Label free microfluidic isolation, characterization and ex vivo expansion of CTCs

项目摘要/摘要 到目前为止，胰腺癌，超过大多数其他癌症，一直被明确地与这个术语联系在一起 “致命的”。这种联系源于这样一个事实：胰腺癌的中位存活率不到6 确诊后几个月，惨淡的5年存活率为3-5%。最常见的胰腺癌 癌症，胰腺导管腺癌(PDAC)，表现出特别侵袭性的生物学特征 对常规疗法和靶向疗法都有抵抗力，因此当患者收到诊断结果时， 这种疾病已经发展到无法治愈的地步。此外，在获得 胰腺癌患者的组织，使肿瘤及其药效学研究变得困难 治疗过程中的反应。这些事实突显了识别存活的致命细胞的尚未满足的挑战 即使在接受治疗后也会茁壮成长，而且很容易复发。确定推动经济增长的机制 疾病及其复发可以刺激新的治疗策略的发展，以改善对 这些病人。一条可能导致准确的预测工具、治疗靶点和 药效学生物标志物信息来自对循环肿瘤细胞(CTCs)的分析。已经过去了 二十年来，研究表明，来自原发实体肿瘤的肿瘤细胞可以在循环中检测到。 这些CTC可能是全身转移的先兆。外周血中CTCs的检测已经完成 被认为是诊断癌症和细胞转移的潜在工具。此外，相对数字 血液中CTCs的数量似乎是几种癌症进展的独立预测因素。 然而，在将四氯化碳作为可靠的生物标记物之前，必须回答以下基本问题 它们的生物学和临床意义。是否所有的CTC都具有增殖、侵袭和转移的能力？是 有没有CTC亚群比其他亚群更具攻击性？如果是这样的话，这些激进的CTC是否携带 特殊的司机特征以及它与原发肿瘤有何不同或相似之处？这些细胞是否能在 治疗并有能力扩散吗？这些问题的答案可以揭示出最早发生转移的细胞-- 启动能力，提供治疗靶点。因此，建立CTC的临床应用 胰腺癌和其他癌症的个性化治疗，迫切需要灵敏、准确的治疗方法 区分启动转移的驾驶员CTC和本质上为“乘客”的CTC的方法。我们会 通过复杂的生物标记物独立微流控平台实现这一目标 不仅能够实现高度敏感的CTCs分离，而且还提供了培养和扩展 隔离的CTC数量较少。大多数CTC分离技术依赖于已知的表面标记 并遭受着低吞吐量的困扰。此外，存在多个CTC亚群(包括那些 对于具有不同转移潜能的EMT表型)需要一种将这些细胞从 不依赖于特定抗体(如EpCAM)的血液。在这份提案中，我们将开发、测试和 优化一种新的抗原非依赖性微流控平台(迷宫)在分离中的效果， 胰腺癌CTCs的特性、体外扩增及治疗靶点检测。迷宫之旅 利用曲线几何中的惯性力根据细胞的大小对细胞进行差异化聚焦以分离CTC 在没有任何生物标志物的帮助下，以2.5ml/分钟(150ml/小时)的高通量一步从全血中提取。 多圈(超过50圈)确保即使是较小的细胞在穿过 因此，WBC和CTCs之间实现了显著的分离，从而获得了高纯度。这个 独特的能力，以无标记的方式以高通量、更高的灵敏度和可靠性分离CTCs 扩展几个CTC为生物、基因组和功能分析打开了令人兴奋的新机会 我们预计这将改变CTCs在临床肿瘤学中的使用范式。

Project Summary/Abstract To date, pancreatic cancer, beyond most other cancers has been categorically associated with the term “lethal”. This association stems from the fact that pancreatic cancer has a median survival rate of less than 6 months after diagnosis, and a bleak 5-year survival rate of 3-5 percent. The most prevalent form of pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), demonstrates a particularly aggressive biology with resistance to both conventional and targeted therapeutics, so that by the time a patient receives the diagnosis, the disease has already advanced to an incurable state. Furthermore, significant challenges exist in obtaining tissue from pancreatic cancer patients, making it difficult to study tumors and their pharmacodynamic responses during treatment. These facts highlight the unmet challenge of identifying the lethal cells that survive and thrive even after treatment and are predisposed to recur. Ascertaining the mechanisms that drive the disease and its recurrence can spur the development of new treatment strategies to improve outcomes for these patients. One avenue that could lead to accurate predictive tools, therapeutic targets, and pharmacodynamic biomarker information comes from the analysis of circulating tumor cells (CTCs). For over two decades, studies have shown that tumor cells from primary solid tumors can be detected in the circulation. These CTCs may be precursors to systemic metastases. The detection of CTCs in peripheral blood has been recognized as a potential tool in the diagnosis of cancer and cell metastasis. Furthermore, the relative number of CTCs in the blood appears to be an independent predictor of progression in several types of cancer. However, before instituting CTCs as a reliable biomarker, one must answer fundamental questions regarding their biological and clinical significance. Are all CTCs capable of proliferation, invasion, and metastasis? Are there subpopulations of CTCs that are more aggressive than the rest? If so, do these aggressive CTCs carry specific driver signature and how it is different or similar to primary tumor? Are these cells persistent through therapy and capable of proliferation? Answers to these questions can reveal the earliest cells with metastasis- initiating capability, providing a therapeutic target. Hence, to establish clinical applications of CTCs for personalized therapy in pancreatic and other cancers, there is a compelling need for sensitive, accurate approaches to distinguish metastasis-initiating driver CTCs from essentially “passenger” CTCs. We will accomplish this goal through a sophisticated biomarker independent microfluidic platform “labyrinth” that not only enables highly sensitive isolation of CTCs but also provides the novel capability to culture and expand the low numbers of isolated CTCs. Most of the CTC isolation technologies depend on the known surface markers and suffer from low throughput. Moreover, the presence of multiple sub-populations of CTCs (including those of an EMT phenotype) with different metastatic potential requires a method for separating these cells from blood that does not depend on specific antibodies such as EpCAM. In this proposal, we will develop, test and optimize the efficacy of a novel antigen-independent microfluidic platform (the labyrinth) in the isolation, characterization, ex vivo expansion and therapeutic targets testing of pancreatic cancer CTCs. Labyrinth takes advantage of inertial forces in curved geometries to differentially focus cells based on their size to isolate CTCs from whole blood at a high throughput of 2.5mL/min (150mL/hr) in a single step without any aid of a biomarker. The multiple turns (more than 50) ensure focusing of even the smaller cells while traversing through the device, and hence achieve a remarkable separation between WBCs and CTCs resulting in high purity. The unique ability to isolate CTCs in a label free manner at a high throughput, with greater sensitivity and reliably expand few numbers of CTCs opens exciting, new opportunities for biologic, genomic, and functional analyses of CTCs, which we expect will change the paradigm for use of CTCs in clinical oncology.