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，其已经作为一种新的脂质微泡出现。 癌症中细胞间通讯的重要途径。我们的团队已经进行了开创性的研究， 用于内脏癌的基因组和转录组学分析的循环外泌体， 纵向取样。具体来说，我们已经证明了核酸货物的显着完整性， （exoDNA和exoRNA），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， 高质量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（CRC）。在目标1中，将仔细评估最合适的方法， 使用梯度超离心从转移性CRC患者中灵敏地分离循环外泌体， 两种最常用的商业试剂盒。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再现性测定， 用于将基于外泌体的“液体活检”转化为临床分子诊断实验室（MDL）。我们将 还对从一组CRC类器官中分离的外来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，以鉴定存在的表面蛋白（“表面组”） CRC衍生的外泌体。表面组数据将作为一个宝贵的工具，丰富癌症- 低体积肿瘤环境中的特异性外泌体，例如微小残留疾病监测。在目标2中，我们 比较接受外科手术的患者中组织样本的靶向突变组和拷贝数谱 转移性（m）CRC的减积，以及相应的血浆exoDNA衍生的谱。的显著 外泌体内exoRNA的保存将使我们能够进行RNA-Seq并比较共有序列 分子亚型（CMS）分类，通过脱落的外来体相对于转移组织获得。最后，在Aim 3，我们将纵向监测手术切除的CRC患者，或两组mCRC患者的队列， 前瞻性临床试验，使用外来体作为识别疾病复发和出现的分子工具 的治疗阻力。第一项试验将评估低频KRAS突变的出现 以及其他对EGFR治疗产生耐药性的次要驱动因素。第二项试验是免疫疗法试验 多价肽疫苗，其中exoRNA数据也将用于预测，然后监测，表达 新抗原的mCRC样本，使用生物信息学工具，我们已经开发了映射转录数据， 癌细胞表面的HLA呈递肽。这些研究的长期目标是产生 成功翻译外来体作为液体活检平台所需的标准和测定的概要 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.