Abstract Background 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 tumor microenvironment (TME). In this study, we investigated the generation of extrachromosomal DNA (ecDNA) in cell lines derived from FGFR2-amplified cancer of unknown primary (CUP). This ecDNA is loaded as cargo inside CUP extracellular vesicles and, once delivered to tumor microenvironment cells, the cargo is functional: FGFR2 DNA is transcribed and changes co-cultured cell morphology and proliferation. Methods Three CUP cell lines (CUP#55A/S, CUP#96) dependent on FGFR2 were established. FGFR2 copy number was measured by ddPCR and the amplification type by FISH, while EVs were purified by ultracentrifugation. ecDNA presence and circularity were validated by DNase treatment, atomic force microscopy, and whole-genome sequencing. Oncogene transfer was tested by exposing gastric cancer cells (NCI-N87), monocytes (THP-1), endothelial cells (HUVECs), and fibroblasts to CUP-EVs, either directly or through coculture. CUP xenograft models were employed to evaluate ecDNA transfer in vivo. Results CUP-derived FGFR2-containing ecDNA, whose circular structure was validated by WGS, 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, demonstrating immunomodulatory and proangiogenic activity. In vivo, xenografts generated with CUP cell lines released circulating FGFR2+ EVs, which mediated the transfer of FGFR2 ecDNA to distant, even non-metastatic, sites. Conclusion Collectively, these findings demonstrate that tumor- derived EVs can propagate and horizontally transfer oncogenic ecDNA both in vitro and in vivo, thereby reshaping the TME and supporting long-range communication that may underlie CUP’s early metastatic potential.
Horizontal transfer of functional extrachromosomal DNA via extracellular vesicles in FGFR2-amplified cancer / Salamon, I., Gallerani, G., Storci, G., Luebeck, J., Fontana, B., Serravalle, S., Valle, F., Brucale, M., Pagano Mariano, M., Soru, A., Pace, I., Cavazzoni, A., Roncarati, R., Santi, S., Bafna, V., Bonafè, M., Ferracin, M.. - (2025), pp. 155-155. (65th Annual Meeting of the Italian Cancer Society. Targeting Cancer Hallmarks ).
Horizontal transfer of functional extrachromosomal DNA via extracellular vesicles in FGFR2-amplified cancer
Fontana B.;Cavazzoni A.;Santi S.;
2025-01-01
Abstract
Abstract Background 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 tumor microenvironment (TME). In this study, we investigated the generation of extrachromosomal DNA (ecDNA) in cell lines derived from FGFR2-amplified cancer of unknown primary (CUP). This ecDNA is loaded as cargo inside CUP extracellular vesicles and, once delivered to tumor microenvironment cells, the cargo is functional: FGFR2 DNA is transcribed and changes co-cultured cell morphology and proliferation. Methods Three CUP cell lines (CUP#55A/S, CUP#96) dependent on FGFR2 were established. FGFR2 copy number was measured by ddPCR and the amplification type by FISH, while EVs were purified by ultracentrifugation. ecDNA presence and circularity were validated by DNase treatment, atomic force microscopy, and whole-genome sequencing. Oncogene transfer was tested by exposing gastric cancer cells (NCI-N87), monocytes (THP-1), endothelial cells (HUVECs), and fibroblasts to CUP-EVs, either directly or through coculture. CUP xenograft models were employed to evaluate ecDNA transfer in vivo. Results CUP-derived FGFR2-containing ecDNA, whose circular structure was validated by WGS, 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, demonstrating immunomodulatory and proangiogenic activity. In vivo, xenografts generated with CUP cell lines released circulating FGFR2+ EVs, which mediated the transfer of FGFR2 ecDNA to distant, even non-metastatic, sites. Conclusion Collectively, these findings demonstrate that tumor- derived EVs can propagate and horizontally transfer oncogenic ecDNA both in vitro and in vivo, thereby reshaping the TME and supporting long-range communication that may underlie CUP’s early metastatic potential.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
