Enhancing Epigenetic Analysis Of Rare Cells With Multi-Phase Microfluidics

项目总结 虽然基因组革命已经确定了几个与癌症有关的重要突变 随着病情的发展，只有少数患者从针对这些改变的治疗中受益。一个 新出现的感兴趣的领域涉及异常的表观遗传修饰，这已被牵连 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记物 通常比基因组标记的频率高得多(例如，超甲基化 在超过90%的前列腺癌原发癌中发现了GSTP1启动子)。因此，在那里 是将精确医学的概念从基因组像差扩展到 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记物的分析缺乏测量许多 临床样本，通常含有相对较低的细胞数量。这种麻木不仁在很大程度上源于 从识别特定表观遗传学所需的DNA/蛋白质复合体的广泛操作 标记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 实现两种保持分子相互作用的技术：1)基于排除的 样品制备(ESP)和2)独有液体驱避性(ELR)。使用电除尘器，分析物 结合到功能化顺磁粒子(PMP)上并通过磁转移 相界面(例如，空气/水、油/水)，以分离与PMP结合的分析物(S)。The Rapid ESP的非稀释性保留了分子相互作用，特别是那些不稳定的分子相互作用 或者是短暂的。ELR利用油中的水滴，这些水滴从表面被排斥(即，它们 保持悬浮并且不接触表面)以使表面衍生的分析物损失最小化(例如， 吸附)，同时也最大限度地减少反应体积(减少无意解离)。 ESP-ELR平台的结合将显著提高ESP-ELR平台的效率 表观遗传学分析，便于在小的临床样本中进行表观遗传学测量(例如， 针吸活组织检查、循环肿瘤细胞)。具体来说，我们将开发、优化和基准 ESP-ELR版本的甲基化分析(其中甲基化DNA结合蛋白(Mbd2)是 用于选择性地捕获甲基化DNA序列)和染色质免疫沉淀 (芯片；其中组蛋白/DNA复合体被分离以询问染色质状态)。 最后，我们将自动化该平台并使用它来执行预期的生物标记物验证 GSTP1的研究为其用于临床试验铺平了道路。在这里，我们关注前列腺癌，因为 一个模型系统，但我们预计一个改进的表观遗传学分析平台将具有广泛的 对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.