Fluorescence Correlation Quantitation of Insulin-like Growth Factor and Insulin R

描述（由申请人提供）：胰岛素受体（IR）和胰岛素样生长因子受体（IGF 1 R）家族是肿瘤学的治疗靶点。 靶向IR/IGF 1 R受体家族的药物的开发基于表明循环水平或胰岛素和/或胰岛素样生长因子1（IGF-1）与癌症风险相关的观察。 此外，以IR-IR和IGF 1 R-IGF 1 R同源二聚体以及IGF 1 R-IR异源二聚体形式存在的任一受体的增加可能降低乳腺癌患者的无复发生存率。 然而，IGF 1 R和IR单体总表达的简单增加并不能充分解释IGF-1对肿瘤细胞的生长促进作用。 相反，似乎这些受体在同源二聚体和异源二聚体之间的分布调节肿瘤细胞对胰岛素和IGF-1的反应，包括细胞增殖和迁移潜力。 目前还没有直接的方法来评估乳腺癌细胞或任何其他正常或癌性组织中这些单个受体种类的浓度。 荧光相关光谱（FCS）和光子计数直方图（PCH）方法将在新开发的4探测器荧光相关系统中实施，为这种直接测量提供了一条途径，并使该项目的三个具体目标成为可能：目标1：开发新的FCS和PCH方法，以直接评估活单细胞上IR同二聚体的实际表面浓度，IGF 1 R同源二聚体和IR-IGF 1 R异源二聚体。 初始实验将涉及由同时表达IR和IGF 1 R的绿色和红色荧光形式（IR-GFP和IGF 1 R-mCherry）的CHO细胞组成的模型系统，每种形式以不同的水平表达。 目的2：研究胰岛素受体（IR）和胰岛素样生长因子1 R（IGF 1 R）同源二聚体及IR-IGF 1 R杂合受体形成的热力学。 这个目标的目的是解释FCS和PCH测量的单独的受体物种的定量模型，涉及受体对和受体单体表达水平的关联常数。 目标3：评估乳腺癌细胞的受体二聚体种类以及胰岛素或IGF-1治疗对乳腺癌细胞的促生长作用，包括人工操纵IR和IGF 1 R表达数量的乳腺癌细胞。 在这个目标的激素效应与各种受体物种的测量水平的相关性将进行探讨。 总的来说，该项目将提供改进的工具和数据，用于定量了解IR和IGF 1 R表达如何共同调节胰岛素和IGF-1在特定细胞中的信号传导。 这些结果将有助于理解 激素反应如何在细胞类型之间变化，并最终通过使用适当调节肿瘤细胞介导的肿瘤细胞增殖的靶向药物来改善患者护理。

DESCRIPTION (provided by applicant): The insulin receptor (IR) and insulin-like growth factor receptor (IGF1R) family is a therapeutic target in oncology. Development of pharmaceuticals that target the IR/IGF1R receptor family has been based on observations suggesting that circulating levels or insulin and/or insulin-like growth factor 1 (IGF-1) are related to cancer ris. In addition, increases in either receptor, present both as IR-IR and IGF1R-IGF1R homodimers and as IGF1R-IR heterodimers may reduce relapse-free survival in breast cancer patients. However, simple increases in the total expression of IGF1R and IR monomers do not adequately explain the growth-promoting effects of IGF-1 on tumor cells. Rather, it seems that distribution of these receptors among homodimers and heterodimers regulates tumor cell responses to insulin and IGF-1 including cell proliferation and migratory potential. There is currently no direct method to evaluate the concentrations of these individual receptor species in breast cancer cells or, for that matter, in any other normal or cancerous tissue. Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) methods, to be implemented in a newly-developed 4-detector fluorescence correlation system, provide a path to such direct measurements and make possible the project's three specific aims: Aim 1: To develop new FCS and PCH methods to directly evaluate, on viable single cells, the actual surface concentrations of IR homodimers, IGF1R homodimers and IR-IGF1R heterodimers. Initial experiments will involve a model system consisting of CHO cells simultaneously expressing green and red fluorescent forms of IR and IGF1R (IR-GFP and IGF1R-mCherry), each expressed at varying levels. Aim 2: To evaluate the thermodynamics of IR and IGF1R homodimer and IR-IGF1R hybrid receptor formation. The goal of this aim is interpret FCS and PCH measurements of separate receptor species in terms of a quantitative model involving association constants for receptor pairs and receptor monomer expression levels. Aim 3: To evaluate receptor dimer species of, and growth-promoting effects of insulin or IGF-1 treatment on, breast cancer cells, including ones where the numbers of IR and IGF1R expressed have been artificially manipulated. In this aim correlation of hormone effects with measured levels of the various receptor species will be explored. Overall, this project will provide improved tools and data for quantitative understanding of how IR and IGF1R expression jointly regulate signaling by insulin and IGF-1 in particular cells. These results will facilitate understanding of how hormone responses can vary among cell types and lead ultimately to improved patient care through the use of targeted pharmaceutical agents that appropriately modulate hormone-mediated tumor cell proliferation.