Exploration of Exosomal Membrane Proteins as Novel Salivary Biomarkers for Early Detection/Disease Monitoring and Reveal of Salivary Exosome-Mediated Mechanisms for Cancer Progression in Oscc

Oral squamous cell carcinoma (OSCC), one of the malignant diseases with high prevalence in Taiwan, frequently results in poor prognosis and mortality because of delay diagnosis, tumor recurrence or metastasis. Therefore, early detection and disease monitoring are very important for disease management in OSCC. Although many candidate biomarkers for oral cancer have been discovered in the past decades, very few of them have been verified and translated into clinical use. However, there are still no biomarkers currently approved by official health agents in the endemic areas for aiding OSCC management. Committed to OSCC-related studies, we have verified four-marker panel in ＞400 saliva samples for Taiwanese OSCC detection, and there is still some room for improvement of increasing early detection rate (84%) [4]. In addition, we also found that the proteome data of 38 paired tissues in Taiwanese OSCC showing a significant proportion of the differentially expressed proteins between OSCC tissues and their adjacent normal counterparts are membrane-associated exosomal proteins, some of which can even be detected in saliva and may also involve in providing the inflammatory environment for tumor progression. Therefore, the membrane fraction of salivary exosome is worth to be comprehensively analyzed for both biomarker discovery and mechanistic study. In this project, we first propose to discover exosomal membrane protein biomarkers which are relatively stable and easily detected (compare to cell-free protein biomarkers) for distinguishing OSCC patients from oral potentially malignant disorder (OPMDs) and normal individuals in pooled salivary samples (10 individuals each group) by using 2D-LC-MS/MS with TMT-labeling strategy. After selecting the 50 potential candidate biomarkers of exosomal membrane proteins, LC-MRM-MS with stable isotope-labeled internal standards spiked in each sample will be used for verification in a retrospective cohort (50 normal, 50 OPMD, and 200 OSCC). Moreover, the latex bead-based, flow cytometry-assisted assays for 5 most promising candidates will also be developed and used for saliva samples. It will be more suitable for clinical usage because of reducing the time consuming of sample pre-processing in flow cytometry-assisted assays compared to MRM-MS assay. We also plan to longitudinally collect salivary samples from 20 OPMD and 20 treated cancer subjects during regular follow-up every 3-6 months for at least 3 years until malignant transformation or recurrence. In the prospective study, we will examine the abilities of selected exosomal membrane-biomarkers for monitoring the treatment response, malignant transformation, and disease recurrence. Besides, the exosome-mediated mechanism will also be dissected in oral squamous cell line (SCC4) and precancer cell line (MSK-LEUK1) models to provide clues for further understanding of OSCC progression. We expect that the success of verification of exosomal membrane biomarkers offer the opportunity to reorganize the four-marker panel we identified previously [4] and improve its performance in detecting OSCC, and exploration of exosome-mediated mechanisms for disease progress can provide a new insight into development of new oral cancer treatment strategy.

Project ID:PC10901-0198
External Project ID:MOST107-2320-B182-028-MY3