A combinatorial EVs-miRNA signature mediates the anti-tumoral activity of NFAT3-regulated extracellular vesicles in aggressive cancers.

Aggressive cancers such as triple-negative breast cancer (TNBC) and pancreatic cancer remain difficult to treat because their malignant behavior is driven by complex gene networks rather than single oncogenic targets. Our study identifies a NFAT3 transcription factor-dependent miRNAs signature that suppresses tumor aggressiveness through extracellular vesicles (EVs). Functional analyses demonstrated that a specific combination of fifteen miRNAs (miR-Comb 15) is required to inhibit cancer cell proliferation and invasion across TNBC and pancreatic cancer models, whereas individual miRNA are insufficient to reproduce the full anti-tumoral effect. To enable therapeutic translation, we have engineered EVs derived from HEK 293T, a non-tumoral and scalable vesicle source. Using an optimized exogenous pH-gradient loading strategy, miR-Comb 15 was efficiently incorporated into EVs without altering their integrity or intrinsic bioactivity. Among multiple delivery platforms tested, miR-Comb 15 loaded HEK 293T EVs consistently exhibited superior anti-tumoral efficacy both in vitro and in vivo. Together, these findings establish a strong mechanistic link between transcriptional regulation and EVs-mediated RNA delivery and demonstrates that rationally designed miRNA combinations delivered by EVs represent a promising therapeutic strategy for aggressive cancers.