Clinical cancer proteomics aims at the identification of markers for early detection and predictive purposes, as well as to provide novel targets for drug discovery and therapeutic intervention. Proteomics-based analysis of traditional sources of biomarkers, such as serum, plasma, or tissue lyzates, has resulted in a wealth of information and the finding of several potential tumor biomarkers. However, many of these markers have shown limited usefulness in a clinical setting, underscoring the need for new clinically relevant sources. Here we present a novel and highly promising source of biomarkers, the tumor interstitial fluid (TIF) that perfuses the breast tumor microenvironment. We collected TIFs from small pieces of freshly dissected invasive breast carcinomas and analyzed them by two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Western immunoblotting, as well as by cytokine-specific antibody arrays. This approach provided for the first time a snapshot of the protein components of the TIF, which we show consists of more than one thousand proteins-either secreted, shed by membrane vesicles, or externalized due to cell deathproduced by the complex network of cell types that make up the tumor microenvironment. So far, we have identified 267 primary translation products including, but not limited to, proteins involved in cell proliferation, invasion, angiogenesis, metastasis, inflammation, protein synthesis, energy metabolism, oxidative stress, the actin cytoskeleton assembly, protein folding, and transport. As expected, the TIF contained several classical serum proteins. Considering that the protein composition of the TIF reflects the physiological and pathological state of the tissue, it should provide a new and potentially rich resource for diagnostic biomarker discovery and for identifying more selective targets for therapeutic intervention. Molecular & Cellular Proteomics 3:327-344, 2004.

It has become clear that growth and progression of breast tumor cells not only depend on their malignant potential but also on factors present in the tumor microenvironment. Of the cell types that constitute the mammary stroma, the adipocytes are perhaps the least well studied despite the fact that they represent one of the most prominent cell types surrounding the breast tumor cells. There is compelling evidence demonstrating a role for the mammary fat pad in mammary gland development, and some studies have revealed the ability of fat tissue to augment the growth and ability to metastasize of mammary carcinoma cells. Very little is known, however, about which factors adipocytes produce that may orchestrate these actions and how this may come about. In an effort to shed some light on these questions, we present here a detailed proteomic analysis, using two-dimensional gel-based technology, mass spectrometry, immunoblotting, and antibody arrays, of adipose cells and interstitial fluid of fresh fat tissue samples collected from sites topologically distant from the tumors of high risk breast cancer patients that underwent mastectomy and that were not treated prior to surgery. A total of 359 unique proteins were identified, including numerous signaling molecules, hormones, cytokines, and growth factors, involved in a variety of biological processes such as signal transduction and cell communication; energy metabolism; protein metabolism; cell growth and/or maintenance; immune response; transport; regulation of nucleobase, nucleoside, and nucleic acid metabolism; and apoptosis. Apart from providing a comprehensive overview of the mammary fat proteome and its interstitial fluid, the results offer some insight as to the role of adipocytes in the breast tumor microenvironment and provide a first glance of their molecular cellular circuitry. In addition, the results open new possibilities to the study of obesity, which has a strong association with type 2 diabetes, hypertension, and coronary heart disease. Molecular & Cellular Proteomics 4: 492-522, 2005.During the last years there have been numerous reports indicating that growth and progression of breast as well as other tumor cells depend not only on their malignant potential but also on stromal factors present in the tumor microenvironment, the insoluble extracellular matrix as well as cell-cell interactions (Refs.

Novel and powerful technologies such as DNA microarrays and proteomics have made possible the analysis of the expression levels of multiple genes simultaneously both in health and disease. In combination, these technologies promise to revolutionize biology, in particular in the area of molecular medicine as they are expected to reveal gene regulation events involved in disease progression as well as to pinpoint potential targets for drug discovery and diagnostics. Here, we review the current status of these technologies and highlight some studies in which they have been applied in concert to the analysis of biopsy specimens. ß

The galectins are a family of beta-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions. Here we report the cloning and expression of a novel member of this family (galectin-7) that correspond to IEF (isoelectric focusing) 17 (12,700 Da; pI, 7.6) in the human keratinocyte protein data base, and that is strikingly down-regulated in SV40 transformed keratinocytes (K14). The cDNA was cloned from a lambda gt11 cDNA expression library using degenerated oligodeoxyribonucleotides back-translated from an IEF 17 peptide sequence. The protein encoded by the galectin-7 clone comigrated with IEF 17 as determined by two-dimensional (two-dimensional gel electrophoresis) analysis of proteins expressed by transiently transfected COS-1 cells, and bound lactose. Alignment of the amino acid sequences with other members of the family showed that the amino acids central to the beta-galactoside interaction are conserved. Galectin-7 was partially externalized to the medium by keratinocytes although it has no typical secretion signal peptide. Immunoblotting as well as immunofluorescence analysis of human tissues with a specific galectin-7 antibody revealed a narrow distribution of the protein which was found mainly in stratified squamous epithelium. The antigen localized to basal keratinocytes, although it was also found, albeit at lower levels, in the suprabasal layers where it concentrated to areas of cell to cell contact. Both, its cellular localization as well as its striking down-regulation in K14 keratinocytes imply a role in cell-cell and/or cell-matrix interactions necessary for normal growth control. The galectin-7 gene was mapped to chromosome 19.

The S100A4 protein, which is involved in the metastasis process, is a member of the S100 superfamily of Ca-binding proteins. Members of this family are multifunctional signaling proteins with dual extra and intracellular functions involved in the regulation of diverse cellular processes. Several studies have established a correlation between S100A4 protein expression and worse prognosis for patients with various malignancies including breast cancer. In this article, we have used specific antibodies in combination with immunohistochemistry (IHC) to identify the cell types that express S100A4 in human breast cancer biopsies obtained from high-risk patients. IHC analysis of 68 tumor biopsies showed that the protein is expressed preferentially by various cell types present in the tumor microenvironment (macrophages, fibroblasts, activated lymphocytes), rather than by the tumor cells themselves. Moreover, we show that the protein is externalized by the stroma cells to the fluid that bathes the tumor microenvironment, where it is found in several forms that most likely correspond to charge variants. Using a specific ELISA test, we detected a significant higher concentration of S100A4 in the tumor interstitial fluid (TIF) as compared to their corresponding normal counterparts (NIF). ' 2007 Wiley-Liss, Inc.Key words: breast cancer; tumor stroma; S100A4The S100 protein family is the largest subgroup within the superfamily of proteins carrying the Ca 21 -binding EF-hand motif. Members of this family are multifunctional signaling proteins that are involved in the regulation of diverse cellular processes such as contraction, motility, cell growth, differentiation, cell cycle progression, transcription, and secretion. 1 Diseases such as cardiomyopathies, neurodegenerative and inflammatory disorders, as well as cancer are associated with altered S100 protein levels. 2 Despite their small molecular size and their conserved functional domain of 2 distinct EF-hands, these proteins exhibit a wide-range of tissue-specific intra and extracellular functions.Similar to the other members of this protein family, S100A4 exhibits diverse functions. 1 Several lines of evidence based either on spontaneous metastasis formation in rodent or experimental models utilizing human cancer cell lines have suggested a causative role for this protein in the metastatic process. 3,4 In particular, experiments with transgenic mouse models of breast cancer have shown that the expression levels of S100A4 correlates with metastasis. 5,6 Moreover, recent studies by Xue et al. have shown that transgenic mice expressing the Polyoma virus middle T antigen when crossed with mice carrying null alleles for S100A4 exhibit a significant decrease in lung metastases. 7 It has been hypothesized that S100A4 stimulates metastatic spread of tumor cells by stimulating motility via interaction with its target proteins, such as nonmuscle myosin. [8][9][10][11][12] S100A4 has also, from studies on renal diseases in mice, been identified as the fibroblast specific protein 1 (...

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Proteomic technologies, such as yeast twohybrid, mass spectrometry (MS), protein/peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, it is not fruitful for the biomedical community to contribute these types of data to centralized repositories. Even more difficult is the annotation and display of pertinent information in the context of the corresponding proteins. Wikipedia, an online encyclopedia that anyone can edit, has already proven quite successful1 and can be used as a model for sharing biological data. However, the need for experimental evidence, data standardization and ownership of data creates scientific obstacles. Here, we describe Human Proteinpedia (http://www.humanproteinpedia.org/) as a portal that overcomes many of these obstacles to provide an integrated view of the human proteome. Human Proteinpedia also allows users to contribute and edit proteomic data with two significant differences from Wikipedia: first, the contributor is expected to provide experimental evidence for the data annotated; and second, only the original contributor can edit their data. Human Proteinpedia's annotation system provides investigators with multiple options for contributing data including web forms and annotation servers. Although registration is required to contribute data, anyone can freely access the data in the repository. The web forms simplify submission through the use of pull-down menus for certain data fields and pop-up menus for standardized vocabulary terms. Distributed annotation servers using modified protein DAS (distributed annotation system) protocols developed by us (DAS protocols were originally developed for sharing mRNA and DNA data) permit contributing laboratories to maintain protein annotations locally. All protein annotations are visualized in the context of corresponding proteins in the Human Protein Reference Database (HPRD)3. Figure 1 shows tissue expression data for alpha-2-HS glycoprotein derived from three different types of experiments. Our unique effort differs significantly from existing repositories, such as PeptideAtlas and PRIDE5 in several respects. First, most proteomic repositories are restricted to one or two experimental platforms, whereas Human Proteinpedia can accommodate data from diverse platforms, including yeast two-hybrid screens, MS, peptide/protein arrays, immunohistochemistry, western blots, coimmunoprecipitation and fluorescence microscopy-type experiments. Second, Human Proteinpedia allows contributing laboratories to annotate data pertaining to six features of proteins (posttranslational modifications, tissue expression, cell line expression, subcellular localization, enzyme substrates and protein-protein interactions;). No existing repository currently permits annotation of all these features in proteins. Third, all data submitted to Human Proteinpedia...

Proteomics provides powerful tools for the study of clinically relevant samples in the context of translational cancer research. Here we briefly review applications of gel-based proteomics for the study of bladder and lung cancer using fresh tissue biopsies. In general, these studies have emphasized the potential of the technology for biomarker discovery, as well as for addressing the issue of cancer heterogeneity.