Longitudinal therapeutic monitoring of colorectal cancer patients using exosome-based liquid biopsies

项目摘要和相关性： Exosome是含有DNA、RNA和蛋白质的双层脂质微囊，它们作为一种 癌症中细胞间通讯的重要途径。我们的团队对公用事业进行了开创性的研究 用于内脏癌基因组和转录图谱的循环外切体，这些内脏癌对 纵向取样。具体地说，我们已经证明了核酸货物的非凡完整性 (exoDNA和exoRNA)包含在exosome中，这使得它们很容易服从于下一代 测序(NGS)。这项建议的目的是建立液体活组织检查的效用，以丰富高 优质外切体作为晚期结直肠癌治疗分层和疾病监测的平台 癌症(CRC)。在目标1中，将仔细评估最适合可重现和 梯度超速离心法对转移性结直肠癌患者外周血中外切体的敏感分离 两个最常用的商业套件。我们将开发执行严谨的生物和技术 对分离的外切体和核酸货物进行重复性分析，以制定必要的标准 用于将基于胞外体的“液体活检”翻译到临床分子诊断实验室(MDL)。我们会 还对从一组CRC有机化合物中分离出来的外切体进行基于质谱学的蛋白质组学分析 模型与对照有机物和细胞系，以确定存在的表面蛋白(“表面蛋白”) 在CRC衍生的外切体上。表面组数据将成为丰富癌症的宝贵工具- 在低体积肿瘤环境中的特定外切体，例如最小残留疾病监测。在目标2中，我们将 比较外科手术患者组织样本的靶向突变面板和拷贝数图谱 对于转移性(M)结直肠癌，与相应的血浆外源DNA衍生谱进行比较。令人瞩目的 Exosome中外源RNA的保存将使我们能够进行RNA-Seq并比较共识 脱落外切体与转移组织的分子亚型(CMS)分类。最后，在AIM中 3，我们将纵向监测手术切除的CRC患者或mCRC患者的队列 使用外切体作为识别疾病复发和复发的分子工具的前瞻性临床试验 分别对治疗抗性进行了研究。第一个试验将评估低频率KRAS突变的出现 以及其他导致对基于EGFR的治疗产生耐药性的次要驱动因素。第二个试验是免疫疗法试验。 其中外源RNA数据也将被用来预测并监测表达 MCRC样本中的新抗原，使用我们开发的生物信息学工具将转录数据映射到 癌细胞表面有多个多肽，呈HL A阳性。这些研究的长期目标是产生 成功地将外切体转化为液体活组织检查平台所需的标准和分析概要 到临床领域。

Project Summary and Relevance: Exosomes are bi-layered lipid microvesicles containing DNA, RNA and proteins, which have emerged as an important avenue for cell-cell communication in cancer. Our team has performed pioneering studies on the utility of circulating exosomes for genomic and transcriptomic profiling of visceral cancers that are challenging to sample longitudinally. Specifically, we have demonstrated the remarkable integrity of the nucleic acid cargo (exoDNA and exoRNA) contained within exosomes, which makes them readily amenable to next generation sequencing (NGS). The objective of this proposal is to establish the utility of liquid biopsies that enrich for high quality exosomes as a platform for therapeutic stratification and disease monitoring in advanced colorectal cancer (CRC). In Aim 1 will meticulously evaluate the most appropriate methodology for reproducible and sensitive isolation of circulating exosomes from metastatic CRC patients using gradient ultracentrifugation versus two of the most commonly used commercial kits. We will develop perform rigorous biological and technical reproducibility assays on the isolated exosomes and nucleic acid cargo that will develop standards necessary for translation of exosomal-based “liquid biopsy” to a clinical molecular diagnostics laboratory (MDL). We will also perform mass spectrometry-based proteomic profiling of exosomes isolated from a panel of CRC organoid models versus control organoids and cell lines, in order to identify the surface proteins (“surfaceome”) present on CRC-derived exosomes. The surfaceome data will serve as an invaluable tool for enrichment of cancer- specific exosomes in low-volume tumor settings, such as minimal residual disease monitoring. In Aim 2, we will compare targeted mutation panel and copy number profiles of tissue samples in patients undergoing surgical debulking for metastatic (m)CRC, with that of corresponding plasma exoDNA-derived profiles. The remarkable preservation of exoRNA within exosomes will allow us to perform RNA-Seq and compare the consensus molecular subtype (CMS) classification obtained by shed exosomes versus metastatic tissues. Finally, in Aim 3, we will longitudinally monitor cohorts of surgically resected CRC patients, or mCRC patients on two prospective clinical trials, using exosomes as a molecular tool for identifying disease recurrence and emergence of treatment resistance, respectively. The first trial will evaluate for emergence of low frequency KRAS mutations and other secondary drivers of resistance to EGFR-based therapies. The second trial is an immunotherapy trial of multivalent peptide vaccine, where exoRNA data will also be used to predict, and then monitor, expressed neoantigens in mCRC samples, using bioinformatics tools we have developed for mapping transcript data to HLA-presented peptides on the cancer cell surface. The long-term goal of these studies is to generate the compendium of standards and assays needed for successful translation of exosomes as a liquid biopsy platform to the clinical realm.