced by matrix composition and spatial heterogeneity. Together, these observations provide a framework for understanding variability in spheroid invasion assays.