Tumor stromal residing cancer-associated fibroblasts (CAFs) are significant accomplices in the growth and development of malignant neoplasms. As cancer progresses, the stroma undergoes a dramatic remodeling and stiffening of its extracellular matrix (ECM). However, exactly how these biomechanical changes influence the CAF behavior and the functional paracrine crosstalk with the neighboring tumor cells in a 3-dimensional (3D) microenvironment remains elusive. Herein, a collagen and alginate interpenetrating network (COAL-IPN) hydrogel system was employed as a 3D in vitro surrogate of the cancerous breast tissue stromal niche. In this study, the mechanical properties of COAL-IPN were precisely fine-tuned with Young's modulus (E) values of similar to 108 and 898 Pa. The results revealed that the 3D polymeric network mechanics and microstructure are critical biophysical determinants of the human breast CAF (b-CAF) morphology, phenotype, and paracrine dialogue with MDA-MB-231 tumoroids. A compliant hydrogel network favors b-CAF spreading, nuclear translocation of the YAP/TAZ mechanosignaling protein, and upregulation of CAF hallmark transcripts. Conversely, a rigid and highly cross-linked hydrogel network imposed a physical entrapment effect on the b-CAFs that limited their spreading and phenotype in a manner that effectively muted their pro-tumorigenic paracrine activity. Collectively, the CoAl-IPN 3D culture system has proven to be a versatile platform in defining the 3D biophysical parameters that could either promote or restrain the protumorigenic activity of b-CAFs and sheds critical mechano-mediated light onto the phenotypic plasticity and corresponding specific bioactivity of b-CAFs in the 3D microenvironment.