AbstractThe stiffness of tumors and their host tissues is much higher than most hydrogels, which are conventionally used to study in vitro cancer progression. The tumoroid assay is an engineered 3D in vitro tumor model that allows investigation of cancer cell invasion in an environment that is biomimetic in terms of extracellular matrix (ECM) composition and stiffness. Using this model, the change in matrix stiffness by epithelial colorectal cancer cells is systematically characterized by atomic force microscopy indentation tests. Less invasive epithelial cancer cells stiffen the tumor microenvironment while highly aggressive epithelial cancer cells show significant softening of the tumor microenvironment. Changes in stiffness are attributed to both cell‐generated active forces as well as ECM degradation and remodeling. The degradation is in part attributed to the enzymatic activity of matrix metalloproteinases (MMPs) as demonstrated by the significant expression of MMP‐2 and MMP‐9 at both gene and protein levels. Targeting MMP activity through broad‐spectrum drug inhibition (BB‐94) reverses the changes in stiffness and also decreases cancer cell invasion. These results promote the idea of using mechano‐based cancer therapies such as MMP inhibition.