Exosomal Biomarkers for the Noninvasive Detection of Colorectal Cancer

结直肠癌（CRC）是癌症相关死亡的第二大原因， 美国的然而，与其他癌症不同的是，大多数早期CRC是可以手术治愈的。因此早期 CRC的检测和前体病变的切除，特别是晚期结直肠腺瘤（A-CRA）， 被认为是降低CRC相关死亡率的最佳方法。然而，目前可用的筛选 程序不切实际。尽管结肠镜检查作为筛查工具的有效性，但其侵入性和 费用往往劝阻个人遵循CRC筛查指南;和粪便为基础的测试不准确 作为一种诊断方法仍然不够。越来越多的证据表明， （miRNAs）存在于所有人类癌症中。作为生物标志物，miRNAs比mRNAs更有弹性，因为它们不太 易于降解，甚至在肿瘤形成的最早阶段也经常被解除管制， 基因改变此外，最近发现癌症主动分泌小的细胞外囊泡， 被称为“外泌体”，进入体循环，给翻译领域带来了额外的热情。 生物标志物研究尽管外泌体由于其结构稳定性和生物相容性而被认为是有希望的， 由于外泌体的分子谱反映了它们的细胞起源，因此在生物标志物研究中利用外泌体受到了阻碍 由于多种原因，包括：i）缺乏用于其分离和纯化的标准化方案; ii）使用 用于生物标志物发现的细胞系来源而非患者来源的标本; iii）缺乏分子特征分析研究 来自匹配组织和血浆样本的癌症来源的外来体，以确定其癌症特异性; iv）尽管认为外泌体-miRNA（exo-miRNA）可能比循环的无细胞miRNA上级， （cf-miRNAs），没有研究进行了直接比较这两种类型的努力，以支持或 否定了任一类型作为临床相关疾病生物标志物的优越性。在本提案中，我们将解决 这些问题通过以下具体目标来解决。目的1：患者来源的外泌体的优化 分离，然后基于RNA-Seq发现循环无细胞（cf-miRNAs）和外泌体miRNAs（exo-miRNAs）。 在从患有A-CRA、CRC和个体的患者收集的匹配组织和血浆样本中， 结肠正常。目的2：开发循环无细胞和外泌体miRNA生物标志物组， 区分A-CRA和CRC患者与健康个体。目标3：临床确认和性能 在大型独立的结直肠癌患者队列中评价优化的cf-miRNAs和exo-miRNAs 肿瘤形成 如果成功，该提议将提供急需的无细胞和外泌体的分子表征。 miRNA生物标志物作为液体活检生物标志物，可以将CRC的早期检测转化为一种稳健的，非特异性的， 侵入性和廉价临床测定。

PROJECT SUMMARY: 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 (exo- miRNAs) 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, non- invasive, and inexpensive clinical assay.