Patient data collection and ethics statement
Medical records were retrieved from a database constructed by the Fudan University Shanghai Cancer Center. Variables including age, sex, tumor location, metastatic status, size, CA19-9, lymphatic metastasis, and overall survival were retrieved. Pancreatic cancers were classified according to the 8th American Joint Committee on Cancer (AJCC) staging system. All pancreatic cancer tissue specimens were obtained from the Fudan University Shanghai Cancer Center, Shanghai, China.
Animal Protocols, Diets, And Treatment
Six-week-old athymic BALB/c nude mice (female) were obtained from Shanghai Jihui Laboratory Animal Co., Ltd. (Shanghai, China) and maintained under special pathogen-free (SPF) facilities with approved experimental protocols. None of the experiments exceeded the limits (2000 mm3) permitted by the IACUC of Fudan University.
Cell Lines And Cell Culture
BxPC-3, SU.8686, and SW1990 cells were purchased from American Type Culture Collection (ATCC). All the cells were kept in DMEM with 4.5 g/liter glucose supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.). Cell lines were maintained in a 5% CO2 incubator at 37˚ C. Cells used for the experimental study were passaged within 10 to 15 passages after reviving from the frozen vials. Cell lines were routinely tested to exclude Mycoplasma contamination.
Immunofluorescence Staining
For cell staining, cells grown on coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) buffer. For specimen staining, formalin-fixed, paraffin-embedded sections of pancreatic cancer tissues were obtained. After blocking with 5% BSA (Yeasen, Shanghai) in PBS, antibodies against O-GlcNAc (Abcam, Inc., ab2739) or CA19-9 (Abcam, Inc., ab3982) at a dilution of 1/200 in 5% BSA were used to detect their expression at 4˚ C overnight. After washing, the samples were stained with AlexaFluor® 488 goat anti-rabbit secondary antibody (Jackson ImmunoResearch, Inc.) or AlexaFluor® 594 goat anti-mouse secondary antibody (Jackson ImmunoResearch, Inc.) at a 1/200 dilution for 1 hour at room temperature. DAPI (Southern Biotech, Inc.) was used as the nuclear counterstain. Fluorescent images were acquired using a Leica confocal laser scanning microscope (Leica, Inc.). Confocal imaging was performed in seven random fields. Mean fluorescence intensity (MFI) was calculated using Image-Pro plus software (version 6.0, Media Cybernetics, Inc.).
Western Blot And Lectin Blot Analyses
Collected cells were incubated in RIPA buffer, 1 mM PMSF, and 1🞨 Proteinase Inhibitor Cocktail (Beyotime, China) for 30 min. Diluted protein samples were added to 5× SDS‒PAGE loading buffer and then heated at 100˚ C for 10 min to denature proteins. Protein samples (10 µg) were loaded on a 10% SDS‒PAGE gel and run at 100 V for 80 min in SDS‒PAGE running buffer. After blocking with 5% skimmed milk in TBST, the blots were incubated with anti-CA19-9 antibody (Abcam, USA) in blocking solution on a shaker at 4˚ C overnight. Blots were incubated with goat anti-mouse IgG (H + L)-HRP or goat anti-rabbit IgG (H + L)-HRP (Proteintech Group, USA) at 1:5,000 in TBST on a shaker at room temperature for 1 hour.
For lectin blot analysis, biotinylated Aleuria aurantia lentin (AAL, 3 µg/ml), biotinylated Datura sodium lectin (DSL, 3 µg/ml), biotinylated Galanthus nivalis lectin (GNL, 3 µg/ml), biotinylated Phaseolus vulgaris leucoagglutinin (PHA-L, 3 µg/ml), biotinylated Phaseolus vulgaris erythroagglutinin (PHA-E, 3 µg/ml), and biotinylated wheat germ agglutinin (WGA, 5 µg/ml; Vector Laboratories, Inc.) were incubated with 3% BSA on a shaker for 30 min and then incubated with 0.1 µg/ml streptavidinHRP conjugate (Vector Laboratories, Inc.) in blocking buffer for 20 min at room temperature [27]. Images were taken using a ChemiScope 3000 mini (CLiNX, Shanghai, China) after the Chemi Signal ECL Plus (CLiNX, China) reaction. The intensity of the blotting signals was measured by densitometric analysis using ImageJ.
Measurement Of Ca19-9 In Fluids
For patients with pancreatic cancer, serum was collected at the time of diagnosis without major treatments. For mouse models, serum was collected at sacrifice. For cultured cells, supernatants were collected at predetermined times of cell culture. The concentration of CA19-9 in fluids was measured using an electrochemiluminescence immunoassay with a CA19-9 assay kit (Roche) on a Roche Cobase 8000 immunoassay analyzer (Roche Diagnostics, Mannheim, Germany) [28].
Subcutaneous Animal Models
The right flanks of mice were injected subcutaneously with 1× 106 pancreatic cancer cells. Mice were randomly assigned to the Don group (750 mg/kg) or the control group (the same volume of saline). Drugs were administered intraperitoneally twice weekly after inoculation. Mice were observed twice weekly after inoculation. The size of xenografts was measured twice a week by measuring tumors with calipers. Tumor volumes were determined using the formula volume = (long axis) × (short axis)2 × π/6. At the indicated times, the tumors were surgically dissected and weighed. Samples were then processed for histopathologic examination.
Lymphatic Metastasis Models
The right nail pad of mice was injected subcutaneously with 1🞨106 pancreatic cancer cells [29]. Mice were randomly assigned to the Don group (750 mg/kg) or the control group (the same volume of saline). Drugs were administered intraperitoneally twice weekly after inoculation. Mice were observed twice weekly after inoculation. At the indicated times, the animals were euthanized, and the popliteal, inguinal, iliac and renal hilus lymph nodes were harvested to examine lymphatic metastasis. Metastatic lymph nodes were confirmed by H&E staining and pathological examination.
Intraperitoneal Mouse Models
Mice were injected intraperitoneally with 1🞨106 pancreatic cancer cells. Mice were randomly assigned to the Don group (750 mg/kg) or the control group (the same volume of saline). Drugs were administered intraperitoneally twice weekly after inoculation. Mice were observed twice weekly after inoculation. At the indicated times, the animals were euthanized, and the intraperitoneal metastases were harvested. Intraperitoneal implantation was confirmed by H&E staining and pathological examination.
Uhplc‒ms-based Quantitative Measurement Of Ca19-9
SU. 8686 and SW1990 pancreatic cancer cells were cultured for 72 hours in L-glutamine-15N2 (Sigma‒Aldrich, 490032). Cells cultured in unlabeled glutamine (Sigma‒Aldrich, Cat.# G3126) were used as controls. A total of 1×107 cells were used for analysis. Before examination, a standard curve was built using 3'-Sialyl Lewis A (Carbosynth, Cat.# OS00745) diluted in methanol/water (1:1) at different concentrations (1, 5, 10, 50, 100, 500, 1000, 5000, 10000 nM). A total of 160 µL of methanol (precooled at -20˚ C) was added to each sample for metabolite extraction. Then, the samples were centrifuged at 18000 g and 4°C for 20 min, and the clear supernatant was transferred to an autosampler vial for UHPLC‒MS/MS analysis. Ultra-performance liquid chromatography coupled to a tandem mass spectrometry system (ACQUITY UPLC I class-Xevo TQ-S system, Waters Corp., Milford, MA, USA) equipped with a Waters ACQUITY UPLC BEH Amide column (100 × 2.1 mm, 1.7 µm) was used to measure the metabolites. Mobile phase A was 10 mM ammonium acetate in water, and mobile phase B was acetonitrile. The elution gradient was set as follows: 0–1.0 min, 1% A; 1.0–3.0 min, 1% − 40% A; 3.0–5.0 min, 40% A; 5.0–5.1 min, 40% − 1% A; 5.1–6 min, 1% A. The injected volume was 5 µL. Data acquisition and metabolite quantification were performed using MassLynx 4.1 software (Waters Corp., Milford, MA, USA).
Label-free Quantitative N-glycosylation Proteomics
Proteins were extracted using SDT lysis buffer (4% SDS, 100 mM DTT, 100 mM Tris-HCl pH 8.0). Protein digestion was carried out using the FASP method described by Wisniewski et al. [30]. Four hundred micrograms of tryptic peptides was transferred to YM-30 filtration units and incubated for 1 hour with a lectin mixture containing 90 µg ConA, 90 µg WGA and 90 µg RCA120 in 36 µl 2X binding buffer [31, 32]. LC‒MS/MS was performed on a Q Exactive Plus mass spectrometer coupled with Easy 1200 nLC (Thermo Fisher Scientific). The MS data were analyzed using MaxQuant software version 1.6.1.0. MS data were searched against the SwissProt human database (20431 total entries, downloaded 10/15/2019). Construction of protein–protein interaction (PPI) networks was also performed using the STRING database with Cytoscape software [33]. The Motif-X algorithm in the MEME Suite (MEME version 5.1.0) was used for N-linked glycosylation motif analysis [34].
Ultrahigh-performance Liquid Chromatography (Uhplc)-ms/ms
For metabolite extraction, 1000 µL of extract solvent (precooled at -20˚ C, acetonitrile-methanol-water, 2:2:1) was added to each sample, and the samples were vortexed for 30 s, homogenized at 45 Hz for 4 min, and sonicated for 5 min in an ice-water bath. UHPLC separation was performed using an Agilent 1290 Infinity II series UHPLC System (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a Waters ACQUITY UPLC BEH Amide column (100 × 2.1 mm, 1.7 µm). An Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with an AJS electrospray ionization (AJS-ESI) interface was used for assay development. Agilent MassHunter Work Station Software (B.08.00, Agilent Technologies) was applied for MRM data acquisition and processing.
Isotope Tracing Untargeted Metabolomics
SU. 8686 pancreatic cancer cells were cultured for 12 hours or 24 hours in L-glutamine-13C5 (2 mM, Cat. #605166, Sigma‒Aldrich) or L-glutamine-15N2 (Sigma‒Aldrich, 490032). Cells cultured in unlabeled glutamine (Sigma‒Aldrich, Cat.# G3126) were used as controls. A total of 1×107 cells were sent for analysis. Samples were prepared as previously described [35]. LC‒MS/MS analyses were performed using a UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm) coupled to a Q Exactive mass spectrometer (Thermo). Mobile phase A was 0.1% formic acid in water for positive mode and 5 mmol/L ammonium acetate in water for negative mode, and mobile phase B was acetonitrile. The elution gradient was set as follows: 0–1.0 min, 1% B; 1.0–8.0 min, 1% − 99% B; 8.0–10.0 min, 99% B; 10.0–10.1 min, 99% − 1% B; 10.1–12 min, 1% B. The flow rate was 0.5 mL/min. The raw data were converted to the mzXML format using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS, for peak detection, extraction, alignment, and integration [36]. Then, an in-house MS2 database (BiotreeDB) was applied for metabolite annotation. The cutoff for annotation was set at 0.3.
Co-immunoprecipitation Coupled To Mass Spectrometry (Co-ip/ms)
Proteins in pancreatic cancer cells were enriched using the Protein A/G PLUS-Agarose (Santa Cruz, USA). The bound proteins were extracted from IP beads using SDT lysis buffer (4% SDS, 100 mM DTT, 100 mM Tris-HCl pH 8.0). Protein digestion was performed with FASP method described by Wisniewski et al. [30]. The peptide was desalted with C18 StageTip for further LC-MS analysis. LC-MS/MS experiments were performed on a Q Exactive Plus mass spectrometer that was coupled to Easy nLC (Thermo Scientific). MS data was acquired using a data-dependent top20 method dynamically choosing the most abundant precursor ions from the survey scan (350–1800 m/z) for HCD fragmentation. A lock mass of 445.120025 Da was used as internal standard for mass calibration. The full MS scans were acquired at a resolution of 70,000 at m/z 200, and 17,500 at m/z 200 for MS/MS scan. The maximum injection time was set to for 50 ms for MS and 50 ms for MS/MS. The MS data were analyzed using MaxQuant software version 1.6.0.16. MS data were searched against the UniProtKB Rattus norvegicus database (36080 total entries, downloaded 08/14/2018). The trypsin was seleted as digestion enzyme. The maximal two missed cleavage sites and the mass tolerance of 4.5 ppm for precursor ions and 20 ppm for fragment ions were defined for database search. Carbamidomethylation of cysteines was defined as fixed modification, while acetylation of protein N-terminal, oxidation of Methionine was set as variable modifications for database searching. The database search results were filtered and exported with < 1% false discovery rate (FDR) at peptide-spectrum-matched level, and protein level, respectively.
Statistical analysis
The statistical analyses were performed using SPSS 19.0 software (IBM Corp.) and Prism statistical software (version 8, GraphPad Software, Inc.). Paired 2-tailed Student’s t tests were used to examine the difference in glutamine concentration between primary pancreatic cancers and their matched metastases (liver or lymph node). Unpaired 2-tailed Student’s t tests were used to evaluate other data. For multiple comparisons, ANOVA combined with Bonferroni was applied. Data are presented as the mean ± standard error of the mean. Spearman correlation analysis was used to determine the correlation between glutamine concentration and other variables. Survival analysis was calculated by the KaplanMeier method, and the survival curves were examined by logrank tests. Significance was defined as a p value < 0.05, unless otherwise stated.