Exosome separation and digital resolution detection of blood-based nucleic acid biomarkers for noninvasive therapeutic diagnostics in cancer

用于癌症无创治疗诊断的血液核酸生物标志物的外泌体分离和数字分辨率检测

• 批准号: 10214617
• 负责人: Brian T. Cunningham
• 金额: $ 50.56万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-15 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10214617
• 关键词: Acetates; Advanced Malignant Neoplasm; Algorithms; Biological Assay; Biological Markers; Biosensor; Blood; Blood specimen; Clinical; Clinical Data; Clinical Trials; Collection; Coupling; Crystallization; DNA Sequence Alteration; Detection; Devices; Diagnosis; Diagnostic; Disease; Drops; Drug Targeting; Effectiveness; Enzymes; Equipment; Fluorescence; Fluorescent Dyes; Generations; Genome; Genomics; Goals; Individual; Laboratories; Link; Liquid substance; Malignant Neoplasms; Malignant neoplasm of prostate; Measurement; Measures; Methods; MicroRNAs; Microfluidics; Microscopy; Molecular; Monitor; Mutation; Nucleic Acid Probes; Nucleic Acids; Oncologist; Optics; Outcome; Patients; Pharmacotherapy; Plasma; Precision therapeutics; Prednisone; Preparation; Protocols documentation; Quantitative Reverse Transcriptase PCR; Reproducibility; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Screening for cancer; Surface; System; Technology; Temperature; Testing; Therapeutic; Thermodynamics; Time; Translations; Treatment outcome; Validation; Variant; Vision; Work; abiraterone; absorption; advanced prostate cancer; assay development; base; cancer biomarkers; cancer genome; cancer therapy; cancer type; castration resistant prostate cancer; chemotherapy; clinical application; clinical diagnostics; cost; design; detection limit; detection method; digital; docetaxel; effective therapy; effectiveness evaluation; exosome; experience; imaging detection; individual patient; innovative technologies; insight; instrument; instrumentation; microRNA biomarkers; nanoGold; nanoparticle; novel; photonics; plasmonics; point of care; portability; predict clinical outcome; predicting response; prospective; rapid technique; rapid test; ratiometric; repository; sample collection; simulation; single molecule; success; technology validation; tumor

Abstract Multiple drug therapies have been approved for treating advanced cancer. However, the effectiveness of each is variable and the ability to monitor or predict efficacy in individual patients is underdeveloped. Our team recently demonstrated (using traditional sequencing-based methods) that expression levels of specific microRNAs (miRNAs) in blood can effectively predict treatment outcomes. The goal of this proposal is to develop innovative technologies that will allow us to measure miRNAs from a patient on a frequent basis, in a way that is convenient and rapid, to enable precise adjustment of therapy. This is currently not achievable using RT-PCR or sequencing-based detection. All cancers are associated with heterogeneous somatic genetic alterations, ushering in a new generation of nucleic-acid-based targeted treatments. The measurement of somatic genome based biomarkers to assess, monitor, and change treatments is needed. Circulating exosomal miRNAs represent one class of highly specific markers of cancer-associated genetic mutations that can be noninvasively sampled from blood, whose quantitation can provide previously-unavailable information to clinicians for generating informed decisions on selection of effective treatments among the wide array of options. In order to make effective routine use of miRNA cancer biomarkers, novel technical approaches will need to be developed that can offer a high degree of multiplexing, quantitation, ultrasensitivity, low cost, simplicity, integrated sample processing, and robust instrumentation suitable for point of care (POC) settings. We link a newly demonstrated form of microscopy, called NanoParticle Photonic Resonator Absorption Microscopy (NP-PRAM) with a simple and effective exosome isolation approach to perform sample preparation that yields exosomal miRNA for detection. Using plasmonic NPs whose resonant wavelength matches a photonic crystal surface, NP-PRAM demonstrates high contrast “digital resolution” precision sensing of exosomal miRNAs. We plan to develop assays for simultaneous detection of 5 miRNA sequences extracted from a single droplet of blood with a rapid assay protocol that does not require fluorescent emitters or enzymatic amplification. We utilize simulation-guided miRNA probe design for ultraspecific hybridization. We will apply NP-PRAM in the context of a panel of clinically validated miRNA biomarkers for advanced prostate cancer. Our approach offers important advantages compared to existing methods for detection of circulating nucleic acid biomarkers: It requires only a ~50 µl droplet of test sample unlike 10-20 ml of blood for RT-PCR based detection methods. NP-PRAM detection produces highly quantified results because nanoparticle tags are not subject to the effects of quenching or background fluorescence that are common to fluorescent dyes. The assay is isothermal, conducted at room temperature, and highly selective, while it does not require enzyme amplification or wash steps. The approach can be applied to quantitative characterization of miRNA biomarkers for all cancer types, although here we specifically focus on a clinically validated set of miRs for prostate cancer.

摘要