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中文摘要
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摘要 早期癌症检测和干预对于长期生存至关重要。 原发性或复发性肿瘤循环肿瘤细胞(CTC)可以被分离用于肿瘤细胞。 癌症的诊断、预后和治疗计划。基因图谱和表达 CTC分析可为准确诊断提供额外的宝贵信息 和预后。此外,CTC的体外培养可用于功能分析, 癌细胞,如药物敏感性试验。为了实现这一目标, 技术必须能够使用具有成本效益的方法, 和非侵入性手术。 在之前的R21 IMAT奖下,我们开发了一种新技术, 肿瘤细胞分离。脂质壳-全氟化碳浮力微泡(MB), 抗EpCAM抗体立即附着于未分级血液中的肿瘤细胞,和 在快速离心步骤后分离细胞(Shi等,PLoS One,2013)。短 本申请书附有一段短片以示范这项技术。最后,我谨指出, MB技术与现有的免疫富集技术相比具有独特的优势 技术:(a)短处理时间可避免RNA和蛋白质降解;(B) 可扩展性(从大体积样品中分离细胞)可以帮助获得大量的细胞。 (c)特异性(白细胞的最小残留)可以提高灵敏度, (d)灵活性(细胞以非常浓缩的方式收集) 体积为5-10升,可用于细胞生长、免疫染色和/或分子标记。 分析. 本提案的R33阶段重点是进一步开发MB隔离 实现从转移性脑癌中高纯度分离CTC的方法 计数以及分子和功能分析。我们设定了以下具体目标 (1)优化MB方法,实现了MB的高速、高纯度收获, 在7- 20 ml血液样品中掺入肿瘤细胞;(2)MB分离的CTC用于 PCR分析和培养;(3)检测MB法的敏感性和特异性, 用CellSearch系统对脑转移瘤患者血液中的CTC计数 作为基准。 简单但强大的CTC分离技术,可产生高质量的CTC 用于下游分析的样本将对临床管理产生变革性影响 以及无症状癌症的早期诊断。
英文摘要
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.
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High quality CTC isolation using microbubbles for downstream molecular analysis
High quality CTC isolation using microbubbles for downstream molecular analysis
Non-invasive sampling of DNA markers for pancreatic cancer screening
Polyomavirus- Associated Human Cancers
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