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

项目摘要和相关性： 外泌体是含有 DNA、RNA 和蛋白质的双层脂质微泡，已作为一种 癌症中细胞间通讯的重要途径。我们的团队对该实用程序进行了开创性研究 循环外泌体用于内脏癌的基因组和转录组分析，这对内脏癌具有挑战性 纵向采样。具体来说，我们已经证明了核酸货物的卓越完整性 （外切DNA和外切RNA）包含在外泌体中，这使得它们很容易适应下一代 测序（NGS）。该提案的目的是建立液体活检的实用性，以丰富高浓度 优质外泌体作为晚期结直肠癌治疗分层和疾病监测的平台 癌症（结直肠癌）。目标 1 将仔细评估最合适的方法，以实现可重复和 使用梯度超速离心法与使用梯度超速离心法从转移性 CRC 患者中灵敏分离循环外泌体 两种最常用的商业套件。我们将开发执行严格的生物和技术 对分离的外泌体和核酸货物进行可重复性测定，这将制定必要的标准 将基于外泌体的“液体活检”转化为临床分子诊断实验室（MDL）。我们会 还对从 CRC 类器官中分离的外泌体进行基于质谱的蛋白质组学分析 模型与对照类器官和细胞系的比较，以识别存在的表面蛋白（“表面组”） CRC 衍生的外泌体。表面组数据将作为富集癌症的宝贵工具 小体积肿瘤环境中的特定外泌体，例如微小残留病监测。在目标 2 中，我们将 比较接受手术的患者组织样本的靶向突变组和拷贝数概况 转移性 (m)CRC 的减灭，以及相应的血浆外切 DNA 衍生图谱的减灭。非凡的 外泌体中外切RNA的保存将使我们能够进行RNA测序并比较共识 通过脱落的外泌体与转移组织获得的分子亚型（CMS）分类。最后，在目标 3、我们将纵向监测手术切除的结直肠癌患者队列，或两个阶段的转移性结直肠癌患者 前瞻性临床试验，使用外泌体作为识别疾病复发和出现的分子工具 分别为治疗耐药性。第一项试验将评估低频 KRAS 突变的出现 以及其他对基于 EGFR 的疗法产生耐药性的次要驱动因素。第二项试验是免疫治疗试验 多价肽疫苗的开发，其中 exoRNA 数据也将用于预测，然后监测表达的 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 HLA 呈递肽位于癌细胞表面。这些研究的长期目标是产生 成功将外泌体转化为液体活检平台所需的标准和测定方法概要 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.