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.

项目总结