3D-Nanostrcutured Substrates for Detection of Circulating Tumor Cells

描述(申请人提供)：这项申请的长期目标是开发一个高灵敏的全血循环肿瘤细胞(CTCs)检测和分子分析的集成技术平台。基于高亲和力纳米接枝底物的独特工作机制使这项新技术具有提高CTC捕获效率/纯度、操作成本低和易于使用的优点。PI的研究小组已经证明，覆盖有硅纳米管(SiNP)的底物，涂有抗EpCAM，当用于从全血样本中分离活的CTCs时，显示出非凡的效率。通过简单的固定装置设置和操作规程，可以将CTCs固定在SiNP底物上，因为SiNP与细胞表面组分之间的地形相互作用增强。这种CTC捕获技术的临床研究已经开始，用于FDA批准的CellSearchTM试验的并列验证。同时，建立了单个癌细胞多参数分子图谱的定量ICC方法，并可直接应用于CTCs的分子分析。这些初步结果为我们提出的研究奠定了坚实的基础。CTC是从原发肿瘤或转移部位(S)分离出来并在外周血液中循环的癌细胞。患者血液中CTCs的计数和特征为检查早期癌症转移、预测患者预后和监测治疗措施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开发了各种能够分离和计数四氯化碳的技术。其中一些技术已在临床环境中得到证明，并允许重复检测患者血液中的CTCs。然而，在提高CTC捕获效率、降低测量成本和对这些细胞进行顺序分子分析方面仍然存在挑战。在此，我们建议首先对基于SiNP的CTC捕获技术进行全面优化，方法包括：(I)探索聚合物纳米管柱的使用；(Ii)改变纳米管柱的尺寸和堆积密度；(Iii)实现捕获更广泛多样性CTCs的能力；(Iv)整合抗生物污染功能；以及(V)集成微流控混沌混合器。同时，我们将对分离的CTCs中包括细胞角蛋白(CK)、CD45、雄激素受体(AR)和CD44在内的4种蛋白质分子进行ICC定量的操作规程的优化。接下来，我们将使用最佳的CTC捕获条件来检测不同阶段前列腺癌患者的全血样本中的CTC。随后，将使用定量ICC方法对底物固定的CTCs进行单细胞多参数分子分析(即CK、CD45、AR和CD44)，以揭示CTCs的分子特性和细胞异质性。 公共卫生相关性：这项应用的长期目标是开发一种新的技术平台，用于检测和表征癌症患者血液中的循环肿瘤细胞(CTCs)。这一新的基于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，因此为转移性癌症患者引入了一种宝贵的护理点工具。

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.