High quality CTC isolation using microbubbles for downstream molecular analysis

描述(由申请人提供)：早期癌症检测和干预对原发或复发肿瘤的长期生存至关重要。循环肿瘤细胞(CTCs)的分离可用于癌症的诊断、预后和治疗计划。CTC的遗传图谱和表达分析可以为准确的诊断和预后提供额外的宝贵信息。此外，CTC的体外培养可用于肿瘤细胞的功能分析，如药物敏感试验。为了实现这一目标，一项变革性的技术必须能够使用具有成本效益的非侵入性程序准确地分离出几个癌细胞。在之前的R21IMAT奖下，我们开发了一种罕见肿瘤细胞分离的新技术。脂壳-涂有抗EpCAM抗体的全氟化碳浮力微泡(MBS)立即附着在未分离血液中的肿瘤细胞上，并在快速离心步骤后分离细胞(Shiet al.，PLoS One，2013)。此应用程序中包含演示该技术的视频短片。总之，MB技术与现有的免疫浓缩技术相比具有独特的优势：(A)处理时间短，可避免RNA和蛋白质的降解；(B)可伸缩性(从大容量样本中分离细胞)可以帮助获得大量的CTCs；(C)特异性(最小限度地携带白细胞)可以提高分子分析的灵敏度和特异性；(D)灵活性(将细胞集中在5-10%的浓度范围内)L收集的细胞，可用于细胞生长、免疫染色和/或分子分析。这项建议的R33阶段的重点是进一步开发MB分离方法，以实现从转移性脑癌中高纯度地分离CTCs，用于计数和分子及功能分析。我们设定了以下具体研究目标：(1)优化MB方法，实现高速、高纯度采集7-20ml血样中添加的肿瘤细胞；(2)应用MB分离的CTCs进行PCR分析和培养；(3)以CellSearch系统为基准，验证MB方法对脑转移瘤患者血液CTC计数的敏感性和特异性。简单但强大的CTC分离技术将为下游分析提供高质量的CTC样本，这将对恶性肿瘤的临床管理和无症状癌症的早期诊断产生革命性影响。

DESCRIPTION (provided by applicant): 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 ae 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.