VERSA: An Integrated, Multi-Endpoint Platform for Circulating Tumor Cell Analysis

VERSA：用于循环肿瘤细胞分析的集成多端点平台

基本信息

批准号：
8720248
负责人：
Scott M Berry
金额：
$ 30.56万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-09 至 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8720248
关键词：
Advanced Malignant Neoplasm Androgen Receptor Antibodies Antineoplastic Agents Binding Biological Biological Assay Biological Models Biopsy Blood Blood specimen Buffers Cell Count Cell Nucleus Cell Separation Cell physiology Cells Centrifugation Clinical Clinical Research Clinical Trials Coupling Cytoplasmic Protein DNA Data Development Devices Dialysis procedure Disease Drug resistance Excision Exclusion Force of Gravity Future Gene Expression Genetic Transcription Genomics Gold Heterogeneity Image In Vitro Individual Liquid substance Malignant neoplasm of prostate Medical Membrane Messenger RNA Methods Microfluidics Molecular Molecular Target Mutate Neoplasm Circulating Cells Nucleic Acids Oils Pain Patients Phase Preparation Procedures Process Protein Analysis Proteins RNA Receptor Gene Research Research Personnel Resistance Reverse Transcriptase Polymerase Chain Reaction Sampling Sequence Analysis Single Nucleotide Polymorphism Staining method Stains Surface Tension Techniques Technology Testing Therapeutic Time Universities Wisconsin aqueous base cancer therapy extracellular flexibility immunocytochemistry in vitro Model interest mRNA Transcript Degradation magnetic beads magnetic field minimally invasive neoplastic cell nuclease overexpression particle prospective public health relevance receptor resistance mechanism response sample fixation success therapeutic target tool treatment strategy tumor validation studies virtual

项目摘要

DESCRIPTION (provided by applicant): Easy access to tumor samples, without the need for painful and expensive tumor biopsies, would allow clinicians and researchers to repeatedly interrogate patient samples to identify mechanisms of therapeutic resistance and personalize subsequent treatment strategies to these continually evolving tumors. The great hope in circulating tumor cell (CTC) research lies in the potential for these rare cells to be accessible va a "fluid biopsy" that would permit frequent, minimally invasive sampling of tumor cells for the same molecular assays performed on traditional biopsies. Furthermore, access to CTCs would enable many critical future studies, increasing our understanding of the metastatic cascade, tumor heterogeneity, drug resistance, etc. In the proposed research, a new analysis technique termed Vertical Exclusion-based Rare Sample Analysis (VERSA) will be developed. The VERSA combines CTC purification with downstream protein analysis, RNA extraction, and DNA extraction, all on a single chip from a single patient sample. The VERSA will enable multi- endpoint analyses on CTCs, providing users with a comprehensive snapshot of CTC function at a molecular level. The fundamental technology underlying the function of the VERSA is exclusion-based sample preparation, a new method for performing isolations in which magnetic beads are used to selectively bind an analyte of interest and then draw the captured analyte through an oil barrier and into a second aqueous liquid. Importantly, since exclusion-based purification is achieved by simply drawing an analyte from the sample to another aqueous buffer, the original sample is never diluted or washed away. This enables the sequential extraction of multiple analytes from a single sample including protein, RNA, and DNA. Development and testing of the VERSA-based platform with patient samples will occur throughout three Specific Aims. In the first Aim, we will optimize and validate two additional readouts, subcellular protein localization and total protein quantification. In Aim 2, we will combine CTC isolation with subsequent isolation of both mRNA (for RT-PCR readouts) and DNA (for SNP and sequencing readouts). In Aim 3, we will integrate and validate the readouts of Aims 1 and 2 into a single chip and use this platform to perform a multi-endpoint prospective clinical trial on 40 patients with prostate cancer. During this trial, we will collect correlated protein, gene expression, and genomic data describing the status of the androgen receptor (AR), the major therapeutic target in prostate cancer. This trial will lay the groundwork for futur biological and clinical studies with the VERSA platform.

描述（由申请人提供）：轻松获取肿瘤样本，无需进行痛苦且昂贵的肿瘤活检，将使临床医生和研究人员能够反复询问患者样本，以确定治疗耐药机制，并针对这些不断发展的肿瘤制定个性化的后续治疗策略。循环肿瘤细胞（CTC）研究的巨大希望在于通过“液体活检”获得这些稀有细胞的潜力，这将允许对肿瘤细胞进行频繁、微创的采样，以进行与传统活检相同的分子测定。此外，获得 CTC 将使许多关键的未来研究成为可能，增加我们对转移级联、肿瘤异质性、耐药性等的理解。在拟议的研究中，将采用一种称为基于垂直排除的稀有样本分析 (VERSA) 的新分析技术。发达。 VERSA 将 CTC 纯化与下游蛋白质分析、RNA 提取和 DNA 提取结合在一起，所有这些都在单个患者样本的单个芯片上进行。 VERSA 将能够对 CTC 进行多端点分析，为用户提供分子水平上 CTC 功能的全面快照。 VERSA 功能的基本技术是基于排除的样品制备，这是一种执行分离的新方法，其中使用磁珠选择性地结合感兴趣的分析物，然后将捕获的分析物通过油屏障吸入第二个水相中。液体。重要的是，由于基于排除的纯化是通过简单地将分析物从样品中抽取到另一种水性缓冲液中来实现的，因此原始样品永远不会被稀释或洗掉。这使得能够从单个样品中顺序提取多种分析物，包括蛋白质、RNA 和 DNA。基于 VERSA 的平台的开发和患者样本测试将在三个具体目标中进行。在第一个目标中，我们将优化和验证两个额外的读数，即亚细胞蛋白质定位和总蛋白质定量。在目标 2 中，我们将把 CTC 分离与随后的 mRNA（用于 RT-PCR 读数）和 DNA（用于 SNP 和测序读数）分离结合起来。在目标 3 中，我们将把目标 1 和 2 的读数集成并验证到单个芯片中，并使用该平台对 40 名前列腺癌患者进行多端点前瞻性临床试验。在此试验期间，我们将收集描述雄激素受体 (AR) 状态的相关蛋白质、基因表达和基因组数据，雄激素受体 (AR) 是前列腺癌的主要治疗靶点。该试验将为未来利用 VERSA 平台进行生物学和临床研究奠定基础。