Advanced Development of An Integrated CTC Enrichment Technology

本研究计划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发展我们高效和特异性的CTC捕获技术，不仅为CTC枚举作为PC的生物标志物更好地预测临床结果铺平道路，而且为连续分子分析提供临床材料来源（即，作为“液体活检”的CTC），可用于指导针对个体患者的适当治疗（即“正确的治疗针对正确的患者”）。我们的联合团队已经展示了一种独特的、相对便宜的细胞亲和力测定方法，能够在PC患者的全血样本中鉴定、枚举和捕获活的（不含防腐剂的）ctc。最初，我们首创了应用抗epcam（上皮细胞粘附分子）涂层的纳米结构表面作为富集ctc的高亲和力底物的概念。通过将高亲和性衬底与能够产生混沌湍流的微流控组件集成，最近由于CTC与衬底之间的碰撞增强，进一步提高了CTC捕获效率（高达99%）。将我们的纳米结构底物与CellSearchTM分析进行了并排分析验证，使用添加了PC细胞系的血液以及从预定阶段的PC患者分离的33份血液样本进行了比较。ctc是一种从原发肿瘤或转移部位脱落并在外周血中循环的癌细胞。CTCs的计数在转移性去势抵抗（CR，即激素难治性）PC患者中已经建立了临床应用，在这些患者中，CTCs是对化疗反应、无病生存和总生存的独立预测因子。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许多有价值的信息。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地富集CTC，并提高血液处理能力，然后使用从预定阶段（例如CRPC， PSA复发）的PC患者收集的血液样本进行全面的分析验证。然后，我们将纯化和分离单个ctc，使用商用实时qPCR系统对16个基因进行定量。我们打算量化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信号轴的标记。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治疗的反应/耐药性的希望。

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.