Advanced Development of An Integrated CTC Enrichment Technology

描述(由申请人提供)：项目概述本研究方案的长期目标是对循环肿瘤细胞(CTCs)浓缩技术进行高级开发和分析验证。我们的目标是进一步开发我们的高效和特定的CTC捕获技术，为CTC计数不仅作为PC的生物标记物更好地预测临床结果铺平道路，而且还作为临床材料的来源(即，CTC作为“液体活检”)，用于连续的分子分析，可用于指导个别患者的适当治疗(即，“正确的患者的正确治疗”)。我们的联合团队已经证明了一种独特的、相对便宜的细胞亲和力分析，该方法能够识别、计数和捕获从PC患者采集的全血样本中活的(不含防腐剂的)CTCs。最初，我们率先提出了将抗上皮细胞黏附分子(EpCAM)涂层的纳米结构表面作为高亲和力基质来浓缩CTCs的概念。通过将高亲和力底物与能够产生混沌湍流的微流体组件相结合，由于CTCs与底物之间的碰撞增强，最近实现了进一步提高CTC捕获效率(高达99%)。我们进行了并排分析验证，以将我们的纳米结构底物与CellSearchTM分析进行比较，该分析使用了添加了PC细胞系的血液以及33个从PC患者预定义阶段分离的血液样本。CTCs是从原发肿瘤或转移部位分离出来并在外周血液中循环的癌细胞。CTCs计数已在转移性、去势耐受(CR；即激素难治性)PC患者中建立了临床实用价值，在这些患者中，CTCs是化疗反应、无病生存期和总生存期的独立预测因子。可以想象，CTCs的分子和功能特征可以为预测患者预后和监测治疗干预和结果提供许多有价值的信息。在这里，我们将首先开发新一代集成CTC芯片，能够高效和特异地浓缩CTC，并提高血液处理能力，然后使用从PC患者预定义阶段(如CRPC、PSA复发)采集的血液样本进行全面的分析验证。然后，我们将提纯和分离单个CTC，用于使用商业实时qPCR系统对16个基因进行量化。我们建议量化16个基因的表达，我们选择这些基因作为分化状态、上皮-间充质转化和AR信号轴的标志。这些基因赋予的分子特征不仅描述了不同的细胞表型，而且提供了预测对抗癌治疗的反应/耐药的前景。

DESCRIPTION (provided by applicant): Project Summary The long-term objective of this research proposal is to perform advanced development and analytical validation of a technology for enrichment of circulating tumor cells (CTCs). The goal is to further develop our highly efficient and specific CTC capture technology to pave the way not only for CTC enumeration to serve as a biomarker for PC to better predict clinical outcomes, but also as a source of clinical material (i.e., CTCs as a "liquid biopsy") for sequential molecular analyses that can be used to direct appropriate therapies for individual patients (i.e., "the right treatment for the right patient"). Our joint team has demonstrated a unique, relatively inexpensive cell affinity assay, which is capable of identification, enumeration and capture of viable (preservative-free) CTCs in whole-blood samples collected from PC patients. Initially, we pioneered the concept of applying anti-EpCAM (epithelial cell adhesion molecule)-coated nanostructured surfaces as a high-affinity substrate for enrichment of CTCs. By integrating the high-affinity substrate with a microfluidic component capable of generating chaotic turbulence, further improved CTC capture efficiency (up to 99%) has recently been achieved as a result of the enhanced collisions between CTCs and the substrate. Side-by-side analytical validation was conducted to compare our nanostructure substrates with CellSearchTM assay using blood spiked with PC cell lines as well as 33 blood samples isolated from in PC patients at predefined stages. CTCs are cancer cells that break away from either the primary tumor or metastatic sites and circulate in the peripheral blood. Enumeration of CTCs has established clinical utility in patients with metastatic, castration-resistant (CR; i.e. hormone refractory) PC, in whom CTCs are an independent predictor for response to chemotherapy, disease free survival and overall survival. It is conceivable that the molecular and functional characterization of CTCs could provide much valuable information for predicting patient prognosis and monitoring therapeutic interventions and outcomes. Herein, we will first develop a new-generation integrated CTC chip capable of highly efficient and specific enrichment of CTCs with improved blood handling capacity, followed by comprehensive analytical validation using blood samples collected from PC patients at predefined stages (e.g., CRPC, PSA recurrence). We will then purify and isolate individual CTCs for quantification of 16 genes using a commercial real-time qPCR System. We propose to quantify expression of 16 genes, which we have chosen as markers of differentiation state, epithelial-mesenchymal transition, and the AR signaling axis. The molecular signatures imparted by these genes not only depict various cellular phenotypes but also offer the promise of predicting response/ resistance to anti-cancer therapeutics.