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Background. The most common oral cancer in the world is squamous cell carcinoma (OSCC), accounting for more than 90% of all cases of cancer in the oral cavity. Thus, the research on molecular markers associated with the development and progress of human diseases has been the subject of intense research. The findings that saliva has molecular profiles indicating systemic diseases urge the study of non-invasive diagnosis using saliva as source of potential diagnosis, prognosis and predictive based on proteomics. Methods. 204 proteotypic peptides were selected previously based on DDA data from our own studies or retrieved from the SRMAtlas. Heavy labeled synthetic peptides were synthetized and SRM method was developed using Skyline v3.6. Saliva samples were collected from patients with lymph node metastasis (n=26) and without lymph node metastasis (n=14). Saliva proteins were digested with trypsin. Samples were randomized in R environment and analyzed in a triple-quadrupole mass spectrometer (Xevo TQ-XS, Waters). A Peptide Retention Time Calibration Mixture was spiked into the samples. Data comparison between groups was performed using Wilcoxon Mann Whitney test in R environment. Results. In the SRM method, we monitored 68 proteins and 24 of them were found decreased in patients with lymph node metastasis in comparison with patients without lymph node metastasis. The data analysis showed that at least one protein has, by itself, a higher relative risk in the development of lymph node metastasis in patients with OSCC. Two other panel of proteins presented the same relative risk described before. Conclusions. This study indicates a panel of potential OSCC marker proteins, which can contribute to the prognostic evaluation, the patient's risk profile and the possibility of recurrence, and guide therapeutic intervention strategies. Note: This abstract was not presented at the meeting. Citation Format: Tatiane De Rossi, Daniela Campos Granato, Ana Carolina Ribeiro, César Rivera, Henry Heberle, Guilherme Pimentel Telles, Carolina Moretto Carnielli, Thais Bianca Brandão, Alan Roger Santos-Silva, Márcio Ajudarte Lopes, Adriana Franco Paes Leme. The use of saliva proteins as biomarkers to predict the risk of lymph node metastasis in oral cancer [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 4542.

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.

Lymph node metastasis is the main prognostic factor in patients with head and neck squamous cell carcinoma (HNSCC), but no molecular markers are currently used in clinical practice. Considering that the invasive front (IF) of the tumor has a prognostic value due to the invasive and metastatic potential of the cells, we explored the mechanisms associated with lymph node metastasis in the primary tumor IF and its paired lymph nodes from HNSCC patients. For that, the proteomic profile was determined for formalin-fixed, paraffin-embedded (FFPE) tissues using mass spectrometry in a 29-patient cohort. Specific cell populations from primary tumor IF (tumor and stroma cells) and lymph nodes (microenvironment cells) were microdissected and HPV genotyping was performed. In total, 27.5% of the cases were positive for HPV16 and 2,154 ± 285 proteins were identified for the three cell populations analyzed. Eighty-one proteins from tumor cells, 36 proteins of stroma cells and 149 proteins of lymph node microenvironment cells were differentially expressed between HNSCC patients positive and negative for lymph node metastasis (Student´s t-test; P-value ≤ 0.05). Specific processes were enriched for the differentially expressed proteins from tumor, stroma and lymph node cells, including post-translational modification, splicing and translation, respectively. Interestingly, differential proteins isolated from primary tumor cells are mainly transported by exosomes. Therefore, our data provide protein signatures related to lymph node metastasis that are expressed by specific cell populations and candidates as prognostic markers in HNSCC. Financial support: FAPESP (Process number 2015/19191-6). Citation Format: Ariane F. Busso-Lopes, César Rivera, Carolina C. Macedo, Barbara P. Mello, Luisa L. Villa, Wilfredo A. González-Arriagada, Adriana F. Paes Leme. Specific cell populations from the primary tumor invasive front and lymph nodes show protein signatures associated with local metastasis in head and neck cancer [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 2700.

More than 300,000 new cases of head and neck cancer occur each year around the world, and although it is not one of the most common tumors in the world, has an elevated morbidity rate (37.8%) after five years of diagnosis. The development and progression of the Oral Squamous Cell Carcinoma (OSCC) involve complex cellular mechanisms which contribute to a tumor progression. Recently extracellular vesicles (EVs) have taken a significant physiological and pathological role in cell-cell communication through interactions mediated by receptors or by transfer of bioactive molecules such as receptors, proteins, lipids, mRNA, microRNA, and DNA, by changing the phenotype of the recipient cell. The tumor cells can transfer information to different cell types using EVs as a vehicle, modifying the characteristics of the tumor microenvironment, and contributing to cancer sustaining. The detection of alterations in the EVs proteome may, therefore, contribute to the understanding the cellular mechanisms modulated in OSCC and help to improve the clinical prognosis and potential therapeutic targets in oral cancer. Thus, this study analyzed the cargo proteins of different EVs from less and high aggressive oral cancer cells to understand their possible role in microenvironment modulation and oral cancer progression. Our results indicate that EVs of aggressive cells have exclusive proteins that may contribute to lymph node metastasis, which is the worse prognostic factor in OSCC and can be considered potential targets in OSCC.FAPESP: 2016/19337-3 Citation Format: Ana K. Oliveira, Ariane F. Busso-Lopes, Jamile Oliveira Sá, César Rivera, Adriana F. Paes Leme. Proteomics analysis of oral cancer cell-derived extracellular vesicles [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 5648.

Oral squamous cell carcinoma (OSCC) is the eighth most prevalent malignant neoplasm and accounts for 2% of all deaths by cancer worldwide. OSCCs have a highly variable clinical course, and because it is often diagnosed only after it has reached an advanced stage, the overall survival rate is less than 50% in 5 years. A better knowledge of the mechanisms that influence OSCC development and its progression is still needed. Particularly, most of the attention has been driven to mechanisms associated with tumor cell invasion and how the surrounding tumoral stroma may influence it. The myoma organotypic model is a novel fully human organotypic invasion model that mimics the tumor microenvironment (TME) (Nurmenniemi et al. 2009). Aiming to investigate the molecular mechanisms related to OSCC development and search for prognostic markers for this tumor, we used the myoma organotypic assay, followed by MS-ESI technique of laser microdissected invaded tissue, to analyze several features related to OSCC invasive vs. non-invasive cells. Our results showed that the HSC-3, an aggressive and invasive oral squamous cell carcinoma cell line, invaded as small islands and cell chords and penetrated deeply in the tissue. Unlike, the less aggressive LN2 cells, formed a well structured epithelium layer, but they invaded less in myoma. The proteomic analysis was able to characterize the differential expressed proteins of these, and revealed a group of proteins exclusively expressed in the cells that deeply invade the myoma, such as HMGA2 (high mobility group), JUP (junction plakoglobin), PLEC (plectin), HIST1H2B (Histone H2B), IQGAP1 (Ras GTPase-activating-like protein), EEF1A1 (elongation factor 1-alpha), HSPA9 (Stress-70 protein, mitochondrial), HADHA (Trifunctional enzyme subunit alpha). Additionally, the TME is a unique ambient created and shaped by the tumor, which orchestrates molecular and cellular events taking place in surrounding tissues. Interestingly, we found a group of high abundant proteins expressed in the TME surrounding the invasive island. Among them are PALLD (palladin), TNC (tenascin C), LMNA (laminin), VIM (vimentin), and COL1A (collagen-type I-alpha I), which have been described to be involved in cell migration and proliferation. Our preliminary data suggests that the myoma organotypic tumor invasion assay is successful in determining new molecules and potential pathways associated with OSCC tumor invasion. We also present here a new insight for head and neck cancer biomarkers research that can, ultimately, contribute to more individualized treatment of patients affected by OSCC. However, validation of such results in clinical sample cohorts is important for a better understanding of the biological events associate with tumor invasion and disease spread (metastasis) in those patients. Citation Format: Nilva K. Cervigne, Carolina Bitu, Bianca A. Pauletti, Adriana F. Paes Leme, Tuula Salo, Ricardo Della Coletta. In-depth analysis of myoma organotypic model can determine potential markers of oral cancer invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4998. doi:10.1158/1538-7445.AM2014-4998