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

描述(申请人提供)：容易获得肿瘤样本，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，以确定治疗耐药机制，并针对这些不断演变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，这些稀有细胞有可能通过“液体活检”获得，这将允许对肿瘤细胞进行频繁、微创的采样，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、耐药性等的了解。在拟议的研究中，将开发一种新的分析技术，称为基于垂直排除的稀有样本分析(反之亦然)。反之，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完成。反之亦然，将实现对四氯化碳的多终点分析，向用户提供分子水平上四氯化碳功能的全面快照。反之作用的基本技术是基于排除的样品制备，这是一种进行分离的新方法，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层屏障吸引到第二水溶液中。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含水缓冲液中来实现的，所以原始样品永远不会被稀释或冲走。这使得能够从单个样品中顺序提取多个分析物，包括蛋白质、RNA和DNA。以患者样本为基础的基于Versa的平台的开发和测试将贯穿三个具体目标。在第一个目标中，我们将优化和验证两个额外的读数，亚细胞蛋白质定位和总蛋白质定量。在目标2中，我们将结合CTC分离和随后的mRNA(用于RT-PCR读出)和DNA(用于SNP和测序读出)的分离。在AIM 3中，我们将把AIMS 1和AIMS 2的读数集成到一个芯片中并进行验证，并使用该平台对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，描述前列腺癌的主要治疗靶点雄激素受体(AR)的状态。这项试验将为未来利用Versa平台进行生物学和临床研究奠定基础。

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.