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Originally published In Press as doi:10.1074/mcp.M600164-MCP200 on July 21, 2006.
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Molecular & Cellular Proteomics 5:2083-2091, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

A Chemical Proteomics Approach for the Identification of Accessible Antigens Expressed in Human Kidney Cancer*,S

Vincent Castronovo{ddagger},§, David Waltregny{ddagger},§, Philippe Kischel{ddagger}, Christoph Roesli, Giuliano Elia, Jascha-N. Rybak,|| and Dario Neri,**

From the {ddagger} Metastasis Research Laboratory, Center of Experimental Cancer Research, University of Liège, 4000 Liège, Belgium and Institute of Pharmaceutical Sciences, ETH Zurich, 8093 Zurich, Switzerland


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivo perfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes.

A promising avenue toward the development of more selective, better anticancer drugs consists in the targeted delivery of bioactive molecules (e.g. cytokines, radionuclides, antibody constant regions, drugs, photosensitizers, procoagulant factors, etc.) to the tumor environment by means of binding molecules (such as recombinant human antibodies and their fragments) specific to tumor-associated markers (1–4). This strategy crucially relies on the availability of good quality tumor antigens and suitable high affinity ligands (e.g. human monoclonal antibodies). Target proteins located around tumor blood vessels and in the stroma are particularly suited for targeted anticancer strategies in view of their accessibility for intravenously administered biopharmaceuticals (5). Recent strategies for the identification of tumor-associated endothelial markers include global profiling of gene transcripts (6), bioinformatics analysis of expressed sequence tags (7), in vivo peptide phage biopanning (8, 9) and the silica bead-based stripping of membrane proteins followed by proteomics analysis (10).

Chemical proteomics, i.e. the use of chemical modification of proteins for sampling of subproteomes, has been used for the reduction of sample complexity prior to mass spectrometric identification (11) and for activity-based protein profiling (12). In principle, chemical modification strategies could also be applied to identify markers of pathology that are accessible from the bloodstream and thus ideally suited for ligand-based biomedical strategies. We have recently described a method for the identification of accessible antigens in murine organs and tumors based on the terminal perfusion of tumor-bearing mice with reactive ester derivatives of biotin (13). This methodology allows the efficient biotinylation of accessible proteins on the membrane of endothelial cells and other structures (e.g. extracellular matrix components) that are readily accessible from the bloodstream. The purification of biotinylated proteins from organ lysates on streptavidin resin followed by a comparative proteomics analysis based on mass spectrometry permitted the identification of hundreds of accessible proteins, some of which were found to be differentially expressed in organs and in tumors. The identification of human tumor markers is of fundamental importance for the treatment of cancer patients. For this reason we have now developed a procedure that allows the biotinylation of accessible antigens in human surgical specimens.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Patients—
This study was started upon approval by the ethical committee of the University Hospital of Liège (Belgium). Criteria adopted for patient selection were as follows: 1) diagnosis of a tumor highly compatible with a clear cell carcinoma of the kidney as assessed by routine ultrasound and abdomen computed tomography scan, 2) a therapeutic indication for a total nephrectomy, and 3) a tumor size and localization that allowed healthy portions of the kidney to be to clearly distinguished to be used as normal controls. Immunohistochemical procedures compatible with the detection of specific proteins without biotin interference were adopted for the diagnostic histopathological analysis. Patient’s informed consent was obtained, and serology for negativity to human immunodeficiency virus and hepatitis A, B, and C was performed. For specific information about the patients see Supplemental Table 1.

Ex Vivo Vascular Perfusion—
Surgery was performed according to a standard procedure, which includes the ligation and section of renal artery, vein, and ureter and subsequent nephrectomy. The renal artery carried a longer suture for immediate identification in the perfusion step. Within 2 min after nephrectomy, the renal artery was cannulated, the renal vein was opened (by removing the suture) to allow outflow of the perfusate, and perfusion via the renal artery was started. Kidneys were first perfused for 7–9 min with 500 ml of a 1 mg/ml solution of sulfo-NHS-LC-biotin (NHS is N-hydroxysuccinimide) in PBS, washing away blood components and labeling accessible primary amine-containing structures with biotin. Immediately afterward, a second perfusion step with 450 ml of PBS containing a 50 mM solution of the primary amine Tris was performed for 8–9 min to quench unreacted biotinylation reagent. All perfusion solutions contained 10% dextran 40 as a plasma expander and were prewarmed to 40 °C. Both perfusion steps were performed with a pressure of 100–150 mm Hg. Successful perfusion was indicated by the wash out of blood during the first minutes of perfusion and subsequent flow of clear perfusate out of the renal vein. After perfusion, the organs were washed with 50 mM Tris in PBS, dried, rubbed with black ink to allow the later pathologic investigation of surgical margins, and cut in half along the sagittal axis. Successful perfusion resulted in a whitish color of the tissue. Specimens from the tumor and from the normal kidney tissue (unaffected by the tumor) were excised (from well perfused, whitish parts) and immediately snap-frozen for proteomics and histochemical analyses or paraformaldehyde-fixed and paraffin-embedded for histochemical analyses. As negative controls, unperfused organs after nephrectomy were cut in half, and specimens were taken as described above from the tumor and from normal kidney tissue. For specific information about the examined organs see Supplemental Table 1.

Histochemical Staining of Tissue Sections with Avidin-Biotinylated Peroxidase Complex—
Sections from paraformaldehyde-fixed, paraffin-embedded tissue specimens were stained with avidin-biotin-peroxidase complex using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA).

Preparation of Protein Extracts for Proteomics Analysis—
Specimens were resuspended in lysis buffer (2% SDS, 50 mM Tris, 10 mM EDTA, Complete EDTA-free protease inhibitor cocktail (Roche Diagnostics) in PBS, pH 7.4) at 40 µl/mg of tissue and homogenized using an Ultra-Turrax T8 disperser (IKA-Werke, Staufen, Germany). Homogenates were sonicated using a Vibra-Cell (Sonics, New Town, CT) followed by 15-min incubation at 99 °C and 20-min centrifugation at 15,000 x g. The supernatant was used as total protein extract. Protein concentration was determined using the BCA protein assay reagent kit (Pierce).

Purification of Biotinylated Proteins—
For each sample, 960 µl of streptavidin-Sepharose (Amersham Biosciences) slurry were washed three times in buffer A (1% Nonidet P-40, 0.1% SDS in PBS), pelleted, and mixed with 15 mg of total protein extract. Capture of biotinylated proteins was allowed to proceed for 2 h at room temperature in a revolving mixer. The supernatant was removed, and the resin was washed three times with buffer A, two times with buffer B (0.1% Nonidet P-40, 1 M NaCl in PBS), and once with 50 mM ammonium bicarbonate. Finally the resin was resuspended in 400 µl of a 50 mM solution of ammonium bicarbonate, and 20 µl of sequencing grade modified porcine trypsin (stock solution of 40 ng/µl in 50 mM ammonium bicarbonate) (Promega, Madison, WI) were added. Protease digestion was carried out overnight at 37 °C under constant agitation. Peptides were desalted, purified, and concentrated with C18 microcolumns (ZipTip C18, Millipore, Billerica, MA). After lyophilization peptides were stored at –20 °C.

Nanocapillary HPLC with Automated On-line Fraction Spotting onto MALDI Target Plates—
Tryptic peptides were separated by reverse phase HPLC using an UltiMate nanoscale LC system and a FAMOS microautosampler (LC Packings, Amsterdam, The Netherlands) controlled by Chromeleon software (Dionex, Sunnyvale, CA). Mobile phase A consisted of 2% acetonitrile and 0.1% TFA in water; mobile phase B was 80% acetonitrile and 0.1% TFA in water. The flow rate was 300 nl/min. Lyophilized peptides derived from the digestion of biotinylated proteins affinity-purified from 1.5 mg of total protein were dissolved in 5 µl of buffer A and loaded on the column (inner diameter, 75 µm; length, 15 cm; filled with C18 PepMap 100; 3-µm, 100-Å beads; LC Packings). The peptides were eluted with a gradient of 0–30% B for 7 min, 30–80% B for 67 min, 80–100% B for 3 min, and 100% B for 5 min; the column was equilibrated with 100% A for 20 min before analyzing the next sample. Eluting fractions were mixed with a solution of 3 mg/ml {alpha}-cyano-4-hydroxycinnamic acid, 277 pmol/ml neurotensin (internal standard), 0.1% TFA, and 70% acetonitrile in water and deposed on a 192-well MALDI target plate using an on-line Probot system (Dionex). The flow of the MALDI matrix solution was set to 1.083 µl/min. Thus, each fraction collected during 20 s contained 361 nl of MALDI matrix solution and 100 nl of sample. The end concentration of neurotensin was 100 fmol/well.

MALDI-TOF/TOF Mass Spectrometry—
MALDI-TOF/TOF mass spectrometric analysis was carried out with the 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). For precursor ion selection, all fractions were measured in MS mode before MS/MS was performed. A maximum of 15 precursors per sample spot were selected for subsequent fragmentation by collision-induced dissociation. Spectra were processed and analyzed by the Global Protein Server Workstation (Applied Biosystems), which uses internal MASCOT (Matrix Science, London, UK) software for matching MS and MS/MS data against databases of in silico digested proteins. The data obtained were screened against a human database downloaded from the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/). Protein identifications, performed by means of the MASCOT software, were considered to be correct calls within the 95% confidence interval for the best peptide ion. Selected hits within the confidence interval between 90 and 95% were verified by manual inspection of the spectra.

Antibodies—
The immunoaffinity-purified rabbit polyclonal anti-periostin antibody was purchased from Biovendor (Heidelberg, Germany), and the monoclonal anti-versican antibody (clone 12C5) was from the Developmental Studies Hybridoma Bank (University of Iowa, Ames, IA).

Antibodies in the single chain Fv format against recombinant fragments of melanoma-associated antigen MG50 and of annexin A4 were selected from the ETH-2-Gold phage display library according to the procedure reported in Ref. 14. As antigens for the biopanning experiments, recombinant protein domains corresponding to sequences 539–632 of melanoma-associated antigen MG50 and 1–318 of annexin A4 were used. These proteins were expressed in Escherichia coli using the pQE12 vector (Qiagen, Hilden, Germany). The corresponding gene segments were PCR-amplified using primers 5'-ACTGGATCCAGAGTCACCCCAGTGTTTG-3' and 5'-ACTGGATC-CGTCAGGAACGTTCACACTGAG-3' for melanoma-associated antigen MG50 and 5'-ATCGGATCCATGGCAACCAAAGGAGGTACTGTCAAA-3' and 5'-TAATTAAGCTTAGTGATGGTGATGGTGATGATCATCTCCTCCACAGGAACAAGCAG-3' for annexin A4 and cloned into the BamHI and BamHI/HindIII sites of pQE12, respectively. Proteins were purified from E. coli lysates using nickel-nitrilotriacetic acid columns (Qiagen).

Immunohistochemical Staining—
Anti-periostin and anti-versican antibodies were used in a dilution of 1:500 to stain sections from paraformaldehyde-fixed, paraffin-embedded tissue specimens and were detected by the immunoperoxidase technique (Vectastain Elite ABC kit, Vector Laboratories) according to standard procedures. Immunohistochemical staining using single chain Fv preparations on sections of freshly frozen tissue samples was performed as described in Refs. 14 and 15 using the alkaline phosphatase/Fast Red TR detection system and counterstaining with hematoxylin.

PCR Analysis—
A human kidney tumor cDNA panel containing cDNAs from clear cell carcinoma, granular cell carcinoma, transitional cell carcinoma, normal adult, and fetal kidney was purchased from BioChain (Hayward, CA). PCR was performed using the Hot Start Taq polymerase kit (Qiagen). PCR conditions were as follows: denaturation at 95 °C for 15 min followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 54 °C for 1 min, and elongation at 72 °C for 1 min. A final step of elongation at 72 °C for 10 min was performed. Primer sequences are available upon request. The products of the PCR were analyzed by 2% agarose gel electrophoresis, stained by ethidium bromide, and imaged using the BioDoc-It imaging system (UVP, Upland, CA).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Ex Vivo Biotinylation of Surgically Resected Kidneys—
Three kidneys were surgically resected from patients with renal cell carcinoma (for detailed information about the patients see Supplemental Table 1). Immediately after nephrectomy, the organs were perfused ex vivo according to the scheme provided in Fig. 1A. Perfusion with biotinylation reagent lasted 7–9 min and was followed by an additional perfusion step with a primary amine-containing solution (Tris) to quench excessive, unreacted biotinylation reagent. The procedure allowed efficient and selective labeling of vascular structures in the tumor portions (Fig. 1C), whereas both vascular and tubular structures were labeled in the normal kidney portions (Fig. 1D).


Figure 1
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  		FIG. 1. Ex vivo biotinylation of human tumor-bearing kidneys. A, schematic representation of the ex vivo kidney perfusion procedure. Within 2 min after nephrectomy, the tumor-bearing kidney is perfused with a reactive ester derivative of biotin, thus washing away blood components and biotinylating accessible proteins. Biotinylated tissue specimens can be cut and processed separately for the purification of biotinylated proteins, yielding tryptic peptides that are separated by nano-HPLC and MALDI-TOF/TOF mass spectrometry. B, tumor-bearing kidney, cut in half, after ex vivo perfusion. Biotinylated structures in tissue sections were detected in the tumor portion (C) and in the normal kidney portion (D) using avidin-horseradish peroxidase-based staining protocols. Vascular structures are preferentially biotinylated in the neoplastic mass. Scale bars, 100 µm.

 
Proteomics Analysis of Biotinylated Specimens—
After ex vivo biotinylation, multiple specimens were excised from various areas of the normal kidney portion and the tumor of each organ and analyzed independently. Samples were homogenized in the presence of SDS and loaded onto streptavidin resin, thus enriching for biotinylated proteins. A subsequent proteolytic digestion followed by nanocapillary HPLC peptide separation and MALDI-TOF/TOF mass spectrometric analysis permitted the identification of 637 proteins cumulated in all specimens (a complete list of proteins identified in all 38 specimens from the three biotinylated organs and in additional control samples from two unperfused organs is provided in Supplemental Table 2).

Table I shows a selection of proteins that were identified either only in normal kidney portions, only in tumor specimens, or in both. As expected, abundant proteins observed in both normal and neoplastic specimens included components of the extracellular matrix such as collagens, laminin, perlecan, lumican, vitronectin, fibronectin, and tenascin. Proteins found exclusively in the normal kidney portions included the kidney-specific cadherin 16, several transporters, apolipoprotein E, and uromodulin. A number of proteins were found exclusively in the tumor specimens.


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  		TABLE I Selection of putative membrane proteins and extracellular matrix proteins identified

Numbers indicate in how many of the three patients, whose kidneys were biotinylated ex vivo, the protein was identified in specimens from the normal kidney portion or the tumor, respectively. For a description of the patients see Supplemental Table 1. A complete list of proteins identified in all 38 specimens from the three biotinylated organs and in additional control samples from two unperfused organs is provided in Supplemental Table 2.

 
Target Validation—
Because the detection of a protein in the tumor specimens might, in principle, reflect not only a preferential pattern of expression but also a differential accessibility to the biotinylation reagent, we further studied selected protein candidates both by immunohistochemistry and by PCR analysis of cDNA libraries.

Some of the putative tumor-associated antigens were found to be present in cDNA libraries of both tumor and normal kidney (Fig. 2), including fibulins, tumor suppressor candidate 3 (N33 protein), and the hypothetical protein DKFZp686K0275. By contrast, a number of interesting markers yielded substantially stronger PCR bands in fetal and tumor specimens, including periostin, versican, carbonic anhydrase IX, TEM-4, melanoma-associated antigen MG50, integrin {alpha}1, thrombospondin 2, putative G-protein-coupled receptor 42, aggrecan, probable G-protein-coupled receptor 37, fibromodulin, solute carrier family 2 member 1, and FLJ00154 (Fig. 2).


Figure 2
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  		FIG. 2. Target validation by semiquantitative PCR analysis of cDNA libraries. *, unlike other proteins, confidence of the assignment of CEACAM3 with the MASCOT software was below 95% for the best peptide ion and is not unambiguous even after visual inspection of the MS-MS spectra.

 
Periostin was the most abundant tumor-associated antigen in our analysis (Table I). The existence of five different splice isoforms of periostin in humans has been reported (16), but sequences of only three isoforms have been published (Swiss-Prot/TrEMBL and NCBI protein databases). We identified six isoform sequences in the PCR analysis of cDNA libraries with different relative abundance in normal, fetal, and tumor kidneys (Fig. 2; see also Supplemental Figs. 1 and 2). An immunohistochemical analysis of normal kidney and clear cell carcinoma specimens exhibited low background staining in the normal kidney samples but revealed a striking overexpression of periostin in eight of eight tumors investigated with a prominent vascular and stromal pattern of staining (Figs. 3A and 4A). Similarly, versican was found to be more abundant in fetal and tumor specimens both by PCR (Fig. 2) and by immunohistochemical analysis. Versican was strongly overexpressed in six of eight tumors but did not stain normal kidneys and other normal tissues (see Figs. 3B and 4B). A tissue microarray analysis of 50 kidney tumors and 23 normal kidney specimens confirmed the preferential expression of these antigens in kidney cancer (data not shown).


Figure 3
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  		FIG. 3. Target validation by immunohistochemistry. Immunohistochemical analysis of normal kidney and tumor sections (formaldehyde-fixed, paraffin-embedded) with antibodies specific to periostin (A) and versican (B) is shown. Staining with anti-periostin antibody exhibited low background staining in the normal kidney samples but revealed a strong overexpression in tumor specimens. Versican was strongly overexpressed in tumor samples but did not stain normal kidneys and other normal tissues. Staining reactions were absent in negative control experiments where the primary antibodies were omitted. Scale bars, 100 µm.

 

Figure 4
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  		FIG. 4. Immunohistochemical detection of identified targets in several patients with renal clear cell carcinoma. Immunohistochemical staining revealed a strong overexpression of periostin (A) in eight of eight tumors investigated and of versican (B) in six of eight tumors. Scale bars, 100 µm in A and 25 µm in B.

 
For most proteins identified in our proteomics analysis, monoclonal antibodies were not commercially available. We produced human monoclonal antibodies to melanoma-associated antigen MG50 and annexin A4 using antibody phage technology (14, 17) with recombinant protein preparations of these antigens. Although the annexin A4 antibody exhibited a diffuse staining pattern not only on vascular structures but also on cancer cells, the MG50-specific antibody preferentially stained vascular structures in cryostat sections of renal cell carcinomas (Fig. 5).


Figure 5
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  		FIG. 5. Different localization of identified antigens within the tumor tissue. Immunohistochemical staining of acetone-fixed tissue sections from freshly frozen kidney tumor specimens revealed a vascular and stromal pattern of staining for melanoma-associated antigen MG50 and a more diffuse staining of cancer cells for annexin A4 (ANX4). Scale bars, 100 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cancer chemotherapy relies on the expectation that anticancer drugs will preferentially kill rapidly dividing tumor cells rather than normal cells. Because a large portion of the tumor cells has to be killed to obtain and maintain a complete remission, large doses of drugs are typically used, resulting in significant toxicity toward proliferating non-malignant cells. Indeed, the majority of pharmacological approaches for the treatment of solid tumors suffers from poor selectivity, which imposes limits on dose escalation (i.e. the doses of drug that are required to kill tumor cells cause unacceptable toxicity to normal tissue). A number of antibody-based fusion proteins are currently in clinical development, including the human antibody L19, which is specific to the extra domain B of fibronectin (a marker of angiogenesis) (18–20). L19 is being tested in patients with cancer both as radioconjugate with iodine-131 (21, 22) and as a recombinant fusion protein with human interleukin-2 (23–25). The discovery of novel accessible and abundant tumor-associated antigens, such as those identified in this study, will facilitate the development of more selective anticancer agents based on the targeted delivery of bioactive molecules to the tumor environment. Furthermore antigens expressed in accessible structures are more likely to be released in circulation upon tissue remodeling and may be detected in the serum (26) or urine of patients with kidney cancer (e.g. for periostin, elevated serum levels in cancer patients have been reported (27)).

Using the ex vivo perfusion of surgically resected human kidneys with clear cell carcinoma with an active ester derivative of biotin we were able to identify an unprecedented number of kidney tumor markers. Biotinylated, accessible proteins, mainly lining vascular structures in the normal kidney and in the solid tumor mass, were purified on streptavidin resin and identified using nano-HPLC and MALDI-TOF/TOF methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens. Our methodology opens the possibility to study human surgical specimens, leading to the identification of marker proteins that are overexpressed around vascular structures. Such antigens accessible from the vasculature are likely to be suitable targets for ligand-based tumor targeting applications.

Indeed, some of the proteins only found in the tumor in this study had previously been reported to be overexpressed in certain neoplastic structures (e.g. carbonic anhydrase IX (28), TEM-4 (6), melanoma-associated antigen MG50 (29), malignant melanoma-associated protein 1 (30), integrin {alpha}1 (31, 32), and ectonucleotide pyrophosphatase/phosphodiesterase 3 (33)). However, the majority of the tumor antigens identified in our analysis represent novel tumor target candidates. Only a small portion (e.g. netrin receptor DCC, solute carrier family 2 member 1, and neural cell adhesion molecule 1) have so far been reported in the "Human Protein Atlas," a genome-wide initiative for the characterization of protein expression patterns in normal tissues and cancer (34).

The protein most abundantly identified exclusively in the tumor was periostin. Periostin represents a particularly interesting marker having been found previously to be up-regulated in epithelial ovarian tumors (35), in breast cancer (36), at the periphery of lung carcinomas (37), and in colorectal cancers and their liver metastases (38). The immunohistochemical analysis with anti-periostin antibody demonstrates that periostin was indeed highly overexpressed in the tumor stroma of renal clear cell carcinoma compared with normal kidney tissue. Thus, periostin is likely to be a useful target for ligand-based tumor targeting strategies. Additionally our PCR data suggest that certain periostin splice variants might represent even more specifically expressed tumor targets.

Another promising target candidate we identified and further validated is versican. Up-regulation of versican protein expression has been reported previously in human pharyngeal squamous cell carcinoma (39), ovarian cancer (40), and node-negative breast cancer (41) and was related to higher tumor recurrence rate and more advanced disease in non-small cell lung cancer (42). In a recent proteomics approach, versican was found to be released by pancreatic cancer cells (43). Our analysis for the first time demonstrates overexpression of versican in kidney cancer and suggests versican to be an accessible target for ligand-based therapy and imaging.

Interestingly our method was capable of identifying different classes of targets for ligand-based targeting of tumors. Although periostin and versican exhibited a stromal expression profile, melanoma-associated antigen MG50 was found to be located around tumor blood vessels, and annexin A4 was found to be on the tumor cells. Until recently, most targeting approaches relied on targets expressed directly on the surface of the cancer cells (see e.g. Ref. 44) to bring toxic payloads in a close proximity to kill the tumor cells. Our group has mainly focused its research activity on vascular and stromal tumor antigens (such as oncofetal isoforms of fibronectin (5, 45) and oncofetal isoforms of tenascin C (14, 15, 46)) because of the accessibility, specificity, stability, and abundance of many antigens belonging to this class.

Among the 637 proteins identified in this analysis, ~20% corresponded to intracellular proteins (see Supplemental Table 2). Although some more abundant intracellular proteins (e.g. actin, tubulin, keratin, and histones) could be recovered either by stickiness to the streptavidin resin or by biotinylating necrotic structures ex vivo, some intracellular proteins have been reported to become accessible on the surface of proliferating endothelial cells (10, 47).

It will take generation of monoclonal antibodies, careful immunofluorescence analysis, and biodistribution studies in appropriate animal tumor models to reveal the real potential of more of the 184 putative tumor-associated antigens identified in this study (see Supplemental Table 2). Interestingly ~20% of these antigens are hypothetical proteins that are mostly completely uncharacterized. The work presented here with the human antibodies specific to melanoma-associated antigen MG50 and annexin A4 indicates that antibody phage technology may represent an ideal avenue for complementing the high throughput information arising from ex vivo proteomics investigations (14, 17, 48). Finally, it is worth mentioning that the experimental approach described here should be applicable to several other pathologies (e.g. atherosclerosis, aneurisms, and chronic inflammatory conditions) as well as to the study of basic physiological and immunological processes.


   ACKNOWLEDGMENTS
 
We are grateful to the Functional Genomics Center Zurich for access to mass spectrometers and support and to Giorgio La Corte and Fabio Ugolini for help in the production of monoclonal antibodies.


  FOOTNOTES
 
Received, May 3, 2006, and in revised form, July 10, 2006.

Published, MCP Papers in Press, July 21, 2006, DOI 10.1074/mcp.M600164-MCP200

* This work was supported by the European Union project STROMA, the Bundesamt für Bildung und Wissenschaft (FLUOR-MMPI and STROMA), the Swiss National Science Foundation, the Gebert-Rüf Foundation, and ETH Zurich. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

§ Both authors contributed equally to this work. Back

|| To whom correspondence may be addressed: ETH Zurich, Inst. of Pharmaceutical Sciences, HCI G390, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland. Tel.: 41-44-63-37354; Fax: 41-44-63-31358; E-mail: jascha.rybak{at}pharma.ethz.ch

** To whom correspondence may be addressed: ETH Zurich, Inst. of Pharmaceutical Sciences, HCI G 396, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland. Tel.: 41-44-63-37401; Fax: 41-44-63-31358; E-mail: neri{at}pharma.ethz.ch


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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J.-N. Rybak, C. Roesli, M. Kaspar, A. Villa, and D. Neri
The Extra-domain A of Fibronectin Is a Vascular Marker of Solid Tumors and Metastases
Cancer Res., November 15, 2007; 67(22): 10948 - 10957.
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