Article pubs.acs.org/jpr
Identification of Glycoprotein Markers for Pancreatic Cancer CD24+CD44+ Stem-like Cells Using Nano-LC−MS/MS and Tissue Microarray Jianhui Zhu, Jintang He, Yashu Liu, Diane M. Simeone, and David M. Lubman* Department of Surgery, University of Michigan Medical Center, Ann Arbor, Michigan 48109, United States S Supporting Information *
ABSTRACT: Pancreatic adenocarcinoma is characterized by late diagnosis due to lack of early symptoms, extensive metastasis, and high resistance to chemo/radiation therapy. Recently, a subpopulation of cells within pancreatic cancers, termed cancer stem cells (CSCs), has been characterized and postulated to be the drivers for pancreatic cancer and responsible for metastatic spread. Further studies on pancreatic CSCs are therefore of particular importance to identify novel diagnosis markers and therapeutic targets for this dismal disease. Herein, the malignant phenotype of pancreatic cancer stem-like CD24+CD44+ cells was isolated from a human pancreatic carcinoma cell line (PANC-1) and demonstrated 4-fold increased invasion ability compared to CD24−CD44+ cells. Using lectin microarray and nano LC−MS/MS, we identified a differentially expressed set of glycoproteins between these two subpopulations. Lectin microarray analysis revealed that fucose- and galactose-specific lectins, UEA-1 and DBA, respectively, exhibit distinctly strong binding to CD24+CD44+ cells. The glycoproteins extracted by multilectin affinity chromatography were consequently analyzed by LC−MS/MS. Seventeen differentially expressed glycoproteins were identified, including up-regulated Cytokeratin 8/CK8, Integrin β1/CD29, ICAM1/CD54, and Ribophorin 2/RPN2 and downregulated Aminopeptidase N/CD13. Immunohistochemical analysis of tissue microarrays showed that CD24 was significantly associated with late-stage pancreatic adenocarcinomas, and RPN2 was exclusively coexpressed with CD24 in a small population of CD24-positive cells. However, CD13 expression was dramatically decreased along with tumor progression, preferentially present on the apical membrane of ductal cells and vessels in early stage tumors. Our findings suggest that these glycoproteins may provide potential therapeutic targets and promising prognostic markers for pancreatic cancer. KEYWORDS: glycoprotein, pancreatic cancer, stem-like cells, CD24, LC−MS/MS, lectin affinity chromatography, tissue microarray
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INTRODUCTION Pancreatic adenocarcinoma is currently the fourth deadliest cancer in the United States, with an overall 5-year survival rate of 1−4% and a median survival period of 4−6 months.1,2 It is typically diagnosed at a late stage, frequently after metastasis, and is notoriously resistant to chemo/radiation therapy.3 Discovery of new early detection markers and efficient therapeutic targets still remains a major challenge. Increasing evidence has verified that solid tumors, including pancreatic cancer, are initiated and sustained by a subset of cancer stem cells (CSCs).4−10 CSCs have characteristic traits such as a distinct surface marker expression profile and the intrinsic capacity to self-renew and recapitulate the phenotype of the original tumor.11 Putative pancreatic CSCs, defined first by the simultaneous expression of CD44, CD24, and epithelialspecific antigen (ESA), are highly tumorigenic and possess the properties of self-renewal and the ability to produce differentiated progeny.9 Further studies on CSCs offer the possibility to find early detection markers and to develop novel © 2012 American Chemical Society
therapeutic targets that can improve therapeutic efficacy for pancreatic cancer. Glycosylation is one of the most common posttranslational modifications in proteins. The alterations in protein glycosylation that occur through varying the heterogeneity of glycosylation sites or changing glycan structure of proteins on the cell surface have been shown to correlate with the development of cancer and other disease states.12,13 Most clinical biomarkers and CSCs surface markers in solid tumors are glycoproteins,11,14 suggesting that altered glycosylation may be important in CSC identification and/or function. Lectin microarrays15,16 coupled with lectin affinity chromatography17 have greatly facilitated the profiling of glycan expression patterns of glycoproteins.18,19 The enriched glycoproteins or glycopeptides can be consequently determined using mass spectrometry, which plays an essential role in the analysis of glycoproteins.20,21 Received: October 22, 2011 Published: February 15, 2012 2272
dx.doi.org/10.1021/pr201059g | J. Proteome Res. 2012, 11, 2272−2281
Journal of Proteome Research
Article
performed in triplicate. Statistical significance was calculated using a student’s t test.
Herein, we present a glycoproteomics comparison of two subpopulations isolated from PANC-1 cells, one of the most aggressive human pancreatic carcinoma cell lines, using the stemness markers CD24 and CD44. We demonstrated that PANC-1 cells were nearly all positive for CD44; therefore, the comparison was investigated between CD24+CD44+ and CD24−CD44+ cells. We utilized matrigel invasion assay to explore the invasion ability of the two subpopulations, and lectin microarrays to detect differences in their glycosylation patterns. The glycoproteins extracted by multilectin affinity chromatography were identified by LC−MS/MS and quantified using a label-free method termed normalized spectral index. Seventeen differentially expressed glycoproteins were identified in CD24+CD44+ cells compared to CD24−CD44+ cells. The differential expression was further validated by Western blot and evaluated by immunohistochemistry on tissue microarrays including 13 normal human pancreas tissues and 53 pancreatic adenocarcinomas to investigate the correlation with clinicopathologic parameters. Our findings may provide promising prognostic markers and potential therapeutic targets for pancreatic cancer.
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Protein Extraction
Cells from both populations were washed twice with PBS and then suspended in ice-cold lysis buffer containing 1% octyl-Dglucopyranoside, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1× protease inhibitor cocktail. The cells were homogenized with 30 strokes in a Dounce glass homogenizer with a tightfitting pestle, and then incubated on ice for 10 min with periodic vortex. The cell lysate was centrifuged at 40000× g for 30 min at 4 °C. The supernatant was collected and the protein concentration was determined by the Bradford method.22 Lectin Microarray
The Lectin microarrays were produced as described previously.17 The SNA-2 and TKA lectins were purchase from E.Y. Laboratories (San Mateo, CA) and the other thirteen lectins were purchased from Vector Laboratories (Burlingame, CA). Their carbohydrate specificities are listed in Supplementary Table S1 (Supporting Information). Lectins were dissolved in PBS at a concentration of 1 mg/mL and spotted on FAST 16 pad nitrocellulose slides (Whatman Inc., Florham Park, NJ) using a piezoelectric noncontact printer (Nano plotter; GESIM). Each lectin was printed in triplicate and each spot contained 2.0 nL of lectin, with spotting of 400 pL for five times. The slides were incubated in a humidity-controlled incubator (>45% humidity) overnight to allow lectin immobilization. The glycoprotein-lectin interaction was explored using a biotin-streptavidin system as described before.15 Four micrograms of protein from cell lysate were labeled with EZ-link iodoacetyl-LC-biotin (Pierce) for 90 min, followed by adding 1 μL of 2-mercaptoethanol (Sigma) to quench the reaction. The slides were blocked with 1% BSA/PBS for 1 h, then the labeled sample was added to lectin blocks and incubated for 1 h. After washing with PBST, the slides were incubated with streptavidinylated fluorescent dye Alexa555 (Invitrogen Biotechnology) for 1 h, followed by three washes with PBST, and then dried by centrifugation prior to imaging. The fluorescence intensity of each spot was explored under a microarray scanner (Genepix 4000A; Axon) and analyzed by Genepix Pro 6.0 software.
MATERIALS AND METHODS
Cell Culture
PANC-1 cells were cultured in DMEM/F12 (Gibco, Invitrogen) supplemented with 10% (v/v) FBS (fetal bovine serum; Invitrogen), 100 units/mL penicillin and 0.1 mg/mL streptomycin. Adherent cells were maintained in standard conditions for less than 8 passages at 37 °C with 5% CO2 and detached using trypsin/EDTA solution (trypsin 0.25% and 1 mM EDTA). FACS
PANC-1 cells were grown to 80−90% confluence prior to flow sorting. The cells were trypsinized for dissociation, washed twice with FACS buffer (PBS containing 1% BSA and 1 mM EDTA), and resuspended in FACS buffer at a concentration of 106 cells/100 μL. Direct-labeled antibodies, anti-CD24-FITC and anti-CD44-PE (BD Bioscience, Pharmingen), were added to the sample at a dilution of 1:40 and incubated with the sample in the dark for 20 min on ice. Subsequently, cells were washed twice with FACS buffer and resuspended in FACS buffer followed by flow cytometry using a FACSAria (BD Immunocytometry Systems, Franklin Lakes, NJ). Side scatter and forward scatter profiles were used to eliminate cell doublets. Cells were reanalyzed for purity, which typically was >96%.
Lectin Affinity Chromatography
The Pierce centrifuge column (Thermo Scientific, IL) was packed with agarose-bound lectins, containing Ulex Europaeus Agglutinin I (UEA-1), Dolichos biflorus Agglutinin (DBA), and Aleuria Aurantia Lectin (AAL), 0.5 mL of slurry each. All lectins were purchased from Vector Laboratories (Burlingame, CA). The column was washed with 3 mL of binding buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM CaCl2). Cell lysates from both populations, containing one milligram of proteins respectively, were diluted four times with ice-cold binding buffer, loaded onto the column and incubated for 15 min. The column was washed with 6 mL of binding buffer to remove nonspecific binding proteins. The captured glycoproteins were then released twice with 2 mL of elution buffer (100 mM L-fucose and 200 mM N-acetylgalactosamine in binding buffer, pH 7.4). The eluted fractions were concentrated using Microcon YM-3 centrifugal filter devices to 200 μL in 50 mM NH4HCO3.
Matrigel Invasion Assay
In vitro invasion assays were performed using 24-well Matrigelcoated transwell chambers with 8 μm pore size (BD Biosciences, Bedford, MA). CD24+CD44+ and CD24−CD44+ cells were isolated by flow sorting in PBS containing 2% FBS. After sorting, cells were washed twice with PBS and seeded in serum-free media at a density of 5 × 104 cells per well on the upper matrigel chambers. Media containing 10% FBS was placed in the lower chamber as a chemoattractant. After 24 h of incubation at 37 °C in a 5% CO2 atmosphere, cells on the upper surface of the filter were removed. The invading cells on the underside were examined under a Nikon inverted microscope at ×200 magnification and counted by choosing five high power fields randomly. All experiments were 2273
dx.doi.org/10.1021/pr201059g | J. Proteome Res. 2012, 11, 2272−2281
Journal of Proteome Research
Article
Tryptic Digestion and PNGase F Treatment
CA). After transfer, the membranes were blocked for 2 h by 1% BSA in PBST, and then incubated with the following primary antibodies: mouse anti-CD24, mouse anti-CD13 and rabbit anti-RPN2 from Abcam (Cambridge, MA), rabbit anti-CD44 from Millipore (Temecula, CA) and rabbit β-Actin from Cell Signaling (Boston, MA). After being washed with PBST three times, the membranes were incubated with peroxidaseconjugated anti-IgG (H+L) for 1 h, washed with PBST three times, and then detected by Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific, IL).
The glycoproteins were reduced with 5 mM TCEP (Thermo) for 30 min at 37 °C, alkylated with 15 mM iodoacetamide (Sigma) in the dark at room temperature for 30 min, and then digested with trypsin (Promega, Madison, WI) at 37 °C overnight. The glycopeptides were deglycosylated by PNGase F (New England Biolabs, Ipswich, MA) at 37 °C for 16 h, and then dried using a SpeedVac concentrator (Thermo Savant, Milford, MA). LC−MS/MS
Tissue Samples
The peptide mixtures were resolubilized in 0.1% formic acid and analyzed by an LTQ mass spectrometer (Thermo Finnigan, San Jose, CA). Chromatographic separation of peptides was explored on a Paradigm MG4 micropump system (Michrom Biosciences Inc., Auburn, CA) with a flow rate of 300 nL/min. Peptides were separated by a C18 separation column (0.1 mm × 150 mm, C18 AQ particles, 5 μm, 120 Å, Michrom Biosciences Inc., Auburn, CA). Both solvents, A (water) and B (acetonitrile), contained 0.1% formic acid. A 90 min linear gradient was applied from 5 to 40% B. The MS instrument was operated in positive ion mode. The spray voltage was set at 1.5 kV, the capillary voltage was 30 V and the capillary temperature was 150 °C. The normalized collision energy was set at 35% for MS/MS. For each cycle of one full mass scan (range of m/z 400−2000), the five most intense ions in the spectrum were selected for tandem MS analysis, unless they appeared in the dynamic or mass exclusion lists. The data were acquired in data-dependent mode using the Xcalibur software (2.1.0 build 1139).
The tissue microarrays (TMAs) of formalin-fixed paraffinembedded pancreatic tumors and normal tissues were purchased from US Biomax Inc. (Catalog No. PA484 and PA1002). Tissue specimens included 13 normal human pancreas tissues (age: from 21 to 50 years, median: 35 years) and 53 cases of pancreatic ductal adenocarcinoma tissues (age: from 34 to 78 years, median: 57 years), identified with clinical stages and pathology grades. The clinical characteristics are listed in Supplementary Table S2 and S3 (Supporting Information). The images of the TMAs stained with hematoxylin-eosin (H&E) were provided on the Web site of US Biomax Inc. The tissue samples originated from different donors and each sample had 2 replicates. Fluorescent Immunohistochemistry
The paraffin-embedded 5 μm tissue microarrays were dewaxed in xylene for 10 min twice and rehydrated through a series of alcohol solutions (100% ethanol twice, 90% ethanol, 70% ethanol, 5 min each) to water. Antigen retrieval was achieved by boiling the arrays in citrate buffer at pH 6.0 (Invitrogen) for 15 min. The TMAs were incubated with 1% BSA in PBS for 1 h at room temperature to block nonspecific staining between the primary antibodies and the tissues. To achieve immunofluorescence staining, mouse anti-CD13 (Abcam, Cambridge, MA) or a mixture of mouse anti-CD24 (R&D Systems Inc., Minneapolis, MN) and rabbit anti-RPN 2 (Abcam, Cambridge, MA) was incubated with the TMAs overnight at 4 °C at a dilution of 1:100. Then DyLight 488 antirabbit IgG (H+L) and DyLight 549 antimouse IgG (H+L) (Vector Laboratories, Burlingame, CA) were diluted (1:200) and incubated with the TMAs for 1 h at room temperature. The nuclei visualization was explored by DAPI counterstaining. Between each step, there were three washes in PBST for 5 min each. Finally, the TMAs were dehydrated in alcohol (70% ethanol, 90% ethanol, 100% ethanol twice, 5 min each), and coverslipped using a CC/Mount permanent mounting medium (Sigma).
Data Analysis
All MS/MS spectra were searched using SEQUEST algorithm 29 incorporated in Proteome Discoverer software version 1.1.0.263 (Thermo Finnigan) against the UniProt human protein database Release 2010_11. The search was performed using the following parameters: (1) Fixed modification, Carbamidomethyl of C; (2) variable modification, oxidation of M and Asn→Asp; (3) allowing two missed cleavages; (4) peptide ion mass tolerance 1.50 Da; (5) fragment ion mass tolerance 1.0 Da; (6) peptide charges +1, +2, and +3. False discovery rate (FDR) was set to be
