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Originally published In Press as doi:10.1074/mcp.M400009-MCP200 on January 30, 2004.
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Molecular & Cellular Proteomics 3:327-344, 2004.
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Proteomic Characterization of the Interstitial Fluid Perfusing the Breast Tumor Microenvironment

A Novel Resource for Biomarker and Therapeutic Target Discovery*

Julio E. Celis{ddagger},§,, Pavel Gromov{ddagger},§, Teresa Cabezón{ddagger},§, José M. A. Moreira{ddagger},§, Noona Ambartsumian{ddagger},||, Kerstin Sandelin{ddagger},**, Fritz Rank{ddagger},{ddagger}{ddagger} and Irina Gromova{ddagger},§

From the {ddagger} 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; || Department of Molecular Cancer Biology, Institute of Cancer Biology, The Danish Cancer Society, DK-2100 Copenhagen, Denmark; ** Department of Breast and Endocrine Surgery, Rigshospitalet, DK-2100 Copenhagen, Denmark; {ddagger}{ddagger} Department of Pathology, The Centre of Diagnostic Investigations, Rigshospitalet, DK-2100 Copenhagen, Denmark


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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)1 proteins and cytokines that modify the biology and activity of the cancer cells (1). In addition, modified stroma cells secrete proteases that facilitate tissue destruction, cancer cell migration, and metastasis (2, 5, 6, 10 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 (11), matrix metalloproteinases (MMPs) such as interstitial collagenases, gelatinases, stromelysins, and membrane-type MMPs (12, 13), urokinase-type plasminogen activator receptor (14, 15), intercellular adhesion molecule-1 (16, 17), E-cadherin (18), transforming growth factor-ß system (19–21), epidermal growth factor (EGF) (22), EGF receptor-2 (HER-2/neu; c-erbB-2) (23–26), insulin growth factor 1 (27–29), hepatocyte growth factor (9, 20, 30), 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 (29, 31–34).

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 (35). 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 (35). 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 (36). 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 (37 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 (38). 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
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 2 invasive breast cancer were selected for this study (38). 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).



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  		FIG. 1. Mastectomy from a high-risk patient. A, tumor. B, small tumor pieces used to collect the TIF as described under "Experimental Procedures."

 

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  		TABLE I Invasive ductal carcinomas

 
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 (39). 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 (40). 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 (41). Immunoblotting was performed as previously described (42).

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 (43). 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 (44). 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 {alpha}-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 (45), 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, RayBioTM 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 [{alpha}-32P]GTP blot overlay assay essentially as previously described (46, 47). Protein samples were subjected to both IEF and NEPHGE 2D PAGE, and the proteins were electro-transferred to nitrocellulose membranes as previously described (42). 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 [{alpha}-32P]GTP (final concentration 1 µCi [{alpha}-32P]GTP/ml). The nitrocellulose membranes were then washed four times, 5 min each, in the same buffer lacking dithiothreitol, ATP, and [{alpha}-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 14-3-3{sigma} 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
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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" (40). 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 {alpha}-enolase (IEF 22, NEPHGE 1) and triosephosphate isomerase (IEF 148, NEPHGE 19), migrated in the IEF and NEPHGE gels (Figs. 2 and 3) (48). 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) (49). 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.



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  		FIG. 2. TIF 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 Table II. Some major proteins exhibiting charge trains are underlined.

 


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  		FIG. 3. TIF 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 Table II. Some major proteins exhibiting charge trains are underlined.

 


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  		FIG. 4. IEF 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 Fig. 2). 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 (50–52) and NAF proteomes (35). 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. 2–4. 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, {alpha}1 antichymotrypsin, {alpha}1 protease inhibitor, {alpha}1 ß 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.



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  		FIG. 5. Protein 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, Fig. 2). 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, Fig. 2). 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 Table II. Positive and negative controls are included (see grid).

 

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  		TABLE II TIF proteins

 
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 (53).



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  		FIG. 6. IHC 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 14-3-3{sigma}. D, tumor 40 stained with a vimentin antibody. Tumor cells as well as adipocytes are indicated for reference.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 (50–52). Classical serum proteins such as albumin, ferritin, {alpha}1 antichymotrypsin, {alpha}1 protease inhibitor, {alpha}1 ß 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 (50–52; 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 [{alpha}-32P]GTP (46, 47). A database of TIF proteins will soon be made available to the scientific community through our newly revised web site (proteomics.cancer.dk).



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  		FIG. 7. 2D 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 [{alpha}-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.3 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 (54), a K19 fragment that is produced by apoptosis-activated caspases (55, 56). The above observations are relevant in the context of recent comments from Liotta and colleagues (57), 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 (50–52), 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 (58, 59 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 (60). Examples of proteins shed by vesicles include matrix-degrading proteinases (59, 61), cathepsins B and D (62–65), BRCA1 (66), IL-1ß (67), fibroblast growth factor-2 (68), and tumor-associated surface antigens (69). 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 (70). 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 (70). 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 14-3-3{sigma} (71), a protein that is externalized to the medium by epithelial cell types (proteomics.cancer.dk) (72). 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 (73) 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 (35, 74). 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 (35), 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 (74).

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 (38, 75). 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 (76–78) and may affect tumor development and progression (79, 80), 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 (81). 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 (38), 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.


   FOOTNOTES
 
Received, January 22, 2004, and in revised form, January 30, 2004.

Published, MCP Papers in Press, January 30, 2004, DOI 10.1074/mcp.M400009-MCP200

1 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. Back

2 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. Back

3 J. E. Celis, T. Cabezon, I. Gromova, and P. Gromov, unpublished data. Back

* This work was supported by the Danish Cancer Society through the budget of the Institute of Cancer Biology and by grants from the Danish Medical Research Council, the Natural Science and Medical Committee of the Danish Cancer Society, Novo Nordisk, and the John and Birthe Meyer Foundation. The support from the Marketing Department at the Danish Cancer Society is also greatly appreciated. We would also like to acknowledge the support and collaboration of Eurogentec. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

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; E-mail: jec{at}cancer.dk


   REFERENCES
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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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