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A Comprehensive Proteomics Analysis Reveals a Secretory Path- and Status-Dependent Signature of Exosomes Released from Tumor Associated Macrophages Yinghui Zhu, Xianwei Chen, Qingfei Pan, Yang Wang, Siyuan Su, Cuicui Jiang, Yang Li, Ningzhi Xu, Lin Wu, Xiaomin Lou, and Siqi Liu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00770 • Publication Date (Web): 27 Aug 2015 Downloaded from http://pubs.acs.org on August 31, 2015

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Journal of Proteome Research

A Comprehensive Proteomics Analysis Reveals a Secretory Path- and Status-Dependent Signature of Exosomes Released from Tumor Associated Macrophages

Yinghui Zhu1, 2, Xianwei Chen1, 2, Qingfei Pan1, 2, Yang Wang1, 2, Siyuan Su1,2, Cuicui Jiang3, Yang Li3, Ningzhi Xu4, Lin Wu1, Xiaomin Lou1*, Siqi Liu1*

1

CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China 2

University of Chinese Academy of Sciences, Beijing, 100049, China 3

4

Beijing Protein Innovation, Beijing, 101318, China

Cancer Institute and Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100021, China

* To whom correspondence should be addressed E-mail: [email protected] E-mail: [email protected] Tel: 86-10-84097465 Fax: 86-10-84097465 Authors Email: First author: [email protected] [email protected],

[email protected],

[email protected], 1

ACS Paragon Plus Environment

[email protected],


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[email protected],

[email protected],

[email protected],

[email protected], [email protected]

2


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[email protected],

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Abstract Exosomes are 30-120 nm sized membrane vesicles of endocytic origin that are released into extracellular environment, and play roles in cell-cell communication. Tumor associated macrophages (TAMs) are important players in tumor microenvironment, so it is critical to study the features and complex biological functions of TAM-derived exosomes. Here, we constructed a TAM cell model from a mouse macrophage cell line, Ana-1, and performed comparative proteomics on exosomes, exosome-free media, and cells between TAMs and Ana-1. Proteomic analysis between exosome and exosome-free fractions indicated that the functions of exosome dominant proteins were mainly enriched in RNA processing and proteolysis. TAMs status dramatically affected the abundances of 20S proteasome subunits and ribosomal proteins in their exosomes. The 20S proteasome activity assay strongly indicated that TAM exosomes possessed higher proteolytic activity. In addition, Ana-1 and TAM-derived exosomes have different RNA profiles, which might result from differential RNA processing proteins. Taken together, our comprehensive proteomics study provides novel views for understanding

the

complicated

roles

of

macrophage-derived

exosomes

in

tumor

microenvironment.

Keywords Exosome, tumor associated macrophage, iTRAQ, protein signature, proteasome, ribosome

3



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Introduction In decade years, a special extracellular organelle aroused great concerns in cell to cell communication studies. Exosomes, 30-120 nm vesicles released from multivesicular bodies fused with plasma membrane into extracellular environment, are able to encapsulate proteins, lipids, RNAs from donor cells to recipient cells.1,2 Distinct from the free proteins in extracellular environment, proteins in exosomes are enclosed by bilayer lipid membrane, and thus protected from the hostile extracellular environment and safely delivered to targeted cells. Similar to many intracellular organelles, exosomes have multiple intrinsic protein composition, but its contents are found in a wide variety, which is highly dependent on cell or tissue type.3,4 For instance, Skogeberg et al applied multiple methods to characterize human thymic exosomes and found that thymic exosomes exhibited some thymus specific features regarding the surface markers (HLA-DR, MEF-E8), tissue restricted antigens and 38 specific miRNAs for human thymus.5 Mathivanan et al adopted proteomics analysis to the exosomes isolated from neoplastic cells, hepatocytes and urine, and revealed that some protein signatures were shared by the exosomes from different origins but certain exosome proteins were associated with the disease states and specific tissues.3,6,7 Macrophages are a population of immune cells with a high degree of phenotypic and functional heterogeneity in the microenvironment of solid tumor. In general, macrophages have been classified into two subsets: the classical M1 (pro-inflammatory) and the alternative M2 (anti-inflammatory). With stimulations of M-CSF, CCL2, IL-4, IL-10 and TGF-β, macrophages are recruited into the tumor local tissue and turned into a polarized population with tumor-supportive properties called tumor associated macrophages (TAMs).8,9 A large 4


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body of evidences emerging recently have demonstrated that the secreted molecules from TAMs possess pro-tumorigenesis activity.10,11,12 For example, TAMs produce several proteolytic enzymes such as MMPs (MMP 2, 7, 9) and cathepsins (cathepsin B, S) that incessantly degrade ECM proteins, thus favoring tumor expansion, motility and invasion. Herbeuval et al reported that TAM-derived IL-6 induced STAT3-mediated IL-10 production in tumor cells, which had been correlated with poor prognosis.13,14 However, TAMs were also reported to suppress growth of lung metastasis through releasing MMP12 into microenvironment.15 Khorana et al analyzed the presence of VEGF-expressing TAMs and found a significant association with favorable outcome in a multivariate analysis.16 These reports are in agreement with a ‘‘mixed’’ M1/M2 status of TAMs and emphasize the intricate interaction between cancer cells and macrophages in tumor microenvironment.17,18,19 Although it has been reported that macrophages could regulate recipient cells through releasing exosomes, the features of macrophage-derived exosomes are still elusive. Aucher et al reported that human macrophages could transfer microRNAs, miR-142 and miR-223, to hepatocarcinoma cells (HCCs) through exosomes, and functionally inhibited proliferation of the HCCs.20 Yang et al found the specific microRNAs carried by IL-4 activated macrophage-derived exosomes could promote breast cancer invasion.21 Two comparative proteomics analysis of exosomes released by macrophages exposed to Leishmania and Mycobacterium avium respectively indicated that the exosomes from infected macrophages displayed unique signatures of immune response in terms of composition and abundances of many inflammatory proteins.22,23 These studies imply that exosomes convey specific information, and only some cellular proteins or RNAs are exported from cells and into 5



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exosomes in response to biological stimuli. Comparison with surveying the exosome composition in static state, studying the exosome behavior under stress conditions will further contribute to understanding exosome exporting mechanisms. TAMs, as a sort of typical heterogeneous macrophages resulted from extracellular stimuli, are mainly reported as pro-tumoral populations. Their exosomes were also considered to be strong candidates to promote tumor viability. However, the alterations of exosome behavior from the original macrophages to TAMs have yet to be explored. Taken together, in order to have a comprehensive understanding of characteristics and potential roles of macrophages in tumor microenvironment, a close investigation on exosomes derived from macrophages and TAMs is urgently required. In present study, we used colon cancer cell cultured media as stimulus for the construction of TAM model, and performed comprehensive comparative proteomics analysis on exosomes, exosome-free media, and cells between TAMs and their original macrophages. The results revealed that macrophage exosome proteins are mainly involved in RNA processing and proteolysis functions, and compared with original macrophages, TAM-derived exosomes exhibited increased proteasome subunits and decreased ribosomal proteins, indicating TAM-derived exosomes had enhanced proteasome activity and reduced RNA binding capacity.

Materials and Methods Cell culture Mouse macrophage cell line Ana-124 and mouse colorectal cancer cell line CT2625 were cultured in RPMI1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL 6


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streptomycin with 5% CO2 at 37°C. When cells reached 70% confluence (about 24 h), CT26 cultured media were harvested and centrifuged at 1,000 g for 5 min at 4°C. Ana-1 (4 x106/10cm2 dish) cells were induced by collected CT26 cultured media for 48 h to become TAMs. The partial cells were harvested for further analysis. After 3 washes in 0.9% normal saline, Ana-1 and TAMs with 70% confluence were cultured in the serum-free RPMI1640 at 37°C for 24 h before collection of both supernatants. Exosome purification The harvested media from Ana-1 and TAMs culture were clear of cells, large debris and shed microvesicles by serial centrifugation (300 g for 10 min and then 16,500 g for 20 min). The supernatants were filtered through 0.22 µM filters (Millipore) to remove large membrane vesicles and ultracentrifuged at 120,000 g for 2 h. The 15.8 ml supernatant per tube (16.8 ml in total) were collected to isolate exosome-free fraction, and the resting 1 ml supernatant at the bottom of the ultracentrifuge tube was transferred to a sterile tube and mixed with 250 µl ExoQuick-TC exosome precipitation solution (SBI) followed by refrigeration overnight (at least 12 h). Finally, exosomes were pelleted at 1,500 g for 30 min. Transmission Electron Microscope (TEM) and Nanoparticle Tracking Analysis (NTA) TEM was employed to visualize exosome preparations as previously reported.26 Briefly, exosomes were resuspended in 2.5% (v/v) glutaraldehyde in PBS at room temperature (RT) for 30 min, and transferred onto 200 mesh copper grids and allowed to stand for drying. The grids were washed three times with distilled water for 3 min each and then negatively stained with 1% (w/v) aqueous uranyl acetate for 5 min. The grids were imaged using a FEI Tecnai G2 Spirit electron microscope operated at 120 kV. Purified Exosomes were examined by NanoSight 7



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LM10 system27,28 (NanoSight, UK) and calibrated at 183 nm/pixel using 100 nm calibration beads. Shutter and gain were kept at the same settings for all samples. Video recordings of 60 s and approximately 1000 tracks were analyzed per sample. Protein preparation The exosome-free supernatant was concentrated by 10 fold by Amicon Ultra centrifugal filters (3K Ultracel, Millipore). The supernatant fraction that remained above the filter was mixed with 3 volumes of acetone, containing 10% (w/v) TCA. After 3 h incubation at -20°C, the mixture was centrifuged at 20,000 g for 30 min at 4°C. The pellet was briefly rinsed with acetone and incubated with 4 volumes of acetone at -20°C overnight. After centrifugation at 20,000 g for 30 min at 4°C, the clean exosome-free proteins were in the pellet. The exosome-free protein pellets, exosomes and cells of Ana-1 and TAM were resolved/lysed in the lysis buffer containing 8M urea, 4% HEPES. After centrifugation at 12,000 g for 30 min at 4°C, the proteins in the supernatants were transferred to new tubes. The protein concentrations were determined using Bio-Rad protein assay (Hercules, CA, USA). Western blotting analysis The proper amount of proteins from exosomes (5 µg) and cells (10 µg) were separated on SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes for 70 min at 350 mA. After blocking with 5% (w/v) BSA in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TTBS) for 1 h, membranes were probed with primary mouse anti-TSG101 (BD Transduction Laboratories; 1:500), mouse anti-Alix (Cell Signaling Technology; 1:1000), mouse anti-HSP90 (Enzo Life Science; 1:1000), mouse anti-ATP synthase β (BD Transduction Laboratories; 1:1000), rabbit anti-PERK (Santa Cruz Biotechnology; 1:1000), mouse 8


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anti-Cathepsin B (Sigma-Aldrich; 1:1000), rabbit anti-Cathepsin D (SantaCruz Biotechnology; 1:1000), mouse anti-PSMB7 (SantaCruz Biotechnology; 1:1000), mouse anti-PSMA2 (Upstate Biotechnology; 1:1000), rabbit anti-RPS6 (Cell Signaling Technology; 1:1000), rabbit anti-RPL10 (Sangon Biotech; 1:500), or rabbit anti-RPL15 (Sangon Biotech; 1:500) for 3 h in blocking buffer followed by incubation with appropriate HRP-conjugated secondary antibodies (1:3000, ZSGB-BIO, China) for 1 h in blocking buffer. All antibody incubations were carried out using gentle orbital shaking at RT. Proteins were visualized by incubating membranes with HRP substrate (GE Healthcare, Pittsburgh, PA, USA) followed by imaging with the ImageQuant ECL (GE Healthcare). Real-time PCR analysis Total RNAs from cells and exosomes were isolated with TriPure reagent (Roche, Indianapolis, USA) according to the manufacturer’s instructions. The quantity, quality and composition of RNA samples were evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). One microgram RNA from Ana-1 and TAM cells was reversely transcribed using TaqMan reverse transcription regents (TransGen Biotech, China) following manufacturer’s instructions. The primers were designed against Gapdh (FP: 5’-CTTTGTCAAGCTCATTTCCTGG-3’, RP: 5’-TCTTGCTCAGTGTCCTTGC-3’), Il-1rn (FP: 5-TCATTGCTGGGTACTTACAAGG-3’, RP:5’-ATCTCCAGACTTGGCACAAG-3’),

Il-10

(FP:

5’-AACATACTGCTAACCGA

CTCC-3’, RP: 5’-CAAATGCTCCTTGATTTCTGGG-3’), Arg-1 (FP: 5’-AAGAATGGAA GAGTCAGTGTGG-3’,

RP:

5’-GGGAGTGTTGATGTCAGTGTG-3’),

5’-CATAGAGGAAGCCCATTACAGG-3’,

RP:

Mmp9:

(FP:

5’-TGTACACCCACATTTGACGTC-3’),

Il-1b (FP: 5’-ACGGACCCCAAAAGATGAAG-3’, RP: 5’-TTCTCCACAGCCACAATGAG 9



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-3’), Il-6 (FP: 5’-GATAAGCTGGAGTCACAGAAGG-3’, RP: 5’-GGA ATGTCCACAAACT GATATGC-3’),

Cxcl10

(FP:

5’-GGTCTAAAAGGGCTCCTTAACTG-3’,

RP:

5’-GACCAAGGGCAATTAGGACTAG-3’), Cd206 (FP: 5’-ATGGATGTTGATGGCTACTG G-3’,

RP:

5’-TTCTGACTCTGGACACTTGC-3’).

The

mRNA

abundances

were

quantitatively determined using a PRISM 7300 System (Applied Biosystem, Foster City, CA). The amplification cycling conditions were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 30 sec, 60°C for 15 sec, 72 °C for 10 sec and 78 °C for 40 sec to collect the fluorescent signals, then 72°C for 10 min to elongate. The dissociation curves were performed with 95°C for 15 sec, 60°C for 15 sec and 95°C for 15 sec. All targets were tested three times and their expression levels were normalized by GAPDH abundance. Peptides fractionation and identification by MS/MS Eighty microgram proteins from exosomes, exosome-free media and 100 µg proteins from cells of Ana-1 and TAM were reduced by 5 mM tris-(carboxyethyl) phosphine and alkylated by 10 mM methyl methanethiosulfonate followed by a 16 h trypsin digestion at 37°C. The tryptic peptides were labeled by the 8-plex iTRAQ reagents (AB Sciex, FosterCity, CA) according to the manufacturer’s protocol. After 2 h labeling reactions, the labeled peptides from same fraction of two cells were pooled together respectively for the further peptide fractionation and identification. Each pool of mixed peptides was lyophilized and dissolved in solution A (2% acetonitrile (ACN) and 20 mM ammonium formate (NH4FA), pH=10). Then, they were loaded onto a RP column (Luna C18, 4.6 X 150 mm, Phenomenex, CA) and eluted by a step linear elution program, 0-10 min equilibrated in 100% solution A, 10-15 min fast elution from 0-12% of solution B (80% ACN, 20 mM NH4FA , pH=10), 15-45 min liner 10


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elution from 12-56% of solution B, and 45-57 min washing elution from 56-100% of solution B. The RP HPLC procedures were manipulated in an LCsolution 20A (Shimadzu, Nakagyo-ku, Kyoto, Japan) with the flow rate at 0.5 ml/min and the peptides were monitored at 214 nm. The fractionated peptides were collected at one tube/min during the linear elution period and further pooled into indicated fractions (10 for exosome, 15 for exosme-free and 18 for cells). Each fraction was analyzed by a Q Exactive mass spectrometer (Thermo Fisher Scientific) coupled with an Easy-nLC 1000 UPLC (Thermo Fisher Scientific) system twice. Peptides were loaded on a pre-column (10 µm-C18 resin, 75 µm * 8 cm) and separated with an analytical column (3 µm-C18 resin, 75 µm * 11 cm, YMC Co., Ltd) using acetonitrile gradients from 5-40% in 65 min at a flow rate of 400 nl/min. Spectra were acquired in a data-dependent mode. The 10 most intense ions of +2, +3 or +4 charge from each full scan (R=70,000) were isolated for HCD MS2 (R=17,500) at 27% normalized collision energy (NCE) with a dynamic exclusion time of 150 sec. Database searches for Peptide, protein identification and quantitative data analysis The raw MS/MS data were converted into MGF format by Proteome Discoverer 1.3 (Thermo Fisher Scientific, Waltham, MA), and the exported MGF files were searched by Mascot 2.3 (Matrix Science, Boston, MA) against the database Uniprot (selected for Mus. unknown version, 16700 entries). An automatic decoy database search was performed. Several parameters in Mascot were set for peptide searching, including iTRAQ 8-plex for quantification, tolerance of one missed cleavage of trypsin, methylthio for cysteine as fixed modification, oxidation for methionine as variable modification. The precursor mass tolerance was 15 ppm, and the product ion tolerance was 0.8 Da. After database searching, the DAT files 11



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were imported into Scaffold v4.3.2 (Proteome Software Inc., Portland, OR). Scaffold was used to organize all data, quantitate proteins and validate peptide identification using the Peptide Prophet algorithm, and unique proteins with at least two unique peptides, with a false discovery rate (FDR)

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