Abstract 3546: Extracellular vesicles and exosome-associated microRNAs in squamous cell carcinoma of the head and neck disrupt dendritic cell differentiation and maturation
Authors: Marti Luciana C. , Silva Elisangela P. , Rizzo Mariana B. , Lopez Rossana V. , Curioni Otavio A. , Moyses Raquel A. , Nunes Fabio D. , Severino Patricia 
Published: August 2018

Abstract
Squamous cell carcinomas (SCCs) remain among the most common malignancies of the head and neck region. Plasma microRNAs (miRNAs) are appealing biomarker candidates for diagnosis or therapy response monitoring since they may be associated with clinical and pathological features of the disease. Increasing evidence also points to a role for these molecules, mostly when transported by extracellular vesicles (EVs), in signaling between cancer and immune cells. The aims of this study are to search for potential miRNA biomarkers for SCCs in plasma, to evaluate the presence of these markers within EVs using SCC-derived cell lines as study models and to study the effect of EVs on monocyte-derived dendritic cells functions. Total RNA was isolated from plasma samples from head and neck SCC patients and healthy donors following optimized protocols. EVs from the culture medium of SCC cell lines were collected by differential centrifugation and visualized by transmission electron microscopy. The expression profile of miRNAs in plasma and EVs was evaluated by qRT-PCR. CD14+ cells from healthy donors were selected from peripheral blood using magnetic selection columns. Monocyte-derived immature dendritic cells were obtained following CD14+ cells treatment with human recombinant IL-4 and GM-CSF for six days and the maturation signal consisted on treatment of immature dendritic cells with LPS for 24 hours. Both treatments were carried out either in the presence or absence of EVs. Immunophenotyping of monocytes and monocyte-derived dendritic cells was based on the detection and expression levels of cell surface markers CD14, CD33, CD45, CD80, CD86, CD209 and HLA-DR by flow cytometry. A subset of miRNAs was consistently detected in SCC patients' plasma and EVs. Among these, miR-24 is of special interest since it has been associated with antigen processing and presentation by dendritic cells. The percentage of CD14+ cells expressing CD33 when cultured in the presence of EVs was significantly lower, and the same effect was seen for the CD209 and CD80 markers, suggesting alterations of antigen presentation functions. The percentage of cells expressing HLA-DR was similar both when CD14+ cells were cultured in the presence or without the addition of EVs, but the level of HLA-DR expression, as measured by fluorescence intensity, was lower when EVs were present, following both differentiation and maturation signals. The detection of miRNAs both in plasma and in association with EVs corroborates their potential as cancer biomarkers but also their possible implication in cell signaling. Differences in surface markers of monocyte-derived dendritic cells in the presence of EVs suggest an effect of EV content, including miRNAs, on differentiation and maturation processes, essential aspects for the proper function of dendritic cells in the tumor microenvironment.
Citation Format: Luciana C. Marti, Elisangela P. Silva, Mariana B. Rizzo, Rossana V. Lopez, Otavio A. Curioni, Raquel A. Moyses, Fabio D. Nunes, Patricia Severino. Extracellular vesicles and exosome-associated microRNAs in squamous cell carcinoma of the head and neck disrupt dendritic cell differentiation and maturation [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 3546.