Extracellular Vesicle Delivery of Functional Extrachromosomal DNA in FGFR2-amplified Cancer of Unknown Primary

The extracellular vesicle (EV) route is essential for cell-to-cell communication. Cancer cells release EVs in the extracellular space, where they can interact with cancer and non-cancer cells, activating specific signaling pathways, modulating tumor microenvironment remodeling and inducing gene expression alterations. We investigated the functional role of EVs released by cancer of unknown primary (CUP), a rare disease (1-3% of novel cancer diagnoses) that presents with metastasis of unknown or uncertain origin and no apparent primary tumor. We derived three CUP cell lines from patient’s tumor cells, CUP#55A and S and CUP#96, both characterized by FGFR2 gene amplification, either in the form of trisomy (CUP#55A), chromosomal homogeneously staining region (CUP#55S) or double minute chromosomes (CUP#96). It has been recently recognized that tumors use extrachromosomal circular DNA (eccDNA) as a way to increase oncogenic amplification, thus conferring resistance to therapy and contributing to a worse survival. We demonstrated that FGFR2 amplification in CUP cell lines is associated with ecDNA generation and that this ecDNA is loaded as cargo inside EVs and exert a functional activity in nearby cells. The full-length FGFR2 DNA was detectable inside both small and large vesicles isolated from cell culture medium and we confirmed the circular nature of a fraction of this FGFR2 ecDNA using DNAse based strategies and visualizing DNA circles in cells and in EVs with atomic force microscopy. The protein surface profile of small and large EV populations revealed the presence of cancer related molecules that mirrored the cells of origin, including EpCAM and CD44, and variable degrees of FGFR2pos EVs. Cancer (NCI-N87) and non-cancer cell exposure (THP1 and HUVEC) to CUP EVs (by direct administration and co-culture) revealed an increase of FGFR2 DNA copies at 24 hours and its functional transcription in FGFR2 mRNA after 48 hours in all exposed cells. In addition to oncogenic delivery, CUP#96 EVs induced a polarization of THP1 cells towards M2 subtype, while CUP#55S EVs exerted a proliferative effect on HUVEC. In conclusion, we identified a model of oncogene amplification and EV-mediated delivery recurrent in cancer of unknown primary, whose DNA content was demonstrated to be functionally active in recipient cells and proportional with extrachromosomal DNA generation. This mechanism could contribute to the high metastatic potential of this cancer type. (The research leading to these results has received funding from AIRC under IG 2021 - ID. 25789 project - P.I. Ferracin Manuela) Irene Salamon, Giulia Gallerani, Gianluca Storci, Beatrice Fontana, Salvatore Serravalle, Francesco Valle, Marco Brucale, Marco Pagano Mariano, Andrea Cavazzoni, Roberta Roncarati, Spartaco Santi, Massimiliano Bonafè, Manuela Ferracin. Extracellular vesicle delivery of functional extrachromosomal DNA in FGFR2-amplified cancer of unknown primary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6569.