3D-Nanostrcutured Substrates for Detection of Circulating Tumor Cells

描述（由申请人提供）：本申请的长期目标是开发一种用于全血循环肿瘤细胞（CTC）的高灵敏度检测和分子分析的集成技术平台。基于高亲和性纳米柱接枝基底的独特工作机制赋予这种新技术增强的CTC捕获效率/纯度、低操作成本和易用性的优点。 PI的研究小组已经证明，涂覆有抗EpCAM的硅纳米柱（SiNP）覆盖的基底在用于从全血样品中分离活的CTC时表现出出色的效率。通过简单的固定装置设置和操作方案，由于SiNP和细胞表面组分之间增强的地形相互作用，CTC可以固定到SiNP基底上。该CTC捕获技术的临床研究已经启动，以与FDA批准的CellSearchTM检测进行并行验证。同时，已经建立了单个癌细胞的多参数分子谱的定量ICC方法，并且可以直接应用于CTC的分子分析。这些初步结果为我们的研究奠定了坚实的基础。 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。患者血液中CTC的计数和表征为检查早期癌症转移、预测患者预后和监测治疗干预和结果提供了有价值的信息。在过去的十年中，基于不同的工作机制，已经开发了各种能够分离和计数CTC的技术。这些技术中的一些已经在临床环境中得到证明，并且允许在患者血液中重复检测CTC。然而，在提高CTC捕获效率、降低测量成本和对这些细胞进行连续分子分析方面仍然存在挑战。 在本文中，我们提出首先通过以下方式对基于SiNP的CTC捕获技术进行全面优化：（i）探索基于聚合物的纳米柱的使用，（ii）改变纳米柱的尺寸和堆积密度，（iii）实现捕获更广泛多样性的CTC的能力，（iv）结合抗生物污染功能，以及（V）集成微流体混沌混合器。与此同时，我们将进行优化的操作方案的ICC定量的4-蛋白质分子，包括细胞角蛋白（CK），CD 45，雄激素受体（AR）和CD 44，在分离的CTC。接下来，我们将使用最佳的CTC捕获条件来检测来自不同阶段前列腺癌患者的全血样本中的CTC。顺序地，单细胞多参数分子分析（即，CK、CD 45、AR和CD 44）的定量分析，以揭示CTC的分子特性和细胞异质性。 公共卫生相关性：本申请的长期目标是开发用于检测和表征来自癌症患者血液的循环肿瘤细胞（CTC）的新技术平台。这种新的基于CTC的诊断平台具有CTC捕获灵敏度高、操作成本低和用户友好的优点，从而为转移性癌症患者提供了一种有价值的即时工具。

DESCRIPTION (provided by applicant): The long-term objective of this application is to develop an integrated technology platform for highly sensitive detection and molecular analysis of circulating tumor cells (CTCs) from whole blood. The unique working mechanism based on the high affinity nanopillar-grafted substrate confers the advantages of enhanced CTC capture efficiency/purity, low operation cost and ease of use to this new technology. The PI's research group has demonstrated that a silicon nanopillar (SiNP)-covered substrate, coated with anti-EpCAM, exhibits outstanding efficiency when employed to isolate viable CTCs from whole blood samples. With a simple stationary device setting and operation protocol, CTCs can be immobilized onto the SiNP substrates because of enhanced topographic interactions between the SiNPs and cell surface components. The clinical studies of this CTC capture technology have been initiated for side-by-side validation with the FDA-approved CellSearchTM assay. In parallel, a quantitative ICC approach for multiparametric molecular profile of individual cancer cells has been established and can be directly applied for molecular analysis of CTCs. These preliminary results constitute a solid foundation for our proposed research. CTCs are cancer cells that break away from either the primary tumor or metastatic site(s) and circulate in the peripheral blood. Enumeration and characterization of CTCs in patient blood provides valuable information for examining early-stage cancer metastases, predicting patient prognosis and monitoring therapeutic interventions and outcomes. Over the past decade, a variety of technologies capable of isolating and counting CTCs have been developed based on different working mechanisms. Some of these technologies have been demonstrated in the clinical setting and allow reproducible detection of CTCs in the patient blood. However, challenges remain in improving CTC capture efficiency, reducing measurement costs and conducting sequential molecular analysis of these cells. Herein, we propose to first perform a comprehensive optimization of the SiNP-based CTC capture technology by (i) exploring the use of polymer-based nanopillars, (ii) altering the dimension and packing density of nanopillars, (iii) enabling a capability to capture a broader diversity of CTCs, (iv) incorporating anti- biofouling function, and (v) integrating a microfluidic chaotic mixer. In parallel, we will carry out optimization of an operation protocol for ICC quantification of 4-protein molecules, including cytokeratin (CK), CD45, androgen receptor (AR) and CD44, in the isolated CTCs. Next, we will use optimal CTC capture conditions to detect CTCs from whole blood samples obtained from prostate cancer patients at different stages. Sequentially, single-cell multiparametric molecular analysis (i.e., CK, CD45, AR and CD44) of the substrate-immobilized CTCs will be carried out using the quantitative ICC approach to unveil the molecular properties and cellular heterogeneity of the CTCs. PUBLIC HEALTH RELEVANCE: The long term objective of this application is to develop a new technology platform for detection and characterization of circulating tumor cells (CTCs) from cancer patient blood. This new CTC-based diagnostic platform offers the advantages of high CTC capture sensitivity, low operation cost and user-friendliness, thus introducing a valuable point-of-care tool for patients with metastatic cancer.