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: 9891982
• 负责人: Siyang Zheng
• 金额: $ 50.51万
• 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/9891982
• 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; Detection; 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; 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.

项目概要 胰腺癌是最致命的实体恶性肿瘤，总体 5 年生存率低于 7%。晚的 分期诊断被认为是导致患者预后不佳的最重要因素。自从 胰腺肿瘤细胞可能需要数十年的时间才能转移，理论上，早期诊断有很大的窗口。 然而，由于缺乏敏感和特异的胰腺生物标志物，目前的早期诊断常常失败。 成像方法的空间分辨率的限制。细胞外囊泡 (EV) 为亚微米级 几乎所有细胞都会释放囊泡，并且正在成为一种有吸引力的非侵入性液体活检目标 临床癌症诊断。与其他液体活检材料相比，EVs 很可能由活细胞释放， 含有比循环肿瘤 DNA (ctDNA) 更长的 DNA 片段，并且比循环肿瘤 DNA 更丰富 循环肿瘤细胞 (CTC) 提供更高的灵敏度和更好的临床实用性。根据我们的初步 使用碳纳米管（CNT）森林作为前所未有的亚微米颗粒隔离材料的研究， 我们提出了一种新型 CNT 集成 EV 分离和基因组富集微器件 (CNT-iEVIGEM)，它是 与碳纳米管森林集成的连续流微流体装置。自调节连续流设计 将我们之前的 CNT 集成设备的样品容量和吞吐量提高了 10 倍，并消除了 堵塞，同时使设备免受局部碳纳米管森林缺陷的影响，并保持高电动汽车捕获效率。 通过在 CNT 上增材制造纳米厚的功能氧化锆，CNT-iEVIGEM 可以有效地净化 EV DNA 原位。对于胰腺癌诊断，我们将开发多重数字液滴 PCR (ddPCR) 检测 检测通过 CNT-iEVIGEM 从血浆分离的 EV 中的 KRAS 突变。 KRAS突变最多 胰腺癌常见突变，发生率超过 90%。它是一个主要的驱动癌基因，并且通常是 胰腺肿瘤发生的最早事件。使用在健康对照中添加的癌症来源的 EV 模型样本 血浆，我们将检测限确定为可检测到的最低突变等位基因分数 明确地。该检测将使用来自 IV 期胰腺癌患者的 75 个血浆样本进行验证 和 75 名年龄/性别匹配的健康捐献者。然后我们将使用该测定来研究胰腺的诊断 通过招募 60 名早期（I + II 期）和 60 名 III 期胰腺癌患者来研究不同阶段的癌症。 不同阶段检测的灵敏度和特异性将根据组织中的突变状态来衡量 样本作为真实值。拟议的研究将为 CNT 的进一步临床验证铺平道路 用于癌症诊断的 iEVIGEM 技术。 EV分离平台还可用于其他DNA突变， 其他 EV 货物和其他癌症类型作为非侵入性液体活检技术。