Proteomic characterization of epithelial-like extracellular vesicles in

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Proteomic characterization of epithelial-like extracellular vesicles in advanced endometrial cancer Javier Mariscal, Patricia Fernández-Puente, Valentina Calamia, Alicia Abalo, Maria Santacana, Xavier Matias-Guiu, Rafael Lopez-Lopez, Antonio Gil-Moreno, Lorena Alonso-Alconada, and Miguel Abal J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00750 • Publication Date (Web): 26 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

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Journal of Proteome Research

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Proteomic characterization of epithelial-like

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extracellular vesicles in advanced endometrial

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cancer

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Javier Mariscal1, Patricia Fernandez-Puente2, Valentina Calamia2, Alicia Abalo1,

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Maria Santacana3, Xavier Matias-Guiu3, Rafael Lopez-Lopez1, Antonio Gil-Moreno4,

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Lorena Alonso-Alconada1, and Miguel Abal1

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(IDIS), University Hospital of Santiago (SERGAS), Santiago de Compostela, Spain

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Investigación Biomédica de A Coruña (INIBIC), University Hospital A Coruña

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(SERGAS), Universidade da Coruña, A Coruña, Spain

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3

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CIBERONC, Lleida, Spain

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4

18

Universitat Autonoma de Barcelona, CIBERONC, Barcelona, Spain

Translational Medical Oncology, CIBERONC, Health Research Institute of Santiago

Proteomics Group-PBR2-ProteoRed/ISCIII, Rheumatology Division, Instituto de

Hospital Universitari Arnau de Vilanova, University of Lleida, IRBLLEIDA,

Biomedical Research Group in Gynecology, Vall Hebron Research Institute (VHIR),

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1 ACS Paragon Plus Environment

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ABSTRACT: Endometrial cancer (EC) is the most frequent gynecological cancer.

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Tumor dissemination affecting around 20% of EC patients is characterized at the primary

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carcinoma by epithelial-to-mesenchymal transition (EMT) associated with myometrial

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infiltration. At distant sites, the interaction of circulating tumor cells (CTCs) with the

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microenvironment results crucial for metastatic colonization, with a participation of the

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extracellular vesicles (EVs). We comprehensively approached these primary and

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secondary sites to study the impact of tumor EVs on the metastatic efficiency of CTCs in

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EC. Tumor EVs in circulation reproduce the epithelial phenotype predominant in the

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primary carcinoma, while CTCs are characterized by an EMT phenotype. We modelled

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this EMT-related clinical scenario in the Hec1A endometrial cell line and characterized

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the epithelial-like EVs in circulation by SILAC proteome analysis. Identification of

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proteins involved in cell-cell and cell-matrix interaction and binding, together with in

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vitro evidences of an improved adhesion of CTC to a functionalized endothelium suggest

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a contribution of the epithelial-like EVs in the homing of CTCs at metastatic sites.

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Accordingly, adhesion protein LGALS3BP was found significantly enriched in

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circulating EVs from a cohort of EC patients with high risk of recurrence by targeted

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proteomics (MRM), highlighting their potential as liquid biopsy in EC.

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KEYWORDS: Endometrial metastasis; EVs proteomics; SILAC; Circulating Tumor

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Cells.


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1. INTRODUCTION

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Endometrial Cancer (EC) constitutes the fourth leading cancer in women with more than

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60,000 new cases and 10,000 deaths estimated in the United States in 2018.1 EC originates

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in the inner layer of the uterus when epithelial cells lining the myometrium start to

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proliferate abnormally and, although most EC are diagnosed early, up to 20% of the

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lesions progress to a high-grade carcinoma.1 Compared to 90% in patients diagnosed with

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confined disease, 5-year survival in advanced disease drops up to 15% as a result of

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primary myometrial infiltration followed by dissemination of aggressive tumor cells.2

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Myometrial invasion results from an epithelial-to-mesenchymal (EMT) process

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characterized by a switch from E-Cadherin-positive epithelial tumor cells, strictly

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organized in glands, transitioning into mesenchymal-like tumor cells able to migrate and

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invade the surrounding myometrium and reach the vasculature.3 The presence of these

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tumor cells in circulation, or CTCs in peripheral blood, has been recently reported by our

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group and others in high risk-EC patients with the FDA-approved CellSearch®

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technology.4–6 Notably, the molecular characterization of these disseminated tumor cells

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has identified an EMT signature characterized by a plasticity phenotype.6 EMT at the

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invasive front of EC is a well described event driving cancer cell aggressiveness, therapy

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resistance and metastasis.7

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Alternatively, tumor-secreted extracellular vesicles (EVs) have been identified as main

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mediators of cell-to-cell communication between tumor and stromal cells both in local

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and distant microenvironments participating in the formation of the pre-metastatic niche

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prior to CTC colonization.8 Tumor exosomes, a subpopulation of 40-150 nm EVs released

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from the tumor cells following the fusion of multivesicular late endosomes / lysosomes

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with the plasma membrane, have been shown to be representative of the tumor of origin9

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and important mediators of intercellular signaling including EMT and the tropism of



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disseminated disease to pre-conditioned tissues.10 In this scenario, EC-derived EVs may

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not only be carriers of molecules of utility for early diagnosis and monitoring of patients,

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but also actively participate in the recruitment of tumor supporting cells and CTCs in a

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pro-metastatic microenvironment. Recent discoveries have reported increased secretion

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of EVs associated with high-grade disease10–12 and the biological relevance of tumor EVs

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in disease progression and metastatic potential.13 However, identification of the cell of

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origin and biological profiling of circulating tumor EVs remains challenging.

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To study this, we comprehensively approached EMT in EC and the contribution of the

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epithelial and mesenchymal components to the tumor EVs shed in circulation. By

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combining EMT modelling and quantitative mass spectrometry analysis by stable isotope

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labeling with amino acids in cell culture (SILAC), we were able to characterize the

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predominant epithelial-like tumor EVs. We further explored by multiple reaction

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monitoring (MRM) the potential of these EVs as biomarkers of disease in plasma from a

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cohort of EC patients with high risk of recurrence.

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2. MATERIALS & METHODS

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2. 1. Histology and Immunohistochemistry analysis & cell culture of EC cell lines.

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EC tissue sections from patients (n= 5) were selected where the non-invasive, the

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superficial invasive, the invasive front and areas with deep invasion could be

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distinguished. Consecutive sections were obtained from tissue blocks, sectioned at a

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thickness of 3 μm, dried for 1h at 65ºC before pre-treatment procedure of

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deparaffinization, rehydration and epitope retrieval in the Pre-Treatment Module, PT-

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LINK (Agilent Technologies-DAKO, Santa Clara, CA, USA) at 95°C for 20 min in 50x

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Tris/EDTA buffer, pH 9. Before staining the sections, endogenous peroxidase was

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blocked. The antibodies used were against Cytokeratin (Ready to Use, clone AE1/AE3,),

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E-cadherin (Ready to Use, clone NHC-38, Agilent Technologies-DAKO), SNAIL (1:100,

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Polyclonal, ABCAM) and HMGA2 (1:300, Polyclonal, Thermo Fisher scientific). After

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incubation, the reaction was visualized with the EnVisionTM FLEX Detection Kit

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(Agilent Technologies-DAKO). Sections were counterstained with hematoxylin.

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Appropriate negative controls including no primary antibody were also tested.

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The human EC cell line Hec1A (ATCC® HTB-122™) and the derivative cell line

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overexpressing ETV5 transcription factor (Hec1A-ETV5) were cultured in McCoy’s 5A

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media (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) FBS and 1% (v/v)

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PenStrep (100 U/mL penicillin - 100 µg/mL streptomycin; Gibco). Cells were maintained

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in humidified atmosphere at 37°C and 5% CO2. ETV5 transfected cells (EX-F080000-

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Lv105, NM_004454; GeneCopoieia, Rockville, MD, USA) were selected in 500 µg/mL

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G-418 (Gibco). This cell line has been previously generated and thoroughly

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characterized.14,15

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2.2.

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in supplemented media until 70% confluence, washed 3 times in PBS and cultured in

EV production and collection. Hec1A and Hec1A-ETV5 cell lines were grown



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FBS-free McCoy’s 5A to produce the EVs. After 48h, small EVs were harvested (cell

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viability at collection >95%) by differential centrifugation as referred by Théry et al.16

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Briefly, conditioned medium was initially cleared of floating (300x g, 10 min) and dead

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cells (2,500x g, 15 min, 4°C) at low speed centrifugation and immediately cleared of cell

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debris and large EVs (10,000x g, 30 min, 4°C, k-Factor: 2567.7) in a SW32Ti swinging

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rotor (Beckman Coulter, Brea, CA, USA). Small vesicles were then collected at high-

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speed ultracentrifugation (100,000x g, 90 min, 4°C, k-Factor: 256.8), washed in 0.22 µm

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Ø-filtered PBS (100,000x g, 90 min, 4°C, k-Factor: 256.8) and resuspended in the

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appropriate buffer. Alternatively, circulating small EVs were purified from human plasma

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by drawing after tourniquet with a 21-gauge needle and 15 mL of peripheral blood

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collected and immediately cleared of blood cells (1,250x g, 15 min, no brake); 12 mL of

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buffer solution (Ca2+ and Mg2+ depleted PBS, 0.1% BSA, 2 mM EDTA) were then added

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and plasma further cleared of cell debris (2,500x g, 15 min, 4°C), and frozen at -80°C

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until EVs purification. When needed, plasma samples were thawed at 4°C, cleared of

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large EVs (30 min, 4°C, k-Factor: 2567.7) and small EVs harvested at 100,000x g (90

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min, 4°C, k-Factor: 256.8). Small EVs used for MRM development were resuspended in

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6M urea/2M thiourea buffer. For retrospective analyses, plasma was processed as referred

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and pelleted EVs directly resuspended in AllPrep® DNA/ RNA/ Protein Mini lysis buffer

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(Qiagen, Hilden, Germany).

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2.3.

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EV batches were routinely assessed by Dynamic Light Scattering (DLS) in a ZetaSizer

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Nano ZS bioanalyzer (Malvern, Worcestershire, UK). 1:10 diluted EVs were measured

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(25°C, 5 runs of 10 seconds per sample, backscattering 173°, attenuator 8) in disposable

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cuvettes. PSD and count rate (kilo counts per second, kcps) are represented as the mean

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value of 3 replicates. Alternatively, EV size and shape were visualized by transmission

Characterization of EVs. Number and Particle Size Distribution (PSD) among


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electron microscopy (TEM, JEOL JEM-2010, 120 KV; Tokyo, Japan). To do so, fresh

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pelleted vesicles were directly deposited onto formvar-coated grids, washed, air dried and

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stained with phosphotungstic acid (5 min, pH 6.8) prior imaging.

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Immunoblot analysis of EVs was performed by direct lysis (25 mM Tris-HCl, pH 7.6,

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150 mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS) of EVs in presence of protease inhibitors

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(4 mM NaF, 2mM NaSOV4, 1x Sigma Protease Inhibitor Cocktail and 1 mM

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phenylmethanesulfonyl fluoride (PMSF)). Protein extracts were quantified by BCA assay

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and 10 µg of protein resolved in SDS-PAGE and probed with the indicated antibodies.

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Antibodies used were CD9 (sc-13118; Santa Cruz, Dallas, TX, USA), CD81 (sc-7637;

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Santa Cruz), PDCD6IP (NB-100-65678; Novus Biologicals, Littleton, CO, USA) and

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HSP90B1 (NBP1-04346; Novus Biologicals).

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2.4. Stable isotope labeling with amino acids in cell culture (SILAC). Hec1A and

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Hec1A-ETV5 cells were cultured in SILAC-DMEM 4.5 g/L glucose deficient in arginine

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(Arg) and lysine (Lys), supplemented with 2 mM L-glutamine (Ref. 280001200), 10%

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dialyzed FBS (dFBS; Ref. 281000800) and 1% PenStrep. Labeling media was

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supplemented with 28 mg/L L-arginine-HCl-13C6 (Arg+6; Ref. 201203902) and 73 mg/L

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L-lysine-HCl-4,4,5,5-D4 (Lys+4; Ref. 211103913) (middle); or L-arginine-HCl-13C615N4

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(Arg+10; Ref. 201603902) and L-lysine

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(Silantes, Munich, Germany). Cells were subcultured until homogeneous labeling (>12

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cell doubling accumulated); normal cellular growth under labeling conditions, and

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complete substitution of the naturally light isotopes (labeling efficiency near 100%), were

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assessed (data not shown). For LC-MALDI-MS analysis of the samples, EV protein

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lysates from both cell lines were mixed in 1:1 (w/w). One replicate of forward (Hec1A

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middle / Hec1A-ETV5 heavy) and reverse labeling (Hec1A heavy / Hec1A-ETV5

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middle), combining technical and biological replicates due to the limited recovery of

13C 15N 6 2

(Lys+8; Ref. 211603902) (heavy)



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sample, were approached. Protein was resolved in 10% SDS-PAGE, stained with 0.1%

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Coomassie G-250 (Thermo Scientific, Waltham, MA, USA) and protein lanes cut into 1

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mm3 pieces. Tryptic digestion and extraction were performed as described by Shevchenko

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et al.17 Briefly, two gel sections were de-stained (50% acetonitrile (ACN) / 50% 25 mM

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NH4HCO3), reduced with 10 mM DTT (45 min, 56°C), alkylated with 50 mM

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iodoacetamide (IAA, 15 min in dark) and in-gel digested with 6 ng/µL sequencing grade

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modified trypsin (16h, 37°C; Promega, Madison, WI, USA). Peptides were then extracted

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(50% ACN, 0.2% TFA), desalted, concentrated in a nu-tipC18 column (Glygen,

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Columbia, MA, USA) and eluted using 70% ACN, 0.1% TFA. Finally, samples were

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dried and resuspended in 7 µL 2% ACN, 0.1% TFA.

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2.5. NanoLC-MALDI-TOF/TOF. Peptide fractions were resolved using RP-LC in a

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Tempo nanoLC system (Eksigent, Dublin, CA, USA). Peptide mixture was injected on a

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C18 precolumn (Michrom, 0.5x2 mm) coupled to a reversed-phase column (Integrafit

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C18, Proteopep II, 75 µm id, 10.2 cm, 5 µm, 300 Å pore size; New Objective, Woburn,

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MA, USA), eluted in a linear gradient of 5-50% ACN (0.35 µL/min, 45 min) in α-cyano

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matrix (4 mg/mL, 1.2 µL/min), and deposited onto a MALDI plate (Sun Collect;

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Sunchrom, Friedrichsdorf, Germany). Chromatograms corresponding to each gel section

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were composed of 180 spots. MS runs for each chromatogram were acquired and

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analyzed in a MALDI-TOF/TOF instrument (4800 Sciex; Framingham, MA, USA) as

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described by Mateos et al.18 A total of 1,500 laser shots were accumulated for each TOF-

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MS spectrum at fixed laser setting (3,600 kV). Selection of precursors was approached

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by Job-Wide interpretation method and peptide fragmentation approached with a laser

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intensity of 4,400 kV and 2,000 shots per spectrum.

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Paragon™ algorithm (ProteinPilot 4.5; Sciex) was used for protein identification by

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searching in the UniProtKB/Swiss-Prot database 2015_05 (http://www.expasy.ch/sprot;


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547,599 sequences, 195,014,757 residues, Homo sapiens). Alternatively, hits were

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contrasted with UniProt_all database for the estimation of contaminants. Search

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parameters included SILAC, IAA alkylation, in-gel trypsin digestion and urea

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denaturalization as specific factors. Proteins identified with ≥2 distinct peptides with 95%

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confidence and a ProtScore ≥1.3 were considered for relative quantification. Exosome

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datasets were manually inspected for proteins identified with one single peptide and

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normalized against 5%-trimmed mean. Finally, abnormal extreme quantifications are

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discarded, and differences were considered significant when mean log2 absolute protein

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ratio ≥ 1.18 and p value ≤0.05 according to a two-tailed t-test.

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2.6. Patient recruitment. Subjects were recruited from June 2013 to May 2016 in Vall

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d’Hebron University Hospital (Barcelona, Spain), University Hospital of Santiago de

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Compostela (Santiago de Compostela, Spain), Arnau de Vilanova Hospital (Lleida,

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Spain) and MD-Anderson Cancer Center Madrid (Madrid, Spain) (supplemental Table

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S1). Samples were collected in EDTA Vacutainer tubes (Becton Dickinson, Franklin

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Lakes, NJ, USA) and processed in less than 48 hours. All participants provided informed

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consent according to the Institutional Ethical Committee (Galicia, Spain). Circulating

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exosomes from age-matched healthy women were included as controls.

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2.7. Multiple Reaction Monitoring of exosome biomarkers. Circulating EVs were

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isolated from human plasma samples as previously described. Protein digestion and

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peptide clean-up was approached as described by Fernandez-Puente et al.19 Briefly,

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protein was reduced in 10 mM DTT, alkylated in 50 mM IAA and in-solution digested

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with trypsin (0.5 µg, 16h, 37°C). Samples were then desalted in C18 stage tips and

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reconstituted in 25 µL of mobile phase buffer A (0.1% formic acid / 2% ACN) for MRM

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development analysis and in a mixture of Stable Isotope-labeled Standard (SIS) peptides

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(7 µL) for MRM validation. SIS or heavy peptides incorporated a fully atom labeled 13C



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and 15N isotope at the C-terminal lysine, (13C6, 15N2-Lys) (K) or arginine (13C6, 15N4-Arg)

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(R) position of each (tryptic) peptide, resulting in a mass shift of +8 or +10 Da

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respectively. SIS peptides (crude purity) were acquired from JPT (JPT Peptide

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Technologies GmnH, Berlin, Germany).

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Protein digests with SIS peptides were analyzed by LC-MS/MS in a nanoLC system

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(TEMPO) coupled to a 5500-QTRAP instrument (Sciex). A label-free EM ER analysis

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(Enhanced Mass / Enhanced Resolution) was performed in EV samples to detect target

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peptides. Initial transitions for each peptide included, MS/MS spectra from the EM ER

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analysis, peptides previously identified by MALDI-TOF/TOF in shotgun experiments

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and sequences frequently observed in the open-source Global Proteome Machine

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Database.20 This analysis involved performing an information-dependent LC/MS/MS

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experiment on the EV extract and searching the acquired data against protein databases

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by Protein Pilot software 4.5 (Sciex). Several candidate peptide precursors and fragment

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ion masses were selected per each protein and analyzed with Skyline 1.3 software

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(MacCoss, Seattle, WA, USA).21 Results were pooled and an optimized set of MRM

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transitions covering a total of 4 and 3 peptides for ANXA2 and LGALS3BP respectively

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were acquired (supplemental Table S3). Mass spectrometer was interfaced with a nano-

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spray source equipped with an uncoated fused silica emitter tip (20 μm inner diameter,

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10 μm tip; New Objective, Woburn, MA) and operated in positive ion mode. MS source

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parameters were set as follows: IS 2,600 V, IHT 150°C, GS2= 0, CUR= 20, GS1= 25 psi

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and high CAD. MS compound parameters were set to 10 for the EP and to 15 for CXP.

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Skyline was used to predict and optimize collision energies for each peptide, both analyte

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and SIS. Q1 and Q3 were set to unit/unit resolution (0.7 Da) and pause between mass

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ranges set to 3 ms. To confirm the identity of the peptides, a MRM Information Dependent

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Acquisition (IDA) experiment was performed for each peptide. Mass spectrometer was


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instructed to switch from MRM to EPI scanning mode when an individual MRM signal

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exceeded 1,000 counts. Each precursor was fragmented a maximum of twice before being

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excluded for 10s and the masses were scanned from 250 to 1,000 Da. The rolling collision

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energy (CE) option was employed to automatically ramp up the CE value in the collision

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cell as the m/z value increased. One replicate was analyzed for each sample. SIS were

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used for relative quantification using one-point calibration to determine basically fold

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changes between the different cancer samples compared to control donors. The

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concentration of the SIS stock solution was 10 fmol/µL for all the peptides except for the

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ASHEEVEGLVEK (LGALS3BP) that was 1 pmol/µL due to the low signal of the peptide

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compared to the other two LGALS3BP targeted peptides. A blank solvent injection was

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run between each sample injection to avoid carry over in the nanoLC-MRM system. The

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linearity, LOD and LOQ were not performed due to the low concentration of the exosome

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samples matrix. The MRM method was developed for research use, which aligns this

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work with a Tier 2 Targeted Measurement as described.22

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Data analysis for the target proteins was performed using Skyline for method refinement,

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optimization and peak integration. Raw files were imported to Skyline and the

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chromatograms were manually inspected to ensure correct peak detection and accurate

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integration. Co-elution of all the transitions of the target or light peptide with the SIS

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peptide was considered accurate MRM signal. Peak area ratios between light and heavy

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peptide was used to calculate the relative peak area ratio of each peptide in the conditions

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of the study. Reports of peak area ratios between the light and heavy peptides of each

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peptide in the serum samples were exported by Skyline to MS Excel. The % CV were

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calculated for the RT of the targeted proteins. The peptide RT observed for LGALS3BP

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and ANXA2 was very reproducible with a %CV

Proteomic characterization of epithelial-like extracellular vesicles in

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