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

项目概要 少数细胞的意外反应可能会对发育、预后和疾病产生巨大影响。 治疗疾病。例如，在癌症的进展中，少数具有耐药性的循环肿瘤细胞 （CTC）导致复发时难治性转移的发生。我们才刚刚开始发现这些 现象和潜在的生物机制，因为它们在整体尺度上是模糊的。检测 罕见的突变可能需要分析数百到数千个单个细胞。我们开发了一个 基于介电电泳 (DEP) 的设备，可在阵列中高产率地选择性捕获单细胞 微室沿分支微流体通道的侧面排列。重要的一点是基于 DEP 选择与标记无关，这一点至关重要，使得这种方法与鉴定 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 在该平台中进行基因检测，以识别突变，该突变是特定的指标 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 通过整合促进芯片上端点聚合酶链的结构和定制材料来实现突变 反应（PCR）。我们技术的一个重要特点是将细胞转移到反应室中（用于 遗传分析）是使用简单的流体和电动组件（无移动部件）完成的。这个 技术发展意义重大，因为 1) 它满足了综合无标记选择的需求， 单细胞的分离和分析，2）该平台足够简单，可广泛应用于临床 实验室，从而能够对从个体患者中分离出的 CTC 进行表征，3) 它将使得未来 黑色素瘤获得性耐药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地获取有关治疗和研究分布的信息 与个体 CTC 中治疗药物的敏感性或耐药性相关的突变。

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.