Interleukin-6 Induced Acute Phenotypic Microenvironment Promote...（二）
As a primary component of circulatory system, serum is the major reservoir of thousands of proteins secreted or “leaked” from a broad spectrum of cells and tissues. Proteome changes in serum well reflect the host response to physiological and pathological perturbation. Therefore, serum is regarded as a good window for systematic investigation of the therapeutic responses using proteomics. However, the extreme complexity and huge dynamic range (over 10 orders of magnitude) of serum significantly restricted its application in proteomics via mass spectrometry [24]. Recently, specific focus on
N-linked serum glycoproteins has been demonstrated as an effective way to reduce serum complexity, maximize the detective dynamic range and improve the efficiency of low abundant proteins measurement [25]. Moreover, numerous evidences support that alteration in glycoprotein abundance and glycan composition are closely associated with aberrant physiological state, such as cancer and other infectious diseases [26-28].
In this study, we applied cryo-thermal therapy on a highly malignant murine 4T1 breast cancer xenograft model. Serum samples underwent therapy or in control were comprehensively analyzed by shotgun proteomics following N-glycopeptides enrichment, iTRAQ-labeling, and off-gel fractionation.Subsequently 23 glycoproteins were further selected for validation using PRM technique in a total of 94 enriched serum samples over 8 time points. We found that cryo-thermal therapy reshaped the tumor chronic inflammatory microenvironment to an “acute” phenotype, which played a key role to hamper immunosuppression and recover the host anti-tumor immunity. Moreover, the magnitude of “acute” and “danger” signals could determine the efficacy of anti-tumor immunity. The study allowed us to dissect the network of protein changes associated with the cryo-thermal therapeutic efficacy thus deepening our understanding of its mechanism at the molecular level, and providing insights and guidance to improve this cancer treatment with better therapeutic effects.
Experimental Procedures
Animal experiments
Cell culture
4T1 mouse mammary tumor cell line was obtained from Shanghai First People’s Hospital (Shanghai, China). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; GE Healthcare, Logan, UT ) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA ), 100 units/ mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified 5% CO2 incubator.
Xenograft breast tumor model and cryo-thermal / cryo- / hyperthermia therapy
Pathogen-free female Balb/c mice (4 weeks old) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd., and housed at constant temperature and humidity with a 12-h light cycle. All animals were allowed food and water ad libitum until ~20 g before experiments (approximately 6-week old). All animal experiments were approved by the Animal Welfare Committee of Shanghai Jiao Tong University.
Tumor was induced by injection of 5×105 4T1 cells subcutaneously into the right dorsal region of mouse and received cryo-thermal therapy 3 weeks later, reaching the size of ~10 mm in diameter. Cryo-thermal therapy was performed using a modified protocol [11]. Briefly, a thermocouple was inserted to the bottom of tumor to monitor the local temperature in real time. Firstly, tumor was rapidly frozen to -20 °C by liquid nitrogen and maintained for 5 min. Subsequently, liquid nitrogen was turned off and frozen tumor was recovered naturally to 8 °C. Finally, radiofrequency (460 kHz) was introduced to heat the tumor (10 min, 50 °C). Afterwards, mice under cryo-thermal or no treatment as controls were kept for survival rate observation. Tumor volume was monitored and measured by the length (L), width (W) and height (H), and calculated as V=π/6×L×W×H. Lungs were harvested and fixed in Bouin’s solution(Sigma-Aldrich, St. Louis, MO). For time course proteome analysis, another group of mice were kept for another 2 h, 2, 5, 8, 11, 14, 21 and 28 days post therapy, respectively and mice left untreated were taken as control with matching time points. In addition, 8-week-old healthy mice were taken as “healthy group”. Plasma from each mouse was taken for proteomic analysis.
For cryotherapy, tumor was rapidly frozen to -20 °C by liquid nitrogen and maintained for 15 min. For hyperthermia therapy, radiofrequency (460 kHz) was introduced to heat tumor (15 min, 50 °C). Sera were collected 2 days post therapy.
Sample preparation for proteomics analysis
Serum N-glycosylated peptides capture
Plasma was left to be clotted (30 min, 25°C) and centrifuged (500 g, 10 min, 4 °C) and the supernatant (serum) was collected. Prior to glycopeptide capture, serum in each condition (n=5~8) was pooled to minimize the individual variance. Serum concentration was determined using BCA assay (Thermo Fisher Scientific, San Jose, CA).
Subsequently, 25 μL of each pooled sample was processed and subjected to glycopeptide capture. Briefly, samples were first diluted to 100 μL with 0.1 M NH4CO3, and denatured with 50% TFE(Trifluoroethanol)/0.1% SDS (30 min, 55 °C). Samples were reduced with 8 mM TCEP (30 min, 55 °C) and alkylated with 12 mM iodoacetamide (30 min, 25 °C) in the dark (all of the reagents were obtained from Sigma-Aldrich, St. Louis, MO). Samples were then diluted with 0.1 M NH4CO3 to a final concentration of 5% TFE and digested overnight at 37 °C with sequencing grade modified trypsin (1/50, w/w; Sigma-Aldrich, St. Louis, MO). After digestion, samples were acidified with 1% formic acid to quench the reaction and desalted on C18 reverse-phase spin columns according to the manufacturer’s instructions (100 mg, Waters, Milford, MA), dried under vacuum to ~ 100 μL before glycopeptide capture.
N-glycopeptide capture procedure was carried out according to a published protocol [25] with a slight modification. Briefly, desalted peptides were treated with 10 mM sodium periodate (30min, 25°C ,dark) to oxidize the cis-diol groups on glycans. The oxidized glycopeptides were then coupled to hydrazide resin (Bio-Rad, Hercules, CA) by the reaction of aldehyde groups. The extensively washes were performed to remove non-glycosylated peptides with three times of 1 mL of 8M urea/0.1% SDS, 1.5 M NaCl, 80% ACN, Milli-Q H2O and 50mM NH4CO3. N-glycopeptides were then released by PNGase F (New England Biolab, Ipswich, MA).
iTRAQ labeling and off-gel electrophoresis
iTRAQ labeling was carried out using iTRAQ reagent 8-plex kit (Sciex, Framingham, MA) according to the manufacturer’s introduction. Cleaved N-glycopeptides were desalted, dried and labeled with different isobaric tags (2 h, 25°C) and quenched by Milli-Q water. Labeled peptides from multiple time points were assigned to three groups and mixed (Additional File 7: Figure S2C). N-glycopeptides enriched from healthy sera (n=8) were labeled with 119 tag and evenly assigned to three groups as global reference (Additional File 7: Figure S2C). Formally N-glycopeptide mixtures were separated by OFFGEL fractionation (24 wells, 3-10 pI strips) in a 3100 OFFGEL fractionator (Agilent Technologies, Santa Clara, CA) according to the manufacture’ s instructions. Afterwards, peptide mixtures were collected from each well and pooled into 8 different fractions (A: 1-3; B:4-6; C:7-9; D:10-12; E:13-15; F:16-18; G: 19-21; H: 22-24). Fractions were desalted on C18 columns (50 mg, Waters, Milford, MA), dried under vacuum and dissolved in 0.1% formic acid prior to data-dependent LC-MS/MS analysis.
LC-MS/MS acquisition
Shotgun data-dependent acquisition (DDA) MS
Approximately 0.5 μg of glycopeptides from each fraction were separated in a 2-h gradient on a 15-cm long (75 μm inner diameter) PicoFrit Self-Pack Column (360 μm OD, 75 μm ID, 15 μm Tip, New Objective, Woburn, MA) packed in-house with 3 μm 200 Å C18 particles (Dr. Maisch GmbH). Reverse phase chromatography was performed with an EASY-nLC 1000 ultra-high-pressure system, which was coupled to an Orbitrap Elite mass spectrometer interfaced with a nanoelectrospray ion source (Thermo Fisher Scientific, San Jose, CA). Peptides were loaded to a trap column (150 μm¡Á 12 mm) at 10 μL/min with solvent A (0.1% (v/v) formic acid) and eluted with a linear 120-min gradient of 2% to 35% solvent B (0. 1% (v/v) formic acid, acetonitrile) at a flow rate of 300 nL/min. After each gradient, the column was flushed with 80% solvent B for 10 min and equilibrated with 2% buffer B for another 20 min. MS data were acquired with a shotgun proteomics method, where in each cycle, a full scan was acquired at a resolution of 30,000 at m/z 400 with a mass range of 380-1600 m/z. Up to 15 most intense precursor ions with charge ≥ 2 were selected with an isolation window of 2 Da and subsequently fragmented by higher-energy collisional dissociation (HCD) with normalized collision energy of 40%. The MS/MS scans were acquired at a resolution of 15000 at m/z 400 with fixed first mass of 100 Da. The precursor ions were dynamically excluded from reselection for 60 s, and the exclusion list size was 500. Peptides in each fraction were analyzed for two technical replicates. Additionally, to evaluate the efficiency of N-glycopeptide capture, prior to the iTRAQ labeling and fractionation, glycopeptides extracted from each pooled sera were analyzed on the Orbitrap Elite mass spectrometer, respectively.
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