A Droplet Microfluidic Approach to Investigate How Fibroblasts Alter Collagen I Spatial Distribution and Endocrine Response in ER ⁺ Breast Cancer

The loss of therapeutic efficacy in breast cancer treatment remains a significant challenge, with increasing evidence that the tumor microenvironment (TME) plays a crucial role in driving resistance. Understanding how stromal cells remodel the TME is critical for advancing cancer treatment strategies and drug development. Among the key cellular components of the TME, fibroblasts are recognized for their pivotal role in cancer progression through extracellular matrix remodeling. To enable in vitro interrogation of tumor-stromal interactions relevant to endocrine response, a microfluidic droplet-based 3D coculture platform was developed to demonstrate the feasibility of uniform breast cancer-fibroblast coculture models. As proof of concept, this system was used to investigate the impact of stromal remodeling by coculturing an estrogen receptor-positive (ER + ) breast cancer cell line with either primary fibroblasts or immortalized lung fibroblasts. Collagen I and Ki67 expressions were evaluated following exposure to endocrine therapy. The 3D coculture of ER + breast cancer cells with primary fibroblasts resulted in two distinct spatial distributions of collagen I, termed “hotspots”. One cohort exhibited a single hotspot with reduced collagen I expression, while the other displayed multiple hotspots with increased collagen I expression. Notably, the stratification of these populations was independent of the spheroid size. Coculture with immortalized fibroblasts produced similar hotspot patterns but with higher overall collagen I levels. Notably, during endocrine response studies, an increase in Ki67 and collagen I expression was found in the coculture spheroids, suggesting that fibroblasts contribute to proliferation and modulate endocrine response. Collectively, this study establishes a versatile 3D microfluidic model for probing tumor-stromal interactions and provides a foundation for future mechanistic studies of ECM-mediated remodeling in breast cancer.