Abstract 4037: The pattern of extracellular vesicles secretion and their role in head and neck squamous cell carcinoma
Authors: Giudice Fernanda S. , Rodrigues Bruna R. , Lacerda Tonielli C. , Cartaxo Rodrigo T. , Baptistella Antuani R. , Dias Marcos V. S. , Kowalski Luiz P. , Martins Vilma R. 
Published: August 2015

Abstract
Head and Neck Squamous Cell Carcinoma (HNSCC) is a keratinocytic neoplasm that is considered to be one of the six most common malignancies worldwide. It represents more than 90% of all cancers that occur throughout the upper aerodigestive tract. Unfortunately, HNSCC is still related to a high rate of morbidity and mortality. The poor prognosis of HNSCC shows remarkable association with local invasion and subsequent metastasis. Recent evidences have shown that the tumor development and the metastatic process are supported by extracellular vesicles (EVs), which are released from the tumor cells to the blood or lymphatic system. The molecular mechanisms involved in EVs biogenesis and secretion are, however, poorly understood. Herein, we sought to evaluate the pathways that govern EVs formation and the crucial mechanism that regulates EVs’ release by HNSCC cells. In addition, we tried to recognize the role of HNSCC EVs upon dysplastic keratinocytes and microenvironment cells such as endothelial cells. EVs derived from three HNSCC cell lines (SCC-9, Cal27 and FaDu) were isolated by ultracentrifugation and quantified using Nanoparticle Tracking Analysis (NTA - NanoSight®). A screen targeting for the components of EVs’ biogenesis in the HNSCC cell lines through western blotting was also performed and lysosomal compartment was followed by immunofluorescence in live cells. Finally, the invasion and angiogenic potential of dysplastic keratinocytes (DOK cell line) and endothelial cells (HUVEC cell line) were evaluated, respectively, after treatment with EVs secreted by HNSCC cell lines. Comparing the three cell lines, FaDu cells presented higher expression of proteins responsible for EVs formation (p&lt;0.05), such as Flotilin-1, Tsg101, VPS36 and also Rab7, a small GTPase that directs vesicles to lysosomes. However, FaDu cells secreted less EVs than SCC-9 and Cal27, as well as it showed lower levels of Vps4, required for formation of multivesicular bodies. Immunofluorescence experiments showed the presence of numerous perinuclear clusters positive for LAMP-1 (lysosome marker) in FaDu and Cal27 cells, while SCC-9 showed LAMP1-positive organelles dispersed throughout its cytoplasm. These findings indicate that the majority of EVs formed by FaDu cells can be driven to lysosome compartment instead of being released to the extracellular milieu. Interestingly, when DOK and HUVEC cells were treated with the same number of EVs derived from HNSCC cell lines, SCC-9 EVs enhanced the invasion and angiogenic potential of keratinocytes and endothelial cells, respectively. Taken together, our data highlights a possible role for Rab7 protein in the negative regulation of EVs secretion by HNSCC cell lines. Moreover, the results provided by the in vitro functional assays suggest the clinical importance of regulating EVs secretion for the controlling of HNSCC tumor aggressivenes.
Citation Format: Fernanda S. Giudice, Bruna R. Rodrigues, Tonielli C. Lacerda, Rodrigo T. Cartaxo, Antuani R. Baptistella, Marcos V. S. Dias, Luiz P. Kowalski, Vilma R. Martins. The pattern of extracellular vesicles secretion and their role in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4037. doi:10.1158/1538-7445.AM2015-4037