Fractionation and Profiling of Heterogeneous Circulating Tumor Cells Using a Hyperuniform- Structured Microchip

标题：异质循环肿瘤细胞的分离和图谱 超均匀结构微芯片 项目摘要/摘要 循环肿瘤细胞(CTC)是高度异质性的，并且是特定的CTC亚群，而不是整个CTC， 对癌症转移负有责任。目前的CTC技术只是简单地分离血液样本中的所有CTC 如果不将它们分解成不同的亚群，阻止研究人员获得对 CTCs的转移潜能。因此，CTC异质性与肿瘤进展的相关性 在很大程度上是未知的。此外，现有的CTC表征方法通常涉及破坏性固定和 渗透方案，这限制了随后进行CTCs和其他表型分析的可能性 下游应用程序。这项建议的目标是通过以下途径了解CTC的转移潜能 四氯化碳亚群的有效分离和分析。我建议隔离，原位鉴定，并有选择地 利用超均匀结构的微芯片回收CTCs。超一致性(HU)是一个新兴的概念 包含局部异质性或随机性和全局规律性或同质性的布局模式。我的 Work将首次将超均匀度的概念集成到基于亲和力的微流控设备中，以实现 CTC隔离。我假设由于局部流动模式的受控差异 超均匀的结构，细胞停滞在微芯片上的不同位置，将需要不同的粘接强度。 此外，这种粘接强度预计与表面标记的类型和密度有关 捕获了CTC，因此，它们的转移特征。具体目标包括(1)设计和表征HU 用于CTC捕获和分析装置内的流态和粘附力的结构化微芯片；(2)捕获和 使用HU芯片鉴定表面标记表达可变的CTC亚群；以及(3) 探索可能将HU微芯片平台纳入临床肿瘤学环境的几个关键因素。 HU微芯片为CTCs的分离提供了一种简单而独特的解决方案，因为它的全球同质性 提供了遵守CTC的同等可能性；并且本地异质性允许同时区分 通过分析单个CTC所需的粘合强度来确定亚群。因此，CTC的亚群 可以仅使用它们在HU芯片上的捕获位置来识别，而不需要额外的捕获后 免疫荧光鉴定。最重要的量化里程碑是达到80%的准确率 关于统计相关性：1)HU芯片上的位置与细胞-岗位相互作用的强度，2)预测 癌细胞混合物(PC3和LNCaP)中的位置与细胞类型，以及3)在识别上达到80%的准确率 在HU微芯片上显示释放的EMT和非EMT细胞的位置，通过免疫染色进行验证。如果 开发成功的HU微芯片可以很容易地集成到研究实验室进行研究 与CTC异质性相关的基础癌症生物学，以及进入临床环境以分析CTC 帮助癌症诊断、预测肿瘤进展和监测治疗效果的亚群。

Title: Fractionation and Profiling of Heterogeneous Circulating Tumor Cells Using a Hyperuniform-structured Microchip Project Summary/Abstract Circulating tumor cells (CTCs) are highly heterogeneous, and specific CTC subpopulations, rather than the whole, are responsible for cancer metastasis. Current CTC technologies simply isolate all CTCs in a blood sample without resolving them into distinct subpopulations, preventing researchers from acquiring true insights into the metastatic potential of CTCs. As a consequence, correlation between CTC heterogeneity and tumor progression is largely unknown. In addition, existing CTC characterization methods often involve destructive fixation and permeabilization protocols, which limit the potential for subsequent phenotypic analysis of CTCs and other downstream applications. The goal of this proposal is to understand the metastatic potential of CTCs through effective fractionation and profiling of CTC subpopulations. I propose to isolate, in-situ identify, and selectively recover CTCs using a microchip with hyperuniform structure. Hyperuniformity (HU) is an emerging concept of a packing pattern which contains local heterogeneity or randomness and global regularity or homogeneity. My work, for the first time, will integrate the concept of hyperuniformity into affinity-based microfluidic devices for CTC isolation. I hypothesize that due to the controlled differences in local flow patterns induced by the hyperuniform structure, cell arrest in different locations on the microchip will require different adhesive strengths. Further, this adhesive strength is anticipated to be related to the types and densities of surface markers on the captured CTCs and therefore, their metastatic character. Specific aims include (1) Design and characterize HU structured microchip for CTC capture and analyze flow pattern and adhesion force in the device; (2) Capture and identify subpopulations of CTCs with variable expression of the surface marker using a HU microchip; and (3) Explore several key factors for potential incorporation of the HU microchip platform into clinical oncology settings. The HU microchip offers a simple and unique resolution for fractionation of CTCs, as its global homogeneity provides equal possibility of CTC adherence; and local heterogeneity allows simultaneous differentiation of subpopulations by analyzing adhesive strength required for individual CTCs. As a result, subpopulations of CTCs can be identified using only their capture locations on the HU chip without requiring additional post-capture immunofluorescence characterization. The most significant quantitative milestones are to achieve 80% accuracy on statistical correlation on: 1) the locations on a HU chip with strength of cell-post interaction, 2) predictions of location vs. cell type in a cancer cell mixture (PC3 and LNCaP), and 3) achieving 80% accuracy on identification of released EMT and non-EMT cells from their locations on a HU microchip, validated by immunostaining. If successfully developed, this HU microchip can be easily integrated into research laboratories to study fundamental cancer biology related to CTC heterogeneity, as well as into clinical settings to profile CTC subpopulations to assist cancer diagnosis, predict tumor progression, and monitor therapeutic efficacy.