Microfluidic Apheresis to Isolate Circulating Tumor Clusters

摘要 循环肿瘤细胞簇（CTC-簇），在20世纪70年代也称为肿瘤栓塞，已知在肿瘤的发生发展中起主要作用。 转移过程例如，已经推断CTC簇的转移性是一个CTC的50倍。它们也不同 在许多方面来自单个CTC，包括具有不同的表观遗传标记。部分原因是它们的细胞 尽管它们与CTCs连接，但证据还表明它们避免失巢凋亡，因此与单个CTCs不同，它们较少凋亡。然而，在这方面， 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 mL血液，这意味着大多数10-20 mL的样本不含CTC簇。将CTC簇与较大的 因此，需要血容量来解锁与这种转移性最强的人群相关的生物标志物的独特储库。 肿瘤细胞虽然已经有许多方法来分离CTC簇，主要是使用微流体，但它们不能分离CTC簇。 主要使用最多10-20 mL的少量血液，因此不能可靠地分选CTC簇以影响 临床决策。为了克服现有方法的缺点，我们建议开发核心技术 使得微流控CTC-簇单采（CTApheresis）系统能够询问20-100%（即，1-5（二）全体 在大多数患者中，在一小时内分离足够的CTC簇。我们将实现我们的目标， CTApheresis通过建立创新的微流体技术，从未稀释的CTC中温和地选择性地分选CTC簇 高通量系统中的血液。在这里，我们将创建用于可扩展CT分离的“功能模块单元”。我们将 将项目分为两个不同的综合目标。在目标1中，我们将创建一个微流控芯片， 在大量的全血中。在aim 2中，我们将把这个排序器集成到一个功能模块化的单元排序器中进行处理 1小时内120 mL全血（通过平行化可放大9倍至1 L/h）。作为一个全面的 在目标2完成后，我们将进行一系列测试，比较处理过的和未处理过的血液， 如果使用所提出的微流体装置处理的CT单采血液成分术血液将安全地返回到患者体内。温柔 从几升全血中分选CTC簇有可能解开转移性肿瘤的一些奥秘， 这不仅是一种新的方法，而且还能够使CTC的离体培养用于癌症患者的“真实的”功能性药物测试。常规 CTC簇基因组、转录组和蛋白质组的取样可能成为侵入性外科手术的替代品。 活组织检查，并最终提供了一个强大的方法，早期发现癌症的高危患者。

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.