Developing a single cell growth monitor for classifying therapeutic response

描述（由申请人提供）：临床肿瘤学和癌症生物学受到缺乏用于测量响应于治疗干预的癌细胞生长动力学变化的测定平台的挑战。细胞生长动力学可以以多种方式测量，例如通过DNA拷贝数、体积、质量、密度、形状或通过特定蛋白质的表达。在这里，我们将生长动力学定义为细胞质量和密度随时间的变化。我们建议开发一种仪器，同时监测单细胞生长动力学和细胞表面蛋白表达。我们假设生长动力学和细胞表面蛋白表达的变化可以用作对途径导向治疗剂的反应的替代。作为对该仪器的验证，我们将监测单个A431细胞中的质量、密度和细胞表面蛋白表达（通过荧光确定），以响应途径靶向治疗吉非替尼（表皮生长因子受体的小分子抑制剂）的干预（EGFR）和适当的对照。单细胞质量和密度将通过先前验证的称为悬浮微通道谐振器（SMR）的装置测量。SMR可以测量哺乳动物细胞的质量，分辨率接近0.01%（1 Hz带宽）。为了实现质量和密度的连续测量，硅柱将用于捕获微通道谐振器的敏感区域内的单个细胞。为了能够通过偶联抗体的荧光同时检测蛋白质表达，SMR将被修饰为在捕获细胞的区域内具有光学透明度。拟议的仪器将利用我们先前与癌症纳米技术卓越中心（CCNE-TR）合作的蛋白质组学工作，该中心专注于治疗反应，我们使用蛋白质组学方法来发现指示治疗反应的细胞表面蛋白质生物标志物。这些蛋白质的丰度的单细胞测量，同时与细胞质量和密度动力学将提供一种新的方法，用于表征和监测细胞对生理和分子水平上的治疗的反应。

DESCRIPTION (provided by applicant): Clinical oncology and cancer biology are challenged by the lack of assay platforms for measuring changes in cancer-cells' growth kinetics in response to therapeutic intervention. Cell growth kinetics can be measured in a number of ways, such as by DNA copy number, volume, mass, density, shape, or by expression of particular proteins. Here we define growth kinetics as changes in a cell's mass and density over time. We propose to develop an instrument for concurrently monitoring single cell growth kinetics and cell surface protein expression. We hypothesize that changes in growth kinetics and in cell surface protein expression can be used as a surrogate for response to pathway-directed therapeutic agents. As a validation of the instrument, we will monitor mass, density and cell-surface protein expression (determined by fluorescence) in single A431 cells in response to intervention with the pathway targeted therapy gefitinib, a small molecule inhibitor of the epidermal growth factor receptor (EGFR), and appropriate controls. Single cell mass and density will be measured by a previously validated device known as the suspended microchannel resonator (SMR). The SMR can measure the mass of a mammalian cell with a resolution near 0.01% (1 Hz bandwidth). In order to achieve a continuous measurement of mass and density, silicon posts will be used for capturing a single cell within the sensitive region of the microchannel resonator. To enable simultaneous detection of protein expression, by fluorescence of coupled antibodies, the SMR will be modified to have optical transparency within the region where the cell is captured. The proposed instrument will leverage our prior proteomics work with the Center for Cancer Nanotechnology Excellence focused on Therapeutic Response (CCNE-TR) where we used proteomic methods to discover cell-surface protein biomarkers indicative of therapeutic response. Single-cell measurement of the abundance of these proteins, concurrently with cell mass and density kinetics will provide a new approach for characterizing and monitoring cell response to therapy on a physiological and molecular level.