LPC focus delta: 4. RoboLPC joint distance: 6 µm. Different software settings are available for microdissection. We either use âCutâ and manual LPC or, ...
Chapter 11 Laser Capture Microdissection Applications in Breast Cancer Proteomics René B.H. Braakman, Theo M. Luider, John W.M. Martens, John A. Foekens, and Arzu Umar Abstract Breast cancer tissues are characterized by cellular heterogeneity, representing a mixture of, e.g., healthy epithelial ducts, invasive or in situ tumor cells, surrounding stroma, infiltrating immune cells, blood vessels, and capillaries. As a consequence, protein extracts from whole tissue lysates also represent a variety of cell types present in the tissues under examination. This, however, seriously hampers the analysis of tumor cell-specific signals, which is of interest when performing biomarker discovery-type of studies. Therefore, laser capture microdissection is a perfect tool to isolate a relatively pure population of cells of interest, such as tumor cells. In this chapter, we describe the use of the PALM MicroBeam system for laser microdissection and pressure catapulting. Protocols are provided for sectioning, staining, microdissection, sample preparation, and mass spectrometric analysis of snap frozen breast cancer tissue. Key words: Breast cancer, Laser capture microdissection, nLC, MALDI, Mass spectrometry, Proteomics, Biomarker discovery
1. Introduction High throughput, state-of-the-art “omics” techniques are becoming widely used in cancer research and biomarker discovery studies (1–3). The secret of success of all these “omics” studies, however, is not merely technology based, but very heavily leans on the source that is being investigated. In cancer research, the basis of all work is the use of (tumor) tissue specimens from individuals of whom clinical follow-up information is available. Although tissue specimens provide a perfect source for translational research,
Graeme I. Murray (ed.), Laser Capture Microdissection: Methods and Protocols, Methods in Molecular Biology, vol. 755, DOI 10.1007/978-1-61779-163-5_11, © Springer Science+Business Media, LLC 2011
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tumor tissues, in particular breast cancer, are very heterogeneous in cell type and composition. Tissue heterogeneity can seriously hamper downstream data interpretation acquired from a mixture of cells. In this respect, development of laser capture microdissection (LCM) technology has filled this gap by providing a means of selecting and enriching for specific cell populations of interest from their natural environment prior to subsequent molecular analysis (4). LCM has put an important step forward in both genomics and proteomics cancer biomarker discovery studies. Two types of LCM-based technologies are commonly used: the thermoplastic film contact based Arcturus system (Molecular Devices) (5) and the noncontact laser pressure catapulting PALM system (Carl Zeiss) (6). Comparative proteomics of LCM-derived breast cancer cells has been performed using 2DE (7, 8), or LC-MS/MS (9) approaches, which has resulted in the identification of proteins involved in breast cancer metastasis (7, 9), and prognosis (8). These studies have shown that proteomics technologies have advanced in such a way that they can contribute to biomarker discovery. However, major drawbacks of these studies are that they either required the use of large amounts of starting material (varying from 42 to 700 mg protein), or they yielded few identifications (50–76 proteins) in the case where little starting material was used. Clinical samples, on the other hand, are usually available in very small quantities. To allow comprehensive analysis of minute amounts of material (
