Parallel Selective Capture of Single Circulating Melanoma Cells and Integrated On-Chip Determination of Mutational Status

项目摘要 少数细胞的意外反应可能对肿瘤的发展、预后和预后产生巨大影响。 疾病的治疗。例如，在癌症的发展过程中，循环肿瘤细胞的耐药少数 在某些情况下，CTC导致复发时难治性转移的发展。我们才刚刚开始发现这些 这些现象和潜在的生物学机制，因为它们在大规模上是模糊的。检测 一个罕见的突变可能需要分析数百到数千个单个细胞。我们已经开发出一种 介电电泳（DEP）为基础的设备，具有高产量的选择性单细胞捕获的阵列， 微室沿分支微流体通道的侧面沿着布置。重要的一点是， 选择是不依赖于标记的，这一点至关重要，使得这种方法特别适用于鉴定 黑色素瘤中的CTC，没有可靠的生物标志物（细胞表面抗原）。我们建议整合 基因分析到这个平台上，用于鉴定突变，该突变是特定 化疗剂是广泛使用的化疗剂。具体来说，我们将创建设备和方法来评估 通过引入结构和定制材料来促进芯片上的终点聚合酶链的突变 反应（PCR）。我们的技术的一个重要特征是将细胞转移到反应室（用于 遗传分析）使用简单的流体和电动部件（没有移动部件）来完成。这 技术发展是重要的，因为1）它解决了对整合的无标记选择的需要， 分离和分析单细胞，2）该平台足够简单，可广泛应用于临床 实验室，从而能够表征从个体患者中分离的CTC，以及3）它将使未来能够 黑色素瘤获得性耐药的分子机制的基础研究。这种创新的组合 检测黑色素瘤突变的无标记选择平台将改变黑色素瘤 治疗和研究，因为它将允许无与伦比的访问信息的分布， 与个体CTC之间对治疗剂的敏感性或抗性相关的突变。

Project Summary An unexpected response from a minority of cells can have a dramatic impact on the development, prognosis, and treatment of disease. For example, in the progression of cancer, a resistant minority of circulating tumor cells (CTCs) lead to the development of refractory metastases at relapse. We are only just beginning to uncover these phenomena and the underlying biological mechanisms because they are obscured at the bulk scale. Detection of a rare mutation can require the analysis of hundreds to thousands of individual cells. We have developed a dielectrophoresis (DEP)-based device with a high yield of selective single-cell capture in an array of microchambers arranged along the sides of branched microfluidic channels. An important point is that DEP-based selection is marker-independent, which critically, makes this approach especially relevant to the identification of CTCs in melanoma, for which there are no reliable biomarkers (cell surface antigens). We propose to integrate genetic assays into this platform for the identification of a mutation that is an indicator for a specific chemotherapeutic agent that is in widespread use. Specifically, we will create devices and methods to assess mutations by incorporating structures and custom materials that facilitate on-chip end-point polymerase chain reaction (PCR). An important feature of our technology is that the transfer of cells into reaction chambers (for genetic analysis) is accomplished using simple fluidic and electrokinetic components (no moving parts). This technological development is significant because 1) it addresses a need for integrated marker-free selection, isolation, and analysis of single cells, 2) the platform is sufficiently simple for broad application in clinical laboratories, thus enabling characterization of CTCs isolated from individual patients, and 3) it will enable future basic research into the molecular mechanisms of acquired resistance in melanoma. This innovative combination of a platform for marker-free selection with detection of mutations in melanoma will transform melanoma treatment and research because it will allow unparalleled access to information regarding the distribution of mutations related to susceptibility or resistance to therapeutic agents among individual CTCs.