Microfluidic Apheresis to Isolate Circulating Tumor Clusters

摘要 循环肿瘤细胞团(CTC-Clusters)在20世纪70年代也被称为肿瘤栓子，已知在 转移过程。例如，据推测，CTC-簇的转移性是一个CTC的50倍。它们也是不同的 在许多方面来自单一的CTC，包括具有不同的表观遗传标记。部分是由于它们完好无损的细胞--细胞 除了连接，证据还表明它们避免了失巢凋亡，因此与单一的CTC不同，它们的凋亡率较低。然而， CTC簇甚至比单个CTC更罕见，在许多情况下，平均只有一个与CTC簇相关的肿瘤细胞 10毫升的血液，这意味着大多数10-20毫升的样本不包含CTC-簇。将CTC集群与更大的集群隔离 因此，需要血液容量来解锁与这种最转移的人群相关的独特的生物标记物储存库。 肿瘤细胞。虽然已经有许多方法来分离CTC-簇，主要使用微流控，但它们 主要使用少量血液，最高可达10-20毫升，因此不能可靠地对CTC簇进行分类以影响 临床决策。为了克服当前方法的缺点，我们建议开发核心技术 启用微流控四氯化碳集群分离(CTApheresis)系统，可以询问整个样品的20%-100%(即1-5%L 在一小时内分离出足够多的CTC簇。我们将达到我们的目标 CTApheresis通过构建一种创新的微流控技术来温和和选择性地从未稀释的CTC簇中分选CTC 高通量系统中的血液。在这里，我们将为可伸缩的CTApheresis创建“功能模块单元”。我们会 将项目分成两个截然不同的、综合的目标。在目标1中，我们将创建一个微流控芯片来浓缩CTC-簇 在大量的全血中。在目标2中，我们将把这个分类器集成到一个功能模块化的单元分类器中进行处理 120毫升全血在1小时内(可通过并行化扩展9倍至1 L/小时)。在整个过程中，作为一个全面的 安全性研究在Aim 2完成后，我们将进行一系列测试，比较经过处理和未处理的血液，以检查 如果CTApheresis，使用所建议的微流控装置处理的血液将安全地返回到患者体内。温和的 从升全血中分离CTC簇有可能解开转移的一些谜团 这一技术不仅可用于体外培养CTCs，还可用于癌症患者的“实时”功能药物检测。例程 CTC簇基因组、转录组和蛋白质组的采样可能成为侵入性外科手术的替代品 活组织检查，最终为高危患者的癌症早期检测提供了一种强有力的方法。

ABSTRACT Circulating tumor cells clusters (CTC-clusters), also called tumor emboli in 1970s, are known to play a major role in the metastatic process. For example, CTC-clusters have been inferred to be 50-fold as metastatic as one CTC. They also differ in many ways from single CTCs, including having distinct epigenetic markers. Due in part to their intact cell-cell junctions, the evidence also suggests they avoid anoikis, hence unlike single CTCs they are less apoptotic. However, CTC-clusters are even rarer than single CTCs, with in many cases an average of just one cluster-associated tumor cell in 10 mL of blood, meaning most samples of 10-20 mL do not contain a CTC-cluster. Isolating CTC-clusters from larger blood volumes is thus required to unlock the unique reservoir of biomarkers linked to this most metastatic population of tumor cells. Although there has been a number of approaches to isolate CTC-clusters, mostly using microfluidics, they primarily use a small amount of blood up to 10-20 mL and hence are not able to sort CTC-clusters reliably to impact clinical-decision making. To overcome the shortcomings of current approaches, we propose to develop core technology enabling a microfluidic CTC-cluster apheresis (CTApheresis) system that can interrogate 20-100% (i.e., 1-5 L) of the entire blood volume within an hour to isolate enough CTC-clusters in a majority of patients. We will reach our goal of CTApheresis by building an innovative microfluidic technology to gently and selectively sort CTC-clusters from undiluted blood in a high-throughput system. Here, we will create the “functional modular unit” for scalable CTApheresis. We will split the project in two distinct, integrated, aims. In aim 1, we will create a microfluidic chip to concentrate CTC-clusters within large volumes of whole blood. In aim 2, we will integrate this sorter into a functional modular unit sorter to process 120 mL of whole blood in 1 hour (scalable by nine-fold by parallelization to 1 L/h). Throughout and as a comprehensive safety study after aim 2 completes, we will run a battery of tests comparing processed and unprocessed blood, to examine if CTApheresis blood processed using the proposed microfluidic device will be safe to return back to the patient. Gentle sorting of CTC-clusters from liters of whole blood has the potential to unlock some of the mysteries of the metastatic process but also to enable ex-vivo culture of CTCs for “real time” functional drug testing for cancer patients. Routine sampling of the CTC-cluster genome, transcriptome and proteome could become a replacement for invasive surgical biopsies, and ultimately provide a powerful approach for early detection of cancer in high-risk patients.