Advanced Development of An Integrated CTC Enrichment Technology

描述（由申请人提供）：项目概述本研究提案的长期目标是对循环肿瘤细胞（CTC）富集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高效和特异性的CTC捕获技术，不仅为CTC计数铺平道路，作为PC的生物标志物，以更好地预测临床结果，而且还作为临床材料的来源（即，CTC作为“液体活组织检查”）用于连续分子分析，其可用于指导个体患者的适当治疗（即，“为正确的病人提供正确的治疗”）。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测定法，该测定法能够识别，计数和捕获从PC患者收集的全血样本中的活的（无抗生素的）CTC。最初，我们开创了将抗EpCAM（上皮细胞粘附分子）涂覆的纳米结构表面作为用于富集CTC的高亲和力基底的概念。通过将高亲和力基底与能够产生混沌湍流的微流体组件整合，由于CTC和基底之间增强的碰撞，最近已经实现了进一步提高的CTC捕获效率（高达99%）。进行并行分析验证以比较我们的纳米结构基底与CellSearchTM测定，使用掺有PC细胞系的血液以及从PC患者中分离的33个预定义阶段的血液样品。CTC是从原发肿瘤或转移部位脱离并在外周血中循环的癌细胞。CTC计数已在转移性去势抵抗性（CR;即激素难治性）PC患者中确立了临床实用性，其中CTC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，CTC的分子和功能表征可以为预测患者预后和监测治疗干预和结果提供非常有价值的信息。在此，我们将首先开发新一代集成CTC芯片，该芯片能够高效和特异性富集CTC，具有改进的血液处理能力，然后使用从PC患者在预定义阶段（例如，CRPC、PSA复发）。然后，我们将使用商业实时qPCR系统纯化和分离单个CTCs以定量16个基因。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-间质转化和AR信号轴的标志物。由这些基因赋予的分子特征不仅描绘了各种细胞表型，而且还提供了预测对抗癌疗法的响应/抗性的希望。

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.