Engineering Multicellular Breast Cancer Spheroids in Decellularized Adipose Tissue Hydrogels Using a Microfluidic Platform to Recapitulate Tumor Microenvironment Complexity.

Three-dimensional (3D) cancer spheroids are critical for recapitulating tumor architecture and microenvironmental cues that govern disease progression and therapeutic response. However, most existing spheroid models lack biomimetic extracellular matrices, controlled fabrication, and stromal complexity representative of the native tumor microenvironment (TME). Here, we report a multifaceted engineering strategy that integrates a decellularized adipose tissue (DAT) hydrogel, a modified microfluidic T-junction platform, and multicellular coculture to generate physiologically relevant breast cancer spheroids. The DAT hydrogel provides a tissue-specific extracellular matrix that closely mimics the biochemical and biophysical properties of breast tissue, while the microfluidic platform enables rapid (30 spheroids per minute), reproducible, and size-controlled fabrication of homogeneous spheroids (360 μm to 600 μm, tunable through controlled flow rate ratios of the continuous and dispersed phases). To recapitulate TME complexity, breast cancer cells (MDA-MB-231) were co-encapsulated with fibroblasts (L929), monocytes (THP-1), and endothelial cells (HUVECs), yielding highly organized multicellular spheroids. The engineered spheroids were maintained for 7 days and extensively characterized for their metabolic activity, viability, morphology, histology and immunohistochemistry, and gene expression profiles. Metabolic activity assessments demonstrated an 8-fold increase across spheroid types from day 1 to day 7, while gene expression analyses showed 2- to 40-fold upregulation in mRNA levels of key TME-associated genes, and drug screening with 5-fluorouracil and doxorubicin demonstrated enhanced chemoresistance with increasing cellular complexity (2-fold and 1.4-fold increases in IC50 values, respectively), highlighting the functional significance of stromal interactions in recapitulating clinically relevant drug response within the model. Collectively, this study establishes a biomaterial-driven microfluidic platform for engineering TME-mimicking breast cancer spheroids, offering a robust and scalable approach for preclinical drug screening and the development of personalized cancer organoid models.