Enhancing Epigenetic Analysis Of Rare Cells With Multi-Phase Microfluidics

利用多相微流体增强稀有细胞的表观遗传分析

基本信息

批准号：
10552039
负责人：
David J Beebe
金额：
$ 62.16万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-03 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10552039
关键词：
Acceleration Address Adsorption Aggressive behavior Air Antibodies Area Automation Benchmarking Binding Binding Proteins Biological Biological Assay Biological Models Biopsy Cell Count Cell Line Cells Chromatin Chromatin Structure Clinic Clinical Clinical Trials Complex Core Biopsy Coupling DNA DNA Binding DNA Methylation DNA Sequence DNA Sequence Alteration DNA-Binding Proteins Data Development Devices Disease Progression Dissociation Ensure Epigenetic Process Exclusion Fine needle aspiration biopsy Force of Gravity Frequencies Future GSTP1 gene Gene Expression Gene Silencing Genes Genetic Transcription Genomics Goals Histone Acetylation Histones Human Hypermethylation Immune Evasion Kinetics Liquid substance Magnetism Malignant Neoplasms Malignant neoplasm of prostate Measurement Measures Methods Methylation Microfluidics Minority Modification Molecular Mutation Nature Needle biopsy procedure Neoplasm Circulating Cells Neoplasm Metastasis Neurosecretory Systems Oils Patients Pattern Phase Phenotype Play Pre-Clinical Model Preparation Primary Neoplasm Procedures Process Proteins Protocols documentation Reaction Reagent Recovery Research Personnel Research Technics Role Sampling Science Solid Neoplasm Speed Surface Surface Tension Techniques Technology Translations Tumor Cell Invasion Variant Wettability aqueous biomarker development biomarker validation bisulfite sequencing cancer therapy cell immortalization chemotherapy chromatin immunoprecipitation diagnostic biomarker epigenetic marker epigenome epigenomics genomic aberrations genomic biomarker histone modification hormonal signals hormone therapy improved interest magnetic field microfluidic technology new therapeutic target novel therapeutic intervention particle precision medicine preservation promoter prospective protein complex stem success targeted treatment therapeutic target therapy resistant tumor tumor DNA tumor heterogeneity tumor progression tumorigenesis validation studies virtual

项目摘要

PROJECT SUMMARY While the genomic revolution has identified several important mutations involved in cancer progression, only a minority of patients benefit from therapies that target these alterations. An emerging area of interest involves aberrant epigenetic modifications, which have been implicated in a broad range of solid tumor malignancies. Importantly, specific epigenetic biomarkers have been identified, often at much higher frequencies than genomic markers (e.g., hypermethylation of the GSTP1 promoter is found in more than 90% of prostate cancer primary tumors). Thus, there is a critical need to extend the concepts of precision medicine beyond genomic aberrations to include epigenomic alterations that drive cancer progression and treatment resistance. Unfortunately, assays to identify epigenetic biomarkers lack the sensitivity to measure many clinical samples, which often contain relatively low cell numbers. Much of this insensitivity stems from the extensive manipulation of DNA/protein complexes required to identify specific epigenetic markers and the associated inadvertent dissociation of these interactions (resulting in analyte loss). Therefore, we aim to improve the state-of-the-art of epigenetic analyses via the implementation of two technologies to preserve molecular interactions: 1) Exclusion-based Sample Preparation (ESP) and 2) Exclusive Liquid Repellency (ELR). With ESP, analytes are bound to functionalized paramagnetic particles (PMPs) and magnetically transferred across phase boundaries (e.g., air/aqueous, oil/aqueous) to isolate the PMP-bound analyte(s). The rapid and non-dilutive nature of ESP preserves molecular interactions, particularly those that are labile or short-lived. ELR utilizes aqueous droplets in oil that are “repelled” from a surface (i.e., they remain suspended and do not contact the surface) to minimize surface-derived analyte loss (e.g., adsorption) while also minimizing reaction volumes (mitigating inadvertent dissociation). Together, the combination of ESP-ELR platform will significantly improve the efficiency of epigenetic analyses, facilitating epigenetic measurements within small clinical samples (e.g., needle biopsies, circulating tumor cells). Specifically, we will develop, optimize, and benchmark ESP-ELR versions of methylation analysis (where a methylated DNA binding protein (MBD2) is employed to selectively capture methylated DNA sequences) and chromatin immunoprecipitation (ChIP; where histone/DNA complexes are isolated in order to interrogate chromatin status). Lastly, we will automate the platform and use it to perform a prospective biomarker validation study of GSTP1 paving the way for it’s use in clinical trials. Here we focus on prostate cancer as a model system, but we expect that an improved platform for epigenetic analysis will have broad impact across the biomedical sciences.

项目概要虽然基因组革命已经确定了与癌症有关的几个重要突变一个令人感兴趣的新兴领域涉及异常的表观遗传修饰，这已被暗示重要的是，特定的表观遗传生物标志物具有广泛的实体瘤恶性肿瘤的作用。已被识别，通常比基因组标记（例如，高甲基化 GSTP1 启动子存在于超过 90% 的前列腺癌原发肿瘤中）。迫切需要将精准医学的概念扩展到基因组畸变之外包括导致癌症进展和治疗耐药的表观基因组改变。不幸的是，识别表观遗传生物标志物的测定缺乏测量许多生物标志物的灵敏度。临床样本中的细胞数量通常相对较低。来自识别特定表观遗传所需的 DNA/蛋白质复合物的广泛操作标记物以及这些相互作用的相关无意解离（导致分析物因此，我们的目标是通过以下方法提高表观遗传学分析的最新水平。实施两种技术来保持分子相互作用：1）基于排除样品制备 (ESP) 和 2) 独有的防液性 (ELR) 分析仪具有 ESP。与功能化顺磁性颗粒 (PMP) 结合并通过磁性转移相边界（例如空气/水、油/水）以分离 PMP 结合的分析物。 ESP 的非稀释性质保留了分子相互作用，特别是那些不稳定的分子相互作用 ELR 利用油中的水滴，这些水滴被表面“排斥”（即它们）。保持悬浮状态且不接触表面）以尽量减少表面衍生的分析物损失（例如，吸附），同时还最大限度地减少反应体积（减少无意的解离）。 ESP-ELR平台的结合将显着提高效率表观遗传分析，促进小临床样本中的表观遗传测量（例如，具体来说，我们将开发、优化和基准测试甲基化分析的 ESP-ELR 版本（其中甲基化 DNA 结合蛋白 (MBD2) 用于选择性捕获甲基化 DNA 序列）和染色质免疫沉淀（ChIP；分离组蛋白/DNA 复合物以询问染色质状态）。最后，我们将使平台自动化并使用它来执行前瞻性生物标志物验证 GSTP1 的研究为其在临床试验中的使用铺平了道路这里我们重点关注前列腺癌。一个模型系统，但我们预计表观遗传分析的改进平台将具有广泛的应用整个生物医学科学的影响。