3D carbon-nanotubes integrated microdevice for extracellular vesicle isolation and in situ sample preparation towards noninvasive pancreatic cancer diagnosis

用于细胞外囊泡分离和原位样品制备的 3D 碳纳米管集成微器件，用于非侵入性胰腺癌诊断

• 批准号: 10231020
• 负责人: Siyang Zheng
• 金额: $ 49.27万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-10 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10231020
• 关键词: 3-Dimensional; Alleles; Biological Assay; Biological Markers; Blood Circulation; Carbon Nanotubes; Cells; Clinical; Cytolysis; DNA; DNA Binding; DNA Sequence Alteration; DNA amplification; DNA purification; Defect; Deposition; Devices; Diagnosis; Diagnostic; Early Diagnosis; Event; Excision; Feasibility Studies; Filtration; Genes; Genome; Genomic DNA; High Prevalence; Immune; In Situ; KRAS2 gene; Malignant Neoplasms; Malignant neoplasm of pancreas; Measures; Membrane Lipids; Microfluidic Microchips; Modeling; Mutation; Mutation Detection; Neoplasm Circulating Cells; Neoplasm Metastasis; Nucleic Acids; Oncogenes; Operative Surgical Procedures; Pancreas; Patient-Focused Outcomes; Patients; Pattern; Plasma; Preparation; Prognostic Marker; Proteins; RNA; Research Personnel; Resolution; Sampling; Sensitivity and Specificity; Solid; Source; Stage at Diagnosis; Technology; Thick; Time; Tissue Sample; Ultrafiltration; Validation; Vesicle; atomic layer deposition; base; cancer biomarkers; cancer diagnosis; cancer type; curative treatments; design; detection assay; detection limit; diagnostic biomarker; digital; ds-DNA; extracellular vesicles; forest; imaging modality; improved; interdisciplinary collaboration; liquid biopsy; microdevice; mutant; mutation assay; mutational status; nanomaterials; nanometer; neoplastic; neoplastic cell; noninvasive diagnosis; nucleic acid purification; operation; pancreatic cancer patients; pancreatic tumorigenesis; particle; recruit; sex; theories; tumor; tumor DNA; vesicular release; zirconium oxide

PROJECT SUMMARY Pancreatic cancer is the deadliest solid malignancy with an abysmal overall 5-year survival less than 7%. Late stage diagnosis is regarded as the most important factor contributing to the bleak patient outcome. Since pancreatic neoplastic cells can take decades to metastasis, in theory, there is a large window for early diagnosis. However, current early diagnosis often fails due to lack of sensitive and specific pancreatic biomarkers and limitations on the spatial resolution of the imaging methods. Extracellular vesicles (EVs) are sub-micrometer vesicles released by almost all the cells and are emerging as an attractive non-invasive liquid biopsy target for clinical cancer diagnosis. Compared with other liquid biopsy materials, EVs are likely released by live cells and contains much longer DNA fragments than circulating tumor DNA (ctDNA), and are more abundant than circulating tumor cells (CTCs) to provide higher sensitivity and better clinical utility. Based on our preliminary study on using carbon nanotube (CNT) forest as an unprecedented material for sub-micrometer particle isolation, we propose a new CNT-integrated EV isolation and genome enrichment microdevice (CNT-iEVIGEM), which is a continuous flow microfluidic device integrated with CNT forest. The self-regulating continuous-flow design boosts the sample capacity and throughput of our previous CNT integrated device by 10 times and eliminates clogging, while making the device immune to local CNT forest defects and maintaining high EV capture efficiency. With additive fabrication of nanometer-thick functional zirconia on CNTs, the CNT-iEVIGEM can efficiently purify EV DNA in situ. For pancreatic cancer diagnosis, we will develop a multiplex digital droplet PCR (ddPCR) assay to detect KRAS mutations from EVs isolated from plasma by CNT-iEVIGEM. KRAS mutations are the most prevalent pancreatic cancer mutations with over 90% occurrence. It is a major driver oncogene and often the earliest event in pancreatic tumorigenesis. Using model samples of cancer-derived EVs spiked in healthy control plasma, we will determine the detection limit as the lowest mutant allele fraction that can be detected unambiguously. The assay will be validated with 75 plasma samples from stage IV pancreatic cancer patients and 75 age-/sex- matched healthy donors. Then we will use this assay to investigate diagnosis of pancreatic cancer at different stages, by recruiting 60 early stage (stage I + II) and 60 stage III pancreatic cancer patients. The sensitivity and specificity of the assay at different stages will be measured with the mutation status in tissue samples as the true values. The proposed study will pave the way for further clinical validation of the CNT- iEVIGEM technology for cancer diagnosis. The EV isolation platform can also be used for other DNA mutations, other EV cargos, and other cancer types as a non-invasive liquid biopsy technology.

项目摘要 胰腺癌是最致命的固体恶性肿瘤，总5年生存率较差，低于7％。晚的 阶段诊断被认为是导致惨淡患者结局的最重要因素。自从 从理论上讲，胰腺肿瘤细胞可能需要数十年的时间转移，有一个很大的早期诊断窗口。 但是，由于缺乏敏感和特定的胰腺生物标志物，当前的早期诊断通常失败 成像方法空间分辨率的局限性。细胞外囊泡（EV）是亚微米 几乎所有细胞释放的囊泡都作为有吸引力的非侵入性液体活检靶标的 临床癌症诊断。与其他液体活检材料相比，电动汽车可能由活细胞释放 与循环肿瘤DNA（CTDNA）相比，包含更长的DNA片段，并且比 循环肿瘤细胞（CTC）提供更高的灵敏度和更好的临床实用性。基于我们的初步 研究使用碳纳米管（CNT）森林作为固定粒子分离的前所未有的材料， 我们提出了一种新的CNT集成的EV隔离和基因组富集微电位（CNT-IVIGEM），它是 与CNT森林集成的连续流微流体设备。自我调节的连续流设计 将以前CNT集成设备的样本容量和吞吐量提高10次，并消除 堵塞，使该设备免疫到局部CNT森林缺陷并保持高EV捕获效率。 通过在CNT上的纳米厚功能锆石的添加剂制造，CNT-IVIGEM可以有效纯化 原位EV DNA。对于胰腺癌诊断，我们将开发多重数字液滴PCR（DDPCR）测定法 从CNT-IVIGEM中检测从等离子体分离的EV的KRAS突变。 KRAS突变是最大的 普遍发生的胰腺癌突变，发生超过90％。这是主要的驱动器癌基因，通常是 胰腺肿瘤发生的最早事件。使用在健康对照中峰值的癌症衍生电动汽车的模型样本 血浆，我们将确定检测极限为可以检测到的最低突变等位基因分数 明确。该测定将通过IV期胰腺癌患者的75个血浆样品进行验证 和75岁/性别匹配的健康捐助者。然后，我们将使用该测定法调查胰腺的诊断 在不同阶段的癌症，通过招募60个早期阶段（I + II期）和60阶段III胰腺癌患者。 测定在不同阶段的敏感性和特异性将通过组织中的突变状态进行测量 示例作为真实值。拟议的研究将为进一步的临床验证铺平道路。 IEVIGEM技术用于癌症诊断。 EV隔离平台也可以用于其他DNA突变， 其他电动汽车和其他癌症类型是一种非侵入性液体活检技术。