Enhancing Epigenetic Analysis Of Rare Cells With Multi-Phase Microfluidics

项目摘要 虽然基因组革命已经确定了几个与癌症有关的重要突变， 然而，随着疾病的进展，只有少数患者受益于针对这些改变的治疗。一个 一个新兴的感兴趣的领域涉及异常的表观遗传修饰， 在广泛的实体肿瘤恶性肿瘤中。重要的是，特定的表观遗传生物标志物具有 被鉴定，通常比基因组标记的频率高得多（例如，超甲基 在超过90%的前列腺癌原发性肿瘤中发现了GSTP 1启动子）。因此 是一个关键的需要，以扩大精准医学的概念超越基因组畸变， 包括驱动癌症进展和治疗抗性表观基因组改变。 不幸的是，鉴定表观遗传生物标志物的测定缺乏测量许多表观遗传生物标志物的灵敏度。 临床样品，其通常含有相对低的细胞数量。这种不敏感的大部分原因是 从DNA/蛋白质复合物的广泛操作，需要确定特定的表观遗传 标记物和这些相互作用的相关无意解离（导致分析物 损失）。因此，我们的目标是通过以下方法来改善表观遗传分析的最新水平： 实施两种技术以保持分子相互作用：1）基于排除的 样品制备（ESP）和2）排斥液体（ELR）。使用ESP，分析物 结合到功能化的顺磁性颗粒（PMP）上， 相边界（例如，空气/水、油/水）以分离PMP结合的分析物。快速 ESP的非稀释性质保留了分子间的相互作用，特别是那些不稳定的相互作用 或是短命的ELR利用从表面“排斥”的油中的含水液滴（即，他们 保持悬浮并且不接触表面）以最小化表面衍生的分析物损失（例如， 吸附），同时还使反应体积最小化（减轻无意的解离）。 总之，ESP-ELR平台的结合将显著提高 表观遗传分析，促进小临床样品内的表观遗传测量（例如， 针活检、循环肿瘤细胞）。具体来说，我们将发展、优化、对标 甲基化分析的ESP-ELR版本（其中甲基化DNA结合蛋白（MBD 2）是 用于选择性捕获甲基化DNA序列）和染色质免疫沉淀 (ChIP其中分离组蛋白/DNA复合物以询问染色质状态）。 最后，我们将自动化平台，并使用它来执行前瞻性生物标志物验证 GSTP 1的研究为它在临床试验中的应用铺平了道路。在这里，我们重点关注前列腺癌， 一个模型系统，但我们预计，一个改进的平台，表观遗传分析将有广泛的 对整个生物医学科学的影响。

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.