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Proteomic Characterization of the Interstitial Fluid Perfusing the Breast Tumor Microenvironment

A Novel Resource for Biomarker and Therapeutic Target Discovery*
  • Julio E. Celis
    Correspondence
    To whom correspondence should be addressed: Danish Centre for Translational Breast Cancer Research, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. Tel.: 45-35257363; Fax: 45- 35257375
    Affiliations
    Danish Centre for Translational Breast Cancer Research, DK-2100 Copenhagen, Denmark;

    Department of Proteomics in Cancer, Institute of Cancer Biology, The Danish Cancer Society, DK-2100 Copenhagen, Denmark;
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  • Pavel Gromov
    Affiliations
    Danish Centre for Translational Breast Cancer Research, DK-2100 Copenhagen, Denmark;

    Department of Proteomics in Cancer, Institute of Cancer Biology, The Danish Cancer Society, DK-2100 Copenhagen, Denmark;
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  • Teresa Cabezón
    Affiliations
    Danish Centre for Translational Breast Cancer Research, DK-2100 Copenhagen, Denmark;

    Department of Proteomics in Cancer, Institute of Cancer Biology, The Danish Cancer Society, DK-2100 Copenhagen, Denmark;
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  • José M.A. Moreira
    Affiliations
    Danish Centre for Translational Breast Cancer Research, DK-2100 Copenhagen, Denmark;

    Department of Proteomics in Cancer, Institute of Cancer Biology, The Danish Cancer Society, DK-2100 Copenhagen, Denmark;
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  • Noona Ambartsumian
    Affiliations
    Danish Centre for Translational Breast Cancer Research, DK-2100 Copenhagen, Denmark;

    Department of Molecular Cancer Biology, Institute of Cancer Biology, The Danish Cancer Society, DK-2100 Copenhagen, Denmark;
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  • Kerstin Sandelin
    Affiliations
    Danish Centre for Translational Breast Cancer Research, DK-2100 Copenhagen, Denmark;

    Department of Breast and Endocrine Surgery, Rigshospitalet, DK-2100 Copenhagen, Denmark;
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  • Fritz Rank
    Affiliations
    Danish Centre for Translational Breast Cancer Research, DK-2100 Copenhagen, Denmark;

    Department of Pathology, The Centre of Diagnostic Investigations, Rigshospitalet, DK-2100 Copenhagen, Denmark
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  • Irina Gromova
    Affiliations
    Danish Centre for Translational Breast Cancer Research, DK-2100 Copenhagen, Denmark;

    Department of Proteomics in Cancer, Institute of Cancer Biology, The Danish Cancer Society, DK-2100 Copenhagen, Denmark;
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      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 death—produced 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.
      Today there is evidence indicating that tumor growth and progression is dependent on the malignant potential of the tumor cells as well as on the multidirectional interactions of local factors produced by all the cell types—tumor, stroma, endothelial cells, and immune and inflammatory cells—present in the local microenvironment (1–9 and references therein). These interactions are most likely unique for any given lesion and may differ both in time and space within the same tumor.
      The available information indicates that tumor cells secrete factors that alter the activity of fibroblasts in the supporting stroma, which in turn secrete extracellular matrix (ECM)
      The abbreviations used are: ECM, extracellular matrix; TIF, tumor interstitial fluid; NAF, nipple aspirate fluid; IHC, immunochemistry; 2D PAGE, two-dimensional polyacrylamide gel electrophoresis; IEF, isoelectric focusing; NEPHGE, nonequilibrium pH gradient electrophoresis; MMP, matrix metalloproteinase; EGF, epidermal growth factor; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid; SN, supernatant; ACN, acetonitrile; PSD, post-source decay; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MIF, metastasis interstitial fluid; NIF, nonmalignant interstitial fluid; FIF, fat interstitial fluid.
      1The abbreviations used are: ECM, extracellular matrix; TIF, tumor interstitial fluid; NAF, nipple aspirate fluid; IHC, immunochemistry; 2D PAGE, two-dimensional polyacrylamide gel electrophoresis; IEF, isoelectric focusing; NEPHGE, nonequilibrium pH gradient electrophoresis; MMP, matrix metalloproteinase; EGF, epidermal growth factor; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid; SN, supernatant; ACN, acetonitrile; PSD, post-source decay; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MIF, metastasis interstitial fluid; NIF, nonmalignant interstitial fluid; FIF, fat interstitial fluid.
      proteins and cytokines that modify the biology and activity of the cancer cells (
      • Leek R.D.
      • Harris A.L.
      Tumor-associated macrophages in breast cancer..
      ). In addition, modified stroma cells secrete proteases that facilitate tissue destruction, cancer cell migration, and metastasis (
      • Liotta L.A.
      • Kohn E.C.
      The microenvironment of the tumour-host interface..
      ,
      • Ahmad S.A.
      • Jung Y.D.
      • Liu W.
      • Reinmuth N.
      • Parikh A.
      • Stoeltzing O.
      • Fan F.
      • Ellis L.M.
      The role of the microenvironment and intracellular cross-talk in tumor angiogenesis..
      ,
      • Quaranta V.
      • Giannelli G.
      Cancer invasion: watch your neighbourhood!.
      ,
      • Bogenrieder T.
      • Herlyn M.
      Axis of evil: Molecular mechanisms of cancer metastasis..
      and references therein). Other non-neoplastic cell types in the tumor microenvironment include endothelial cells and their supporting cells (pericytes), inflammatory cells (neutrophils, macrophages, eosinophils, and mast cells), immune cells (lymphocytes, dendritic cells), smooth muscle cells, myoepithelial cells, and adipocytes, all of which are believed to have a profound influence on the biological potential of a lesion (1, 2 and references therein). So far, several proteins have been implicated in the regulation of the tumor ecosystem in breast cancer: these include the estrogen and progesterone receptors (
      • Doisneau-Sixou S.F.
      • Sergio C.M.
      • Carroll J.S.
      • Hui R.
      • Musgrove E.A.
      • Sutherland R.L.
      Estrogen and antiestrogen regulation of cell cycle progression in breast cancer cells..
      ), matrix metalloproteinases (MMPs) such as interstitial collagenases, gelatinases, stromelysins, and membrane-type MMPs (
      • Duffy M.J.
      • Maguire T.M.
      • Hill A.
      • McDermott E.
      • O’Higgins N.
      Metalloproteinases: Role in breast carcinogenesis, invasion and metastasis..
      ,
      • Fata J.E.
      • Werb Z.
      • Bissell M.J.
      Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes..
      ), urokinase-type plasminogen activator receptor (
      • Choong P.F.
      • Nadesapillai A.P.
      Urokinase plasminogen activator system: a multifunctional role in tumor progression and metastasis..
      ,
      • Magdolen V.
      • Kruger A.
      • Sato S.
      • Nagel J.
      • Sperl S.
      • Reuning U.
      • Rettenberger P.
      • Magdolen U.
      • Schmitt M.
      Inhibition of the tumor-associated urokinase-type plasminogen activation system: Effects of high-level synthesis of soluble urokinase receptor in ovarian and breast cancer cells in vitro and in vivo..
      ), intercellular adhesion molecule-1 (
      • O’Hanlon D.M.
      • Fitzsimons H.
      • Lynch J.
      • Tormey S.
      • Malone C.
      • Given H.F.
      Soluble adhesion molecules (E-selectin, ICAM-1 and VCAM-1) in breast carcinoma..
      ,
      • Kostler W.J.
      • Tomek S.
      • Brodowicz T.
      • Budinsky A.C.
      • Flamm M.
      • Hejna M.
      • Krainer M.
      • Wiltschke C.
      • Zielinski C.C.
      Soluble ICAM-1 in breast cancer: clinical significance and biological implications..
      ), E-cadherin (
      • Berx G.
      • Van Roy F.
      The E-cadherin/catenin complex: An important gatekeeper in breast cancer tumorigenesis and malignant progression..
      ), transforming growth factor-β system (
      • Kretzschmar M.
      Transforming growth factor-β and breast cancer: Transforming growth factor-β/SMAD signaling defects and cancer..
      ,
      • Pollard J.W.
      Tumour-stromal interactions. Transforming growth factor-β isoforms and hepatocyte growth factor/scatter factor in mammary gland ductal morphogenesis..
      ,
      • Fearon E.R.
      Connecting estrogen receptor function, transcriptional repression, and E-cadherin expression in breast cancer..
      ), epidermal growth factor (EGF) (
      • Normanno N.
      • Ciardiello F.
      EGF-related peptides in the pathophysiology of the mammary gland..
      ), EGF receptor-2 (HER-2/neu; c-erbB-2) (
      • Yarden Y.
      • Sliwkowski M.X.
      Untangling the ErbB signalling network..
      ,
      • Baselga J.
      • Hammond L.A.
      HER-targeted tyrosine-kinase inhibitors..
      ,
      • Ross J.S.
      • Fletcher J.A.
      • Linette G.P.
      • Stec J.
      • Clark E.
      • Ayers M.
      • Symmans W.F.
      • Pusztai L.
      • Bloom K.J.
      The Her-2/neu gene and protein in breast cancer 2003: Biomarker and target of therapy..
      ,
      • Earp 3rd, H.S.
      • Calvo B.F.
      • Sartor C.I.
      The EGF receptor family—Multiple roles in proliferation, differentiation, and neoplasia with an emphasis on HER4..
      ), insulin growth factor 1 (
      • Helle S.I.
      • Lonning P.E.
      Insulin-like growth factors in breast cancer..
      ,
      • Gray S.G.
      • Stenfeldt Mathiasen I.
      • De Meyts P.
      The insulin-like growth factors and insulin-signalling systems: An appealing target for breast cancer therapy?.
      ,
      • Gross J.M.
      • Yee D.
      The type-1 insulin-like growth factor receptor tyrosine kinase and breast cancer: Biology and therapeutic relevance..
      ), hepatocyte growth factor (
      • Haslam S.Z.
      • Woodward T.L.
      Host microenvironment in breast cancer development: Epithelial-cell-stromal-cell interactions and steroid hormone action in normal and cancerous mammary gland..
      ,
      • Pollard J.W.
      Tumour-stromal interactions. Transforming growth factor-β isoforms and hepatocyte growth factor/scatter factor in mammary gland ductal morphogenesis..
      ,
      • Elliott B.E.
      • Hung W.L.
      • Boag A.H.
      • Tuck A.B.
      The role of hepatocyte growth factor (scatter factor) in epithelial-mesenchymal transition and breast cancer..
      ), as well as several other factors. Some of these proteins represent important candidates for cancer therapy targeting the complex and dynamic network of interactions that modulate the biology and activity of tumor cells (
      • Gross J.M.
      • Yee D.
      The type-1 insulin-like growth factor receptor tyrosine kinase and breast cancer: Biology and therapeutic relevance..
      ,
      • Shao W.
      • Brown M.
      Advances in estrogen receptor biology: Prospects for improvements in targeted breast cancer therapy..
      ,
      • Ulrich A.
      Molecular targets in cancer therapy and their impact on cancer management..
      ,
      • Shawver L.K.
      • Slamon D.
      • Ullrich A.
      Smart drugs: Tyrosine kinase inhibitors in cancer therapy..
      ,
      • Erickson A.C.
      • Barcellos-Hoff M.H.
      The not-so innocent bystander: the microenvironment as a therapeutic target in cancer..
      ).
      With the advent of enabling technologies within proteomics, it is now feasible to undertake a systematic characterization of the proteins that are released to the interstitial space by all the cell types resident in the tumor microenvironment. The main challenge, however, remains the application of these technologies to clinically relevant samples in a well-defined clinical and pathological framework. Toward this aim, efforts have been made to characterize the protein composition of the nipple aspirate fluid (NAF), which contains proteins directly secreted by the ductal and lobular epithelium, in patients with breast cancer using proteomic technologies (
      • Varnum S.M.
      • Covington C.C.
      • Woodbury R.L.
      • Petritis K.
      • Kangas L.J.
      • Abdullah M.S.
      • Pounds J.G.
      • Smith R.D.
      • Zangar R.C.
      Proteomic characterization of nipple aspirate fluid: Identification of potential biomarkers of breast cancer..
      ). This study identified 64 proteins, some of which, like cathepsin D and osteopontin, had previously been found to be deregulated in serum or tumor tissue from women with breast cancer (
      • Varnum S.M.
      • Covington C.C.
      • Woodbury R.L.
      • Petritis K.
      • Kangas L.J.
      • Abdullah M.S.
      • Pounds J.G.
      • Smith R.D.
      • Zangar R.C.
      Proteomic characterization of nipple aspirate fluid: Identification of potential biomarkers of breast cancer..
      ). NAF has also been analyzed by surface-enhanced laser desorption ionization time-of-flight, and protein signatures have been discovered that appear to differentiate breast cancer fluid from healthy controls (
      • Paweletz C.P.
      • Trock B.
      • Pennanen M.
      • Tsangaris T.
      • Magnant C.
      • Liotta L.A.
      • Petricoin 3rd., E.F.
      Proteomic patterns of nipple aspirate fluids obtained by SELDI-TOF: Potential for new biomarkers to aid in the diagnosis of breast cancer..
      ). Other proteins detected in NAF include carcinoembryonic antigen, prostate-specific antigen, lactate dehydrogenase, basic fibroblast growth factor, vascular endothelial growth factor, and c-erbB-2 (
      • Shao Z.M.
      • Nguyen M.
      Nipple aspiration in diagnosis of breast cancer..
      and references therein).
      In our laboratories, we are interested in identifying novel diagnostic biomarkers as well as more selective targets for therapeutic intervention in breast cancer using clinically relevant samples and cutting-edge technologies from proteomics, functional genomics, and cellular and molecular biology (
      • Celis J.E.
      • Gromov P.
      • Gromova I.
      • Moreira J.M.
      • Cabezon T.
      • Ambartsumian N.
      • Grigorian M.
      • Lukanidin E.
      • Thor Straten P.
      • Guldberg P.
      • Bartkova J.
      • Bartek J.
      • Lukas J.
      • Lukas C.
      • Lykkesfeldt A.
      • Jaattela M.
      • Roepstorff P.
      • Bolund L.
      • Orntoft T.
      • Brunner N.
      • Overgaard J.
      • Sandelin K.
      • Blichert-Toft M.
      • Mouridsen H.
      • Rank F.E.
      Integrating proteomic and functional genomic technologies in discovery-driven translational breast cancer research..
      ). To achieve these goals, however, it is first necessary to identify sources of potential biomarkers that mirror the in vivo situation as accurately as possible, and that are amenable to multifactorial analysis. Toward this aim, we present here a novel and potentially highly promising source of biomarkers, the tumor interstitial fluid (TIF) that perfuses the tumor microenvironment in invasive ductal carcinomas of the breast. Besides providing the first overview of the TIF proteome, our results open the possibility for the systematic search of diagnostic biomarkers and targets for therapeutic intervention using this novel resource.

      EXPERIMENTAL PROCEDURES

       Patient Selection

      Access to large tumors was deemed essential, as the chosen approach required substantial amounts of tissue material. Consequently, women with primary operable high-risk
      The criteria for high-risk cancer applied by the Danish Cooperative Breast Cancer Group are age below 35 years old, and/or tumor diameter of more than 20 mm, and/or histological malignancy 2 or 3, and/or, negative estrogen and progesterone receptor status, and/or positive axillary status.
      invasive breast cancer were selected for this study (
      • Celis J.E.
      • Gromov P.
      • Gromova I.
      • Moreira J.M.
      • Cabezon T.
      • Ambartsumian N.
      • Grigorian M.
      • Lukanidin E.
      • Thor Straten P.
      • Guldberg P.
      • Bartkova J.
      • Bartek J.
      • Lukas J.
      • Lukas C.
      • Lykkesfeldt A.
      • Jaattela M.
      • Roepstorff P.
      • Bolund L.
      • Orntoft T.
      • Brunner N.
      • Overgaard J.
      • Sandelin K.
      • Blichert-Toft M.
      • Mouridsen H.
      • Rank F.E.
      Integrating proteomic and functional genomic technologies in discovery-driven translational breast cancer research..
      ). All 16 patients involved had no previous surgery to the breast and did not receive preoperative treatment. Patients underwent mastectomy (Fig. 1A), including axillary dissection. All tumors were diagnosed as invasive ductal carcinomas. Data concerning age of the patient, size of the tumor, histological grade, HER-2/neu status, axillary nodal status, and estrogen and progesterone receptor status are given in Table I. The project was approved by the Scientific and Ethical Committee of the Copenhagen and Frederiksberg Municipalities (KF 01-069/03).
      Figure thumbnail gr1
      Fig. 1Mastectomy from a high-risk patient.A, tumor. B, small tumor pieces used to collect the TIF as described under “Experimental Procedures.”
      Table IInvasive ductal carcinomas
      Patient numberAgeSizeHistological gradeHer-2/neu status
      Determined using Hercep Test™ (Dako).
      Axillary nodal status
      Positive/total.
      Receptor status
      ER
      ER, estrogen receptor.
      PGR
      PGR, progesterone receptor.
      mm
      274720200 /15ER+PGR+
      285324333 /12ER+PGR+
      29451320NDER+PGR+
      306926231 /6ER+PGR+
      318922116 /15ER+PGR+
      336821231 /11ER+PGR+
      346923213 /12ER+PGR+
      364850238 /18ER+PGR+
      378016100 /1ER+PGR−
      386225222 /13ER+PGR−
      397330120 /13ER+PGR−
      405230220 /14ER+PGR+
      416835331 /18ER−PGR−
      425421325 /10ER−PGR−
      444270217 /11ER+PGR+
      456528124 /14ER+PGR+
      a Determined using Hercep Test™ (Dako).
      b Positive/total.
      c ER, estrogen receptor.
      d PGR, progesterone receptor.

       Sample Collection and Handling

      Tissue biopsies were collected from the Pathology Department at Righshospitalet 20–30 min after surgery and were rapidly transported on ice to the Institute of Cancer Biology for further processing. A fraction of each sample was stored as archival material.

       TIF Collection

      About 0.25 g of clean fresh tissue biopsies were cut into small pieces (1–3 mm3) (Fig. 1B), washed carefully in 5 ml of phosphate buffered saline (PBS), and placed in a 10-ml conical plastic tube containing 0.8 ml of PBS. Samples were incubated for different periods of time (0–24 h) at 37 °C in a humidified CO2 incubator. TIFs used in this study were collected after 1 h of incubation. Thereafter, the samples were centrifuged at 1,000 rpm for 2 min and the supernatant was aspirated with the aid of an elongated Pasteur pipette. Samples were further centrifuged at 5,000 rpm for 20 min in a refrigerated centrifuge (4 °C). The final supernatant, with a protein concentration that ranged from 1 to 4 mg/ml, was freeze-dried and resuspended in 0.5 ml of lysis solution (
      • O’Farrell P.H.
      High resolution two-dimensional electrophoresis of proteins..
      ). A fraction of the TIF was kept at –20 °C for antibody array-based analysis.

       Two-dimensional Gel Electrophoresis and Immunoblotting

      Freeze-dried fluids resuspended in lysis solution were subjected to both isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) two-dimensional polyacrylamide electrophoresis (2D PAGE) as previously described (
      • Celis J.E.
      • Trentemølle S.
      • Gromov P.
      Gel-based proteomics: High-resolution two-dimensional gel electrophoresis of proteins. Isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE).
      ). Between 20 and 35 μl of sample were applied to the first dimension, and at least three IEF and NEPHGE gels were run for each sample. Proteins were visualized using a silver staining procedure compatible with mass spectrometry analysis (
      • Gromova I.
      • Celis J.E.
      Protein detection in gels by silver staining: A procedure compatible with mass-spectrometry.
      ). Immunoblotting was performed as previously described (
      • Celis J.E.
      • Gromov P.
      High-resolution two-dimensional gel electrophoresis and protein identification using western blotting and ECL detection..
      ).

       Protein Identification by Mass Spectrometry

       In-gel Digestion Protocol—

      Protein bands were excised from the dry gels followed by rehydration in water for 30 min at room temperature. The gel pieces were detached from the cellophane film, rinsed twice with water, and cut into about 1-mm2 pieces with subsequent additional washes. Proteins were “in-gel” digested with bovine trypsin (unmodified, sequencing grade; Roche Diagnostics, Mannheim, Germany) for 12 h as described by Shevchenko and colleagues (
      • Shevchenko A.
      • Wilm M.
      • Vorm O.
      • Mann M.
      Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels..
      ). The reaction was stopped by adding trifluoroacetic acid (TFA, up to 0.4%) followed by shaking for 20 min at room temperature to increase peptide recovery. In most cases, peptides were analyzed using the supernatant (SN). In the few cases where the amounts of peptides were too low, or when no conclusive identification was achieved by peptide fingerprinting using the SN, the remaining amount of SN (∼10 μl) as well as the peptides additionally extracted from the gel pieces with 1% TFA and 50% acetonitrile (ACN) were concentrated on micro columns containing C18-based 3-mm Empore plugs (
      • Rappsilber J.
      • Ishihama Y.
      • Mann M.
      Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics..
      ). Peptides were eluted from the column with 50% ACN/0.2% TFA directly on the target and co-crystallized with cyano matrix (2 mg/ml cyano-4-hydroxycinnamic acid in 0.5% TFA/ACN, 1.2 v/v). The extraction procedure strongly increased the amount of peptides, thus allowing direct sequence analysis of low-intensity peptides.

       Probe Preparation and Acquisition of the MALDI-TOF Spectra—

      Samples were prepared for analysis by applying 0.8 μl of digested supernatant or microcolumn-eluted material on the surface of a 400/384 AnchorChip target (Bruker Daltonik, Billerica, MA), followed by co-crystallization with 0.3-μl α-cyano matrix. After drying, the droplets were washed twice with 2% TFA to remove contamination from the samples.
      Mass spectrometry was performed using a Reflex IV MALDI-TOF mass spectrometer equipped with a Scout 384 ion source. All spectra were obtained in positive reflector mode with delayed extraction, using an accelerating voltage of 28 kV. Each spectrum represented an average of 100–200 laser shots, depending on the signal-to-noise ratio. The resulting mass spectra were internally calibrated by using the auto-digested tryptic mass values (805.417/906.505/1153.574/1433.721/2163.057/2273.160) visible in all spectra. Calibrated spectra were processed by the Xmass 5.1.1 and BioTools 2.1 software packages (Bruker Daltonik). All spectra were analyzed manually.
      Spectra originating from parallel protein digestions were compared pairwise to discard common peaks derived either from trypsin auto-digestion or from contamination with keratins. Only unique peptides present in the spectra were used in the first search. Database searching was performed against a comprehensive nonredundant database using the MASCOT 1.8 software (
      • Perkins D.N.
      • Pappin D.J.
      • Creasy D.M.
      • Cottrell J.S.
      Probability-based protein identification by searching sequence databases using mass spectrometry data..
      ), without restriction on the protein molecular mass and taxonomy. Because proteins were recovered from dried gels, a number of fixed modifications (acrylamide modified cystein, i.e. propionamide/carbamidomethylation) as well as variable ones (methionine oxidation and protein N terminus acetylation), were included in the search parameters. The peptide tolerance did not exceed 50 ppm and as a maximum only one missed cleavage was allowed. Only protein identifications with score greater than p < 0.05 were considered to be positive. Additionally, peptide mass fingerprinting analysis was performed using the MS-Fit program (ProteinProspector; UCSF Mass Spectrometry Facility, London, UK). We also used the Find-Mod software (ProteinProspector) to check any unmatched peptides for potential protein post-translational modifications. The second search was performed for all identifications as follows: 1) the predicted peptide digest was compared with the experimental one to reveal additional peptides present within the spectra; 2) the unmatched molecular mass values from the initial search were applied for extra search with the same reproducibility requirements for identification of the second and the third proteins in the spot. In all cases in which the intensity of the peptides allowed sequence analysis (either SN or extracted material), post-source decay (PSD) was performed as an additional mean to confirm the identity of the proteins identified by post-translational modifications. The following PSD search parameters were used: peptide tolerance 50 ppm and MS/MS tolerance 1 Da without any restriction on the protein molecular mass and taxonomy. Because the amount of peptides extracted from the silver-stained gels did not yield overall peaks intensities high enough to allow multiple peptide sequencing (prerequirement for conclusive PSD analysis), the identification of proteins was never made solely based on PSD analysis. Positive protein identification was achieved in 80% of the cases with an average sequence coverage of ∼33%.

       Antibody Arrays—

      Detection of multiple cytokines present in TIFs was done using array-based technology. For this purpose, RayBio™ Cytokine Antibody Arrays 5.1 were purchased from RayBiotech, Inc. (Atlanta, GA). Each array was incubated with 0.25 ml of TIF at 4 °C overnight, and bound cytokines were detected according to the manufacturer’s instructions. The sensitivity of the cytokine antibody array ranges from 1 to 2,000 pg/ml.

       GTP-binding Proteins—

      2D gel protein profiling of small GTP-binding proteins was carried out using the [α-32P]GTP blot overlay assay essentially as previously described (
      • Gromov P.S.
      • Celis J.E.
      Several small GTP-binding proteins are strongly down regulated in SV40 transformed human keratinocytes and may be required for the maintainance of the normal phenotype..
      ,
      • Gromov P.
      • Celis J.E.
      Blot overlay assay for small GTP-binding proteins identification.
      ). Protein samples were subjected to both IEF and NEPHGE 2D PAGE, and the proteins were electro-transferred to nitrocellulose membranes as previously described (
      • Celis J.E.
      • Gromov P.
      High-resolution two-dimensional gel electrophoresis and protein identification using western blotting and ECL detection..
      ). The nitrocellulose filters were rinsed twice with a solution containing 50 mm Tris-HCl, pH 7.6, 10 μm MgCl2, and 0.3% Tween 20 and were incubated for 60 min in the same buffer, but containing 100 mm dithiothreitol, 100 μm ATP, and 1 nm [α-32P]GTP (final concentration 1 μCi [α-32P]GTP/ml). The nitrocellulose membranes were then washed four times, 5 min each, in the same buffer lacking dithiothreitol, ATP, and [α-32P]GTP. Air-dried membranes were subjected to phosphorimaging (FLA3000; Fuji, Tokyo, Japan) and/or exposed for autoradiography at −70 °C with an intensifying screen.

       Antibodies—

      Anti-peptide antibodies against thioredoxin, the tumor controlled protein, and 14-3-3σ were prepared by Eurogentec (Brussels, Belgium). Antibodies against metastasin (Prolifia Inc., Tucson, AZ), neutrophils (neutrophil elastase; Dako, Glostrup, Denmark), macrophages (CD68; Dako), mast cells (mast cell tryptase; Dako), B cells (CD20 cy; Dako), MMPs (Oncogene Research Products, San Diego, CA), and albumin (Sigma, St. Louis, MO) were obtained from commercial sources. Antibodies against annexins I and II, cathepsin D, galectin 1, and Cu-Zn superoxide dismutase were kindly provided by B. Pepinsky (Biogen Research Corporation, Cambridge, UK), R. Raclons (Münster University, Münster, Germany), R. Joubert-Caron (Unité de Formation et de Recherche Sante, Bobigny, France), and B. Basse (Aarhus University, Aarhus, Denmark), respectively.

       Immunohistochemistry (IHC)—

      Fresh tumor blocks were immediately placed in formalin fixative and paraffin-embedded for archival use. Five-micrometer sections were cut from the paraffin-embedded tissue blocks and mounted on Super Frost Plus slides (Menzel-Gläser, Braunschweig, Germany), baked at 60 °C for 60 min, deparaffinized, and rehydrated through graded alcohol rinses. Heat-induced antigen retrieval was performed by immersing slides in 10 mm citrate buffer (pH 6.0) and microwaving in a 750-W microwave oven for 10 min. The slides were then cooled at room temperature for 20 min and rinsed abundantly in tap water. Nonspecific staining of slides was blocked (10% normal goat serum in PBS buffer) for 15 min, and endogenous peroxidase activity was quenched using 0.3% H2O2 in methanol for 30 min. Antigen was detected with a relevant primary antibody, followed by a suitable secondary antibody conjugated to a peroxidase complex (horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibody; Dako). Finally, color development was done with 3,3′-diaminobenzidine (Pierce, Rockford, IL) as a chromogen to detect bound antibody complex. Slides were counterstained with hematoxylin. Standardization of the incubation and development times allowed an accurate comparison of expression levels in all cases.

      RESULTS

       Characterization of the TIF

      TIF collected from 16 high-risk patients with invasive ductal carcinomas was analyzed by 2D PAGE as described under “Experimental Procedures” (
      • Celis J.E.
      • Trentemølle S.
      • Gromov P.
      Gel-based proteomics: High-resolution two-dimensional gel electrophoresis of proteins. Isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE).
      ). Silver-stained 2D gels of acidic (IEF) and basic (NEPHGE) TIF proteins recovered from a representative tumor (tumor 41, grade 3) are shown in Figs. 2 and 3, respectively. A total of 1,147 polypeptides (842 in IEF and 305 in NEPHGE) were detected in these reference gels using PDQUEST 7.1 software (BioRad, San Francisco, CA); of these, about 200 proteins, including α-enolase (IEF 22, NEPHGE 1) and triosephosphate isomerase (IEF 148, NEPHGE 19), migrated in the IEF and NEPHGE gels (Figs. 2 and 3) (
      • Celis J.E.
      • Leffers H.
      • Rasmussen H.H.
      • Madsen P.
      • Honore B.
      • Gesser B.
      • Dejgaard K.
      • Olsen E.
      • Ratz G.P.
      • Lauridsen J.B.
      • Basse B.
      • Andersen A.H.
      • Walbum E.
      • Brandstrup B.
      • Celis A.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      The master two-dimensional gel database of human AMA cell proteins: Towards linking protein and genome sequence and mapping information (update 1991)..
      ). Visual inspection of the protein profiles of TIFs collected from all 16 tumors studied showed the absence of keratins, a family of cytoskeletal proteins that are abundantly represented in whole lyzates of invasive ductal carcinomas (compare Figs. 2 and 4A) (
      • Moll R.
      Cytokeratins as markers of differentiation in the diagnosis of epithelial tumors..
      ). In addition, TIFs lack the majority of the nuclear proteins that are ubiquitously present in tumor cells as well as in other cell types present in the tumor microenvironment (not shown, but see proteomics.cancer.dk), arguing against cellular lysis as a main source of the protein spots detected in the 2D gels.
      Figure thumbnail gr2
      Fig. 2TIF proteins from tumor 41 separated by IEF 2D PAGE and stained with silver nitrate. The numbers indicated in the gels correspond to the gel numbers given in . Some major proteins exhibiting charge trains are underlined.
      Figure thumbnail gr3
      Fig. 3TIF proteins from tumor 41 resolved by NEPHGE 2D PAGE and stained with silver nitrate. The numbers indicated in the gels correspond to the gel numbers given in . Some major proteins exhibiting charge trains are underlined.
      Figure thumbnail gr4
      Fig. 4IEF 2D PAGE of whole tumor lyzates, serum, and externalized proteins from breast tissues.A, selected 2D gel area of whole-protein lyzates from tumor 41 stained with silver nitrate. The position of the keratins is indicated for reference. B, 2D gel of serum proteins. A few major proteins are indicated for reference. C, TIF proteins collected from tumor 38. Arrows indicate examples of proteins that are differentially expressed as compared with TIF 41 (see ). D, 2D gel of externalized proteins from axillary nodal metastases 42 (MIF 42). E, 2D gel of externalized proteins from nonmalignant breast epithelial tissue 41 (NIF 41). F, 2D gel of externalized proteins from fat tissue 27 (FIF 27). The positions of actin and apolipoprotein A-I are indicated as a reference in B–F.
      TIF proteins resolved by 2D PAGE were identified using one or a combination of procedures that included MALDI-TOF MS (Fig. 5A), Western immunoblotting (Fig. 5B), and comparison with the master gels of the keratinocyte 2D PAGE database (proteomics.cancer.dk). Cytokine-specific antibody arrays were also used to detect a well-defined set of cytokines and growth factors using unfractionated TIFs (Fig. 5C). In general, identifications were performed using TIFs collected from several tumors. Two hundred sixty-seven primary translation products have been identified to date, and these are listed in alphabetical order in Table II, together with the method(s) of identification, accession number, protein number in the IEF and NEPHGE 2D gels presented in Figs. 2 and 3, as well as presence in the plasma/serum (
      • Adkins J.N.
      • Varnum S.M.
      • Auberry K.J.
      • Moore R.J.
      • Angell N.H.
      • Smith R.D.
      • Springer D.L.
      • Pounds J.G.
      Toward a human blood serum proteome: Analysis by multidimensional separation coupled with mass spectrometry..
      ,
      • Pieper R.
      • Su Q.
      • Gatlin C.L.
      • Huang S.T.
      • Anderson N.L.
      • Steiner S.
      Multi-component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome..
      ,
      • Anderson N.L.
      • Polanski M.
      • Pieper R.
      • Gatlin T.
      • Tirumalai R.S.
      • Conrads T.P.
      • Veenstra T.D.
      • Adkins J.N.
      • Pounds J.G.
      • Fagan R.
      • Lobley A.
      The human plasma proteome: A nonredundant list developed by combination of four separate sources..
      ) and NAF proteomes (
      • Varnum S.M.
      • Covington C.C.
      • Woodbury R.L.
      • Petritis K.
      • Kangas L.J.
      • Abdullah M.S.
      • Pounds J.G.
      • Smith R.D.
      • Zangar R.C.
      Proteomic characterization of nipple aspirate fluid: Identification of potential biomarkers of breast cancer..
      ). Several of the proteins identified by 2D gel/mass spectrometry exhibited post-translational modifications, and these accounted for at least an additional 150 spots in the 2D gels. Given space limitations, proteins showing major charge trains are underlined in Figs. 24. Proteins identified to date include, but are not limited to, polypeptides involved in cell proliferation, invasion, angiogenesis, metastasis, inflammation, protein synthesis, energy metabolism, oxidative stress, the actin cytoskeleton assembly, protein folding, and transport. As expected, TIF contained some major serum proteins, but its overall 2D PAGE protein profile was remarkably different to that of serum (compare Figs. 2 and 4B). Abundant serum proteins such as albumin, ferritin, α1 antichymotrypsin, α1 protease inhibitor, α1 β glycoprotein, haptoglobins 1 and 2, and immunoglobulin light and heavy chains were readily detected (Table II, Fig. 2), although a few classical serum proteins like apolipoproteins C-III and J (indicated in Fig. 4B) were not present, at least at the levels normally observed in serum.
      Figure thumbnail gr5
      Fig. 5Protein identification.A, MALDI-TOF MS. The left panel (a) shows an example of protein identification performed by peptide fingerprinting and PSD analysis of a spot showing strong intensity in silver-stained gels (IEF 96, ). A total of 0.8 μl of SN was used for the analysis. The right panel (b) shows an example of protein identification performed by peptide fingerprinting and PSD analysis of a weakly stained spot (IEF 44, ). Peptide fingerprinting as well as PSD analysis was performed by using extracted peptides concentrated on a microcolumn. Dots in the spectra indicate peaks derived from trypsin auto-digestion. B, Western immunoblotting of TIF proteins reacted with various antibodies. A mixture of several antibodies was applied to the blot shown in a. C, cytokine-specific antibody arrays incubated with TIFs collected from tumors 44 and 45, respectively. Antibodies present in the grid are listed above the arrays, as well as in the . Positive and negative controls are included (see grid).
      Table IITIF proteins
      Protein nameMethod of identification
      MS, Identification of proteins separated by 2D gels using MALD-TOF MS; CKD, identification of 2D gel spots by matching the protein patterns to the master 2D gel keratinocyte database (proteomics.cancer.dk); IB, protein identification by Western 2D gel immunoblotting; AA, protein identification using cytokine-specific antibody arrays.
      Spot number in reference 2D gels
      The position of the identified proteins in the 2D gel is indicated with either an IEF (Fig. 2) or a NEPHGE number (Fig. 3). Proteins migrating in both directions are indicated with both an IEF and a NEPHGE number. A number of proteins exhibited multiple forms and some of them are underlined in Figs 2 and 3.
      Presence reported in plasma/serum
      Presence in the plasma/serum proteome (52): 1, identified by only one approach; 2, confirmed by identification using at least two approaches; 3, occurring in all four sets.
      Presence reported in NAF
      Presence in the NAF proteome (35).
      Accession number
      The Swiss-Prot accession number is given for all identified proteins whenever the same peptides are recognized both in Swiss-Prot and NCBInr. Splice variants could not be distinguished in most cases due to the low amounts of starting material.
      MALDI-TOF MS
      For MALDI-TOF MS scores (Score) obtained from Mascot searches, the number of peptides (Peptides) recognized are indicated together with the mass and position of the peptides that were used for PSD analysis (PSD). The peptide sequence coverage is listed as a percentage of the entire protein sequence (Coverage).
      ScorePeptides and PSDCoverage
      %
      6-phospho-gluconolactonase (6PGL)MSIEF 1O953361501047
      14-3-3 protein βMS, CKDIEF 2YesP319461201145
      14-3-3 protein εMSIEF 3P4265598838
      14-3-3 protein ηMS, CKDIEF 4Q049171301042
      14-3-3 protein σMS, CKD, IBIEF 5Yes (1)P31947100940
      14-3-3 protein ζ/σMS, CKDIEF 6Yes (1)P293121201548
      1-309, T lymphocyte-secreted protein I-309AAP22362
      37/67 laminin receptor, 40S ribosomal proteinMSIEF 7P0886580722
      AAA ATPase p97MS, CKDIEF 8P55072921930
      Actin-like protein 3, actin-related protein 3MSIEF 9P3239118514 (PSD: 1214.56 (266–275))31
      Acylamino-acid-relising enzyme, oxidized protein hydrolaseMSIEF 10P13798801112
      Acylaminoacyl-peptidaseMSIEF 11gi|2118063901115
      Acyl-CoA-binding protein (ACBP), diazepam binding inhibitor (DBI)MSP071081087 (PSD: 1535.70 (1–13 N-Acetyl))40
      Actin regulatory protein (CAP-G)MSP40012112011 (PSD: 11389.74 (307–319))37
      AdenosylhomocysteinaseMSIEF 12Yes (1)P235261551735
      AlbuminMS, IBIEF 13Yes (2)YesP027681451285
      Alcohol dehydrogenaseMSIEF 14P145501201028
      Aldehyde dehydrogenase 1MSIEF 15Yes (1)gi|2183299998 (PSD: 1540.71 (411–425))15
      Aldehyde dehydrogenase 9MSIEF 16P49189801030
      α 1 antichymotrypsinMSIEF 17Yes (3)YesP010111001022
      α 1 protease inhibitorMSIEF 18Yes (1)P01009120816
      α 1-B glycoproteinMSIEF 19Yes (2)P0421785614
      α-actinin 4MSIEF 20Yes (1)O437071001011
      α-centractinMSP42024931027
      α soluble SNFattachment protein alpha (SNAP-α)MSIEF 21P549201701450
      α-enolase, enolase 1MS, CKDIEF 22, NEPHGE 1P067331021125
      α-tubulin 1 chainMS, CKDIEF 23P0520994818
      Aminoacylase-1MSIEF 24Q031541001130
      AngiogeninAAYes (1)P03950
      Annexin IIB, CKDIEF 25P04083
      Annexin IIIBIEF 26, NEPHGE 2Yes (1)P07355
      Annexin IVCKDIEF 27P09525
      Annexin VCKDIEF 28Yes (1)P08758
      Apolipoprotein A-IMSIEF 29Yes (3)P026471501538
      ATP synthase, α chainCKDNEPHGE 3P25705
      ATP synthase, β chainCKDIEF 30Yes (2)P06576
      ATP-dependent DNA helicase II, Ku autoantigen protein p86MSIEF 31P130101201916
      B lymphocyte chemoattractant (CXC chemokine BLC)AAO43927
      β actinMS, CKDIEF 32Yes (2)YesP02570802235
      β tubulinCKDIEF 33P07437
      BPNT1 proteinMSIEF 34gi|173895331411142
      Brain derived neurotrophic factor (BDNF)AAP23560
      Carbonic anhydrase 1MSIEF 38Yes (2)P009151381050
      Cathepsin BIBP07858
      %
      Cathepsin DIB, MSYes (2)YesP073398810>22
      CCG1-interacting factor BMSIEF 39Q96|U4807 (PSD: 1593.79 (9–22))27
      Chaperonin containing TCP 1, subunit β (CCT-β)MS, CKDIEF 40P783711811535
      Chaperonin containing TCP 1, subunit ε (CCT-ε)CKDIEF 41P48643
      Chaperonin containing TCP 1, subunit θ (CCT-θ)CKDIEF 42P50990
      Chloride intracellular channel protein 1MSIEF 43O00299110739
      Chromosome 7 open reading frame 24MSIEF 44gi|13129018745 (PSD: 1253.71 (35–45))21
      Chemokine β 8-1AAP55773
      Coactosin-like proteinMSIEF 45Q14019129750
      CofilinMS, CKDNEPHGE 4Yes (1)P2352895841
      Collagen α 1 (VI) chainMSIEF 46Yes (1)YesP121091171520
      Coronin like protein p57MSIEF 47Yes (1)P31146861020
      Cyclin A/CDK2-associated protein p19MSIEF 48BP34991139950
      Cyclophilin AMSNEPHGE 5P05092110637
      Cyclophilin BMSNEPHGE 6P232841261243
      Cytosol aminopeptidase, leucine aminopeptidaseMSIEF 49gi|126433941161125
      E-CadherinMSIEF 50Yes (1)P12830907 (PSD: 1114.61 (216–224))9
      Elastase inhibitorCKDIEF 51P30740
      Elongation factor 1 α 1MS, CKDNEPHGE 7P04720931020
      Elongation factor 1 βMSIEF 52P2453488732
      Elongation factor 2 (EF-2)MS, CKDIEF 53Yes (1)P136392252630
      Elongation factor 1-γ (EF-1-γ)MSP266411391228
      EndoplasminCKDIEF 54Yes (1)P14625
      Eosinophil lysophospholipaseMSIEF 55Q05315556 (PSD: 1026.60 (135–142))20
      Eotaxin precursorAAP51671
      Eotaxin-2AAYes (1)O00175
      Eotaxin-3AAQ9Y258
      Epidermal growth factor (EGF)AAYesP01133
      Epithelial neutrophil-activating protein 78 (ENA-78)AAYes (1)P42830
      Esterase DMSIEF 56P1076893723
      F-actin cappling protein α-1 subunit, CapZ α-1MSIEF 57P5290770525
      F-actin cappling protein β subunit, CapZ βMSIEF 58P4775680721
      Fatty acid binding protein 5, epidermal (E-FABP)MSIEF 59Q014691501457
      Fatty acid-binding protein, adipocyte (A-FABP)MSIEF 60P1509080625
      Fatty acid-binding protein, heart (H-FABP), mammary-derived growth inhibitor (MDGI)MSIEF 61Yes (1)P0541380637
      Ferritin light chain, L-subunitMSIEF 62Yes (1)P02792655 (PSD: 1491.7 (40–52))30
      Fibrinogen β chain precursor (Contains: Fibrinopeptide B)MSYes (1)P0267516113 (PSD: 1032.33 (420–427))33
      Fibroblast growth factor-4 (FGF-4)AAP08620
      Fibroblast growth factor-6 (FGF-6)AAP10767
      Fibroblast growth factor-7 (FGF-7)AAP21781
      Fibroblast growth factor-9 (FGF-9)AAP31371
      Filamin 1 (C-terminal part, degradation product)MSIEF 63P2133316018 (PSD: 1533.75 (442–455))26
      Flt-3 ligand, Fms-related tyrosine kinase 3 ligandAAP49771
      FractalkineAAP78423
      Fructose-bisphosphate aldolase AMSNEPHGE 8Yes (1)P040751001245
      Fumaryl acetoacetaseMSIEF 64Yes (1)P16930968 (PSD: 1273.66 (132–142))25
      Galectin-1MS, IBIEF 65P093821001177
      %
      G-β 1 (Transducin β chain 1)CKDIEF 66P04901
      G-β 2 (Transducin β chain 2)CKDIEF 67P11016
      Gelsolin isoform bMSIEF 68Yes (1)Yesgi|38044288798 (PSD: 1244.58 (1–10))9
      Gelsolin precursor, plasma (degradation product)MSIEF 69Yes (3)P063961071225
      Glia maturation factor β (GMF-β)MS, AAIEF 70Yes (1)P17774102850
      Glial cell derived neurotrophic factor (GDNF)AAP39905
      Glucose-6-phosphate 1-dehydrogenaseMSIEF 71Yes (1)P114131161135
      Glucose-regulated protein 75 (grp 75)MS, CKDIEF 72P386461331216
      Glucose-regulated protein 78 (grp 78)MS, CKDIEF 73Yes (1)P110211401372
      Glutathione S-transferase PMS, CKDIEF 74P092111971366
      Glyceraldehyde 3-phosphate dehydrogenaseMS, CKDNEPHGE 9P003541751441
      Glyoxalase 1 (GLO1)MSIEF 75Q0476094826
      Granulocyte chemoattractant protein 2 (GCP-2)AAP80162
      Granulocyte colony stimulating factor (GCSF)AAYes (1)Q99062
      Granulocyte-macrophage stimulating factor (GM-CSF)AAYes (1)P04141
      Growth-regulated oncogene (GRO)AAP09341
      Haptoglobin 1MSIEF 76Yes (3)P007391702040
      Haptoglobin 2 (N-terminal part)MSIEF 77Yes (1)P0073870715
      Heat shock 70-kDa protein 4 isoformMSIEF 78Yes (1)P349329812 (PSD: 1495.69 (20–33))13
      Heat shock protein 27 (hsp 27)MS, CKDIEF 79P04792109731
      Heat shock protein 60 (hsp 60)MS, CDKIEF 80Yes (2)P108091231119
      Heat shock protein 70 protein 2 (hsp 70)CDKIEF 81Yes (1)P54652
      Heat shock protein 90 (hsp 90)MS, CDKIEF 82Yes (1)P079001992130
      Heme binding protein 1MSIEF 83gi|20336761117641
      Hemoglobin β chainMSIEF 84Yes (3)P020232121384
      Hepatocyte growth factor (HGF)AAYes (2)P14210
      Human epithelial mucin, Muc 1IBP15941
      Immunoglobulin G light chain, κIBIEF 85, NEPHGE 10Yes (1)Yes
      Immunoglobulin G heavy chainIBIEF 86, NEPHGE 11Yes (1)Yes
      Importin β-1 subunit, Importin 90MSIEF 87Q149741011215
      Initiation factor 3 subunit 2, (eIF3 p36)CKDIEF 88Q13347
      Initiation factor 4D, eIF-4 DCKDIEF 89P10159
      Insulin growth factor 1 (IGF-1)AAYesP01343
      Insulin growth factor binding protein 1 (IGFBP-1)AAYes (1)P08833
      Insulin growth factor binding protein 2 (IGFBP-2)AAYes (1)P18065
      Insulin growth factor binding protein 3 (IGFBP-3)AAYes (2)YesP17936
      Insulin-like growth factor binding protein-4 (IGFBP-4)AAP22692
      Interferon γ (IFN-γ)AAP01579
      Interferon-induced protein 10AAYes (1)P02778
      Interleukin 1-α (IL-1 α)AAP01583
      Interleukin 1-β (IL-1 β)AAYes (1)P01584
      Interleukin 2 (IL-2)AAYes (1)P01585
      Interleukin 3 (IL-3)AAP08700
      Interleukin 4 (IL-4)AAYes (1)P05112
      Interleukin 5 (IL-5)AAYes (1)P05113
      Interleukin 6 (IL-6)AAYes (2)P08887
      Interleukin 7 (IL-7)AAYes (1)P13232
      %
      Interleukin 8 (IL-8)AAYes (1)P10145
      Interleukin 10 (IL-10)AAYes (1)P22301
      Interleukin 12 (IL-12)AAYes (2)P29459
      Interleukin 13 (IL-13)AAYes (1)P35225
      Interleukin 16 (IL-16)AAYes (1)Q14005
      Isocitrate dehydrogenaseMSIEF 90O758741281124
      KAT proteinMSIEF 91gi|21636046112543
      Keratin 19 (N-terminal part)MSIEF 92P0872790716
      Keratin 10 (N-terminal part)MSIEF 93P13645801017
      Lactate dehydrogenase B chainMSIEF 94Yes (2)YesP071952041638
      Lactate dehydrogenase M chainMS, CKDNEPHGE 12P003381401525
      LeptinAAYes (1)P41159
      Leukemia inhibitory factor (LIF)AAYes (1)P15018
      LIGHT, tumor necrosis factor ligand superfamily member 14AAO43557
      LIM and SH protein 1MSIEF 95Yes (1)Q148471981648
      L-plastinMS, CKDIEF 96Yes (1)P137962442542
      Lyase adenylosuccinateMSIEF 97P305661081023
      Lysophospholipase isoform 1MSIEF 98gi|5453722504 (PSD: 1271.66 (134–146))17
      Lysolecithin acylhydrolaseMSIEF 99Q053151006 (PSD: 1439.32 (61–72))28
      Macrophage colony stimulating factor (MCSF)AAYes (1)P09603
      Macrophage inflammatory protein 1 βAAP13236
      Macrophage inflammatory protein 1d (MIP-1d)AAQ16663
      Macrophage inflammatory protein 3-α (MIP-3 α)AAP78556
      Macrophage-derived chemokine (MDC)AAO00626
      Malate dehydrogenaseMSIEF 100P409251311033
      MaspinMSIEF 101P369521271031
      Matrix metalloproteinase-1 (MMP-1)IBP03956
      Matrix metalloproteinase-2 (MMP-2)IBYes (1)P08253
      Matrix metalloproteinase-3 (MMP-3)IBP08254
      Matrix metalloproteinase-9 (MMP-9)IBYes (1)P14780
      Mesoderm inducing factor (MIF)AAP14174
      Metastasin (S100 calcium-binding protein A4)IBP26447
      Metastasis inhibition factor nm23, Nucleoside diphosphate kinase AMSIEF 102P15531927 (PSD: 1344.77 (7–18))40
      MIG, γ interferon induced monokineAAQ07325
      Monocyte chemoattractant protein 1 (MCP-1)AAP13500
      Monocyte chemoattractant protein 2 (MCP-2)AAP80075
      Monocyte chemoattractant protein 3 (MCP-3)AAP80098
      Monocyte chemotactic protein 4 (MCP-4)AAQ99616
      Neurotrophin 3AAP20783
      Neurotrophin 4AAP34130
      Neutrophil activating protein 2 (NAP-2)AAP02775
      Neutrophil defensin 3 precursorMSIEF 103Yes (1)P59666824 (PSD: 1117.54 (80–88))20
      NG-dimethylarginine dimethylaminohydrolase 2MSIEF 104968651731352
      Nuclear matrix protein NMP200MSIEF 105gi|7657381100815
      Nucleosome assembly protein 1-like 4MSIEF 106Q99733828 (PSD: 1336.64 (84–93))23
      Nucleoside diphosphate kinase BMSNEPHGE 13P22392991061
      Oncostatin MAAP13725
      %
      OsteoprotegerinAAO00300
      PAI I (Endothelial plasminogen activator inhibitor)IBYes (1)P05121
      Peroxiredoxin 1, natural killer-enhancing factor AMSIEF 107Q06830102828
      Peroxiredoxin 2, thioredoxin peroxidase 1MSIEF 108Yes (1)P321192401445
      Peroxiredoxin 3, antioxidant protein 1MSIEF 109Yes (2)P30048796 (PSD: 1206.68 (197–207))19
      Peroxiredoxin 6MSIEF 110P300411098 (PSD: 1409.66 (41–52))37
      Phosphoglycerate kinase (PGK)MS, CDKNEPHGE 14Yes (1)P00558811227
      Phosphoglycerate mutase isomerase B (PGAM-B)MSIEF 111Yes (2)P186691501245
      PIGFAAQ07326
      Plasminogen activator inhibitor 2 (PAI-2)CKDIEF 112Yes (1)P05120
      Platelet-derived growth factor BB (PDGF-BB)AAYes (1)P01127
      Profilin IMS, CKDNEPHGE 15P07737142973
      Progesterone membrane binding protein, steroid receptor protein DG6MSIEF 113O1517385622
      Proteasome subunit alpha type 2 (C3)MSNEPHGE 16Yes (1)P2578794823
      Proteasome activator 28-α (PA28 α)MSIEF 114Q0632310710 (PSD: 971.47 (191–198))40
      Proteasome activator 28-β (P28 β)MSIEF 115O9UL46118930
      Proteasome θ chainMSIEF 116P49720103815
      Protein disulfide isomerase A3 precursorMSIEF 117Yes (2)P301011501327
      Protein disulfide isomerase (PDI)MS, CKDIEF 118Yes (1)P072372351831
      Psoriasin (S100A7)IBIEF 119Yes (2)P31151
      Pulmonary and activation-regulated chemokine (PARC)AAP55774
      Pyruvate kinase M2 isoformCKDNEPHGE 17YesP14786
      Rab GDI βMSIEF 120P503951361436
      Rab18MSIEF 121Q9NP72555 (PSD: 1216.62 (1–10 N-Acetyl))17
      Rac 1CKDNEPHGE 18P15154
      Raf kinase binding proteinMSIEF 122, IEF 123P30086100745
      RantesAAYes (1)P15501
      Retinoblastoma DEAE box proteinMSIEF 124Q92499741117
      Retinoblastoma binding protein 4, retinoblastoma-binding protein p48MSIEF 125Q0902821361536
      Retinoic acid-binding protein II, CRABP-IIMSIEF 126P293731901169
      Rho GDP-dissociation inhibitor 1, Rho GDI 1MSIEF 127P52565103729
      Rho GDP-dissociation inhibitor 2, Rho GDI 2MSIEF 128P52566103933
      Ribonuclease/angiogenin inhibitor (RAI)MSIEF 129P134891481231
      Ribosome binding protein 1, ribosome receptor protein (C-terminal part)MSIEF 130Q962E91453042
      Rotamase, p59 proteinMSIEF 131Q027901091228
      Selenium binding protein 1MSIEF 132gi|163065501091123
      Serine/threonine phosphatase 2A, catalytic subunit αMSIEF 133P0532381622
      Sertolli cell factor (SCF)AAP21583
      SH3 domain-binding glutamic acid-rich-like proteinMSIEF 135O75368148967
      Smooth muscle protein 22-α, SM22-αMSIEF 135Q019951701041
      Spermidine synthaseMSIEF 136P1962385818
      Stress induced phosphoprotein 1 (STI1)MSIEF 137P319481011528
      %
      Stromal cell-derived factor 1-α (SDF-1α)AAYes (1)P48061
      Superoxide dismutase [Cu-Zn]MS, IBIEF 138Yes (1)P004412081256
      Superoxide dismutase [Mn]MS, CKDIEF 139P04179100522
      Synthetase glutamine-tRNAMSIEF 140P47897711216
      ThioredoxinMSIEF 141P1059988858
      Thrombopoetin (TRO)AAP40225
      Thymidine phosphorylase precursor (TdRPase), platelet-derived endothelial cell growth factor (PD-ECGF)MSIEF 142P199711601221
      Thymus and activation regulated chemokine (TARC)AAQ92583
      Thyroid protein p24MSIEF 143Q139382081460
      Tissue inhibitor of metalloproteinases-1 (TIMP-1)AAYes (1)P01033
      Tissue inhibitor of metalloproteinases-2 (TIMP-2)AAYes (1)P16035
      TransferrinMSIEF 144Yes (1)YesP027861301311
      Transforming growth factor β 1 (TGF-β 1)AAP01137
      Transforming growth factor β 2 (TGF-β 2)AAP08112
      Transforming growth factor β 3 (TGF-β 3)AAP10600
      TransketolaseMSIEF 145P294011001021
      Translationally controlled tumor protein (TCTP)MS, CKDIEF 146P13693855 (PSD: 1241.66 (101–110))21
      Transthyretin precursor, prealbuminMSIEF 147Yes (3)P02766757 (PSD: 1394.58 (56–68))44
      Triosephosphate isomerase (TIM))MS, CKDIEF 148, NEPHGE 19P601741701969
      Tropomyosin 1 α chain (N-terminal part)MSIEF 149P0949380930
      Tropomyosin α 3 chainMS, CKDIEF 150Yes (1)P067532321440
      Tropomyosin α 4 α chainMSIEF 151Yes (1)P072262402046
      Tropomyosin 3/spliced isoform 2MS, CDKIEF 152PO6753–012401850
      Tumor necrosis factor-α (TNF-α)AAYes (1)P01375
      Tumor necrosis factor-β (TNF-β)AAYes (1)P01374
      UbiquitinMS, CKDIEF 153P022481771085
      Ubiquitin carboxyl-terminal hydrolase 14MSIEF 154Yes (1)P54578991526
      Uroporphyrinogen decarboxylaseMSP061321721430
      Vacuolar ATP synthase, catalytic subunit AMSIEF 155P3860690914
      Vascular endothelial growth factor (VEGF)AAYes (1)P15692
      Vimentin (degradation product)MSYes (1)P086703493347
      WD-repeat protein 1, acting interacting protein 1MSIEF 156O7508370914
      XAG-2, secreted cement gland homologMSNEPHGE 20gi|5453541104741
      a MS, Identification of proteins separated by 2D gels using MALD-TOF MS; CKD, identification of 2D gel spots by matching the protein patterns to the master 2D gel keratinocyte database (proteomics.cancer.dk); IB, protein identification by Western 2D gel immunoblotting; AA, protein identification using cytokine-specific antibody arrays.
      b The position of the identified proteins in the 2D gel is indicated with either an IEF (Fig. 2) or a NEPHGE number (Fig. 3). Proteins migrating in both directions are indicated with both an IEF and a NEPHGE number. A number of proteins exhibited multiple forms and some of them are underlined in Figs 2 and 3.
      c Presence in the plasma/serum proteome (52): 1, identified by only one approach; 2, confirmed by identification using at least two approaches; 3, occurring in all four sets.
      d Presence in the NAF proteome (35).
      e The Swiss-Prot accession number is given for all identified proteins whenever the same peptides are recognized both in Swiss-Prot and NCBInr. Splice variants could not be distinguished in most cases due to the low amounts of starting material.
      f For MALDI-TOF MS scores (Score) obtained from Mascot searches, the number of peptides (Peptides) recognized are indicated together with the mass and position of the peptides that were used for PSD analysis (PSD). The peptide sequence coverage is listed as a percentage of the entire protein sequence (Coverage).
      Comparison of the TIF proteomes from the 16 different tumors revealed striking similarities in their overall protein profiles, although important differences were observed in the relative levels of several proteins as illustrated by the comparison of the protein profiles of TIFs collected from tumors 41 (Fig. 2) and 38 (Fig. 4C). As shown, the levels of several proteins, including those indicated in Fig. 4C, are significantly different between the two TIFs, most likely reflecting differences in the cell composition of the tumor microenvironment of the two tumors. For example, the level of IgG light and heavy chains (spots 85 and 86, respectively) is much higher in TIF 41 (Fig. 2) as compared with TIF 38 (Fig. 4C), a fact that can be tentatively explained by the occurrence of large focal clusters of B cells in tumor 41 (compare Fig. 6, A and B). Alternatively, the elevated levels of immunoglobulins may be due to the production of antibodies by the tumor cells themselves as recently suggested by Qiu and colleagues (
      • Qiu X.
      • Zhu X.
      • Zhang L.
      • Mao Y.
      • Zhang J.
      • Hao P.
      • Li G.
      • Lv P.
      • Li Z.
      • Sun X.
      • Wu L.
      • Zheng J.
      • Deng Y.
      • Hou C.
      • Tang P.
      • Zhang S.
      • Zhang Y.
      Human epithelial cancers secrete immunoglobulin g with unidentified specificity to promote growth and survival of tumor cells..
      ).
      Figure thumbnail gr6
      Fig. 6IHC of tumors reacted with various antibodies.A, tumor 41 reacted with an antibody (CD20 cy) specific for B cells. B, same as A but tumor 38. C, tumor 41 stained with an antibody specific for 14-3-3σ. D, tumor 40 stained with a vimentin antibody. Tumor cells as well as adipocytes are indicated for reference.

      DISCUSSION

      We have reported the isolation of a novel biological fluid, the interstitial fluid that perfuses the tumor microenvironment of invasive ductal carcinomas of the breast. Proteomic analysis of this fluid, which we have termed TIF, provided us with a snapshot of its proteome, which we show harbors more than 1,000 proteins—either secreted, shed by membrane vesicles, or externalized due to cell death—produced by the complex network of cell types that make up the tumor microenvironment.
      As expected, TIF contained several serum proteins, although its overall protein composition was remarkably different to that of serum as judged both by their 2D gel protein profiles and by comparison with the catalogs of normal human plasma/serum proteins recently published (
      • Adkins J.N.
      • Varnum S.M.
      • Auberry K.J.
      • Moore R.J.
      • Angell N.H.
      • Smith R.D.
      • Springer D.L.
      • Pounds J.G.
      Toward a human blood serum proteome: Analysis by multidimensional separation coupled with mass spectrometry..
      ,
      • Pieper R.
      • Su Q.
      • Gatlin C.L.
      • Huang S.T.
      • Anderson N.L.
      • Steiner S.
      Multi-component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome..
      ,
      • Anderson N.L.
      • Polanski M.
      • Pieper R.
      • Gatlin T.
      • Tirumalai R.S.
      • Conrads T.P.
      • Veenstra T.D.
      • Adkins J.N.
      • Pounds J.G.
      • Fagan R.
      • Lobley A.
      The human plasma proteome: A nonredundant list developed by combination of four separate sources..
      ). Classical serum proteins such as albumin, ferritin, α1 antichymotrypsin, α1 protease inhibitor, α1 β glycoprotein, haptoglobins 1 and 2, and IgG light and heavy chains were highly represented in TIF, but other proteins like apolipoproteins C-III and J were not detected, or at least were not present at the levels observed in serum. Interestingly, of the 267 proteins identified in the TIF, 97 are listed in the plasma/serum proteome (
      • Adkins J.N.
      • Varnum S.M.
      • Auberry K.J.
      • Moore R.J.
      • Angell N.H.
      • Smith R.D.
      • Springer D.L.
      • Pounds J.G.
      Toward a human blood serum proteome: Analysis by multidimensional separation coupled with mass spectrometry..
      ,
      • Pieper R.
      • Su Q.
      • Gatlin C.L.
      • Huang S.T.
      • Anderson N.L.
      • Steiner S.
      Multi-component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome..
      ,
      • Anderson N.L.
      • Polanski M.
      • Pieper R.
      • Gatlin T.
      • Tirumalai R.S.
      • Conrads T.P.
      • Veenstra T.D.
      • Adkins J.N.
      • Pounds J.G.
      • Fagan R.
      • Lobley A.
      The human plasma proteome: A nonredundant list developed by combination of four separate sources..
      ; see also Table II). At this point, it is difficult to estimate what may be the total number of proteins that compose the TIF proteome, although we believe these may reach the thousands as, with the exception of the proteins revealed by the cytokine-specific antibody arrays, the gel-based studies detected mainly medium and high-abundance proteins. In an effort to enrich for low-abundance proteins, we are currently evaluating the use of classical fractionation procedures as well as removal of major serum proteins using commercial kits. We are also applying overlay procedures using various radioactive ligands in order to investigate groups of functionally related proteins known to be associated with cancer. As an example, Fig. 7 shows autoradiograms of TIF proteins separated by 2D PAGE, blotted onto nitrocellulose, and reacted with [α-32P]GTP (
      • Gromov P.S.
      • Celis J.E.
      Several small GTP-binding proteins are strongly down regulated in SV40 transformed human keratinocytes and may be required for the maintainance of the normal phenotype..
      ,
      • Gromov P.
      • Celis J.E.
      Blot overlay assay for small GTP-binding proteins identification.
      ). A database of TIF proteins will soon be made available to the scientific community through our newly revised web site (proteomics.cancer.dk).
      Figure thumbnail gr7
      Fig. 72D gel-based profiling of small GTP-binding proteins present in TIF. TIF proteins were resolved by NEPHGE and IEF 2D PAGE, electroblotted to nitrocellose membranes, and overlaid with [α-32P]GTP as described under “Experimental Procedures.”
      Notably, with the exception of actin, filamin, gelsolin, haptoglobin 1, keratin 10 and 19, the ribosome binding protein 1, tropomyosin 1, and vimentin, all of the proteins identified by mass spectrometry, immunoblotting, or by comparison with the master images of the keratinocyte 2D PAGE database (proteomics.cancer.dk) appeared intact as judged by their apparent molecular masses and pIs displayed in 2D gels. This observation is remarkable, particularly in the case of albumin, as this highly abundant protein showed no signs of major degradation even by 2D PAGE immunoblotting (Fig. 5, Bd). We have previously shown that albumin is partially degraded in biopsy specimens derived from both bladder and colon tumors, yielding numerous protein spots displaying molecular masses in the range between 65 and 10 kDa.
      J. E. Celis, T. Cabezon, I. Gromova, and P. Gromov, unpublished data.
      Thus, on the whole the above observations argue against degradation being an important source of proteins spots in the 2D gels of TIF proteins. Analysis of TIFs collected after up to 24 h of incubation still showed no sign of major degradation, although we observed the appearance of several degradation products of keratin 19 that migrated very close to the intact protein (data not shown). We also observed a faint protein spot in the position expected for CYFRA 21 (
      • Dohmoto K.
      • Hojo S.
      • Fujita J.
      • Yang Y.
      • Ueda Y.
      • Bandoh S.
      • Yamaji Y.
      • Ohtsuki Y.
      • Dobashi N.
      • Ishida T.
      • Takahara J.
      The role of caspase 3 in producing cytokeratin 19 fragment (CYFRA21–1) in human lung cancer cell lines..
      ), a K19 fragment that is produced by apoptosis-activated caspases (
      • Ku N.O.
      • Liao J.
      • Omary M.B.
      Apoptosis generates stable fragments of human type I keratins..
      ,
      • Sheard M.A.
      • Vojtesek B.
      • Simickova M.
      • Valik D.
      Release of cytokeratin-18 and -19 fragments (TPS and CYFRA 21-1) into the extracellular space during apoptosis..
      ). The above observations are relevant in the context of recent comments from Liotta and colleagues (
      • Liotta L.A.
      • Ferrari M.
      • Petricoin E.
      Clinical proteomics: written in blood..
      ), who hypothesized that degradation and cleavage of the proteins that perfuse the tumor microenvironment may serve as a source of low molecular mass biomarkers, including peptides, found in the blood. They inferred that some of these peptides might enter the circulation either passively or actively, where carrier proteins transport and amplify them.
      In line with recent reports concerning the protein composition of the human plasma/serum proteome (
      • Adkins J.N.
      • Varnum S.M.
      • Auberry K.J.
      • Moore R.J.
      • Angell N.H.
      • Smith R.D.
      • Springer D.L.
      • Pounds J.G.
      Toward a human blood serum proteome: Analysis by multidimensional separation coupled with mass spectrometry..
      ,
      • Pieper R.
      • Su Q.
      • Gatlin C.L.
      • Huang S.T.
      • Anderson N.L.
      • Steiner S.
      Multi-component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome..
      ,
      • Anderson N.L.
      • Polanski M.
      • Pieper R.
      • Gatlin T.
      • Tirumalai R.S.
      • Conrads T.P.
      • Veenstra T.D.
      • Adkins J.N.
      • Pounds J.G.
      • Fagan R.
      • Lobley A.
      The human plasma proteome: A nonredundant list developed by combination of four separate sources..
      ), our studies revealed proteins known to be secreted, as well as polypeptides that lack signal sequences that target them to the classical endoplasmic reticulum-Golgi-plasma membrane secretory pathway. Presently, there is a wealth of information in the literature indicating that shedding of membrane vesicles from the cell surface to the microenvironment serve as an important mechanism by which normal and tumor cells release proteins to the exterior (
      • Taylor D.D.
      • Black P.H.
      Shedding of plasma membrane fragments. Neoplastic and developmental importance..
      ,
      • Vittorelli M.L.
      Shed membrane vesicles and clustering of membrane-bound proteolytic enzymes..
      and references therein). Shed proteins have been shown to affect the tumor microenvironment and play an important role in cell-cell and cell-matrix interactions, invasion, angiogenesis, metastasis, as well as in evasion of immune surveillance. Tumor cells have also been shown to acquire proteins associated with vesicles, a passive process by which neoplastic cells take up proteins associated with the plasma membrane (
      • Tabibzadeh S.S.
      • Kong Q.F.
      • Kapur S.
      Passive acquisition of leukocyte proteins is associated with changes in phosphorylation of cellular proteins and cell-cell adhesion properties..
      ). Examples of proteins shed by vesicles include matrix-degrading proteinases (
      • Vittorelli M.L.
      Shed membrane vesicles and clustering of membrane-bound proteolytic enzymes..
      ,
      • Taraboletti G.
      • D’Ascenzo S.
      • Borsotti P.
      • Giavazzi R.
      • Pavan A.
      • Dolo V.
      Shedding of the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP as membrane vesicle-associated components by endothelial cells..
      ), cathepsins B and D (
      • Montcourrier P.
      • Mangeat P.H.
      • Salazar G.
      • Morisset M.
      • Sahuquet A.
      • Rochefort H.
      Cathepsin D in breast cancer cells can digest extracellular matrix in large acidic vesicles..
      ,
      • Rozhin J.
      • Sameni M.
      • Ziegler G.
      • Sloane B.F.
      Pericellular pH affects distribution and secretion of cathepsin B in malignant cells..
      ,
      • Sameni M.
      • Elliott E.
      • Ziegler G.
      • Fortgens P.H.
      • Dennison C.
      • Sloane B.F.
      Cathepsin B and D are localized at the surface of human breast cancer cells..
      ,
      • Koblinski J.E.
      • Dosescu J.
      • Sameni M.
      • Moin K.
      • Clark K.
      • Sloane B.F.
      Interaction of human breast fibroblasts with collagen I increases secretion of procathepsin B..
      ), BRCA1 (
      • Jensen R.A.
      • Thompson M.E.
      • Jetton T.L.
      • Szabo C.I.
      • van der Meer R.
      • Helou B.
      • Tronick S.R.
      • Page D.L.
      • King M.C.
      • Holt J.T.
      BRCA1 is secreted and exhibits properties of a granin..
      ), IL-1β (
      • MacKenzie A.
      • Wilson H.L.
      • Kiss-Toth E.
      • Dower S.K.
      • North R.A.
      • Surprenant A.
      Rapid secretion of interleukin-1β by microvesicle shedding..
      ), fibroblast growth factor-2 (
      • Taverna S.
      • Ghersi G.
      • Ginestra A.
      • Rigogliuso S.
      • Pecorella S.
      • Alaimo G.
      • Saladino F.
      • Dolo V.
      • Dell’Era P.
      • Pavan A.
      • Pizzolanti G.
      • Mignatti P.
      • Presta M.
      • Vittorelli M.L.
      Shedding of membrane vesicles mediates fibroblast growth factor-2 release from cells..
      ), and tumor-associated surface antigens (
      • Dolo V.
      • Adobati E.
      • Canevari S.
      • Picone M.A.
      • Vittorelli M.L.
      Membrane vesicles shed into the extracellular medium by human breast carcinoma cells carry tumor-associated surface antigens..
      ). Using protein tagging of whole cells, Jang and Hanash recently identified several proteins in the cell surface of leukemia cells that have previously been shown to occur only in the endoplasmic reticulum (
      • Jang J.H.
      • Hanash S.
      Profiling of the cell surface proteome..
      ). These proteins, which included PDI, grp 78, hsp 70, grp 75, hsp 60, calreticulin, and calnexin, were also detected in TIF (Table II), suggesting that the phenomenon is not restricted to cells in suspension. The mechanism(s) by which endoplasmic reticulum proteins reach the cell surface remains unknown, although Jang and Hanash hypothesized that hsps may become associated with the plasma membrane by accompanying misfolded proteins or peptides via a nonclassical pathway (
      • Jang J.H.
      • Hanash S.
      Profiling of the cell surface proteome..
      ). It seems likely that many of the TIF proteins are externalized to the microenvironment by means of membrane-shed vesicles, and this possibility will be the subject of further studies.
      From the inventory of proteins listed in Table II, it is apparent that TIF contains polypeptides that are derived from many, if not all, of the cell types that compose the tumor local microenvironment. At the moment, however, it is not possible to estimate what proportion of the TIF proteome is derived from each of these cell types as we lack specific, externalized protein markers to assess their contribution using gel-based proteomic procedures. For tumors cells, however, we have evidence indicating that their contribution to TIF is most likely substantial. We have arrived at this conclusion by analyzing the expression by tumor 41 of the epithelial-specific marker 14-3-3σ (
      • Leffers H.
      • Madsen P.
      • Rasmussen H.H.
      • Honore B.
      • Andersen A.H.
      • Walbum E.
      • Vandekerckhove J.
      • Celis J.E.
      Molecular cloning and expression of the transformation sensitive epithelial marker stratifin. A member of a protein family that has been involved in the protein kinase C signalling pathway..
      ), a protein that is externalized to the medium by epithelial cell types (proteomics.cancer.dk) (
      • Moreira J.M.A.
      • Gromov P.
      • Celis J.E.
      Expression of the tumor suppressor protein 14-3-3σ is down regulated in invasive transitional cell carcinomas of the urinary bladder undergoing epithelial to mesenchymal transition..
      ). As shown in Fig. 6C, the antibody decorates the tumor cells specifically and the protein is present in TIF 41 2D gels at levels that are rather high (Fig. 2, spot 2) if one considers their relative ratio to the major classical serum proteins.
      Analysis of TIFs using cytokine-specific antibody arrays revealed similarities as well as differences in the expression of various cytokines and growth factors as exemplified in Fig. 5C. These preliminary findings, even though still in a pilot phase, have opened the possibility of searching for multifactorial signatures that may characterize a given tumor microenvironment. Currently, this possibility is being pursued systematically in our laboratory by combining cytokine-specific antibody array data with gel-based profiles and IHC images generated using a battery of antibodies specific for different cell types, such as macrophages, neutrophils, mast cells, B cells, endothelial cells, and others that populate the tumor microenvironment. In the long run, these studies are expected to elucidate the interplay between the complex network of cytokines, growth factors, signaling factors, and cytoskeletal components that affect tumor behavior, as well as to provide unique signatures or features that may characterize the social interactions in the tumor microenvironment. It should be stressed that the local microenvironment may be different in various areas of the tumor reflecting intra-tumor and intra-stroma heterogeneity, and that new and more sensitive detection technologies in combination with tissue microdissection (
      • Emmert-Buck M.R.
      • Bonner R.F.
      • Smith P.D.
      • Chuaqui R.F.
      • Zhuang Z.
      • Goldstein S.R.
      • Weiss R.A.
      • Liotta L.A.
      Laser capture microdissection..
      ) may be necessary to gain a better understanding of the biological events taking place in the local surroundings.
      The protein concentration of TIF recovered as described here is such that it is now feasible to undertake a search for diagnostic biomarkers and more selective targets for therapeutic intervention using the armamentarium of proteomic technologies currently available. The presence of multiple proteins in this fluid, as well as their multiple interactions, provides not only with a rich source for discovering more specific diagnostic biomarkers, but also offers a model system to generate new therapeutic strategies to target the tumor microenvironment and to understand breast cancer progression. We believe that TIF offers a rich source for generating biomarkers and targets, and that these can be unraveled through the systematic comparison of the proteomes of interstitial fluids collected from different tumors and their normal counterparts (see below). The main challenge will be to find specific markers amid the thousands of proteins that may be present in these fluids. NAF, the breast ductal and lobular fluid, is also a potential source of biomarkers and has gained much attention as a noninvasive procedure to study the local microenvironment associated with the development and progression of breast tumors (
      • Varnum S.M.
      • Covington C.C.
      • Woodbury R.L.
      • Petritis K.
      • Kangas L.J.
      • Abdullah M.S.
      • Pounds J.G.
      • Smith R.D.
      • Zangar R.C.
      Proteomic characterization of nipple aspirate fluid: Identification of potential biomarkers of breast cancer..
      ,
      • Kuerer H.M.
      • Goldknopf I.L.
      • Fritsche H.
      • Krishnamurthy S.
      • Sheta E.A.
      • Hunt K.K.
      Identification of distinct protein expression patterns in bilateral matched pair breast ductal fluid specimens from women with unilateral invasive breast carcinoma. High-throughput biomarker discovery..
      ). Comparison of the TIF and NAF proteomes (Table II), however, indicates that the protein composition of the latter may not reflect the various physiological activities taking place in the tumor microenvironment. TIF and NAF share only a few proteins in common, and some of these corresponded to traditional serum proteins (Table II). So far, only a few components of the NAF proteome have been identified using non-gel-based proteomics (
      • Varnum S.M.
      • Covington C.C.
      • Woodbury R.L.
      • Petritis K.
      • Kangas L.J.
      • Abdullah M.S.
      • Pounds J.G.
      • Smith R.D.
      • Zangar R.C.
      Proteomic characterization of nipple aspirate fluid: Identification of potential biomarkers of breast cancer..
      ), although several studies are currently underway to define its proteome. Gel-based proteomic technologies have revealed fewer proteins, many of which corresponded to glycosylated variants rather than primary translation products (
      • Kuerer H.M.
      • Goldknopf I.L.
      • Fritsche H.
      • Krishnamurthy S.
      • Sheta E.A.
      • Hunt K.K.
      Identification of distinct protein expression patterns in bilateral matched pair breast ductal fluid specimens from women with unilateral invasive breast carcinoma. High-throughput biomarker discovery..
      ).
      Currently, we are pursuing several lines of research in an effort to mine the TIF. First, we have started a methodical comparison of the TIF proteomes from tumors as well as from similar fluids collected from axillary nodal metastasis (MIF) and nonmalignant breast epithelial tissue (NIF) as a part of a large prospective study involving 500 high-risk patients. As shown in Fig. 4D, the protein composition of the MIF is remarkably similar to that of TIF, although we have observed interesting differences in the levels of a number of proteins. The NIF protein profile is also similar to TIF, but the relative levels of most proteins with respect to the major serum proteins are much lower (Fig. 4E). The latter observation may be due in part to the low ratio of glands to connective tissue that we have often observed in mastectomies of elderly women. Second, we plan to study the effect of TIF components on cell proliferation TIF using three-dimensional cultures of nonmalignant breast tissue (
      • Celis J.E.
      • Gromov P.
      • Gromova I.
      • Moreira J.M.
      • Cabezon T.
      • Ambartsumian N.
      • Grigorian M.
      • Lukanidin E.
      • Thor Straten P.
      • Guldberg P.
      • Bartkova J.
      • Bartek J.
      • Lukas J.
      • Lukas C.
      • Lykkesfeldt A.
      • Jaattela M.
      • Roepstorff P.
      • Bolund L.
      • Orntoft T.
      • Brunner N.
      • Overgaard J.
      • Sandelin K.
      • Blichert-Toft M.
      • Mouridsen H.
      • Rank F.E.
      Integrating proteomic and functional genomic technologies in discovery-driven translational breast cancer research..
      ,
      • Hammond S.L.
      • Ham R.G.
      • Stampfer M.R.
      Serum-free growth of human mammary epithelial cells: Rapid clonal growth in defined medium and extended serial passage with pituitary extract..
      ). These experiments will be complemented using interstitial fluid collected from fresh fat tissue (FIF), as the latter plays a role in maintaining the energy balance in the body (
      • MacDougald O.A.
      • Lane M.D.
      Adipocyte differentiation. When precursors are also regulators..
      ,
      • Friedman J.M.
      Obesity in the new millennium..
      ,
      • Saltiel A.R.
      Muscle or fat? Rho bridges the GAP..
      ) and may affect tumor development and progression (
      • Claffey K.P.
      • Wilkison W.O.
      • Spiegelman B.M.
      Vascular endothelial growth factor. Regulation by cell differentiation and activated second messenger pathways..
      ,
      • Manabe Y.
      • Toda S.
      • Miyazaki K.
      • Sugihara H.
      Mature adipocytes, but not preadipocytes, promote the growth of breast carcinoma cells in collagen gel matrix culture through cancer-stromal cell interactions..
      ), as it is often found very close to the tumor cells (Fig. 6D). As shown in Fig. 4F, the overall protein composition of FIF is quite different to that of TIF as it contains much fewer proteins and displays very high levels of the adipocyte fatty acid binding protein as well as annexin V (Fig. 4F). Surprisingly, these proteins have not been reported as being up-regulated in the secreted protein fraction of 3T3-L1 preadipocytes undergoing differentiation to adipocytes (
      • Kratchmarova I.
      • Kalume D.E.
      • Blagoev B.
      • Scherer P.E.
      • Podtelejnikov A.V.
      • Molina H.
      • Bickel P.E.
      • Andersen J.S.
      • Fernandez M.M.
      • Bunkenborg J.
      • Roepstorff P.
      • Kristiansen K.
      • Lodish H.F.
      • Mann M.
      • Pandey A.
      A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes..
      ). Third, we would like to use TIF to reveal novel protein interactions among its components, as these may be prove to be instrumental for functional studies as well as for discovering more selective targets for therapeutic intervention. Fourth, we are also interested in detecting tumor-specific autoantibodies in TIF using Western immunoblotting in combination with IHC. These studies will be complemented by TIF protein arrays reacted with serum collected from the same breast cancer patient. Finally, we are currently attempting to recover TIF from other breast tumor types as well as of other cancers in an effort to facilitate the identification of breast tumor-specific biomarkers.
      In conclusion, our studies have provided a rich source of proteins for biomarker and target discovery. Even though the identity of many proteins still remain to be determined, the biological activities of the proteins identified so far have provided us with a glance of the biological processes taking place in the tumor microenvironment. Together with data currently being generated in whole-tumor lyzates concerning signaling pathways and components affected in breast cancer (
      • Celis J.E.
      • Gromov P.
      • Gromova I.
      • Moreira J.M.
      • Cabezon T.
      • Ambartsumian N.
      • Grigorian M.
      • Lukanidin E.
      • Thor Straten P.
      • Guldberg P.
      • Bartkova J.
      • Bartek J.
      • Lukas J.
      • Lukas C.
      • Lykkesfeldt A.
      • Jaattela M.
      • Roepstorff P.
      • Bolund L.
      • Orntoft T.
      • Brunner N.
      • Overgaard J.
      • Sandelin K.
      • Blichert-Toft M.
      • Mouridsen H.
      • Rank F.E.
      Integrating proteomic and functional genomic technologies in discovery-driven translational breast cancer research..
      ), the data presented here may prove invaluable in the search for biomarkers and targets for cancer therapy, as well as for furthering our understanding of the molecular mechanisms underlying breast cancer development and progression.

      Acknowledgments

      We are indebted to Kitt Christensen, Gitte Lindberg Stott, Dorrit Lützhøft, Hanne Nors, Michael Radich Johansen, and Signe Trentemøller for expert technical assistance.

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