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

标题：使用流式细胞仪对异质循环肿瘤细胞进行分级和分析 超均匀结构微芯片 项目总结/摘要 循环肿瘤细胞（CTC）是高度异质性的，并且特定的CTC亚群，而不是整体， 是癌症转移的原因目前的CTC技术简单地分离血液样品中的所有CTC 没有将它们分解为不同的亚群，阻止研究人员获得真正的见解， CTC的转移潜力。因此，CTC异质性与肿瘤进展之间的相关性 基本上是未知的。此外，现有的CTC表征方法通常涉及破坏性固定， 透化方案，其限制了随后CTC和其他细胞的表型分析的可能性。 下游应用。该提案的目标是通过以下途径了解CTC的转移潜力： CTC亚群的有效分级和谱分析。我建议隔离，就地鉴定，有选择地 使用具有超均匀结构的微芯片回收CTC。超均匀性（HU）是一个新兴的概念， 一种包含局部异质性或随机性和全局规则性或同质性的布局模式。我 这项工作将首次将超均匀性的概念整合到基于亲和力的微流体装置中， CTC分离。我假设，由于局部流动模式的控制差异引起的， 由于微芯片具有超均匀的结构，因此在微芯片上的不同位置的细胞停滞将需要不同的粘合强度。 此外，预期该粘合强度与粘合剂上的表面标记物的类型和密度有关。 捕获的CTC，因此，它们的转移特性。具体目标包括：（1）设计和表征HU 结构化微芯片用于CTC捕获并分析装置中的流动模式和粘附力;（2）捕获和 使用HU微芯片鉴定具有表面标志物的可变表达的CTC亚群;和（3） 探索将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CTC的分级提供了简单而独特的分辨率，因为其整体均匀性 提供了同等的CTC依从性可能性;并且局部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。结果，CTC的亚群 可以仅使用它们在HU芯片上的捕获位置来识别，而不需要额外的后捕获 免疫荧光表征。最重要的量化里程碑是达到80%的准确率 统计相关性：1）HU芯片上的位置与细胞-后相互作用的强度，2）预测 癌细胞混合物（PC 3和LNCaP）中的位置与细胞类型，以及3）实现80%的识别准确度 从HU微芯片上的位置释放的EMT和非EMT细胞，通过免疫染色验证。如果 成功开发后，这种HU微芯片可以很容易地集成到研究实验室中进行研究 与CTC异质性相关的基本癌症生物学，以及临床环境中分析CTC 在某些实施方案中，可以使用细胞亚群来辅助癌症诊断、预测肿瘤进展和监测治疗功效。

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.