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

Background The extracellular vesicle (EV) pathway plays a critical role in cell-to-cell communication. Cancer cells release EVs into the extracellular space, where they interact with both cancerous and noncancerous cells, activating signaling pathways and remodeling the tumor microenvironment (TME). In this study, we investigated the functional role of EVs released by FGFR2-amplified cancers of unknown primary (CUPs), which generate extrachromosomal circular DNA (ecDNA) to enhance oncogenic amplification. Methods FGFR2 copy number was quantified using droplet digital PCR (ddPCR) and visualized using fluorescent in situ hybridization (FISH). EVs were isolated via ultracentrifugation, and the circular nature of FGFR2 ecDNA was assessed using Plasmid-Safe ATP-dependent DNase treatment and atomic force microscopy (AFM). Oncogene transfer through EVs was evaluated by administering isolated CUP-derived EVs to recipient NCI-N87 cells or by using a coculture system that facilitated EV transfer to THP-1, HUVEC, and fibroblast cell lines. Results CUP-derived FGFR2-containing ecDNA, which is partially circular, was packaged within EVs and exhibited functional activity upon delivery to TME cells. When cancer (NCI-N87, THP-1) and noncancer (HUVECs, fibroblasts) cells were exposed to CUP-derived EVs—either via direct administration or coculture—they internalized FGFR2 DNA, which was subsequently transcribed, leading to altered cell morphology and increased proliferation, depending on ecDNA type. CUP-derived EVs induced THP-1 polarization toward the M2 macrophage subtype and promoted HUVEC proliferation. Conclusion This mechanism of oncogene transfer can contribute to TME remodeling, potentially explaining the early metastatic potential of CUP.