Abstract Oral squamous cell carcinoma (OSCC) exhibits high incidence and morbidity, representing 300,000 new cases and 145,000 deaths per year. Moreover, the 5-year survival rate is 50% and this is directly associated with regional lymph node metastases. To explore how the extracellular vesicles (EVs) play a significant role on OSCC lymph node metastasis, we characterized the EVs released by tumor primary site cells and their paired lymph node metastasis cells using nanoparticle tracking analysis, high-resolution microscopy (Cryo-EM), EVs uptake by HUVEC, BJ-5ta and gingival primary fibroblasts recipient cells and the proteins, lipids, metabolites and miRNAs contents. Both cells-derived EVs showed a similar size, morphology, and the presence of annexin-2 and flotillin-1 surface markers. Interesting, only gingival primary recipient cells were more likely to uptake primary OSCC-derived EVs compared with lymph node-derived EVs. This effect is probably associated with the differential abundance of 490 proteins, 316 miRNAs, 63 lipids and 17 metabolites between the oral cancer cells-derived EVs. Markedly, multilevel omic data integration indicates that metabolic processes were over-represented mainly in primary OSCC-derived EVs, suggesting that these EVs carry specific cargo molecules associated with a more aggressive pattern compared with lymph node OSCC-derived EVs. Financial support: FAPESP (Process number 2016/50005-7). Citation Format: Ariane F. Busso-Lopes, Carolina M. Carnielli, Flávia Winck, Diego M. Riaño-Pachón, Ana K. Oliveira, Rute A. Costa, César Rivera, Camila Caldana, Jay W. Fox, Adriana F. Paes Leme. Multi-omics data indicate that primary and lymph node oral cancer cells-derived extracellular vesicles carry cargo molecules with a specific aggressive pattern [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5649.