Exosomal Biomarkers for the Noninvasive Detection of Colorectal Cancer

项目摘要：结直肠癌(CRC)是全球第二大癌症相关死亡原因 美国。然而，与其他癌症不同的是，大多数早期癌是可以通过手术治愈的。因此，很早 癌的检测和前驱病变的切除，特别是晚期结直肠腺瘤(A-CRA)，是 被认为是降低与儿童权利有关的死亡率的最佳办法。然而，目前可用的筛查 程序不切实际。尽管结肠镜检查是一种有效的筛查工具，但它的侵袭性和 费用经常劝阻个人遵循结直肠癌筛查指南；以及粪便检测的不准确性 仍然不足以作为一种诊断手段。越来越多的证据表明，microRNAs的调控失调 (MiRNAs)存在于所有人类癌症中。作为生物标志物，miRNAs比mRNAs更具弹性，因为它们更少 容易退化，甚至在肿瘤的最早阶段也经常被解除管制 基因改变。此外，最近发现癌症主动排泄细胞外小泡， 被称为“exosome”，进入全身循环，为翻译领域带来了额外的热情 生物标志物研究。尽管外切体被认为很有前途，因为它们的结构稳定性和 反映其细胞起源的分子图谱，外切体在生物标志物研究中的应用受到阻碍 由于多种原因，包括：一)缺乏分离和提纯的标准化方案；二)使用 用于发现生物标记物的细胞系样本，而不是患者样本；iii)缺乏分子图谱研究 从匹配的组织和血浆样本中提取癌症来源的外切体，以确定它们的癌症特异性； 四)尽管认为外体miRNAs(exo-miRNAs)可能优于循环中的无细胞miRNAs (cf-miRNAs)，还没有研究致力于直接比较这两种类型，以支持或 否定任何一种类型作为临床相关疾病生物标志物的优越性。在这项提案中，我们将解决 这些关切通过承担以下具体目标来实现。目标1：优化患者来源的外切体 分离，然后基于RNA-Seq发现循环中的无细胞(cf-miRNAs)和外体miRNAs(exo-siRNA)。 从A-CRA、CRCs和个体采集的配对组织和血浆标本中的miRNAs) 有正常的结肠。目标2：开发循环中无细胞和外体miRNA生物标志物面板 将A-CRA和CRC患者与健康人区分开来。目标3：临床验证和绩效 优化的cf-miRNAs和exo-miRNAs在大型、独立的结直肠癌患者队列中的评估 肿瘤。 如果成功，这项提议将提供急需的无细胞和外切体的分子特征 MiRNA生物标记物作为液体活检生物标记物，可能会将结直肠癌的早期检测转变为强大的、非 侵入性的、廉价的临床化验。

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.