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Identification of Core Proteins Carrying the Sialyl Lewis a Epitope in Pancreatic Cancers | OMICS International
ISSN-2155-9929
Journal of Molecular Biomarkers & Diagnosis

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Identification of Core Proteins Carrying the Sialyl Lewis a Epitope in Pancreatic Cancers

Yoshitoshi Hirao1, Satoshi Ogasawara1, Akira Togayachi1, Yu-ki Matsuno1, Makoto Ocho1, Keishi Yamashita2, Masahiko Watanabe2, Shoji Nakamori3, Yuzuru Ikehara1 and Hisashi Narimatsu1*

1Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan

2Department of Surgery, Kitasato University School of Medicine, Kitasato 1-15-1, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan

3Department of Surgery, National Hospital Organization Osaka National Hospital, 2-1-14 Hoenzaka, Chuo-ku, Osaka 540-0006, Japan

*Corresponding Author:
Hisashi Narimatsu
AIST Tsukuba Central 2, 1-1-1 Umezono
Tsukuba, Ibaraki 305-8568, Japan
Phone: +81-861-3200
Fax: +81-861-3201
E-mail: [email protected]

Received date: November 30, 2011; Accepted date: March 16, 2012; Published date: March 23, 2012

Citation: Hirao Y, Ogasawara S, Togayachi A, Matsuno Y, Ocho M, et al. (2012) Identification of Core Proteins Carrying the Sialyl Lewis a Epitope in Pancreatic Cancers. J Mol Biomark Diagn 2:124. doi:10.4172/2155-9929.1000124

Copyright: © 2012 Hirao Y , et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Identification of core proteins carrying the CA19-9 (carbohydrate antigen, sialyl Lewis a) epitope from various tissues will improve the diagnosis of pancreatic cancer in terms of specificity and sensitivity. In this study, we attempted to identify sialyl Lewis a-carrier proteins specifically expressed in pancreatic cancer. Pancreatic cancer is difficult to detect in the early stages of the disease, resulting in a high level of mortality. Therefore, in order to determine the correct course of treatment, it is vital to distinguish cancer from obstruction of the bile duct or other diseases. Our strategy to identify the carrier proteins was as follows: glycoproteins carrying sialyl Lewis a antigen were enriched from pancreatic cancer cell lines using anti-sialyl Lewis a antibody and then subjected to Peptide Mass Fingerprinting analysis. Based on these studies we identified nine glycoproteins carrying the sialyl Lewis a epitope. We evaluated candidate molecules by biochemical analyses of culture supernatants and human sera. In particular, we focused on one candidate molecule carrying a sialyl Lewis a epitope, Galectin-3BP/MAC2BP; M2BP, which was analyzed in detail. These results verified that our candidate molecule is a core protein carrying the sialyl Lewis a epitope. Furthermore, we demonstrated sandwich ELISA, which showed that the glycoprotein was able to detect CA19-9 antigen in culture supernatants. Our approach facilitated the identification of the core protein carrying the sialyl Lewis a epitope. We believe our approach will enable future developments in cancer glycobiomarker identification.

Keywords

CA19-9; Glycosylation; Pancreatic cancer; Sialyl Lewis a

Introduction

Protein glycosylation is highly heterogeneous and comprises approximately 50% of the secreted proteome [1]. Glycosylation is highly sensitive to the biochemical environmental and plays an important role in various biological processes [2-5]. Tumor-specific alternation of glycan structures could be a potential target for cancer diagnosis. Indeed, CA19-9, CEA and CA125 are already established as pancreatic cancer markers [6-8]. The core proteins of these cancer markers have been identified except for CA19-9 [9,10]. CA19-9 is the most widely used marker for pancreatic cancer [6,11,12]. The CA19- 9 marker was identified by Koprowski using a monoclonal antibody 1116NS19-9 obtained through immunizing a mouse with a cell line SW1116 derived from an adenocarcinoma of the human colon in 1979 [11]. CA19-9 showed that the carbohydrate antigen recognizes a carbohydrate determinant called sialyl Lewis a (sLea). However, CA19-9 is also used as a marker for diseases other than pancreatic cancer, such as esophageal cancer, bile duct cancer, stomach cancer, liver cancer, lung cancer and colon cancer and the level of CA19-9 is known to increase at biliary inflammation [7,13,14]. Therefore, there is a serious problem with false positives because a number of similar proteins carrying the sLea epitope are associated with other cancers or disease states. Detecting the core protein specific for each tissue should increase the accuracy of the CA19-9 test in identifying pancreatic cancer. In this study, we tried to establish a strategy to identify the core proteins carrying the sLea epitope in pancreatic cancer.

Of all the cancers of the digestive system, pancreatic cancer has the worst prognosis with a 5-year survival rate of less than 20% [15,16]. A major factor for the poor prognosis of pancreatic cancer is that early detection has proved difficult. The resulting delay in treatment often leads to secondary cancers caused by metastasis. In the USA, pancreatic cancer is the fourth largest cause of death for both men and women and the mortality rate has increased every year from poor diagnosis [17]. Moreover, it is often difficult to accurately judge whether the pancreatic tumor is benign or malignant [18,19]. Mutation of K-ras and a genetic defect of p53 were analyzed as potential biological markers but these were found to be poor indicators for the early diagnosis of this disease [20,21]. The CA19-9 test is often used for the diagnosis of pancreatic cancer, although the sensitivity of this test is only about 80% and specificity is even less because of the high number of false positives [22]. Furthermore, for clinical treatment of pancreatic cancer, anticancer drugs and irradiation or surgical operation are often used. However, obstruction of the bile duct requires a different course of treatment. Therefore, it is important to distinguish between pancreatic cancer and obstruction of the bile duct or other benign diseases including inflammation. One way to circumvent this problem is to identify the core proteins carrying the sLea epitope, which will be invaluable for differentiating between pancreatic cancer and other diseases. Although CA19-9 is a well established carbohydrate antigen as tumor maker, analysis of the corresponding carrier proteins is still lacking. Indeed, it would be useful to identify and analyze the carrier proteins of sLea epitope not only for pancreatic cancer but also for other diseases in which CA19-9 is used as a biomarker. This is because identification of the carrier protein should increase the sensitivity and specificity of techniques such as the sandwich ELISA system.

In this study, we established a methodology to identify the core proteins carrying the sLea epitope using a glycoproteomics strategy. This methodology identified core proteins carrying sLea epitope in pancreatic cancer using human cultured supernatants and human sera. Furthermore, we constructed the sandwich ELISA system using CA19-9 and candidate molecule. Candidate molecules were evaluated as useful markers by analyzing the culture supernatant of pancreatic cancer cells. Our results indicate that this methodology is a useful approach for developing valuable markers in benign diseases as well as other cancers.

Materials and Methods

Cell lines and human samples

Human Pancreatic Epithelial Cells (PE cell) and Human Pancreatic Stromal Cells (PS cell) were obtained from Applied Cell Biology Research Institute (Kirkland, WA). Human pancreatic cancer cell lines (Capan-1, Capan-2, PANC-1, PL45, MIA PaCa-2, BxPC-3, HPAF2 and PSN1) were obtained from American Type Culture Collection (Rockville, MD). The cells were cultured until 70-80% confluent and the cells were then washed using serum-free RPMI medium after removing the supernatant. The cells were further cultured for 48 hours with serum-free RPMI medium. Supernatants were filtered through a MILLEX-HV 0.45 μm filter device (Millipore, Bedford, MA) and then concentrated using an Amicon Ultra-15 (10 K cutoff; Millipore) at 2,000g for 10 min at room temperature. Clinical samples were obtained from National Institute of Advanced Industrial Science and Technology (AIST), National Hospital Organization Osaka National Hospital and Kitasato University Hospital (Table 1). The institutional ethics committees at AIST, National Hospital Organization Osaka National Hospital and Kitasato University Hospital approved this study and informed consent for the use of clinical specimens was obtained from all individuals.

M.W. of SDS-PAGE Gel piece Score Identified protein (Access key of SwissProt) Predicted M.W. (kDa)
  Over 250 kDa 1 110 Agrin precursor (AGRIN_HUMAN) 214
150-250 kDa 1
2
3
4
84
85
-
-
Agrin precursor (AGRIN_HUMAN)
Agrin precursor (AGRIN_HUMAN)
not identified
not identified
214
214
-
-
  100-150 kDa   1
2
43
87
Agrin precursor (AGRIN_HUMAN)
Agrin precursor (AGRIN_HUMAN)
214
214
75-100 kDa 1
2
3
4
87
56
83
56
56
Galectin-3-binding protein precursor (LG3BP_HUMAN)
T-complex protein 1 subunit eta (TCPH_HUMAN)
Ezrin (EZRI_HUMAN)
Neutrophil cytsol factor 2 (NCF2_HUMAN)
Necdin (NECD_HUMAN)
65
59
69
59
36
50-75 kDa 1
2
3
4
127
269
224
82
Transforming growth factor-beta-induced protein ig-h3 precursor (BGH3_HUMAN)
Transforming growth factor-beta-induced protein ig-h3 precursor (BGH3_HUMAN)
Transforming growth factor-beta-induced protein ig-h3 precursor (BGH3_HUMAN)
Transforming growth factor-beta-induced protein ig-h3 precursor (BGH3_HUMAN)
74
74
74
74
  Under 50 kDa   1
2
3
4
100
82
75
114
Tissue-type plasminogen activator precursor (TPA_HUMAN)
Tissue-type plasminogen activator precursor (TPA_HUMAN)
Cathepsin D (CATD_HUMAN)
Cathepsin D (CATD_HUMAN)
62
62
44
44

Table 1: Clinical features of normal healthy volunteers and pancreatic cancer patients.

Peptide Mass Fingerprinting (PMF) analysis

10 mg/ml of anti-sLea, 1116NS19-9 antibody [11] (clone 1116NS19-9, Fujirebio, Tokyo, Japan) was immobilized onto an NHSSepharose column (GE Healthcare, Waukesha, WI) according to the manufacturer’s instructions. The concentrated Capan-2 supernatant was then applied to the column. Captured Capan-2 supernatant was eluted with phosphate buffer (pH 13). The eluate was then analyzed by SDS-PAGE and the resulting bands on the gel were visualized by silver staining. The gels were cut into 6 pieces and destained using a Mass Spectrometry-Compatible reagent (Invitrogen, Carlsbad, CA). Each gel piece was treated with 100 μl of acetonitrile (ACN) and then incubated for 10 min at room temperature before drying the gel. The proteins were then in-gel digested with 50 ng/10 μl of trypsin (Promega, Madison, WI) in 50 mM NH4HCO3 overnight at 37°C. A 50 μl mixture of 50% ACN and 5% TFA was added and incubated for 30 min at room temperature. The washing procedure was then repeated once more. Recovered solution was freeze dried and dissolved in 10 μl of 0.1% TFA. Samples were demineralized with a ZipTip (Millipore) and analyzed by MALDI-TOF-MS (Bruker Daltonics, Billerica, MA). The matrix used for the analysis was 2, 5-dihydroxybenzonic acid and the samples were run in positive-ion mode. Proteins were identified by Mascot analysis (Matrix Science). Glycosylation sites of identified proteins were analyzed by CBS Prediction servers (http://www.cbs.dtu.dk/services/).

Western Blotting

SDS-PAGE was performed using a 10% polyacrylamide gel. After electrophoresis, the gels were stained with silver (ATTO, Tokyo, Japan) or transferred to a PVDF membrane (GE Healthcare). Western blotting was performed as described in a previous study [23]. The membranes were then incubated with anti-Galectin-3BP/MAC-2BP; M2BP (R&D systems, Minneapolis, MN), anti-Cathepsin D (R&D systems), anti-Ezrin (abcam, Cambridge, UK), anti-β ig-h3 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-tPA (Santa Cruz Biotechnology), anti-Agrin (Santa Cruz Biotechnology), 1116NS19-9 antibody or antisLec, KM231 antibody [24] (clone KM231, Kyowa Medex, Tokyo, Japan). The membranes were then incubated with a secondary antibody, either anti-goat IgG HRP antibody (Dako, Glostrup, Denmark), antimouse IgG HRP antibody (GE Healthcare) or anti-rabbit IgG HRP antibody (GE Healthcare). Finally, the membranes were exposed to X-ray film. Clinical samples (NHS-1 to 5 and Cancer-1 to 5) were used in this Western blotting procedure (Table 1).

Immunoprecipitation

Protein G Sepharose (GE Healthcare) was prepared as a 50% slurry. 100 μl of culture supernatant, or 5 μl of serum from a normal healthy volunteer or pancreatic cancer patient, was treated with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Samples were mixed with a 20 μl aliquot of Protein G Sepharose and gently agitated on a rotating wheel. 1 μg of antibody (anti-M2BP, Agrin, Cathepsin D, Ezrin) was added to the mixture and incubated overnight on a rotating wheel at 4°C The captured immunoprecipitated samples were treated with 20 μl of washed Protein G Sepharose and incubated for 1 hour at 4°C with rotation. Finally, 20 μl of elution buffer (2% SDS in PBS-T) was added to the washed Protein G Sepharose and the sample was heated to 98°C for 5 min. This elution procedure was then repeated once more.

Digestion of N-glycan

PNGase F (Peptide-N4-[N-acetyl-β-glucosaminyl] asparagine amidase) was purchased from TaKaRa Bio (Otsu, Japan). The immunoprecipitated proteins were subjected to treatment with PNGase F according to the manufacturer’s instructions. Samples were then separated by SDS-PAGE and analyzed by Western blotting.

Immunohistochemistry

The tissue sections were deparaffinized and rehydrated. Then, endogenous peroxidase was blocked by incubating the sections in methanol containing 0.3% hydrogen peroxide. The tissue sections were blocked with blocking buffer (PBS containing 1.5% horse or goat serum) and then incubated with primary antibody in blocking buffer overnight at 4°C. Immunohistochemistry was carried out as described in detail in a previous study [23].

Sandwich ELISA

For the target glycoprotein-carbohydrate epitope sandwich ELISA, M2BP (0.2 μg/well) in PBS was coated onto a flat-bottomed 96-well microtiter plate (Nunc International, Rochester, NY) for 6 hours at room temperature. The plate was washed with PBS-T and overlaid with 3% BSA overnight at 4°C. Then the plate was washed again and incubated with concentrated supernatants (100 μl/well) or human sera (5 μl/well) in PBS containing 3% BSA and RPMI medium supplemented with 0.1% Tween-20 for 2 hours at 37°C. The plate was next washed and incubated with 1116NS19-9 (0.2 μg/well) in PBS containing 3% BSA for 2 hours at 37°C. The plate was washed again and incubated with 100 μl/well of 1:5000 diluted solution of anti-mouse IgG HRP for 1 hour at room temperature. Then, 100 μl of the substrate 3,3’5,5’-tetramethylbenzidine solution (Thermo Fisher Scientific, Waltham, MA) was added to each well. The enzyme reaction was stopped by adding 50 μl of 1 M sulfuric acid and the optical density (OD) was measured at 450 nm. All experiments were performed in duplicate and the median was used as the final value for each sample. Clinical samples (Cancer-1 to 14) were used in this sandwich ELISA (Table 1). The coefficient correlation of the standard curve was calculated using 4-parameter fit [25].

Results

Identification of glycoproteins carrying the sialyl Lewis a epitope by PMF analysis

To identify carrier proteins, we selected the cell line that expresses CA19-9 antigen to a high level. Initially, cultured supernatants of eight pancreatic cancer cell lines were analyzed for expression of the CA19-9 antigen by Western blotting. The results showed that Capan-2 expressed the CA19-9 antigen at high levels in both the culture supernatant and cell lysate (Figure 1A and 1B). The culture supernatant of Capan-2 was then enriched by an affinity column procedure using immobilized 1116NS19-9 antibody. The enriched supernatant was analyzed by SDS-PAGE and the gel was silver stained (Figure 1C). The relative band intensities are shown in the form of a histogram. The stained gel was cut into 6 pieces and analyzed by PMF. PMF analysis indicated the presence of candidate molecules; Agrin, M2BP, T-complex protein 1 subunit, Ezrin, Neutrophil cytosol factor 2, Necdin, â ig-h3, tPA and Cathepsin D (Table 2). The corresponding score values of the Mascot search by MATRIX SCIENCE are given in Table 2. Next, we checked that the candidate molecules were actually glycoproteins as well as the likely location of the glycosylation site. This was achieved by analyzing potential glycosylation sites using CBS Prediction servers (Supplemental Figure 1). Commercially available antibodies (Agrin, M2BP, Ezrin, â ig-h3, tPA and Cathepsin D) were used in this study.

molecular-biomarkers-diagnosis-pancreatic-cancer

Figure 1: Selection of a pancreatic cancer cell line that express the sLea antigen to a high level. Expression of sLea antigen in a pancreatic cancer cell line together with PMF analysis. A, B) Western blotting analysis using the culture supernatant of 8 pancreatic cancer cell lines and expression of sLea antigen in the cell lysates. C) Capan-2 culture supernatant enriched by 1116NS19-9 antibody was subjected to SDS-PAGE and the resulting bands were visualized by silver staining. Relative band intensities of “1 μg” lane were analyzed and are depicted in the form of a histogram. The gel was cut into six pieces as highlighted by rectangles. Proteins extracted from each slice were analyzed by PMF. Black arrows show the peak of band intensities.

M.W. of SDS-PAGE Gel piece Score Identified protein (Access key of SwissProt) Predicted M.W. (kDa)
  Over 250 kDa 1 110 Agrin precursor (AGRIN_HUMAN) 214
  150-250 kDa 1
2
3
4
84
85
-
-
Agrin precursor (AGRIN_HUMAN)
Agrin precursor (AGRIN_HUMAN)
not identified
not identified
214
214
-
-
100-150 kDa 1
2
43
87
Agrin precursor (AGRIN_HUMAN)
Agrin precursor (AGRIN_HUMAN)
214
214
75-100 kDa 1
2
3
4
87
56
83
56
56
Galectin-3-binding protein precursor (LG3BP_HUMAN)
T-complex protein 1 subunit eta (TCPH_HUMAN)
Ezrin (EZRI_HUMAN)
Neutrophil cytsol factor 2 (NCF2_HUMAN)
Necdin (NECD_HUMAN)
65
59
69
59
36
50-75 kDa 1
2
3
4
127
269
224
82
Transforming growth factor-beta-induced protein ig-h3 precursor (BGH3_HUMAN)
Transforming growth factor-beta-induced protein ig-h3 precursor (BGH3_HUMAN)
Transforming growth factor-beta-induced protein ig-h3 precursor (BGH3_HUMAN)
Transforming growth factor-beta-induced protein ig-h3 precursor (BGH3_HUMAN)
74
74
74
74
Under 50 kDa 1
2
3
4
100
82
75
114
Tissue-type plasminogen activator precursor (TPA_HUMAN)
Tissue-type plasminogen activator precursor (TPA_HUMAN)
Cathepsin D (CATD_HUMAN)
Cathepsin D (CATD_HUMAN)
62
62
44

44

Table 2: Identified candidate molecules carrying sLea epitope by PMF analysis.

Analysis of candidate molecules using human cell line culture supernatant

Selected candidate molecules were analyzed to identify the core protein carrying sLea. Our procedure to identify the core proteins was as follows: commercial antibodies were available for Western blotting and immunoprecipitation and candidate molecules were subsequently detected using the monoclonal antibody 1116NS19-9. Two primary cultured pancreatic cells (PE cell and PS cell) and eight pancreatic cancer cell lines (BxPC-3, Capan-1, Capan-2, HPAF2, MIA PaCa- 2, PANC1, PL45 and PSN1) were analyzed by Western blotting. The antibodies, with the exception of those against â ig-h3 and tPA, could be used for Western blotting analysis. Indeed, Western blotting using antibodies against M2BP, Agrin, Cathepsin D or Ezrin showed positive signals in the supernatants of pancreatic cancer cell lines (data not shown).

Immunoprecipitation resulted in the isolation of M2BP, Cathepsin D, Ezrin and Agrin. However, Agrin is a high molecular weight molecule that is difficult to analyze by SDS-PAGE. Therefore, immunoprecipitates of M2BP, Cathepsin D and Ezrin were analyzed by Western blotting analysis using 1116NS19-9 antibody. Cathepsin D was expressed in only Capan-2 and Ezrin was not expressed in any of the cell lines tested in this study. From these results, M2BP was selected to be the final candidate. M2BP could be immunoprecipitated from all of the cell line supernatants (Figure 2A). The immunoprecipitated samples were subjected to Western blotting analysis using anti- M2BP antibody and 1116NS19-9 antibody (Figure 2A). The results of this analysis show that the sLea epitope can be detected in the immunoprecipitated M2BP from the culture supernatant of BxPC- 3, Capan-1, Capan-2, HPAF2 and PL45. These findings suggested that M2BP has the sLea epitope. Moreover, KM231 antibody, which recognizes not only sLea antigen but also sialyl Lewis c (sLec) antigen, was used to perform Western blotting analysis. The positive signals in culture supernatants of BxPC-3, Capan-1, Capan-2, HPAF2, PL45 and PSN1 were detected with KM231 antibody (Figure 2A).

molecular-biomarkers-diagnosis-immunoprecipitated

Figure 2: M2BP was immunoprecipitated from the culture supernatant and human sera. A) Western blotting analysis of culture supernatant was performed using anti-M2BP and 1116NS19-9 and KM231 antibodies. B) Immunoprecipitated M2BP proteins were digested with PNGase F to remove the N-glycan. Western blotting analysis was then performed on the digested samples using anti-M2BP and 1116NS19-9 antibody. Closed arrowhead indicates the N-glycosylated M2BP and open arrowhead indicates the non-(N-)glycosylated M2BP. This figure was prepared from the results of two images. C) Western blotting analyses were performed using anti-M2BP and 1116NS19-9 antibody with sera of normal healthy volunteers and pancreatic cancer patients. Arrowhead indicates molecular weight of M2BP. D) Western blotting analyses were performed using anti-M2BP, 1116NS19-9 and KM231 antibodies with sera of normal healthy volunteers and pancreatic cancer patients.

Digest of glycosylation to confirm the presence of sLea epitope on N-glycan or O-glycan of M2BP.

We determined whether the sLea epitope was present as an N-glycan or O-glycan in M2BP. Immunoprecipitated samples were digested using PNGase F to remove the N-glycan of M2BP (Figure 2B). Subsequent Western blotting analysis of the digested M2BP indicated that all the N-glycan had been removed. The same samples were also analyzed by Western blotting using 1116NS19-9 antibody. The positive bands, which could be readily detected before PNGase F-digestion, were no longer detectable for BxPC-3, Capan-1, Capan-2, HPAF2 and PL45.

Validation of M2BP for the core protein carrying the sLea epitope in human sera.

M2BP was a candidate core protein carrying the sLea epitope in the culture supernatant. We therefore attempted to validate M2BP using human sera. We performed Western blotting analysis with crude sera of normal healthy volunteers and pancreatic cancer patients (Figure 2C). The results of this experiment showed that M2BP proteins were expressed in all sera samples. However, a smear of positive bands of higher molecular weight than M2BP was also observed by Western blotting using 1116NS19-9 antibody. Indeed, an intense region of smeared bands was observed for crude sera, indicating the expression of many glycoproteins bearing sLea epitopes. Moreover, the positive smear of bands was markedly upregulated in sera of pancreatic cancer patients compared to those of healthy individuals. Western blotting analysis was also performed using the KM231 antibody, which recognizes both sLea and sLec antigen (Figure 2D). The KM231 antibody detected a signal from sera of both normal healthy volunteers and patients with pancreatic cancer.

Observation of the expression pattern by an immunohistochemical approach with pancreatic tissues.

Serial pancreatic tissue sections from pancreatic cancer patients were stained using Hematoxylin-Eosin (HE) and anti-M2BP antibody and 1116NS19-9 antibody (Figure 3). M2BP antibody stained most cells and 1116NS19-9 antibody stained epithelial cells of the pancreatic duct from normal pancreas and ductal cells excluding acinar and islet cells (Figure 3A and 3B). By contrast, M2BP and 1116NS19-9 antibodies stained the cancer cells from pancreatic cancer (Figure 3C and 3D). Moreover, positive signals of M2BP and 1116NS19-9 were detected in the same cells (Figure 3C and 3D). This result suggested that M2BP proteins carrying sLea antigens were produced in pancreatic cells stained by both the M2BP and 1116NS19-9 antibodies.

molecular-biomarkers-diagnosis-Tissues-stained

Figure 3: Tissues were stained by hematoxylin-eosin (HE) and anti-M2BP antibody (M2BP) and 1116NS19-9 antibody (CA19-9). Tissue samples from healthy patients (A, B) and pancreatic cancer patients (C, D). The region enclosed by a rectangle is magnified in (B, D). Scale bar shows: A and C = 20 μm, B and D = 5 μm.

Validation of core protein with M2BP/1116NS19-9 Sandwich ELISA

A validation assay for checking candidate markers was performed by sandwich ELISA using culture supernatant and human sera. In this study, anti-M2BP and 1116NS19-9 antibodies were used for the sandwich ELISA. CA19-9 antigen levels on M2BP were evaluated by sandwich ELISA using culture supernatant (Figure 4). Capan-2 and PANC1 were used as a positive and negative control, respectively. The OD450 for PANC1 and medium for the blank indicated values of ~0.15 for PANC1 and ~1.2 for Capan-2. Measurements were made on progressively diluted samples of the supernatant from Capan-2. A correlation was evident between the OD450 and dilution ratio (Figure 4A). We calculated the coefficient correlation of the standard curve shown in Figure 4A. The result indicated a value of 0.998. Furthermore, we validated other culture supernatant using pancreatic cancer cells (Figure 4B). The OD450 for MIA PaCa-2 and PANC-1 and medium for the blank indicated values of ~0.1. However, BxPC-3, Capan-1, Capan-2, HPAF-II, PL45 and PSN1 gave OD450 values of ~0.2. These results clearly correlated with those from Western blotting using CA19- 9 (Figure 2A). We also attempted to evaluate samples of pooled serum from normal healthy volunteers and 11 samples of sera from pancreatic cancer patients. Sera of pancreatic cancer patients and obstructive icterus patients with known CA19-9 values were used (Table 1). Sera displaying the highest CA19-9 value of over 10000.0 U/ml showed almost the same level of OD value as that of medium as background (or PANC1 as negative control). The OD450 for the sera gave values of 0.05-0.1. Furthermore, we attempted to evaluate immunoprecipitated M2BP from pancreatic cancer patients (Cancer-7, 9, 10 and 11). However, the OD450 for the sera gave values of about 0.1 as background (data not shown). These results show that our sandwich ELISA system is currently unable to detect sLea epitopes on M2BP antigen in samples of sera.

molecular-biomarkers-diagnosis-Sandwich-ELISA

Figure 4: Sandwich ELISA assay using a combination of anti-M2BP and 116NS19-9 antibody. Medium was used for the blank sample. (A) Capan-2 and PANC1 were used as positive and negative control, respectively. Capan-2 supernatant was serial diluted to 1/2-1/512. The background level (negative control) OD450 was ~0.2. (B) Eight cultured supernatants of pancreatic cancer cell lines were validated with sandwich ELISA. The background level (negative control) OD450 was ~0.1.

Discussion

Our aim in this study was to establish a methodology to identify the glycoproteins carrying the sLea epitope associated with pancreatic cancer. There are many ways to discovering a biomarker. Our group has investigated procedures to identify glycobiomarkers [26-29]. Ideally, to maximize sensitivity our candidate serum glycobiomarker will be: 1] on a glycosylated core protein that is secreted and is tissue (cancer cell)-specific protein 2] on a glycosylated protein having many glycans 3] present at high levels in the serum. However, it is often difficult to identify a tissue specific glycobiomarker because candidate biomarkers in the serum can be derived from many different tissues. In this study, we tried to identify the sLea carrier proteins associated with pancreatic cancer. CA19-9 is the most useful marker for the diagnosis of pancreatic cancer [6,11,12]. Indeed, CA19-9 is a cancerrelated carbohydrate antigen that is well established for recognizing the sLea epitope. However, there are problems with this approach such as a high frequency of false positives and inadequate sensitivity and/ or specificity [7,13]. Sensitivity and specificity of positive diagnosis rate is about 80% using CA19-9 for pancreatic cancer [22]. This is because a number of proteins are thought to carry the sLea epitope. Furthermore, CA19-9 is used as a marker for diseases other than pancreatic cancer (such as cancer of the bile duct, lung, liver, stomach and colon) [7,13,14]. Therefore, CA19-9 cannot clearly distinguish between tissue types. Hence, we attempted to develop a procedure for identifying the core protein carrying the sLea epitope that is associated with pancreatic cancer as a first step to discovering the glycobiomarker. Our strategy was as follows; 1] development of a methodology to identify candidate molecules, 2] evaluation of these candidates using culture supernatant and human sera, 3] assessment of candidate molecules as useful markers. Initially, we attempted to identify the core proteins carrying the sLea epitope using culture supernatants. We enriched the sLea-carrying proteins using the 1116NS19-9 antibody column procedure. PMF analysis, which is a method of identifying proteins by mass spectrometry [30,31], was used to determine the sLea carrying proteins. Our analyses identified nine glycoproteins as candidate sLea-carrying proteins using cultured supernatant. Moreover, for selection of candidate molecules, potential glycosylation sites of candidate molecules were analyzed. A large number of glycosylation sites are thought to be important for this type of analysis. Furthermore, we used commercially available antibodies for Western blotting and immunoprecipitation. These analyses showed that Agrin, Cathepsin D and Ezrin were unsuitable candidate molecules. Hence, these molecules were not investigated any further. However, M2BP was identified as a potentially useful glycobiomarker. This is because M2BP is a secreted glycoprotein that also interacts with other extracellular proteins such as collagen IV, V, VI fibronectin and nidogen [32-34]. The serum level of M2BP is about 11 ìg/ml, but is elevated in patients with some types of tumor or viral infection and also has a strong correlation with liver metastasis [35,36]. M2BP is thought to have seven N-glycosylation sites [37,38]. From these characters of M2BP, we finally selected M2BP for most useful candidate molecule in this study. Moreover, we immunoprecipitated M2BP from the culture supernatant and from human sera of normal individuals and pancreatic cancer patients. Western blotting analysis using 1116NS19- 9 antibody revealed that M2BP has the sLea epitope on their glycan. In order to investigate whether the sLea epitopes on the M2BP exist on N-glycan or O-glycan, we attempted to digest the N-glycan of M2BP. Our results clearly showed that the molecular weight of M2BP after treatment with PNGase F had decreased and the sLea epitope was no longer detectable. This result clearly indicates that sLea epitope on the M2BP is present as the N-glycan. M2BP was immunoprecipitated from a normal healthy volunteer and from pancreatic cancer sera. There was almost the same band intensity between normal healthy volunteers and cancer patients. By contrast, however, there was a marked difference between these two groups when Western blotting was performed using antibody 1116NS19-9. The band on the Western blotting was barely detectable for the five normal healthy volunteers, but was 3-7 fold more intense for patients with pancreatic cancer. Furthermore, M2BP was immunoprecipitated and Western blotted with anti-M2BP and 1116NS19-9 antibodies using pancreatic cancer sera in which the level of CA19-9 values had already been determined. Unfortunately, however, there was no correlation in band intensity between M2BP and 1116NS19-9 (data not shown). These results suggested that the amount of M2BP is same levels but the amount of sLea epitope on M2BP is thought to be different in normal healthy volunteers and pancreatic cancer patients. M2BP is thought to have seven N-glycosylation sites [37,38]. Our results suggested that not all of N-glycosylation sites on M2BP keep the sLea epitope. And sLea epitope on M2BP is different at the individual level.

From an immunohistochemical approach (Figure 3), the positive signals of M2BP and 1116NS19-9 were observed in almost all pancreatic cancer cells. This study showed that the normal pancreatic duct is stained with 1116NS19-9 antibody. Indeed, previous reports agree with these observations [39,40]. Hence, CA19-9 antigen is weakly expressed in the sera of normal individuals, but sLea levels are highly elevated in the sera of pancreatic cancer patients (Figure 2D). Our results suggest that M2BP is the carrying protein of the sLea epitope and is upregulated in pancreatic cancer patients.

ELISA is often a useful technique for diagnosis using a serum biomarker. Here, we constructed a sandwich ELISA using 1116NS19- 9 and anti-M2BP antibodies. The sandwich ELISA worked well for the culture supernatants. From this result, we confirmed that M2BP carrying sLea epitope were expressed in several cell lines. Next, we evaluated the sandwich ELISA system using human pancreatic cancer and obstructive icterus sera. However, in this case, M2BP could not be detected in the serum, since detection limit of the sandwich ELISA system was insufficient for evaluating these kinds of samples. M2BP expressed in many tissues such as colon, lung, stomach and pancreas [37,38]. A large number of M2BP thought to be expressed in blood and M2BP derived from pancreas thought to be little. Moreover, the amount of M2BP carrying sLea epitope from pancreatic cancer is considered to be even less. Therefore, in order to make our sandwich ELISA system useful for such samples, we need to improve sensitivity of sandwich ELISA system and/or develop an alternative detection system for target glycoproteins. We are currently focusing our efforts in this direction. Furthermore, tissue specific core proteins should be selected as a suitable biomarker for a given disease. In the future, it is necessary to select more of these tissue specific molecules.

In conclusion, as the first step to developing a serum glycobiomarker, we established a methodology to identify sLea carrier proteins using culture supernatant from pancreatic cancer cells. Using this methodology we identified M2BP as a sLea-carrier protein. This approach will give useful information for the identification of core proteins.

Acknowledgements

We thank Ms. M. Sogabe and Y. Tsunoda and T. Fukuda and Drs. H. Sawaki and J. Iwaki for technical assistance and helpful discussion. This work was supported by “Medical Glycomics: MG” project in New Energy and Industrial Technology Development Organization (NEDO) in Japan.

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