Abstract
The identification of proteins that are preferentially expressed on the membrane of metastatic tumor cells is of fundamental importance in cancer research. Here, we report the systematic comparison of the membrane proteome of two closely related murine teratocarcinoma cell lines (F9B9 and F9DR), of which only one (F9DR) is capable of forming liver metastases in vivo. The proteomic methodology used in this study featured the surface protein biotinylation on tumor cells followed by protein purification on streptavidin resin and relative quantification of corresponding tryptic peptides by mass spectrometric procedures. The study allowed the identification of 998 proteins and the determination of their relative abundance. Proteins previously known to be associated with metastatic spread were found to be either up-regulated (e.g., synaptojanin-2) or down-regulated (e.g., Ceacam1) in F9DR cells. A dramatic increase in abundance at the cell membrane was observed for a broad variety of proteins (e.g., high-mobility group protein B1), which were mainly thought to reside in intracellular compartments, a finding that was confirmed using confocal laser scanning microscopy and immunochemical analysis of cell cultures. Furthermore, we showed by microautoradiographic analysis that certain target proteins can readily be reached by intravenously administered radiolabeled antibodies. Finally, we showed that the most promising antigens for antibody-based pharmacodelivery approaches are strongly and selectively expressed on the surface of tumor cells in three different syngeneic mouse models of liver metastases. Taken together, our results indicate that the expression of intracellular proteins on the membrane of metastatic cells is a feature much more common than previously expected. [Cancer Res 2009;69(13):5406–14]
Introduction
In the last decade, DNA microarrays have become a standard tool for the molecular analysis of cancer, providing global profiles of transcription that reflect the origin (1), stage of development (2), drug sensitivity (3), and metastatic potential of tumor cells (4). The ability to complement these transcriptomic approaches with methods that analyze the proteome (5, 6) is crucial for the identification of proteins that may serve as targets for the development of antibody-based therapeutic strategies. Such proteomic research activities are particularly needed for the characterization of gene products contributing to the metastatic potential of cancer cells, considering the fact that the majority of cancer-related deaths are associated with the metastatic spread of the disease (7).
Monoclonal antibodies (mAb) represent promising and highly versatile tools for the development of more selective anticancer therapeutic strategies. The activity of intact immunoglobulins may rely on the inhibition of functional proteins, the induction of signaling events, complement activation, and/or the recruitment of immune cells at the tumor site (8). By contrast, antibody derivatives can be used for the targeted delivery of bioactive moieties to cancer sites in vivo (9, 10). Because mAbs need to recognize accessible antigens to display a therapeutic activity, proteomic technologies that allow the identification and relative quantification of membrane proteins, secreted proteins, and extracellular matrix components are particularly important for the development of innovative antibody-based anticancer strategies. However, conventional proteomic approaches that rely on two-dimensional gel electrophoresis encounter difficulty analyzing membrane-associated and low abundance proteins (11). On the other hand, recent gel-free proteomic methods either are inherently not quantitative (e.g., MudPit; ref. 12) or may impose stringent requirement on the homogenous chemical modification of target proteins (e.g., ICAT or iTRAQ; ref. 13).
To address these limitations, we have developed a proteomic technology (termed two-dimensional peptide mapping; refs. 6, 14–16) for the identification and relative quantification of accessible proteins in closely related cell culture models. This methodology features the covalent biotinylation of accessible proteins using reactive ester derivatives of biotin followed by sample lysis, protein capture on streptavidin resin, proteolytic digestion, and comparative analysis of the resulting peptides based on nano-high-performance liquid chromatography (HPLC) and mass spectrometric (MS) procedures. This methodology can be applied to the study of accessible proteins in organs and tumors using in vivo (17–19) or ex vivo (20, 21) perfusion procedures with reactive biotin ester solutions.
In this article, we report the comparative analysis of the cell surface proteome of two closely related murine tumor cell lines (F9B9 and F9DR; ref. 22), of which only the latter is capable of forming liver metastases in immunocompetent Sv129Ev mice after intravenous injections of tumor cells. Our findings reveal substantial changes in the abundance of proteins that have previously been associated with metastatic spread of cancer. However, surprisingly, we have discovered that certain intracellular proteins become exposed on the cell surface of the aggressive F9DR cell line. This unexpected property was confirmed to occur also in vivo, evidenced by the immunofluorescence study of four different syngeneic mouse models of liver metastasis, and by the in vivo targeting experiments with radiolabeled antibody preparations. In summary, we have observed that the expression of intracellular proteins on the membrane of metastatic cells is a feature much more common than previously expected.
Materials and Methods
Cell culture and surface biotinylation. The two murine teratocarcinoma cell lines F9B9 (nonmetastatic cell line) and F9DR (metastatic cell line; ref. 23), which have been kindly provided by Dario Rusciano (SIFI), were propagated on gelatin-coated (F9B9) or uncoated (F9DR) cell culture flasks. Cell were cultured in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) and 1% antibiotic-antimycotic solution (Life Technologies; containing 10,000 units/mL penicillin, 10,000 μg/mL streptomycin, and 25 μg/mL amphotericin B).
For each cell type, five 300 cm2 cell culture flasks containing confluent F9B9 and F9DR teratocarcinoma cells were used for cell surface biotinylation as described (16). All solutions used for biotinylation were cooled to 4°C. Cells were washed once with PBS followed by incubation with PBS containing 880 μmol/L EZ-link sulfo-NHS-SS-biotin (Pierce) for 5 min at room temperature on a shaker. To terminate the biotinylation reaction, Trizma base (Fluka) was added to a final concentration of 8.8 mmol/L. Cells were detached into a 10 mmol/L EDTA/PBS solution containing 1.33 mmol/L oxidized glutathione (Fluka). After centrifugation, cell pellets were washed once with PBS containing 0.66 mmol/L oxidized glutathione.
Isolation, elution, and digestion of biotinylated proteins. For each 300 cm2 cell culture flask, cells were lysed for 30 min on ice with 2 mL lysis buffer [2% (w/v) NP-40 substitute (Fluka), 0.2% (w/v) SDS, 1× complete E protease inhibitor (Roche Diagnostics), 10 mmol/L EDTA, and 108 μmol/L oxidized glutathione in PBS]. The cell lysate was centrifuged for 10 min at 16,100 × g and the cleared supernatant was used for purification of biotinylated proteins on streptavidin-Sepharose high-performance resin (GE Healthcare). Five individual samples of F9B9 and F9DR cells were prepared, containing 2.5 mg total protein each (determined by BCA protein assay; Pierce). Then, 400 μL streptavidin-Sepharose slurry was washed three times with wash buffer A [1% (w/v) NP-40 substitute, 0.1% (w/v) SDS, and 5 mmol/L oxidized glutathione in PBS] before the cleared lysates were added. The total SDS concentration was adjusted to 2% (w/v). The samples were tumbled for 2 h at room temperature before removal of unbound proteins by washing three times with buffer A, twice with buffer B [10% (w/v) SDS and 5 mmol/L oxidized glutathione in PBS], twice with buffer C [2 mol/L NaCl, 0.1% (w/v) SDS, and 5 mmol/L oxidized glutathione in PBS, 40°C], and four times with 50 mmol/L Tris-HCl (pH 7.5) containing 5 mmol/L oxidized glutathione. Captured proteins were eluted from the streptavidin-Sepharose by incubation with 400 μL of 5% 2-mercaptoethanol in PBS for 30 min at 30°C. The elution step was repeated three times; eluates were pooled and split in two. Both samples were precipitated by addition of 100 μL of 100% (w/v) TCA and incubated for 30 min on ice. The precipitates were pelleted by centrifugation (16,100 × g for 5 min), washed with ethanol/ether (1:1), and air-dried. Each precipitate was dissolved in 200 μL digestion buffer [50 mmol/L Tris-HCl (pH 8.0) and 1 mmol/L CaCl2], and 1.6 μg sequencing-grade modified trypsin (Promega) was added. The digestion was carried out over night at 37°C in a thermomixer (Eppendorf) at 1,000 rpm. Resulting tryptic peptides were desalted, purified, and concentrated with C18 microcolumns (ZipTip C18; Millipore) according to the manufacturer's instructions. The two split samples were pooled, lyophilized, and stored at −20°C.
Reverse-phase HPLC. Tryptic peptides were separated by nano-reverse-phase HPLC using an UltiMate nanoscale LC system and a FAMOS microautosampler (LC Packings) controlled by the Chromeleon software (Dionex). Mobile phase A consisted of 2% acetonitrile and 0.1% trifluoroacetic acid in water, and mobile phase B consisted of 80% acetonitrile and 0.1% trifluoroacetic acid in water. The flow rate was set to 300 nL/min. Each of the 10 samples was dissolved in 16.7 μL buffer A, and 5 μL were loaded on the column (15 cm × 75 μm i.d., C18 PepMap 100, 3 μm, 100 Å; LC Packings). The peptides were eluted with a gradient of 0% buffer B for 3 min, 0% to 60% buffer B for 81 min, 60% to 100% buffer B for 10 min, and 100% buffer B for 5 min; the column was equilibrated with 100% buffer A for 20 min before the next sample was processed. The eluted fractions were mixed with a solution of 3 mg/mL α-cyano-4-hydroxycinnamic acid, 277 pmol/mL of each of the three internal standard peptides (neurotensin, angiotensin I, and adrenocorticotropic hormone fragment 1-17; all from Sigma), 70% acetonitrile, and 0.1% trifluoroacetic acid in water and were deposited on a 192-well matrix-assisted laser desorption/ionization (MALDI) target plate using the online Probot system (Dionex). The flow of the MALDI matrix solution was set to 1,083 nL/min. Thus, fractions collected each 20 s contained 361 nL MALDI-matrix solution and 100 nL sample. The final concentration of each of the three standard peptides was 100 fmol/spot.
MALDI-time of flight/time of flight. MALDI-time of flight (TOF) and MALDI-TOF/TOF MS analyses were carried out with the 4700 Proteomics Analyzer (Applied Biosystems). Peptide masses were acquired over a range from 750 to 4,000 m/z, with a focus mass of 2,000 m/z. MS spectra were summed from 2,000 laser shots from a Nd:YAG laser operating at 355 nm and 200 Hz. An automated plate calibration was done using five peptide standards (masses 900-2,400 m/z; Applied Biosystems) in six calibration wells. This plate calibration was used to update the instrument default mass calibration, which was applied to all MS and tandem MS spectra. Furthermore, an internal calibration of each MS spectrum using the three internal standard peptides added to the MALDI matrix was done. A maximum of 15 precursors per sample well with a signal-to-noise ratio of >120 was automatically selected for subsequent fragmentation by collision-induced dissociation. Tandem MS spectra were summed from 2,500 to 4,000 laser shots. Spectra were processed and analyzed by the Global Protein Server Workstation (Applied Biosystems), which uses internal MASCOT (Matrix Science) software for matching MS and tandem MS data against databases of in silico digested proteins. The MASCOT search parameters were (a) mouse database downloaded from the European Bioinformatics Institute homepage3
in September 2006; (b) allowed number of missed cleavages: 1; (c) variable post-translational modifications: methionine oxidation; (d) peptide tolerance: ±15 ppm; (e) tandem MS tolerance: ±0.3 Da; and (f) peptide charge: +1. A 95% confidence interval was used in protein identification.Two-dimensional peptide mapping. The MALDI-TOF MS data were used to establish two-dimensional peptide maps with the Spectational software (16), which plots HPLC fractions on the Y axis and the m/z ratios of the measured peptides on the X axis. In addition, the software translates the logarithmic values of the normalized intensities to a grayscale. The normalization is done with respect to the internal standard peptide neurotensin. ASCII spectrum files for the creation of two-dimensional peptide maps were produced using Data Explorer V4.6 software (Applied Biosystems).
Relative quantification with nQuant. MALDI-TOF MS data combined with results from the Global Protein Server Workstation database search were used for relative quantification using the newly developed nQuant software. At first, this software normalizes each peak in a MS spectrum to the internal standard neurotensin. Then, nQuant sums up the normalized peak intensities from a peptide eluting in several HPLC fractions and compares it with the same peptide in another sample. The ratio of these two normalized peak intensity sums is used as relative quantity. A comprehensive description of this technology will be published soon.
Immunocytochemistry/immunofluorescence microscopy. Immunofluorescence analysis was done on methanol fixed cells grown on 0.8 cm2 Lab-Tek chamber slides (Nunc) using anti-adenylate kinase 2 (Ak2), anti-dipeptidyl peptidase 4 (Dpp4), anti-Syk (Santa Cruz Biotechnology), anti-high-mobility group protein B1 (Hmgb1; Abcam), anti-complement regulatory protein Crry (Crry; BD Pharmingen), and anti-α-actinin-4 (Actn4; Invitrogen) antibodies. The corresponding secondary antibodies were labeled with Alexa Fluor 546 (Invitrogen). Nuclei were counterstained with Hoechst 33342 (Invitrogen). Slices were mounted with Glycergel mounting medium (Dako) and analyzed with an Axiovert S100 TV microscope (Zeiss).
Immunocytochemistry/confocal laser scanning microscopy. Confocal laser scanning microscopy was done on methanol-fixed cells grown on 4.2 cm2 Falcon cell culture inserts (BD Biosciences) using the antibodies indicated above. Secondary antibodies were labeled with Alexa Fluor 546, nuclei were counterstained with 1 μg/mL 4′,6-diamidino-2-phenylindole, and Alexa Fluor 660 phalloidin (all from Invitrogen) was used for probing F-actin. Slices were mounted with mounting medium [70% (v/v) glycerol, 30 mmol/L Tris-HCl (pH 9.5), 5% (w/v) n-propyl-gallate (Sigma)] and analyzed with a LSM 510 META from Zeiss. Images were further processed using the IMARIS software (Bitplane).
Microautoradiography. In vivo targeting performance of four mouse mAbs against Hmbg1, Syk, Actn4, and ovalbumin (all obtained from Abcam; ovalbumin was taken as a negative control) was evaluated by microautoradiography as described previously (24). Briefly, mAbs in IgG format were radiolabeled with 125I using the Iodogen method and injected into the tail vein of immunocompetent 129SvEv mice bearing F9 liver metastases (10 μg IgG; 10 μCi). Seventy-two hours after the intravenous injection of the radiolabeled antibodies, mice were sacrificed and tumors were embedded and frozen in OCT medium (MICROM). Ten micrometer sections were cut and fixed with ice-cold acetone. Sections were then coated with NBT Kodak autoradiography emulsion, dried, and stored at 4°C in the dark for ∼10 weeks. The autoradiography emulsions were developed (Kodak Developer D-19) for 4 min and fixed (Kodak EASTMAN Fixer) for 5 min. Finally, slides were rinsed with deionized water and counterstained with hematoxylin (Sigma).
Mouse models of liver metastases. All animal experiments were approved by either the Swiss or the Italian Veterinary Office and done in accordance with national and international laws and regulations. The intravenous injection of F9DR cells into male Sv129Ev mice (Charles River Laboratories) was done as described in ref. 19. The intrasplenic injection of Colon38 and SL4 cells into female C57BL/6J mice (Harlan) was done as described in ref. 25. The subcutaneous injection of M5076 cells into female C57BL/6J mice (Harlan) was done as described in ref. 26.
Immunofluorescence/confocal laser scanning microscopy. Confocal laser scanning microscopy was done on acetone-fixed cryostat sections (10 μm) of freshly frozen tissue specimens of liver metastases deriving from the intravenous injection of F9DR cells, the subcutaneous injection of M5076 cells, and the intrasplenic injection of SL4 and C38 cells. Immunofluorescent staining with anti-Hmgb1 and anti-Syk antibodies (indicated above) was done according to standard protocols with an additional blocking step using goat anti-mouse IgG F(ab) fragment (Jackson ImmunoResearch). As secondary antibody, goat anti-mouse Alexa Fluor 488 (Invitrogen) was used. Nuclei were counterstained with 1 μg/mL 4′,6-diamidino-2-phenylindole (Invitrogen). Slides were mounted with fluorescent mounting medium (DAKO) and analyzed with a LSM 510 META from Zeiss. Images were further processed using the ImageJ software.4
Results
Two-dimensional peptide mapping analysis of F9B9 ad F9DR cell cultures. F9DR cells represent a subline of the murine teratocarcinoma cell line F9B9 (23), which has acquired a striking potential to form liver metastases following intravenous administration into syngeneic immunocompetent Sv129Ev mice (ref. 22; Fig. 1A, and B, insets). We performed surface biotinylation of parallel cultures of F9B9 and F9DR cells (in each case, 5 parallel cultures) using published methodologies (14, 16). The resulting biotinylated proteins were recovered by cell lysis in the presence of strong detergents and captured on streptavidin resin. After elution, biotinylated proteins were subjected to tryptic digestion, yielding peptides that were separated by nano-reverse-phase HPLC and analyzed by MALDI-TOF in the presence of internal standards for quantification and by MALDI-TOF/TOF for identification (Fig. 1A and B). The resulting two-dimensional peptide maps were used for the computer-assisted identification of proteins, which were consistently up-regulated or down-regulated in F9DR cells. Figure 1C and D shows the reproducibility of nano-reverse-phase HPLC chromatographic profiles for the five tryptic digests for each cell as well as representative MALDI-TOF spectra in triplicate for peptides, which exhibited differential surface expression in the two cell lines. A more detailed analysis of two nonregulated, three F9B9 up-regulated, and three F9DR up-regulated proteins is shown in Supplementary Fig. S1.
Two-dimensional peptide maps of metastatic (F9DR; A) and nonmetastatic (F9B9; B) teratocarcinoma cells, reproducibility of chromatographic separation of tryptic digests of F9DR and F9B9 cells, and representative normalized MALDI-TOF spectra. Intravenous injection of F9DR cells leads to the formation of metastases in the liver (A, inset), whereas animals remain healthy after intravenous injection of F9B9 cells (B, inset). C, an overlap of all five HPLC runs (rep 1-5) of F9DR and F9B9 tryptic digests. D, top sequence, three replicates of the normalized MALDI-TOF spectra of an Actn4 peptide (VGWEQLLTTIAR) are presented for both cell lines. A clear difference in normalized signal intensity between F9DR and F9B9 can be seen. Bottom sequence, three replicates of the normalized MALDI-TOF spectra of a Syk peptide (FLIRAR) for both cell lines. The peptide is clearly up-regulated in F9B9 samples.
Two-dimensional peptide maps of metastatic (F9DR; A) and nonmetastatic (F9B9; B) teratocarcinoma cells, reproducibility of chromatographic separation of tryptic digests of F9DR and F9B9 cells, and representative normalized MALDI-TOF spectra. Intravenous injection of F9DR cells leads to the formation of metastases in the liver (A, inset), whereas animals remain healthy after intravenous injection of F9B9 cells (B, inset). C, an overlap of all five HPLC runs (rep 1-5) of F9DR and F9B9 tryptic digests. D, top sequence, three replicates of the normalized MALDI-TOF spectra of an Actn4 peptide (VGWEQLLTTIAR) are presented for both cell lines. A clear difference in normalized signal intensity between F9DR and F9B9 can be seen. Bottom sequence, three replicates of the normalized MALDI-TOF spectra of a Syk peptide (FLIRAR) for both cell lines. The peptide is clearly up-regulated in F9B9 samples.
In the course of this proteomic investigation, a total of almost 1,000 proteins was identified, corresponding to 695 peptide sets, of which 520 peptide sets matched to a single protein (Supplementary Tables S1 and S2).
Table 1 shows a list of selected proteins displaying differential abundance in the two-dimensional peptide mapping analysis. This list features the down-regulation in F9DR cells of proteins that have been associated with inhibition of metastatic spread [e.g., tyrosine protein kinase SYK (Syk)] or to participate in cell-cell interactions (e.g., Dpp4). Similarly, we observed the up-regulation in F9DR cells of proteins that have previously been associated with an invasive phenotype of cancer (e.g., Hmgb1, Ak2 homologue, and Actn4).
List of selected proteins identified in the comparative proteomic analysis
SwissProt accession no. . | Protein name . | Ratio B9/DR* . | SD† . | Subcellular localization‡ . | Type of membrane protein§ . | No. samples . | . | No. identified peptides∥ . | . | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
. | . | . | . | . | . | B9 (total = 4) . | DR (total = 5) . | B9 . | DR . | |||||||||
Proteins identified only in DR samples | ||||||||||||||||||
Q9D2G5 | Synaptojanin-2 | 0.16 | PM | IM | 1 | 1 | ||||||||||||
O35499 | Nuclear autoantigenic sperm protein | 0.27 | IN | 3 | 1 | |||||||||||||
Q8CEW7 | Ethanol induced 6 | 0.28 | IN | 2 | 1 | |||||||||||||
P63158 | Hmgb1 | 0.29 | IN | 5 | 1 | |||||||||||||
Q8C7I9 | Ak2 homologue | 0.33 | IN | 1 | 1 | |||||||||||||
Q9DBG3 | AP-2 complex subunit β-1 | 0.37 | PM | MA | 1 | 1 | ||||||||||||
Q8VBV0 | Protein tyrosine phosphatase, receptor type, δA | 0.43 | PM | IM | 1 | 1 | ||||||||||||
Q61177 | Casein kinase IIα subunit | 0.45 | IN | 2 | 1 | |||||||||||||
O35685 | Nuclear migration protein nudC | 0.47 | 0.01 | IN | 1 | 2 | ||||||||||||
P97855 | Ras GTPase-activating protein-binding protein 1 | 0.48 | 0.02 | IN | 4 | 2 | ||||||||||||
Proteins identified in both cell lines | ||||||||||||||||||
Q61937 | Nucleophosmin | 0.24 | 0.08 | IN | 4 | 5 | 4 | 5 | ||||||||||
O35662 | P68 RNA helicase | 0.37 | IN | 1 | 3 | 1 | 1 | |||||||||||
P57780 | Actn4 | 0.38 | 0.09 | IN | 1 | 5 | 1 | 5 | ||||||||||
P17182 | α-Enolase | 0.94 | 0.18 | IN | 4 | 5 | 7 | 6 | ||||||||||
Q5EBQ0 | Voltage-dependent anion channel 3 | 1.00 | 0.16 | OM | IM | 4 | 5 | 6 | 8 | |||||||||
Q8BKC5 | Importin β-3 | 1.01 | 0.17 | IN | 4 | 5 | 3 | 5 | ||||||||||
P21995 | Embigin precursor | 1.04 | 0.21 | PM | IM | 1 | 4 | 2 | 4 | |||||||||
Q5U647 | Solute carrier family 1, member 5 | 1.06 | 0.40 | PM | IM | 4 | 5 | 4 | 4 | |||||||||
Q5SV72 | A disintegrin and metalloprotease domain 23 | 1.97 | PM | IM | 3 | 1 | 1 | 1 | ||||||||||
Q925F2 | Endothelial cell-selective adhesion molecule | 2.17 | 0.06 | PM | IM | 4 | 2 | 2 | 1 | |||||||||
P52795 | Ephrin-B1 precursor | 2.21 | PM | IM | 3 | 1 | 1 | 1 | ||||||||||
P57787 | Monocarboxylate transporter 4 | 2.35 | 0.40 | PM | IM | 4 | 4 | 4 | 3 | |||||||||
P17809 | Solute carrier family 2, facilitated glucose transporter member 1 | 2.35 | 0.64 | PM | IM | 4 | 5 | 5 | 3 | |||||||||
P48025 | Syk | 2.59 | PM | MA | 1 | 2 | 1 | 1 | ||||||||||
Q3UDE2 | Hypothetical tubulin-tyrosine ligase containing protein | 3.05 | EX | 2 | 1 | 1 | 1 | |||||||||||
Proteins identified only in B9 samples | ||||||||||||||||||
Q3TLF6 | MYB binding protein 1a | 0.46 | 0.01 | IN | 1 | 2 | ||||||||||||
P39038 | Cadherin-4 precursor | 2.15 | PM | IM | 1 | 1 | ||||||||||||
P40240 | CD9 antigen | 2.57 | PM | IM | 3 | 1 | ||||||||||||
Q91ZX7 | Low-density lipoprotein receptor-related protein 1 | 2.61 | 0.95 | PM | IM | 2 | 6 | |||||||||||
P28843 | Dpp4 | 3.36 | PM | IM | 1 | 1 | ||||||||||||
Q64735 | Crry | 3.62 | PM | IM | 1 | 1 | ||||||||||||
P21956 | Lactadherin precursor | 4.97 | 0.79 | EX | 3 | 3 | ||||||||||||
P31809 | Carcinoembryonic antigen-related cell adhesion molecule 1 | 5.05 | PM | IM | 3 | 2 |
SwissProt accession no. . | Protein name . | Ratio B9/DR* . | SD† . | Subcellular localization‡ . | Type of membrane protein§ . | No. samples . | . | No. identified peptides∥ . | . | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
. | . | . | . | . | . | B9 (total = 4) . | DR (total = 5) . | B9 . | DR . | |||||||||
Proteins identified only in DR samples | ||||||||||||||||||
Q9D2G5 | Synaptojanin-2 | 0.16 | PM | IM | 1 | 1 | ||||||||||||
O35499 | Nuclear autoantigenic sperm protein | 0.27 | IN | 3 | 1 | |||||||||||||
Q8CEW7 | Ethanol induced 6 | 0.28 | IN | 2 | 1 | |||||||||||||
P63158 | Hmgb1 | 0.29 | IN | 5 | 1 | |||||||||||||
Q8C7I9 | Ak2 homologue | 0.33 | IN | 1 | 1 | |||||||||||||
Q9DBG3 | AP-2 complex subunit β-1 | 0.37 | PM | MA | 1 | 1 | ||||||||||||
Q8VBV0 | Protein tyrosine phosphatase, receptor type, δA | 0.43 | PM | IM | 1 | 1 | ||||||||||||
Q61177 | Casein kinase IIα subunit | 0.45 | IN | 2 | 1 | |||||||||||||
O35685 | Nuclear migration protein nudC | 0.47 | 0.01 | IN | 1 | 2 | ||||||||||||
P97855 | Ras GTPase-activating protein-binding protein 1 | 0.48 | 0.02 | IN | 4 | 2 | ||||||||||||
Proteins identified in both cell lines | ||||||||||||||||||
Q61937 | Nucleophosmin | 0.24 | 0.08 | IN | 4 | 5 | 4 | 5 | ||||||||||
O35662 | P68 RNA helicase | 0.37 | IN | 1 | 3 | 1 | 1 | |||||||||||
P57780 | Actn4 | 0.38 | 0.09 | IN | 1 | 5 | 1 | 5 | ||||||||||
P17182 | α-Enolase | 0.94 | 0.18 | IN | 4 | 5 | 7 | 6 | ||||||||||
Q5EBQ0 | Voltage-dependent anion channel 3 | 1.00 | 0.16 | OM | IM | 4 | 5 | 6 | 8 | |||||||||
Q8BKC5 | Importin β-3 | 1.01 | 0.17 | IN | 4 | 5 | 3 | 5 | ||||||||||
P21995 | Embigin precursor | 1.04 | 0.21 | PM | IM | 1 | 4 | 2 | 4 | |||||||||
Q5U647 | Solute carrier family 1, member 5 | 1.06 | 0.40 | PM | IM | 4 | 5 | 4 | 4 | |||||||||
Q5SV72 | A disintegrin and metalloprotease domain 23 | 1.97 | PM | IM | 3 | 1 | 1 | 1 | ||||||||||
Q925F2 | Endothelial cell-selective adhesion molecule | 2.17 | 0.06 | PM | IM | 4 | 2 | 2 | 1 | |||||||||
P52795 | Ephrin-B1 precursor | 2.21 | PM | IM | 3 | 1 | 1 | 1 | ||||||||||
P57787 | Monocarboxylate transporter 4 | 2.35 | 0.40 | PM | IM | 4 | 4 | 4 | 3 | |||||||||
P17809 | Solute carrier family 2, facilitated glucose transporter member 1 | 2.35 | 0.64 | PM | IM | 4 | 5 | 5 | 3 | |||||||||
P48025 | Syk | 2.59 | PM | MA | 1 | 2 | 1 | 1 | ||||||||||
Q3UDE2 | Hypothetical tubulin-tyrosine ligase containing protein | 3.05 | EX | 2 | 1 | 1 | 1 | |||||||||||
Proteins identified only in B9 samples | ||||||||||||||||||
Q3TLF6 | MYB binding protein 1a | 0.46 | 0.01 | IN | 1 | 2 | ||||||||||||
P39038 | Cadherin-4 precursor | 2.15 | PM | IM | 1 | 1 | ||||||||||||
P40240 | CD9 antigen | 2.57 | PM | IM | 3 | 1 | ||||||||||||
Q91ZX7 | Low-density lipoprotein receptor-related protein 1 | 2.61 | 0.95 | PM | IM | 2 | 6 | |||||||||||
P28843 | Dpp4 | 3.36 | PM | IM | 1 | 1 | ||||||||||||
Q64735 | Crry | 3.62 | PM | IM | 1 | 1 | ||||||||||||
P21956 | Lactadherin precursor | 4.97 | 0.79 | EX | 3 | 3 | ||||||||||||
P31809 | Carcinoembryonic antigen-related cell adhesion molecule 1 | 5.05 | PM | IM | 3 | 2 |
Ratio calculated by nQuant.
SD of the nQuant calculated ratio.
Abbreviations: EX, extracellular; IN, intracellular; OM, organelle membrane; PM, plasma membrane.
Abbreviations: IM, integral membrane; MA, membrane associated.
The number of identified peptides is the total number of different peptides identified in all samples from one cell line.
Analysis of the putative subcellular localization of the proteins corresponding to the 84 regulated peptide sets revealed, as expected, a strong enrichment in plasma membrane proteins and extracellular space components in F9B9 cells (71%; Supplementary Fig. S2A). By contrast, we observed a striking enrichment for proteins with a putative nuclear and cytoplasmic localization in F9DR cells (74%; Supplementary Fig. S2B).
Characterization of subcellular localization of selected regulated proteins. To confirm that the surface display of intracellular proteins in F9DR cultures was a real event and not merely an artifact of cell lysis before the in vitro biotinylation procedure, we performed a confocal laser scanning microscopic analysis of F9DR and F9B9 cells using fluorescence-labeled antibodies specific to proteins that were found to be up-regulated (Actn4, Hmgb1, and Ak2) or down-regulated (Syk, Dpp4, and Crry) on the surface of F9DR cells.
Figure 2 (left and middle columns) shows a two-color fluorescence microscopic analysis of F9DR and F9B9 cells, revealing as expected an up-regulation of Actn4, Hmgb1, and Ak2 and a down-regulation of Syk, Dpp4, and Crry in F9DR cells. To confirm protein localization on the plasma membrane, we analyzed optical sections of the confocal microscopic images, revealing a presence not only in the cytoplasm but also on the tumor cell membrane for all six proteins (Fig. 2, right column; Supplementary Fig. S3A-F). Two of them (Actn4 and Ak2) have previously been postulated to be located exclusively in cytoplasmic or mitochondrial compartments, respectively.
Immunofluorescence analysis and characterization of the subcellular localization of the six selected differentially regulated proteins. The proteins Actn4, Hmgb1, and Ak2 were identified in the proteomic analysis to be up-regulated in F9DR-cells, which was confirmed by immunofluorescence (left and middle columns). By contrast, the proteins Syk, Dpp4, and Crry have been found to be up-regulated in F9B9 cells. Bar, 100 μm. Two-color confocal laser scanning microscopy (CLSM) images (red, antibody against selected protein; blue, 4′,6-diamidino-2-phenylindole) displaying x-y (big display), x-z (below), and y-z (on the right side) optical sections of representative areas (right column). Alongside, the corresponding three-color images (actin stained green, nuclei stained blue, and listed proteins stained red) are presented. Antibodies against Actn4, Hmgb1, and Ak2 were used to stain F9DR cells; antibodies against Syk, Dpp4, and Crry were used to stain F9B9 cells. All proteins are found, among other things, in the plasma membrane. Bar, 15 μm.
Immunofluorescence analysis and characterization of the subcellular localization of the six selected differentially regulated proteins. The proteins Actn4, Hmgb1, and Ak2 were identified in the proteomic analysis to be up-regulated in F9DR-cells, which was confirmed by immunofluorescence (left and middle columns). By contrast, the proteins Syk, Dpp4, and Crry have been found to be up-regulated in F9B9 cells. Bar, 100 μm. Two-color confocal laser scanning microscopy (CLSM) images (red, antibody against selected protein; blue, 4′,6-diamidino-2-phenylindole) displaying x-y (big display), x-z (below), and y-z (on the right side) optical sections of representative areas (right column). Alongside, the corresponding three-color images (actin stained green, nuclei stained blue, and listed proteins stained red) are presented. Antibodies against Actn4, Hmgb1, and Ak2 were used to stain F9DR cells; antibodies against Syk, Dpp4, and Crry were used to stain F9B9 cells. All proteins are found, among other things, in the plasma membrane. Bar, 15 μm.
To further investigate whether proteins overexpressed in the F9DR cells are also surface-expressed in vivo (and thus accessible to mAbs), we performed a microautoradiography study. To this end, we labeled four different mAbs with 125I and injected them intravenously into immunocompetent 129SvEv mice bearing F9 liver metastases. The microautoradiographic analysis of liver metastasis sections, obtained 72 h post-injection, confirmed the accumulation of the antibodies against Hmgb1 and Syk (Fig. 3) around nests of tumor cells. By contrast, antibodies specific to Actn4 (found to be up-regulated in liver metastases) and ovalbumin (used as negative control) exhibited no preferential accumulation in metastases (Fig. 3). Finally, to assess the generality of our observation, we studied liver metastases of M5076 reticulosarcoma cells and the two colon carcinoma cell lines Colon38 and SL4, developed by either subcutaneous (M5075) or intrasplenic (Colon38 and SL4) injection of tumor cells (Fig. 4). In addition to F9 liver metastases, we observed a strong membranous staining of metastatic cells in the liver lesions of colorectal cancer origin (Colon38 and SL4) and for the reticulum cell sarcoma (M5076). Whereas Hmgb1 and Syk were clearly detectable in all four models of liver metastasis, Hmgb1 was found to be more strongly expressed in F9 and M5076 lesions, whereas Syk was also abundant in C38 metastases.
Microautoradiography of F9 liver metastases 72 h after intravenous injection of 125I-labeled anti-Hmgb1 (A), anti-Syk (B), anti-Actn4 (C), and anti-ovalbumin (D) antibodies. Bar, 50 μm.
Microautoradiography of F9 liver metastases 72 h after intravenous injection of 125I-labeled anti-Hmgb1 (A), anti-Syk (B), anti-Actn4 (C), and anti-ovalbumin (D) antibodies. Bar, 50 μm.
Immunofluorescence analysis of two selected differentially regulated proteins on four different mouse liver metastasis models. Antibodies against Hmgb1 (left, −, omitting the primary antibody; +, using the primary antibody) and Syk (right) were used to stain metastases derived from the intravenous injection of F9DR teratocarcinoma cells (F9), the intrasplenic injection of the two colon carcinoma cell lines C38 and SL4, and the subcutaneous injection of M5076 reticulosarcoma cells (green, antibody against selected protein; blue, 4′,6-diamidino-2-phenylindole). Two bottom rows, tumor-liver boarders in the M5076 model. Bar, 50 μm. M, metastasis.
Immunofluorescence analysis of two selected differentially regulated proteins on four different mouse liver metastasis models. Antibodies against Hmgb1 (left, −, omitting the primary antibody; +, using the primary antibody) and Syk (right) were used to stain metastases derived from the intravenous injection of F9DR teratocarcinoma cells (F9), the intrasplenic injection of the two colon carcinoma cell lines C38 and SL4, and the subcutaneous injection of M5076 reticulosarcoma cells (green, antibody against selected protein; blue, 4′,6-diamidino-2-phenylindole). Two bottom rows, tumor-liver boarders in the M5076 model. Bar, 50 μm. M, metastasis.
Discussion
Metastatic spread of cancer is responsible for the majority of cancer-related deaths (7). The identification of accessible proteins, which are up-regulated in the metastatic process, may facilitate the development of mAbs for pharmacodelivery strategies. We have performed a comparative proteomic analysis of nonmetastatic (F9B9) and metastatic (F9DR) teratocarcinoma cell lines, applying two-dimensional peptide mapping procedure (6, 14–16) and a newly developed software (nQuant), which facilitates the relative protein quantification by comparing MALDI-TOF signal intensities with those of internal standard peptides. In Table 2, we summarized some features of selected proteins that exhibited a different expression in F9B9 and F9DR cells and that have previously been implicated in cancer growth and metastatic spread. Unexpectedly, we found several intracellular proteins to be displayed on the surface of tumor cells with metastatic potential and we confirmed this finding in vivo in four different models of liver metastasis.
Summary of literature findings for selected regulated proteins indentified in the comparative proteomics analysis
SwissProt accession no. . | Protein name . | Ratio B9/DR . | Literature findings . | Ref. . |
---|---|---|---|---|
Q9D2G5 | Synaptojanin-2 | 0.16 | Critical function in the migration and invasion of glioma cells, potentially mediated by its role in formation of lamellipodia and invadopodia | 35 |
O35499 | Nuclear autoantigenic sperm protein | 0.27 | Substantially up-regulated during tumor progression, role of the protein remains to be clarified | 36 |
Q8CEW7 | Ethanol induced 6 | 0.28 | Found to be up-regulated in cells with high metastatic potential in a suppression subtractive hybridization study of mouse hepatocarcinoma cell lines | 37 |
P63158 | Hmgb1 | 0.29 | Plays a role in the invasion and metastasis, proposed to act intracellular (transcription regulation) and extracellular (receptor binding) | 38 |
Q8C7I9 | Ak2 homologue | 0.33 | Up-regulated in metastatic pancreatic endocrine neoplasms based on microarray data | 39 |
Q61177 | Casein kinase IIα subunit | 0.45 | Predictor of outcome in patients with squamous cell carcinoma in the lung; protein up-regulation correlates with a significant reduction of overall survival | 40 |
Q61937 | Nucleophosmin | 0.24 | Frequently overexpressed, mutated, rearranged, and deleted in cancer; mainfold functions, contributes to oncogenesis through several distinct mechanisms | 41 |
P57780 | Actn4 | 0.38 | Actively increases cell motility and promotes lymph node metastasis in colorectal cancer; most significantly up-regulated in dedifferentiated cancer cells at the invasive front | 42 |
Q9R1V7 | ADAM 23 | 1.97 | Hypermethylated in advanced stages of head and neck cancer | 43 |
P31809 | Carcinoembryonic antigen-related cell adhesion molecule 1 | 5.05 | Down-regulated in various cancer types; inverse correlation between CEACAM1 expression and clinical grades of prostate cancer suggested | 44 |
Q64735 | Crry | 3.62 | Up-regulated on some tumor cells; tumor-specific inhibition of complement regulatory proteins using bi-mAbs shown to significantly improve mAb-mediated immunotherapy | 45 |
P28843 | Dpp4 | 3.36 | Loss of Dpp4 hypothesized to reduce tumor cell adhesiveness and thereby to promote detachment of cells from primary tumors initiating metastasis | 46 |
Q91ZX7 | Pro-low-density lipoprotein receptor-related protein 1 | 2.61 | Likely to be required for the inhibition of invasion mediated by trombospondin-2 through an antiangiogenic effect | 47 |
P40240 | CD9 antigen | 2.57 | Expression level varying during tumor progression (low to absent in normal ovarian surface endothelium, increased in low-grade ovarian tumors, lost in ovarian tumors with a malignant phenotype) | 48 |
P39038 | Cadherin-4 precursor | 2.15 | Reported to be one of the genes most frequently repressed by promoter methylation in human gastrointestinal tumors (suggested tumor suppressor gene) | 49 |
P48025 | Syk | 2.59 | Clinical studies revealed a correlation between reduced Syk expression and increased risk for metastasis formation | 50 |
SwissProt accession no. . | Protein name . | Ratio B9/DR . | Literature findings . | Ref. . |
---|---|---|---|---|
Q9D2G5 | Synaptojanin-2 | 0.16 | Critical function in the migration and invasion of glioma cells, potentially mediated by its role in formation of lamellipodia and invadopodia | 35 |
O35499 | Nuclear autoantigenic sperm protein | 0.27 | Substantially up-regulated during tumor progression, role of the protein remains to be clarified | 36 |
Q8CEW7 | Ethanol induced 6 | 0.28 | Found to be up-regulated in cells with high metastatic potential in a suppression subtractive hybridization study of mouse hepatocarcinoma cell lines | 37 |
P63158 | Hmgb1 | 0.29 | Plays a role in the invasion and metastasis, proposed to act intracellular (transcription regulation) and extracellular (receptor binding) | 38 |
Q8C7I9 | Ak2 homologue | 0.33 | Up-regulated in metastatic pancreatic endocrine neoplasms based on microarray data | 39 |
Q61177 | Casein kinase IIα subunit | 0.45 | Predictor of outcome in patients with squamous cell carcinoma in the lung; protein up-regulation correlates with a significant reduction of overall survival | 40 |
Q61937 | Nucleophosmin | 0.24 | Frequently overexpressed, mutated, rearranged, and deleted in cancer; mainfold functions, contributes to oncogenesis through several distinct mechanisms | 41 |
P57780 | Actn4 | 0.38 | Actively increases cell motility and promotes lymph node metastasis in colorectal cancer; most significantly up-regulated in dedifferentiated cancer cells at the invasive front | 42 |
Q9R1V7 | ADAM 23 | 1.97 | Hypermethylated in advanced stages of head and neck cancer | 43 |
P31809 | Carcinoembryonic antigen-related cell adhesion molecule 1 | 5.05 | Down-regulated in various cancer types; inverse correlation between CEACAM1 expression and clinical grades of prostate cancer suggested | 44 |
Q64735 | Crry | 3.62 | Up-regulated on some tumor cells; tumor-specific inhibition of complement regulatory proteins using bi-mAbs shown to significantly improve mAb-mediated immunotherapy | 45 |
P28843 | Dpp4 | 3.36 | Loss of Dpp4 hypothesized to reduce tumor cell adhesiveness and thereby to promote detachment of cells from primary tumors initiating metastasis | 46 |
Q91ZX7 | Pro-low-density lipoprotein receptor-related protein 1 | 2.61 | Likely to be required for the inhibition of invasion mediated by trombospondin-2 through an antiangiogenic effect | 47 |
P40240 | CD9 antigen | 2.57 | Expression level varying during tumor progression (low to absent in normal ovarian surface endothelium, increased in low-grade ovarian tumors, lost in ovarian tumors with a malignant phenotype) | 48 |
P39038 | Cadherin-4 precursor | 2.15 | Reported to be one of the genes most frequently repressed by promoter methylation in human gastrointestinal tumors (suggested tumor suppressor gene) | 49 |
P48025 | Syk | 2.59 | Clinical studies revealed a correlation between reduced Syk expression and increased risk for metastasis formation | 50 |
Antibody-based pharmacodelivery strategies have thus far mainly focused on membrane proteins as target antigens of choice (27). Alternatively, certain intracellular proteins (e.g., histones) have been found to be abundant at sites of necrosis and thus potentially amenable for targeted delivery applications (28), in consideration of the fact that necrosis is a rare event in the healthy adult but a characteristic feature of aggressive solid tumors. More recently, we and others have focused our attention to components of the modified extracellular matrix (e.g., splice isoforms of fibronectins and tenascins) as suitable targets for antibody-based pharmacodelivery applications (9, 29). Indeed, as these targets are often found in the subendothelial extracellular matrix, we have extensively shown, both in animal models of cancer and in patients with metastatic disease, that markers of angiogenesis such as alternatively spliced domains of fibronectin and of tenascin-C may be efficiently targeted by human mAbs in a variety of different malignancies (19, 30). Although we have investigated a variety of different antibody functionalization strategies in preclinical models of cancer (29, 30), the most striking therapeutic results have thus far been observed with radiolabeled antibodies (31) and with immunocytokines (32). For this reason, derivatives of the L19 antibody (specific to the extra-domain B of fibronectin) and the F16 antibody (specific to the extra-domain A1 of tenascin-C) have been moved to clinical trials in patients with cancer, with promising therapeutic results (33).
The structural and functional study of intracellular proteins such as Hmgb1, α-enolase, and nucleolin (34) indicates that certain proteins may be expressed in different cell compartments in healthy cells and cancer cells, with distinct functional roles. Based on surface protein biotinylation and immunofluorescence data, this study reveals that export of intracellular proteins to the cell surface may be a much more frequent process yet restricted to certain pathologic situations. Proteins may reach the cell surface either through active export mechanisms (by the endoplasmic reticulum/Golgi-dependent pathway), endoplasmic reticulum/Golgi-independent protein secretion (e.g., mediated by caspase-1 or heparin sulfate proteoglycans), or by association with the membrane of neighboring cells after apoptotic or necrotic processes.
The surface accessibility of proteins such as Hmgb1 was shown not only in vitro using confocal laser scanning microscopy but also in vivo using injection of radiolabeled antibody and microautoradiographic analysis. The most promising antigens for pharmacodelivery applications (Hmgb1 and Syk) were found to be selectively and strongly expressed in 4 of 4 syngeneic mouse models of liver metastasis, thus reinforcing the concept that surface expression of certain intracellular proteins may be a general process in metastatic spread. These markers may represent ideal targets for antibody-based drug delivery strategies (8, 9). A comparative biodistribution analysis in the same animal models (24)5
B. Borgia et al. A chemical proteomic approach for the identification of accessible markers of liver metastasis, submitted for publication.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Acknowledgments
Grant support: ETH, Swiss National Science Foundation, Bundesamt für Bildung und Wissenschaft (EU Project STROMA), European Union (EU Projects Immuno-PDT and ADAMANT), Oncosuisse, Swissbridge, and Stammbach Foundation. C. Schliemann receives a bursary from the Deutsche Krebshilfe (German Cancer Aid).
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.
We thank the Functional Genomics Center Zurich for access to instrumentation and technical support, Dr. Patrick Pedrioli for help in the development of the nQuant software, and Prof. André W. Brändli for the helpful discussion.