Stromal cell-laden 3D hydrogel microwell arrays as tumor microenvironment model for studying stiffness dependent stromal cell-cancer interactions

Tumor properties such as growth and metastasis are dramatically dependent on the tumor microenvironment (TME). However, the diversity of the TME including the stiffness and the composition of the extracellular matrix (ECM), as well as the involvement of stromal cells, makes it extremely difficult to establish proper in vitro models for studying tumor growth and metastasis. In this research, we fabricated a stromal cell-laden microwell array system with tunable stiffness ranging from 200 Pa up to 3 kPa, which covers the stiffness range of normal and cancerous mammary tissues, to study the effect of ECM stiffness on stromal-cancer interaction. Our results showed that, tumor spheroids closely interacted with the pre-adipocyte stromal cells encapsulated within the microwell array, influencing their differentiation and maturation degree in a stiffness related manner. They inhibited adipogenesis in high stiffness tissue constructs that were at breast cancer stiffness range, while the inhibition effect diminished in the low stiffness tissue constructs that were at normal human breast tissue range. Furthermore, the 3D structure of tumor spheroids was shown to be important for the inhibition of the adipogenesis, as conditioned media from monolayer culture of cancer cells did not show any significant effect. These results show, for the first time in literature, that stromal-cancer interactions are highly dependent on ECM stiffness. The biomimetic TME platform developed here is a powerful organ-specific cancer model for studying the involvement of stromal cells in early mammary tumorigenesis and metastasis, and could be powerful platform for high-throughput drug discovery.