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

描述（由申请人提供）：轻松获取肿瘤样本，不需要痛苦和昂贵的肿瘤活检，将使临床医生和研究人员能够反复询问患者样本，以确定治疗耐药性的机制，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，这些罕见的细胞有可能通过“液体活检”获得，这种方法将允许对肿瘤细胞进行频繁的、微创的采样，以进行与传统活检相同的分子分析。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。在本研究中，将开发一种新的分析技术，称为基于垂直排斥的稀有样品分析（VERSA）。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。VERSA将支持对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功能的基础技术是基于排斥的样品制备，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并进入第二含水液体。重要的是，由于基于排斥的纯化是通过简单地将分析物从样品中提取到另一个含水缓冲液中来实现的，因此原始样品永远不会被稀释或冲走。这使得从单个样品中连续提取多种分析物，包括蛋白质，RNA和DNA。基于versa的患者样本平台的开发和测试将贯穿三个特定目标。在第一个目标中，我们将优化和验证两个额外的读数，亚细胞蛋白定位和总蛋白定量。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Aim 3中，我们将把Aims 1和Aims 2的读数整合并验证到单个芯片中，并使用该平台对40例前列腺癌患者进行多终点前瞻性临床试验。在这项试验中，我们将收集描述雄激素受体（AR）状态的相关蛋白、基因表达和基因组数据，雄激素受体是前列腺癌的主要治疗靶点。该试验将为VERSA平台未来的生物学和临床研究奠定基础。

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.