Rapid unbiased isolation and in situ RNA analysis of circulating tumor cells using a magnetic micropore-based diagnostic chip

摘要： 胰腺癌是美国癌症相关死亡的第四大常见原因。 五年存活率只有4%。我们最近已经证明了循环 在两种疾病周期开始时，都可以在血液中检测到胰腺细胞(CPC) 老鼠和人类。这些发现为开发非侵入性诊断提供了机会。 对于胰腺癌，这有巨大的潜力来改善患者的预后。海流 循环肿瘤细胞(CTC)检测的金标准未能测量到极稀疏的 和异质CPC。即使是下一代微流控技术，它也不依赖于 上皮标记物，由于微流控技术本身的低通量和 检测超罕见细胞所需的大样本量血液(V&GT；10毫升)。至 为了应对这些挑战，我们正在开发一种新的稀有细胞检测方法，使用 磁性微孔，结合了微尺度分选的优点和极快的速度 流速(比典型的微流控方法2、5-7快100倍)。除了改进了它的 吞吐量，我们的技术对未经处理的临床样本具有很强的抵抗力，允许罕见的CPC 和直接从全血中分离的CPC簇。此外，我们正在整合这一点 采用超快速芯片上RNA荧光原位杂交(RNA FISH)的方法，使 单个稀有细胞中的单分子、多重RNA分析。我们的设备，它结合了 将上述功能转化为一种自成一体的、自动化的格式，旨在提取RNA 单个CPC在临床环境中的表达。

ABSTRACT: Pancreatic cancer is the fourth most common cause of cancer related death in the United States, with a five year survival rate of only 4%. We have recently shown that circulating pancreatic cells (CPCs) can be detected in the blood at the onset of the disease cycle in both mice and humans. These findings present an opportunity to develop a non-invasive diagnostic for pancreatic cancer that has enormous potential to improve patient outcomes. The current gold-standard for circulating tumor cell (CTC) detection fails to measure the extremely sparse and heterogeneous CPCs. Even next generation microfluidic technologies, which do not rely on epithelial markers, have limited utility due to the inherently low-throughput of microfluidics and the large sample volumes of blood (V > 10 mL) necessary for ultra-rare cell detection. To address these challenges, we are developing a new approach to rare cell detection, using magnetic micropores, which combines the benefits of micro-scale sorting with extremely fast flow rates (100x faster than typical microfluidic approaches2,5-7). In addition to its improved throughput, our technique is robust against unprocessed clinical samples, allowing rare CPCs and CPC clusters to be isolated directly from whole blood. Moreover, we are integrating this approach with ultra-rapid on-chip RNA fluorescence in-situ hybridization (RNA FISH), enabling single molecule, multiplexed RNA analysis in individual rare cells. Our device, which combines the above features into a self-contained, automated format, is designed to extract the RNA expression of single CPCs in clinical settings.