Detection of Metastatic Human Breast Cells by SECM

描述(由申请人提供)： 本RO_3提案将探索扫描电化学的使用 显微镜(SECM)作为检测转移性乳腺肿瘤的影像工具 培养和动物模型中的细胞。所采用的检测原理 SECM是建立在电子受体和供体的平衡基础上的 (“氧化还原”)存在于给定的细胞类型中。SECM的优势在于其 一)检测和量化单个电池的氧化还原状态的能力；二) 生成单个细胞的氧化还原图，该图标识具有最高 氧化还原活性；以及iii)扫描一组非转移细胞以寻找 检测一个或多个转移细胞的目的。这是一个最初的目标 该项目是为了优化检测和图像分析的方法 单个细胞和一组非转移细胞中的变异细胞 (具体目标1)。SECM将被用来评估蛋白质的作用 正常和非小细胞肺癌转移潜能中的蛋白激酶Cα 转移性培养的人乳腺细胞。这种氧化还原活性酶是受调节的 通过氧化剂和抗氧化剂，其在肿瘤中的过度表达可能 为转移潜能提供了机制上的联系。经过精心设计 PKCα突变体的过度表达将被测试它们对 SECM测量的细胞氧化还原状态(特定目标2)。因为 使用PKCα抗血清进行的初步免疫组织化学分析表明 这种酶在转移性人类乳腺肿瘤中升高，但在 与患者匹配的正常组织，更广泛的肿瘤免疫组织化学 将通过高通量分析来演示统计 PKCα表达升高的意义(特定目标#3)，因此 建立该酶作为乳腺癌早期检测的分子标志物。 SECM将用于分析一小部分新鲜肿瘤的微转移 将从SCID小鼠身上获得的靶组织将被注射 荧光标记的转移细胞(特定目标4)。敏感度和 SECM获得的信号的保真度将通过参考 由靶中标记的转移细胞定义的荧光图案 组织。对准程度将表明SECM是否可能 人体组织活检的诊断潜力。

DESCRIPTION (provided by applicant): This RO3 proposal will explore the use of the scanning electrochemical microscope (SECM) as an imaging tool for detection of metastatic human breast cells in culture and in an animal model. The principle of detection employed with the SECM is based on the balance of electron acceptors and donors ("redox") that exist in a given cell type. Advantages of the SECM lie in its ability i) to detect and quantify the redox status of an individual cell; ii) to generate a redox map of an individual cell identifying areas having highest redox activity; and iii) to scan a field of non-metastatic cells for the purpose of detecting one or more metastatic cells. An initial goal of this project is to optimize the methodology of detection and image analysis of individual cells, and of aberrant cells in a field of non-metastatic cells (Specific Aim #1). The SECM will be harnessed to evaluate the role of protein kinase C alpha (pKC alpha) in the metastatic potential of normal and metastatic cultured human breast cells. This redox-active enzyme is regulated by both oxidants and anti-oxidants, and its overexpression in tumors may provide the mechanistic link to metastatic potential. Engineered overexpression of PKC alpha mutants will be tested for their impact on the cellular redox state as measured by the SECM (Specific Aim #2). Because preliminary immunohistochemical analysis using PKC alpha antisera suggests that this enzyme is elevated in metastatic human breast tumors but not in patient-matched normal tissue, more extensive immunohistochemistry of tumors will be carried out by high-throughput analysis to demonstrate the statistical significance of elevated PKC alpha expression (Specific Aim #3), and thus to establish this enzyme as a molecular marker for early breast cancer detection. The SECM will be used to analyze micro metastases in a small sample of fresh target tissue to be obtained from SCID mice that will have been injected with fluorescently-tagged metastatic cells (Specific Aim #4). The sensitivity and fidelity of the signal obtained by SECM will be determined by reference to the fluorescent pattern defined by the tagged metastatic cells in the target tissue. The degree of alignment will indicate whether the SECM could have diagnostic potential with human tissue biopsies.