Towards a more objective and high-throughput spheroid invasion assay quantification method

This study presents a high-throughput, objective framework for quantifying 3D invasion of multicellular spheroids embedded in collagen hydrogels. Using nuclear fluorescence imaging and a pixel-based computational pipeline, the method enables automated measurement of invasion area, distance, and directional bias independent of spheroid size or morphology. Mechanical boundary constraints introduced by static culture geometry revealed contact-guided invasion patterns that were captured using principal component analysis. The approach was validated across multiple cell types, including cancer spheroids, demonstrating its utility for mechanobiology-driven studies of collective cell invasion in 3D extracellular matrices.