Microfluidic Devices for Molecular Characterization of Cancer Cells at the Single

描述（由申请人提供）：本提案的目标是开发用于生物医学实验室中有效单细胞分析的微流体装置，而无需专业知识。缺乏在单细胞水平上进行生物化学和分子表征的能力是癌症研究和诊断的关键障碍。我们已经开发了用于有效的大规模单细胞分析的微流控设备，以克服这一障碍。该技术在癌症研究和诊断中具有广泛的应用，用于稀有细胞的鉴定、计数和表征，包括1）隐匿性转移中的循环肿瘤细胞（CTC）; 2）T调节细胞; 3）肿瘤特异性T细胞毒性细胞; 4）癌症干细胞; 5）通过激光捕获显微切割（LCM）捕获的恶性细胞;以及6）针吸活检中的可疑恶性细胞。 已经开发了分离方法来从血液和其他组织中捕获罕见的癌细胞用于癌症诊断。然而，仍然缺乏稳健的方法来在单细胞水平上对这些捕获的细胞进行生物化学和分子表征。在这里，我们建议开发一种廉价的集成微流体装置，可以在3小时内同时分析100个单细胞。所提出的装置可以在一般实验室中制造和使用，而不需要专门的专业知识，并且具有比当前批量测定高5倍的mRNA到cDNA效率。使用我们的微流控设备，现在可以在单细胞水平上进行多种生化分析，目标细胞数量有限，例如上面列出的罕见癌细胞。我们建议优化我们的设备，用于我们的膜过滤器捕获的CTC的单细胞生物化学表征。这些捕获的CTC的表征可能导致分子治疗靶点的鉴定。在使用该R21提案开发和优化设备后，将提交R33提案，以应用该设备在单细胞水平上对从癌症患者血液中捕获的CTC进行分子表征。

DESCRIPTION (provided by applicant): The goal of this proposal is to develop microfluidic devices for effective single-cell analysis in biomedical laboratories without the requirement of specialized expertise. The lack of ability to perform biochemical and molecular characterization at the single-cell level is a critical obstacle in cancer research and diagnosis. We have developed microfluidic devices for efficient large-scale single-cell analysis to overcome this obstacle. The technology has broad applications in cancer research and diagnosis for identification, enumeration and characterization of the rare cells including 1) circulating tumor cells (CTC) in occult metastases; 2)T-regulatory cells, 3) tumor-specific T-cytotoxic cells; 4) cancer stem cells; 5) malignant cells captured by laser capture microdissection (LCM); and 6) suspicious malignant cells in needle biopsy. Isolation methods have been developed to capture rare cancer cells from blood and other tissues for cancer diagnosis. However, there is still a lack of robust methods to perform biochemical and molecular characterization on these captured cells at the single-cell levels. Here, we propose to develop an inexpensive and integrated microfluidic device that can simultaneously profile 100 single-cells within 3 hours. The proposed device can be fabricated and used in general laboratories without the need of specialized expertise, and has a 5 fold higher mRNA-to-cDNA efficiency than current bulk assays. With our microfluidic devices, multiple biochemical analyses can now be performed at the single-cell levels with a limited number of target cells such as the rare cancer cells listed above. We propose to optimize our devices for single-cell biochemical characterization of CTC captured by our membrane filters. Characterization of these captured CTC is likely to lead to the identification of molecular therapeutic targets. After developing and optimizing the device with this R21 proposal, a R33 proposal will be submitted to apply this device for molecular characterization of CTC captured from bloods of cancer patients at the single-cell level.