A simple assay system for rapid detection of circulating tumor cells

描述（申请人提供）：原发肿瘤细胞的转移或播散是癌症患者死亡的主要原因。血液中的循环肿瘤细胞（CTCs）是转移性肿瘤形成的关键因素。最近的研究表明，患者血液中存在的ctc数量与癌症的进展、复发和生存率直接相关。CTCs的准确检测将为癌症患者的正确治疗提供关键信息。目前，CellSearch(R)检测是fda批准的唯一检测癌症患者血液中ctc的方法。然而，与所有其他抗体介导的方法类似，该测试需要多个步骤进行样品制备和细胞标记，这可能导致血液样本中ctc的丢失和损伤，并对测试的灵敏度和准确性产生不利影响。此外，多步骤的要求费时费力。在拟议的研究中，我们的目标是克服这些技术障碍，通过整合新的肿瘤细胞活化适体报告系统（TCAR）和创新的旋转圆盘成像技术（iSDI），开发一种简单的即时检测方法。与目前的抗体介导的多步骤测试相比，TCAR系统将能够在几分钟内使用单步反应通过全血中的自然细胞过程选择性地突出ctc。此外，TCAR将不提供背景或脱靶信号，也不需要标记后细胞清洗。iSDI技术将以高速方式检测ctc并记录高分辨率图像，类似于蓝光光盘播放器读取光盘上的比特。与传统的自动荧光显微镜相比，iSDI系统在灵敏度、速度和操作成本上都有优势。我们假设该分析将能够通过单步反应在几分钟内检测到全血样本中ctc的准确数量，这将消除样本中ctc的潜在损失和损害。为了验证我们的假设，提出了三个目标。我们将优化含有EpCAM特异性适配体探针的TCAR系统，以选择性地突出全血中的癌肿瘤细胞（目标1），同时构建并验证iSDI系统，在几分钟内完全不需要手动对细胞进行涂覆、扫描和成像（目标2）。在Aim 3中，我们将整合TCAR和iSDI系统，开发一种快速检测ctc的即时检测方法，并使用正常人类血液样本中的肿瘤细胞验证其性能。我们预测这种简单的分析将在几分钟内一步提供高清晰度的ctc形态及其荧光特性。

DESCRIPTION (provided by applicant): Metastasis or dissemination of primary tumor cells is the major cause of mortality in cancer patients. Circulating tumor cells (CTCs) in the bloodstream are key players in the establishment of metastatic tumors. Recent studies demonstrated that the number of CTCs present in patient blood directly correlate with cancer progression, recurrence, and survival rate. Accurate detection of CTCs will provide critical information for proper management of cancer patients. Currently, the CellSearch(R) test is the only FDA-approved method for detection of CTCs in cancer patient blood. However, similar to all other antibody-mediated methods, this test requires multiple steps for sample preparation and cell labeling, which can cause loss of and damage to CTCs in blood samples and adversely affect the sensitivity and accuracy of the test. In addition, the requirement of multiple steps is time- and labor-consuming. In the proposed study, we aim to overcome these technical obstacles and develop a simple point-of-care assay by integrating a novel Tumor Cell-activation Aptamer Reporting system (TCAR) and innovative Spinning Disc Imaging Technology (iSDI). In contrast to current antibody-mediated multi-step tests, the TCAR system will be able to selectively highlight CTCs through natural cellular processes in whole blood within minutes using a single-step reaction. In addition, TCAR will provide no background or off-target signals and will not require a post-labeling cell wash. The iSDI technology will detect CTCs in a high-speed manner and record high-resolution images, similar to a Blu-ray disc player reading bits on disc. Compared to conventional automated fluorescence microscopes, the iSDI system is superior in sensitivity, speed, and operates at a lower cost. We hypothesize that this assay will be able to detect an accurate number of CTCs within minutes in whole blood samples through a single-step reaction, which will eliminate potential loss of and damage to CTCs in the samples. To test our hypothesis, three aims are proposed. We will optimize the TCAR system containing aptamer probes specific for EpCAM to selectively highlight carcinoma tumor cells in whole blood (Aim 1) while constructing and validating the iSDI system for coating, scanning, and imaging cells completely hands-free in minutes (Aim 2). In Aim 3, we will integrate the TCAR and iSDI systems, develop a point-of-care assay for rapid detection of CTCs, and validate the performance using tumor cells in normal human blood samples. We predict that this simple assay will provide high-definition morphology of CTCs along with their fluorescence identity in one step within minutes.