| Metadata |
| datasetIdentifier | PASS01614 |
| datasetType | MSMS |
| submitter | Juan Lopez <jalopez@cnic.es> |
| submitter_organization | CNIC |
| lab_head_full_name | Jesus vazquez |
| lab_head_email | jesus.vazquez@cnic.es |
| lab_head_organization | CNIC |
| lab_head_country | Spain |
| datasetTag | CAV1_Exosomal |
| datasetTitle | ECM deposition is driven by caveolin 1-dependent regulation of exosomal biogenesis and cargo sorting |
| publicReleaseDate | 2020-09-07 00:00:00 |
| finalizedDate | 2020-07-30 03:49:11 |
| summary | The composition and physical properties of the extracellular matrix (ECM) critically influence tumor progression, but the molecular mechanisms underlying ECM layering are poorly understood. Tumor-stroma interaction is critically dependent on cell-cell communication mediated by exosomes, small vesicles generated within multivesicular bodies (MVBs). Here, we show that caveolin-1 (Cav1) is a central modulator of both exosome biogenesis and exosomal protein cargo sorting through the regulation of cholesterol content at the endosomal compartment/MVBs. Quantitative proteomics profiling revealed that Cav1 is required for exosomal sorting of ECM protein cargo subsets including tenascin-C (TnC), and for fibroblast-derived exosomes to efficiently depose ECM and promote tumor cell invasiveness. Cav1-driven exosomal ECM deposition not only promotes local stromal remodeling, but also the generation of distant ECM-enriched stromal niches. These results support a model by which Cav1 is a central regulatory hub for tumor-stroma interactions through a novel exosome-dependent ECM deposition mechanism. |
| contributors | Lucas Albacete-Albacete, Inmaculada Navarro-Lérida, Juan Antonio López, Inés Martín-Padura, Alma M. Astudillo, Alessia Ferrarini, Michael Van-Der-Heyden, Jesús Balsinde, Gertraud Orend, Jesús Vázquez, and Miguel Ángel del Pozo |
| publication | ECM deposition is driven by caveolin1-dependent regulation of exosomal biogenesis and cargo sorting. Journal of Cell Biology, under revision |
| growth | |
| treatment | |
| extraction | Exosomes were isolated from cultured fibroblasts grown in exosome-free culture medium. To remove detached cells, conditioned medium was collected and centrifuged at 300 g for 10 min at 4ºC. The supernatant was collected and centrifuged at 2000 g for 20 min at 4ºC. The supernatant was then centrifuged at 10000 g at 4ºC for 30 min to completely remove contaminating apoptotic bodies, microvesicles and cell debris. The clarified medium was then ultracentrifuged at 110,000 g at 4ºC for 70 min to pellet exosomes. The supernatant was carefully removed, and crude exosome-containing pellets were washed in ice-cold PBS. After a second round of ultracentrifugation, the resulting exosome pellets were resuspended in the desired volume of PBS.
For further purification, exosomes were ultracentrifuged on a discontinuous sucrose gradient including sucrose concentrations of 0.25M, 0.5M, 0.8M, 1.16M, 1.3M and 2M in 20 mM HEPES, pH 7.4. Exosome samples were laid on the bottom of the gradient in the 2M sucrose fraction, followed by centrifugation at 200,000 g for 18 h. Eleven individual 1 ml gradient fractions were manually collected. Fractions were diluted in PBS and centrifuged at 110,000 g for 1 h at 4ºC, and the resulting pellets were resuspended and analysed. Alternatively, an equal volume of cold acetone was added to each fraction and the proteins precipitated for 2 h at -20ºC. Protein pellets were collected by centrifugation at 16,000 g for 10 min and air dried to eliminate acetone traces. The protein precipitates were monitored by western blot for the expression of the exosomal markers Alix and Tsg101.
Samples were dissolved in 50 mM Tris-HCl pH 8.5, 4% SDS and 50 mM DTT, boiled for 10 min, and centrifuged. Protein concentration in the supernatant was measured with the Direct Detect® Spectrometer (Millipore). |
| separation | |
| digestion | About 100 μg (fibroblasts) or 30 μg (exosomes) of protein were diluted in 8 M urea in 0.1 M Tris-HCl pH 8.5 (UA) and loaded onto 30 kDa centrifugal filter devices (FASP Protein Digestion Kit). The denaturation buffer was removed by washing three times with UA. Proteins were then alkylated by incubation in 50 mM iodoacetamide in UA for 20 min in the dark, and excess alkylation reagents were eliminated by washing three times with UA and three additional times with 50 mM ammonium bicarbonate. Proteins were digested overnight at 37ºC with modified trypsin (Promega) in 50 mM ammonium bicarbonate at a 50:1 protein:trypsin (w/w) ratio. The resulting peptides were eluted by centrifugation with 50 mM ammonium bicarbonate (twice) and 0.5M sodium chloride. Trifluoroacetic acid (TFA) was added to a final concentration of 1% and the peptides were finally desalted onto C18 Oasis-HLB cartridges and dried-down for further analysis.
Multiplexed isobaric labelling. For the quantitative analysis, tryptic peptides were dissolved in triethylammonium bicarbonate (TEAB) buffer, and the peptide concentration was determined by measuring amide bonds with the Direct Detect system. Equal amounts of each peptide sample were labelled using the 4-plex iTRAQ Reagents Multiplex Kit (Applied Biosystems). Briefly, each peptide solution was independently labelled at room temperature for 1 h with one iTRAQ reagent vial (mass tag 114, 115, 116 or 117) previously reconstituted with ethanol. After incubation at room temperature for 1 h, the reaction was stopped with diluted TFA and peptides were combined. Samples were concentrated in a Speed Vac, desalted onto C18 Oasis-HLB cartridges, and dried-down for mass spectrometry analysis. |
| acquisition | Digested peptides were loaded into the LC-MS/MS system for on-line desalting onto C18 cartridges and analysed by LC-MS/MS using a C-18 reversed phase nano-column (75 µm I.D. x 25 cm, 2 µm particle size, Acclaim PepMap RSLC, 100 C18; Thermo Fisher Scientific) in a continuous acetonitrile gradient consisting of 0-30% B for 240 min and 50-90% B for 3 min (A= 0.5% formic acid; B=90% acetonitrile, 0.5% formic acid). A flow rate of 200 nl/min was used to elute peptides from the RP nano-column to an emitter nanospray needle for real time ionization and peptide fragmentation in an Orbitrap QExactive mass spectrometer (Thermo Fisher). During the chromatography run, we examined an enhanced FT-resolution spectrum (resolution=70,000) followed by the HCD MS/MS spectra from the 20 most intense parent ions. Dynamic exclusion was set at 40 s. For increased proteome coverage, labelled samples were also fractioned by cation exchange chromatography (Oasis HLB-MCX columns); fractions were desalted and analysed using the same system and conditions described before. |
| informatics | Protein identification and quantification. All spectra were analysed with Proteome Discoverer (version 1.4.0.29, Thermo Fisher Scientific) using SEQUEST-HT (Thermo Fisher Scientific). The Uniprot database, containing all mouse sequences (March 03, 2013), was searched with the following parameters: trypsin digestion with 2 maximum missed cleavage sites; precursor and fragment mass tolerances of 2 Da and 0.03 Da, respectively; methionine oxidation as a dynamic modification; and carbamidomethyl cysteine and N-terminal and Lys iTRAQ modifications as fixed modifications. Peptides were identified using the probability ratio method (Martinez-Bartolome et al., 2008), and false discovery rate (FDR) was calculated using inverted databases and the refined method (Navarro et al., 2014) with an additional filtering for a precursor mass tolerance of 15 ppm (Bonzon-Kulichenko et al., 2015). Identified peptides had a FDR equal to or lower than 1% FDR.
Proteins were quantified from reporter ion intensities and quantitative data analysed with QuiXoT, based on the WSPP statistical model (Garcia-Marques et al., 2016). In this model, protein log2-ratios are expressed as standardized variables, i.e. in units of standard deviation according to their estimated variances (Zq values). Functional class enrichment was assessed using the Enrichr package (https://amp.pharm.mssm.edu/Enrichr/) and network depiction of annotated relationships among hits was retrieved from the STRING v.11 resource). |
| instruments | Thermo Scientific Orbitrap QExactive |
| species | MOUSE |
| massModifications | static: C+57.021464,K+144.102063,N-term+144.102063, variable: M+15.994915 |
