Proteomics--tissue and protein microarrays and antibody array: what information is provided?
(Development and progression)
Lung cancer (Genetic aspects)
Lung cancer (Analysis)
DNA microarrays (Usage)
Gene expression (Research)
Protein-protein interactions (Research)
Popper, Helmut H.
|Publication:||Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2008 College of American Pathologists ISSN: 1543-2165|
|Issue:||Date: Oct, 2008 Source Volume: 132 Source Issue: 10|
|Topic:||Event Code: 310 Science & research|
Traditionally, genetic aberrations provide the basis of our
understanding of lung tumor development and progression. RNA expression
provides the information about potential aberrations, but proteins and
protein-protein interactions, for example by
phosphorylation/dephosphorylation, produce the end results of the
aberrations. Proteomics aims to characterize the information flow within
a cell through protein pathways and networks. Therefore, different
proteomic methods have been developed to discover new biomarkers and
therapeutic targets, among them 2-color, 2-dimensional gel
electrophoresis and mass spectrometry (matrix-assisted laser desorption/
ionization time-of-flight, surface-enhanced laser desorption/ionization
time-of-flight). Both methods require special, expensive laboratory
equipment, experienced investigators, and considerable time (see also
the article by J. Wisniewski in this special section). A more
cost-effective proteomic method available to most large pathology
laboratories is tissue microarray. (1) Other proteomic methods are in
development, such as protein extraction from formalin-fixed and
paraffin-embedded tissues (FFPET) in association with protein and
The excellent work of Bubendorf et al (1) has provided a major step forward in pathology research with subsequent potential utility in diagnostics, prognostics, and oncologic pathology. Small cores of tissues are punched from paraffin blocks and transferred into recipient blocks. Using these small cores, samples from up to 200 different tumors can be arrayed in 1 paraffin block. From a single array block up to 200 sections can be made and analyzed by immunohistochemistry (2) (Figure 1), in situ hybridization, or immunofluorescence. This technique can complement other proteomic methods and has been used to identify new prognostic markers. But tissue microarray has also an advantage over other proteomic methods: The pattern of protein expression can be studied with respect to cell compartments (nuclear, cytoplasmic, membranous) and the distribution of proteins in tumor cells, stroma, and adjacent normal parenchyma, including normal bronchial and alveolar epithelium.
For some investigations the evaluation of tissue microarray can be automated or semiautomatic; in others the evaluation has to be done manually. (3) Data analysis for the evaluation of tissue microarrays is available, even for large studies using several antibodies. Some of these instruments are commercially available and expensive; others are shareware programs. (4) A relationship among the different antibody reactions can be studied, and so new functional networks can be identified. By thousands of statistical tests, "nearest neighbors" (statistically significant interactions/ associations) can be identified. As a result, proteins of previously unknown function may be functionally associated with known protein signaling pathways (Figure 2). By comparing protein expression in malignant pleural mesotheliomas from long-term versus short-term survivors we were able to identify differences in the regulatory mechanisms of protein pathways, which differentially promote growth and antiapoptosis signaling. The epidermal growth factor receptor pathway was more highly activated in long-term survivors, whereas platelet-derived growth factor receptor activation was more abundant in short-term survivors. (4) This might open new therapeutic options for the treatment of this deadly disease. In a similar investigation, protein differences among all 4 major types of lung carcinoma were identified.
PROTEIN EXTRACTION FROM FFPET
Proteins can be easily extracted from fresh frozen tissues. However, it is much more difficult to extract proteins and peptides from FFPET. The primary problem with extraction from FFPET is to reverse cross-linking of the proteins induced by formalin fixation. A proposal has been published to change the type of fixative used for specimens, but it has not been proven that the stability of proteins, DNA, and RNA remains during the years and that the quality of the tissues is preserved as with formalin-fixed specimens. (5-7) Recent work in several laboratories has shown that extraction of protein from FFPET is feasible (8-10) (Figure 3).
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In these studies, the tissues were deparaffinized and manually dissected from the slides and the proteins were solubilized in a buffer system. By using a commercially available extraction kit, Becker et al (8) succeeded in extracting nondegraded soluble proteins from FFPET blocks and used them on Western blot and reverse-phase protein microarrays.
REVERSE-PHASE PROTEIN MICROARRAY
Glass slides coated with nitrocellulose are available commercially. These provide a 3-dimensional structure in which proteins are noncovalently but irreversibly bound, similar to immunoblots. Protein lysates are printed onto these glass slides. Unspecific binding sites are blocked, for example by milk powder, bovine serum albumin, or casein, depending strongly on the nature of the experiment. The slides are then incubated with antibodies and subsequently washed to remove unbound antibodies. The detection is done either by direct labeling of the antibodies or by incubation with labeled secondary antibodies. Fluorescent, isotopic, and chemiluminescent horseradish peroxidase-luminol systems can be used for detection, similar to those used for Western blots (Figure 4).
Another way of analyzing signaling pathways in lung cancer samples is by antibody array. This is especially suitable in those situations in which nothing is known about the relevant signaling pathways. Proteins are extracted from cancer tissue. Tumor proteins are compared with those of normal adjacent tissues or to standard protein lysates. Protein lysates from tumor and normal tissue are labeled with fluorochromes Cy3 and Cy5, respectively, and cohybridized for 1 hour to commercially or self-made antibody arrays. The antibodies can be spotted either on nitrocellulose membranes or on glass slides but have to be immobilized to avoid washing off during processing. After the washing steps, the antibody-antigen reaction can be visualized by a standard microarray scanner system. Fluorescent signal intensity is compared for tumor versus normal sample. The evaluation can be done similar to a gene expression microarray experiment.
[FIGURE 3 OMITTED]
Several companies offer commercial antibody arrays; specific sets, such as kinase sets, apoptosis sets, or large sets covering as much as 700 different proteins, are available.
APPLICATIONS FOR PROTEIN AND ANTIBODY ARRAYS
Both methods mentioned previously complement each other. If one focuses on specific protein expression profiles, for example, to extract information about a possible response to targeted therapy, an antibody array might be the preferred method. By this method the expression pattern for a specific kinase such as epidermal growth factor receptor can be evaluated for several adenocarcinomas simultaneously. If multiple targets are to be affected by a combined treatment, a protein array of the tumor will allow one to hybridize several antibodies at once and therefore get information about different signaling pathway activation in a single investigation. By this method, failure of tumor response to targeted therapy drugs can be evaluated, for example one can identify probable alternatively activated pathways by which a tumor can circumvent therapeutic inhibition.
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Accepted for publication March 27, 2008.
(1.) Bubendorf L, Kononen J, Koivisto P, et al. Survey of gene amplifications during prostate cancer progression by high-throughout fluorescence in situ hybridization on tissue microarrays [published correction appears in Cancer Res. 1999;59:1388]. Cancer Res. 1999;59:803-806.
(2.) Ullmann R, Morbini P, Halbwedl I, et al. Protein expression profiles in adenocarcinomas and squamous cell carcinomas of the lung generated using tissue microarrays. J Pathol. 2004;203:798-807.
(3.) Haedicke W, Popper HH, Buck CR, Zatloukal K. Automated evaluation and normalization of immunohistochemistry on tissue microarrays with a DNA microarray scanner. Biotechniques. 2003;35:164-168.
(4.) Kothmaier H, Quehenberger F, Halbwedl I, et al. EGFR and PDGFR differentially promote growth in malignant epithelioid mesothelioma of short- and long-term survivors. Thorax. 2008;63:345-351.
(5.) Ikeda K, Monden T, Kanoh T, et al. Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections. J Histochem Cytochem. 1998;46:397-403.
(6.) Stanta G, Mucelli SP, Petrera F, Bonin S, Bussolati G. A novel fixative improves opportunities of nucleic acids and proteomic analysis in human archive's tissues. Diagn Mol Pathol. 2006;15:115-123.
(7.) Uneyama C, Shibutani M, Masutomi N, Takagi H, Hirose M. Methacarn fixation for genomic DA analysis in microdissected, paraffin-embedded tissue specimens. J Histochem Cytochem. 2002;50:1237-1245.
(8.) Becker KF, Schott C, Hipp S, et al. Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis. J Pathol. 2007;211:370-378.
(9.) Hood BL, Conrads TP, Veenstra TD. Unravelling the proteome of formalin-fixed paraffin-embedded tissue. Brief Funct Genomic Proteomic. 2006;5:169-175.
(10.) Lemaire R, Desmons A, Tabet JC, Day R, Salzet M, Fournier I. Direct analysis and MALDI imaging of formalin-fixed, paraffin-embedded tissue sections. J Proteome Res. 2007;6:1295-1305.
Helmut H. Popper, MD; Hannelore Kothmaier, MS
From the Laboratories for Molecular Cytogenetics, Environmental and Respiratory Pathology, Institute of Pathology, Medical University of Graz, Graz, Austria.
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: Helmut H. Popper, MD, Laboratories for Molecular Genetics, Environmental and Respiratory Pathology, Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, Graz A-8036, Austria (e-mail: email@example.com).
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