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检测平台的挑战，以测量癌细胞对治疗干预的生长动力学变化。细胞生长动力学可以通过多种方式测量，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。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。我们建议开发一种同时监测单细胞生长动力学和细胞表面蛋白表达的仪器。我们假设生长动力学和细胞表面蛋白表达的变化可以作为对途径导向治疗剂反应的替代。作为仪器的验证，我们将监测单个A431细胞的质量、密度和细胞表面蛋白表达（通过荧光测定），以响应途径靶向治疗吉非替尼（一种表皮生长因子受体（EGFR）的小分子抑制剂）和适当的对照。单细胞质量和密度将由先前验证过的装置测量，称为悬浮微通道谐振器（SMR）。SMR可以测量哺乳动物细胞的质量，分辨率接近0.01% （1hz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将被修改为在捕获细胞的区域内具有光学透明度。该仪器将利用我们之前与癌症纳米技术卓越中心（CCNE-TR）的蛋白质组学研究成果，我们使用蛋白质组学方法发现指示治疗反应的细胞表面蛋白质生物标志物。单细胞测量这些蛋白的丰度，同时结合细胞质量和密度动力学，将为在生理和分子水平上表征和监测细胞对治疗的反应提供一种新的方法。

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.