Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. Yet, unlike other cancers, the majority of early stage CRCs are surgically curable. Therefore, early detection of CRCs and removal of precursor lesions, particularly advanced colorectal adenomas (A-CRA), is considered as the best approach in reducing CRC-associated mortality. However, current available screening procedures are impractical. In spite of the efficacy of colonoscopy as a screening tool, its invasive nature and expense often dissuade individuals to follow CRC screening guidelines; and inaccuracy of fecal-based tests remains insufficient as a diagnostic modality. Accumulating evidence indicates that dysregulation of microRNAs (miRNAs) occurs in all human cancers. As biomarkers, miRNAs are more resilient than mRNAs as they are less prone to degradation, and are frequently deregulated even in the earliest stages of neoplasia compared to genetic alterations. Furthermore, the recent discovery that cancers actively excrete small extracellular vesicles, called “exosomes”, into systemic circulation, has brought additional enthusiasm to the field of translational biomarker research. Even though exosomes are considered promising due to their structural stability and molecular profiles reflecting their cell-of-origin, utilization of exosomes in biomarker research has been hampered due to multiple reasons, including: i) lack of standardized protocols for their isolation and purification; ii) use of cell line-derived, not patient-derived specimens for biomarker discovery; iii) lack of molecular profiling studies on cancer-derived exosomes from matched tissues and plasma specimens to establish their cancer specificity; iv) albeit the perception that exosomal-miRNAs (exo-miRNAs) may be superior to circulating cell-free miRNAs (cf-miRNAs), no studies have undertaken an effort to directly compare these two types, in order to support or negate the superiority of either type, as clinically-relevant disease biomarkers. In this proposal, we will address these concerns by undertaking the following Specific Aims. Aim 1: Optimization of patient-derived exosome isolation, followed by RNA-Seq based discovery of circulating cell-free (cf-miRNAs) and exosomal miRNAs (exomiRNAs) in matched tissue and plasma specimens collected from patients with A-CRAs, CRCs and individuals with normal colon. Aim 2: Development of circulating cell-free and exosomal miRNA biomarker panels that distinguish patients with A-CRAs and CRCs from healthy individuals. Aim 3: Clinical validation and performance evaluation of optimized cf-miRNAs and exo-miRNAs in large, independent patient cohorts with colorectal neoplasia. If successful, this proposal will provide much needed molecular characterization of cell-free and exosomal miRNA biomarkers as liquid biopsy biomarkers, which may transform early-detection of CRC into a robust, noninvasive, and inexpensive clinical assay.