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

描述（由申请人提供）：容易获得肿瘤样本，而不需要痛苦和昂贵的肿瘤活检，将允许临床医生和研究人员反复询问患者样本，以确定治疗耐药的机制，并为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稀有细胞通过“液体活检”获得的潜力，这将允许频繁，微创地对肿瘤细胞进行采样，用于在传统活检中进行相同的分子测定。此外，获得CTC将使许多关键的未来研究，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，一种新的分析技术，称为垂直排除为基础的罕见样本分析（VERSA）将被开发。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，所有这些都在来自单个患者样本的单个芯片上进行。VERSA将能够对CTC进行多终点分析，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除的样品制备，这是一种用于执行分离的新方法，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种水性液体中。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提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连续提取多种分析物，包括蛋白质、RNA和DNA。将在三个特定目标中使用患者样本开发和测试基于VERSA的平台。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，我们将联合收割机CTC分离与随后分离mRNA（用于RT-PCR读出）和DNA（用于SNP和测序读出）相结合。在目标3中，我们将把目标1和目标2的读数整合并验证到一个芯片中，并使用这个平台对40名前列腺癌患者进行多终点前瞻性临床试验。在这项试验中，我们将收集相关的蛋白质，基因表达和基因组数据，描述雄激素受体（AR）的状态，前列腺癌的主要治疗靶点。该试验将为VERSA平台的未来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.