Detection of Metastatic Human Breast Cells by SECM

SECM 检测转移性人乳腺细胞

• 批准号: 6335320
• 负责人: Susan A. Rotenberg
• 金额: $ 7.7万
• 依托单位: QUEENS COLLEGE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6335320
• 关键词: SCID mouse; bioimaging /biomedical imaging; breast neoplasm /cancer diagnosis; breast neoplasms; diagnosis design /evaluation; enzyme activity; enzyme inhibitors; immunocytochemistry; lung neoplasms; metastasis; mutant; oxidation reduction reaction; protein kinase C; scanning electron microscopy; site directed mutagenesis

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.

描述（由申请人提供）： 该RO 3提案将探索使用扫描电化学 显微镜（SECM）作为检测转移性人乳腺癌的成像工具 细胞培养和动物模型中。所采用的检测原理 与SECM是基于电子受体和供体的平衡 （“氧化还原”）。 SECM的优势在于其 i）检测和量化单个电池的氧化还原状态的能力; ii） 为了生成单个电池的氧化还原图， 氧化还原活性;和iii）扫描非转移性细胞的区域，以检测氧化还原活性。 检测一个或多个转移细胞的目的。 这个项目的最初目标是 项目是优化检测和图像分析的方法， 单个细胞和非转移性细胞场中的异常细胞 （具体目标#1）。 SECM将被用来评估蛋白质的作用， 激酶C α（pKC α）在正常和 转移性培养的人类乳腺细胞。 这种氧化还原活性酶受到调节， 氧化剂和抗氧化剂，它在肿瘤中的过度表达可能 提供转移潜能的机制联系。 工程化 PKC α突变体的过度表达将被测试其对 通过SECM测量的细胞氧化还原状态（特定目标#2）。 因为 使用PKC α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 患者匹配的正常组织，更广泛的肿瘤免疫组织化学 将进行高通量分析，以证明统计 PKC α表达升高的意义（具体目标#3），因此， 建立这种酶作为早期乳腺癌检测分子标记。 SECM将用于分析小样本新鲜肿瘤中的微转移。 靶组织从已经注射了以下物质的SCID小鼠获得： 荧光标记的转移性细胞（特定目标#4）。 的敏感性和 通过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限定的荧光图案 组织. 对准程度将表明SECM是否可能具有 人体组织活检的诊断潜力。