Abstract Oral squamous cell carcinoma (OSCC) is the most prevalent malignant tumor among head and neck cancers, with 300,000 new cases and 145,000 deaths around the world per year, with less than 50% survival rate of advanced cases after five years of diagnosis. The molecular mechanisms involved in immunosurveillance and oral cancer development are poorly understood and, neither molecular markers nor immunotherapy response predictors are available. However, recent findings in molecular biology and immunology of cancer cell have shown that tumor-stroma communication promotes intense suppressive signaling in the microenvironment, which may contribute to failure of therapy. Tumor-stroma communication is not an unidirectional process, driven only by cancer cells. Other resident cells, like cancer-associated fibroblasts (CAFs), immune cells as macrophages and lymphocytes, endothelial cells and tumor stem cells, which are in an intense cross-talk by cell-cell contact, secreted factors and other components of the extracellular matrix (ECM), can alter and be altered by the microenvironment. In this context, the cancer-derived extracellular vesicles (EVs) acquire a significant pathological role in cell-cell communication by changing the phenotype of the recipient cells, promoting tumor growth and metastasis. Here, we explored the role of three different source of EVs, originated from normal gingival keratinocyte (HMK) and from less (SCC9) and high (HSC3) aggressive OSCC cell lines in the modulation of monocyte-derived macrophage M1 or M2 phenotypes, and their ability to contribute to the suppressive phenotype in the tumor microenvironment. CD163 expression, a marker of M2 macrophages, was significantly increased in CD14+ monocytes stimulated by HSC3-EVs, in comparison with non-stimulated monocytes, HMK- and SCC9-EVs-stimulated cells. In addition, CD64+/CD86+/CD80+ M1 macrophage subset, differentiated by GM-CSF/IFN-gamma, showed a decreased expression of the T-cell co-stimulatory molecule, CD86 after stimulus with HSC3-EVs. Furthermore, the mass spectrometry analysis of HSC3-EV protein contents identified a set of up- and down-regulated candidate proteins that may be involved in immunoregulation and suppressive pathways. Since the M2 macrophage phenotype is associated with increased angiogenesis, immunological tolerance and development of neoplastic cells, the M1 subset, the classically activated macrophages, may recognize and eliminate cancer cells. Thus, our results indicate that EVs from aggressive cells HSC3 may contribute to immunosuppressive tumor microenvironment in oral cancer by deviating macrophages into a tumor-promoting instead of a tumor-controlling phenotype. Citation Format: Ana Karina Oliveira, Thiago Andrade Patente, Rodrigo Nalio Ramos, Ariane Busso Lopes, Romênia Ramos Domingues, Mariane Tami Amano, Eliana Blini Marengo, Jose Alexandre Barbuto, Adriana Franco Squina. Oral cancer cell-derived extracellular vesicles can modulate an immunosuppressive microenvironment through M2 phenotype polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2800.