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

项目摘要 少数细胞的意想不到的反应可能对发育、预后和 疾病的治疗。例如，在癌症的发展过程中，循环中的少数耐药肿瘤细胞 (CTCs)在复发时会导致难治性转移。我们才刚刚开始发现这些 现象和潜在的生物机制，因为它们在大范围内是模糊的。检测 一种罕见的突变可能需要对数百到数千个单个细胞进行分析。我们已经开发出一种 基于介电泳法(DEP)的设备，在阵列中具有高选择性单细胞捕获 沿分支微流控通道的侧边布置的微室。重要的一点是，以DEP为基础的 选择是不依赖于标记的，这一点至关重要，这使得这种方法与识别 黑色素瘤中的CTCs，没有可靠的生物标记物(细胞表面抗原)。我们建议整合 基因分析进入这个平台，用于识别突变，该突变是一种特定的 广泛使用的化疗药物。具体来说，我们将创建设备和方法来评估 通过结合促进芯片终点聚合酶链的结构和定制材料进行突变 反应(PCR)。我们技术的一个重要特点是将细胞转移到反应室(用于 遗传分析)是使用简单的流体和电动组件(没有移动部件)完成的。这 技术发展意义重大，因为1)它满足了对集成的无标记选择的需求， 2)该平台操作简单，可广泛应用于临床 实验室，从而能够表征从个别患者分离的CTC，以及3)它将使未来 黑色素瘤获得性耐药分子机制的基础研究。这一创新的组合 检测黑色素瘤突变的无标记选择平台将转化黑色素瘤 治疗和研究，因为它将允许无与伦比的访问关于分布的信息 与个体CTC对治疗药物的敏感性或耐药性有关的突变。

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.