High quality CTC isolation using microbubbles for downstream molecular analysis

摘要 早期癌症检测和干预对其中任何一种患者的长期生存至关重要 原发或复发肿瘤。循环肿瘤细胞(CTCs)可以被分离出来用于 癌症的诊断、预后和治疗计划。遗传图谱和表达 CTC的分析可以为准确的诊断提供额外的宝贵信息 和预后。此外，CTC的体外培养可用于细胞的功能分析。 癌细胞药敏试验等。为了实现这一目标，一个变革性的 技术必须能够以经济高效的方式准确地分离出几个癌细胞 和非侵入性手术。 在之前的R21 IMAT奖下，我们为REARE开发了一种新技术 肿瘤细胞分离。脂壳-全氟碳浮力微泡(MBS)包覆 抗EpCAM抗体立即附着在未分离血液中的肿瘤细胞上，以及 在快速离心步骤后分离细胞(Ship等人，PLoS One，2013)。一小段 演示此技术的视频剪辑包含在此应用程序中。总之， MB技术与现有的免疫浓缩技术相比具有独特的优势 技术：(A)处理时间短可避免RNA和蛋白质降解；(B) 可伸缩性(从大容量样本中分离细胞)可以帮助获得大量 CTCs；(C)特异性(最低限度的白细胞残留)可提高敏感性和 分子分析的特异性；(D)灵活性(细胞以非常集中的方式收集 体积为5-10？L，可用于细胞生长、免疫染色和/或分子 分析。 该提案的R33阶段的重点是进一步开发甲基溴隔离 获得高纯度脑转移癌CTCs的方法 列举以及分子和功能分析。我们设定了以下具体目标 研究内容：(1)优化MB采收方法，实现高速、高纯度采集 肿瘤细胞在7-20毫升血液样本中激增；(2)MB分离的CTCs应用于 (3)验证MB法的敏感性和特异性。 CellSearch系统对脑转移瘤患者外周血CTC计数的研究 作为基准。 简单但强大的CTC隔离技术，可实现高质量的CTC 下游分析样本将对临床管理产生革命性影响 恶性肿瘤和无症状癌症的早期诊断。

ABSTRACT Early cancer detection and intervention are crucial for long-term survival of either primary or recurrent tumors. Circulating tumor cells (CTCs) could be isolated for the diagnosis, prognosis, and treatment planning of cancer. Genetic profiling and expression analysis of CTC could provide additional invaluable information for accurate diagnosis and prognosis. Moreover, in vitro culture of CTC can be applied for functional analysis of cancer cells, such as drug sensitivity test. To achieve this goal, a transformative technology must be able to unmistakably isolate a few cancer cells using cost-effective and non-invasive procedures. Under the previous R21 IMAT award, we developed a novel technology for rare tumor cell isolation. Lipid shell-perfluorocarbon buoyant microbubbles (MBs) coated with anti-EpCAM antibody immediately attached to tumor cells in unfractionated blood, and isolated the cells after a quick centrifugation step (Shi et al., PLoS One, 2013). A short video clip to demonstrate this technology is included in this application. In conclusion, MB technique offers unique advantages over the existing immuno-enrichment technologies: (a) Short processing time can avoid RNA and protein degradation; (b) Scalability (isolation of cells from large volume sample) can help obtain large numbers of CTCs; (c) Specificity (minimal carryover of leukocytes) can improve sensitivity and specificity of molecular analysis; (d) Flexibility (cells are collected in a very concentrated volume of 5-10 ¿l and could be used for cell growth, immunostaining and/or molecular analysis. The R33 phase of this proposal is focused to further develop MB isolation method to achieve high purity isolation of CTCs from metastatic brain cancers for enumeration and molecular and functional analyses. We set the following specific aims of the study: (1) Optimize MB method to achieve high speed, high purity harvesting of tumor cells spiked in 7-20ml blood sample; (2) Applications of MB isolated CTCs for PCR analysis and culturing; (3) Validate the MB method for sensitivity and specificity of CTC enumeration from blood of metastatic brain tumor patients using CellSearch system as a benchmark. Simple but robust CTC isolation technologies that result in high quality CTC sample for downstream analysis will have transformative impact on clinical management of malignancies and early diagnostics of asymptomatic cancers.