A smart 3D microfluidic tumor spheroid-vessel co-culture model for studying exosomal HSP-mediated tumor invasion and angiogenesis

Abstract

Breast cancer is one of the most prevalent malignant tumors in women, primarily due to their metastasis and recurrence. Deciphering the molecular mechanisms underlying breast cancer metastasis and recurrence remains a major challenge. Herein, we developed a microfluidic chip-based 3D co-culture system that integrates tumor spheroids, vascular endothelial cells, and extracellular matrix to model metastasis dynamics. This system enables real-time monitoring of tumor invasion and angiogenesis through immunofluorescence staining of zinc finger transcription factor (ZEB1) and platelet–endothelial cell adhesion molecule (CD31), coupled with vascular endothelial growth factor (VEGF) quantification. Then we employed this platform to investigate the role of exosomal hot shock proteins (HSPs) in breast cancer metastasis, elucidating that breast cancer-derived exosomes significantly promoted tumor invasion and angiogenesis in a dose-dependent manner. At an exosome concentration of 10¹² particles per mL, ZEB1 expression increased by 2.06-fold and VEGF secretion elevated by 3.92-fold. Conversely, HSP-depleted exosomes (Exosome^{HSP del}) reversed these effects, confirming that exosomal HSPs serve as critical mediators of tumor invasion and angiogenesis. This microfluidic model provides a physiologically relevant tool for studying metastatic mechanisms and screening therapeutic targets, highlighting exosomal HSPs as a promising intervention point.