Extracellular vesicles deliver functional extrachromosomal DNA in FGFR2-amplified cancer of unknown primary

Cancer cells actively release extracellular vesicles (EVs) into the tumor microenvironment, where they interact with both malignant and non-malignant cells, activating signaling pathways and reshaping the microenvironment. In this study, we investigated EVs secreted by FGFR2 -amplified cancers of unknown primary (CUPs), which generate extrachromosomal circular DNA (ecDNA) as a mechanism of oncogene amplification. We found that FGFR2 -containing ecDNA is packaged into both small and large EVs, horizontally transferred to recipient cells, and remains functionally active. Upon exposure to CUP-derived EVs—either by direct administration or co-culture—cancer (NCI-N87, THP1) and non-cancer (HUVEC, fibroblasts) cells internalized FGFR2 ecDNA, which was subsequently transcribed and translated to some extent. Functionally, CUP-derived EVs polarized THP1 cells toward an M2-like phenotype and promoted HUVEC proliferation. In vivo , xenografts generated from CUP cell lines released circulating FGFR2 ⁺ EVs, which mediated the systemic transfer of FGFR2 ecDNA to distant organs. Collectively, these findings demonstrate that tumor-derived EVs can propagate and horizontally transfer oncogenic ecDNA both in vitro and in vivo , providing a possible mechanistic basis for the high metastatic potential of this tumor type.