Melanoma-Derived Extracellular Vesicles as Mediators of Stromal Reprogramming: Functional Analysis in a Biofabricated 3D Skin Model

Metastasis, driven by complex tumor-stroma communication, remains the primary cause of melanoma mortality. This study developed an advanced in vitro platform to model the systemic dialogue underpinning early metastatic conditioning. A physiologically relevant, three-dimensional full-thickness skin model has been engineered using a gelatin methacryloyl (GelMA) hydrogel, co-cultured with dermal fibroblasts, microvascular endothelial cells, and stratified keratinocytes. A melanoma-invaded variant was created by integrating VMM15 tumor spheroids. Melanoma-derived extracellular vesicles (mel-EVs) were isolated, characterized, and applied to healthy dermal constructs, inducing a pro-inflammatory secretome and a phenotypic shift toward a cancer-associated fibroblast (CAF)-like state. Finally, a custom multi-tissue perfusion platform was implemented to dynamically connect tissues under flow. This system revealed that perfusion enhances melanoma invasiveness and enables the long-range transfer of tumor-derived components to downstream healthy skin, effectively modeling pre-metastatic niche formation. This integrated approach provides a powerful tool to dissect the flow-mediated, EV-driven crosstalk critical for early melanoma dissemination.