Abstract 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. Citation Format: Irene Salamon, Giulia Gallerani, Jens Luebeck, Gianluca Storci, Simone Spandau, Beatrice Fontana, Alessia Soru, Mattia Riefolo, Marco Pagano Mariano, Ilaria Pace, Andrea Cavazzoni, Vineet Bafna, Massimiliano Bonafe', Manuela Ferracin. Horizontal transfer of functional extrachromosomal DNA via extracellular vesicles in FGFR2-amplified cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3345.
Salamon et al. (Fri,) studied this question.