Detection of Metastatic Human Breast Cells by SECM

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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.