Circulating melanoma exosomes as diagnostic and prognosis biomarkers

Circulating melanoma exosomes as diagnostic and prognosis biomarkers

Estibaliz Alegre a ,Leyre Zubiri b ,Jose Luis Perez-Gracia b ,María González-Cao c ,Lourdes Soria b ,Salvador Martín-Algarra b ,Alvaro González a ,?

a Laboratory of Biochemistry,University Clinic of Navarra,Spain

b Department of Medical Oncology,University Clini

c of Navarra,Spain

c

Translational Cancer Research Unit,Instituto Oncológico Dr Rosell,Quirón Dexeus University Hospital,Barcelona,Spain

a b s t r a c t

a r t i c l e i n f o Article history:

Received 16November 2015

Received in revised form 11December 2015Accepted 21December 2015

Available online 24December 2015Background:Malignant melanoma is an aggressive cancer with an increasing incidence.Exosomes are actively se-creted microvesicles,whose characteristics re ?ect those of the cell they are originated in.The aim of this study was to identify and evaluate the presence of the melanoma biomarkers MIA,S100B and tyrosinase-related pro-tein 2(TYRP2)in exosomes and their potential clinical utility.

Methods:Serum samples were obtained from stage IV melanoma patients,melanoma-free patients and healthy controls.Exosomes were precipitated and TYRP2,MIA and S100B concentrations were quanti ?ed in serum,exosomes,and exosome-free serum.

Results:Both MIA and S100B were detected in exosomes and correlated signi ?cantly with serum concentrations (S100B:r =0.968;MIA:r =0.799;p b 0.001).MIA and S100B concentrations in exosomes were signi ?cantly higher in melanoma patients than in healthy controls and disease-free patients.However,TYRP2concentrations in exosomes did not differ between these three groups.ROC curves analysis rendered AUCs for MIA of 0.883(p b 0.01)and of 0.840for S100B (p b 0.01).Patients with exosome MIA concentration higher than 2.5μg/L showed shorter median survival related to those with lower level (4versus 11months;p b 0.05).

Conclusions:MIA and S100B can be detected in exosomes from melanoma patients and their quanti ?cation pre-sents diagnostic and prognostic utility.

?2015Elsevier B.V.All rights reserved.

Keywords:

Melanoma inhibitory activity S100B Exosomes Melanoma

1.Introduction

Metastatic melanoma is a very aggressive cancer whose incidence is increasing worldwide.The prognosis is generally poor,although new treatments have improved recently overall survival.S100B,Melanoma Inhibitory Activity (MIA)and Lactate Dehydrogenase (LDH)are the most widely used tumor markers for prognosis and follow-up in ad-vanced melanoma [1,2].MIA is a small soluble protein of 11kDa secret-ed by malignant melanoma cells.S100is a 21kDa dimeric protein composed of 2subunits,αor β,being the αβheterodimer expressed by melanoma cells [3].LDH concentrations higher than reference range can classify patients with metastatic melanoma in a more ad-vanced stage (M1c)[4].Both MIA and S100B serum concentrations are elevated in advanced melanoma and their measurement can be use-ful as prognostic factors and to monitor the disease in stages III and IV

[1,5].However they show some limitations of speci ?city and sensitivity,especially LDH and therefore,they are not widely used [2,6,7].

Recently,other biomarkers have been investigated,such as cell-free nucleic acids and exosomes.BRAF mutations in cell-free DNA have also shown to be useful to monitor melanoma patients treated with BRAF in-hibitors [8,9].Exosomes are actively secreted microvesicles derived from the cellular endosomal membrane with sizes ranging from 30to 200nm [10].Cancer cells,and particularly melanoma cells,can release large quantities of exosomes [11],in contrast to normal melanocytes [12].These exosomes derived from cancer cells participate in tumor progression with immune-suppressive functions [13],contributing to angiogenesis [14],drug resistance [15]and cell migration [16].As a re-sult,exosomes are now considered to play a pivotal role in tumor devel-opment and progression [17].

The composition of nucleic acids and proteins cargo of exosomes re-?ects the cells they originate from [18].In fact,Peinado et al.[17]found that exosomes isolated from stage IV melanoma patients characteristi-cally contain the proteins tyrosinase-related protein 2(dopachrome tautomerase,TYRP2),Very Late Antigen 4,Heat Shock Protein 70,HSP90isoform,and MET oncoprotein [17].In addition,melanoma exosomes seem to play a role in the metastasis to lymph nodes.Partic-ularly,TYRP2expression in exosomes has been associated with meta-static progression in advanced melanoma [17].

Clinica Chimica Acta 454(2016)28–32

Abbreviations:AUC,Area Under Curve;LDH,Lactate Dehydrogenase;MIA,Melanoma Inhibitory Activity;ROC,Receiver Operating Characteristics;TYRP2,tyrosinase-related protein 2,Dopachrome tautomerase.

?Corresponding author at:Laboratory of Biochemistry,University Clinic of Navarra,Av.Pio XII 36,Pamplona 31008,Spain.

E-mail address:agonzaleh@unav.es (A.

González).

https://www.360docs.net/doc/9214082795.html,/10.1016/https://www.360docs.net/doc/9214082795.html,a.2015.12.031

0009-8981/?2015Elsevier B.V.All rights

reserved.

Contents lists available at ScienceDirect

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Tumor-derived exosomes have been detected in several biological ?uids and can carry tumor speci?c antigens,such as carcinoembryogenic antigen in ascitic exosomes from colon carcinoma[19],prostate speci?c antigen in urinary exosomes from prostate cancer[20],or CA125in ascitic exosomes from ovarian carcinoma[21].The expression of tumor-speci?c antigens by exosomes can render these particles useful for cancer diagno-sis and monitoring.For this reason,the aim of the present work was to study the presence of melanoma biomarkers S100B and MIA in exosomes and to compare its utility with their determination in serum.

2.Material&methods

2.1.Sample collection

Peripheral blood samples were collected after obtaining informed consent from53advanced melanoma patients(mean age:55years; males:54%),18melanoma disease-free patients(mean age:54years; males:43%)and from25healthy volunteers(mean age:41years; males:29%)used as age and sex matched control groups.Blood samples were centrifuged and serum was isolated and kept at?80°C until anal-ysis.The study was approved by our institution's Ethical Review Board.

M8and UMBY melanoma cell lines were cultured in RPMI1640me-dium supplemented with2mmol/L L-glutamine,10%exosome-free heat inactivated fetal bovine serum,penicillin G100U/mL,geniticin 1mg/mL,streptomycin100μg/mL(Gibco,Grand Island,NY,USA),and were cultured in a37°C,5%CO2humidi?ed incubator.

2.2.Exosome isolation

Exosomes were obtained from serum with ExoQuick precipitation solution(System Biosciences,Mountain View,CA,USA)according to manufacturer's instructions.Brie?y,250μL,of serum were mixed with 63μL of ExoQuick?solution and incubated for1h at4°C.After centri-fugation at1500g for30min,pellets were suspended in100–200μL of PBS or in lysis buffer.

Exosomes from cell cultures were collected by ultracentrifugation at 110,000g for2h at4°C after precleaning the media by two successive centrifugations and?ltration through a0.22μm?lter(EMD Millipore, Billerica,MA,USA).Pellets were resuspended in PBS or in lysis buffer.

2.3.Exosome size analysis

Isolated exosomes were diluted in PBS and particle size was ana-lyzed using Malvern Zetasizer Nano-ZS(Malvern Instruments,UK) and its corresponding software(Zetasizer Ver.7.03).

2.4.Protein concentration

Protein concentration in exosomes was estimated through absor-bance at280nm with a Nanodrop spectrophotometer(Thermo Fisher Scienti?c,Wilmington,USA).A correction factor was introduced to cor-rect sample volume reduction from250to200μL.

2.5.Western blot

Proteins(20μg)were denatured at100°C for5min in loading buffer containing125mM Tris(pH 6.8),4%SDS,30%glycerol,5%β-mercaptoethanol and0.4%bromophenol.Proteins were subjected to 10%polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE),with subsequent electroblotting transfer onto a nitrocellu-lose membrane.The membrane was blocked with5%skimmed milk in PBS-Tween0.1%during30min at room temperature,and then incubat-ed during1h with anti-CD63antibody(Santa Cruz Biotechnology, Texas,USA)diluted1:1000in PBS-Tween0.1%.Immunoblot analysis was performed using an HRP-conjugated anti-mouse antibody (1:5000;Amersham Biosciences,Uppsala,Sweden),and developed using the ECL kit(Amersham Biosciences).

2.6.Immuno assays

MIA was determined by a manual commercial enzyme-linked im-munosorbent assay(Roche,Mannheim,Germany)and S100B was ana-lyzed by an electrochemiluminescence assay automatized in an e602 Module from a C8000(Roche Diagnostics,Mannheim,Germany)[1]. Tyrosinase-related protein2concentration was measured using a com-mercial ELISA kit(Cloud-Clone Corp,Houston,USA).This is a sandwich enzyme immunoassay that uses two antibodies speci?c for TYRP2.A correction factor was introduced to correct sample volume reduction.

The concentrations of biomarker in exosome-free serum were calcu-lated by subtracting the measured concentration in exosomes to the serum concentration.

2.7.Statistical analysis

Concentrations were expressed as median and25th–75th percentile after determining their non-Gaussian distribution with the Kolmogo-rov–Smirnov and Shapiro–Wilk's tests.The non-parametric Kruskal–Wallis test and Dunn's multiple comparison test were applied to com-pare the levels of the tumor markers.Correlation analysis was per-formed using the Pearson correlation test.Overall survival was measured from the time of blood drawing to death or last follow-up and it was analyzed by the Kaplan–Meier method and compared by the Gehan–Breslow–Wicoxon test.Receiver operating characteristic (ROC)curves were constructed to determine the sensitivity and speci-?city of the assays.A two-tailed p-value b0.05was considered to be sta-tistically signi?cant.Statistical analysis was performed with GraphPad Prism version6.07(La Jolla,CA,USA).

3.Results

3.1.Identi?cation of serum exosomes

The mean size of the microvesicles obtained by precipitation with ExoQuick and by ultracentrifugation was lower than200nm,similar to that described for exosomes[10](Fig.1A).Exosomes isolated from plasma were characterized by Western blot using anti-CD63and,as ex-pected,we observed the predicted band at53kDa(Fig.1B).In conclu-sion,we could identify these microvesicles obtained by precipitation as exosomes.

The concentration of exosomes isolated using the ExoQuick reagent was estimated by measuring the protein concentration.The median concentration of proteins in exosomes from melanoma patients was 8μg/L(Q1–Q3:6–10μg/L),very similar to the control group(median: 9μg/L;Q1–Q3:8–10μg/L).These results indicate that patients with ad-vanced melanoma do not show signi?cant differences in serum exosome levels,as compared to melanoma-free patients and healthy controls.

3.2.Biomarkers analysis in exosomes from melanoma patients

S100B and MIA were measured in serum(srm-S100B and srm-MIA respectively),exosomes(exo-S100B and exo-MIA respectively)and exosome-free serum(sn-S100B and sn-MIA respectively)(Table1). There was a signi?cant correlation between biomarker concentrations in exosomes and serum(S100B:r=0.968;MIA:r=0.799;p b0.001 in both cases)(Fig.2).However,while there was a good correlation of S100B concentration between exosomes and supernatant(r=0.943; p b0.001)this association was very poor in the case of MIA(r= 0.189;p=0.138).

As expected,srm-S100B and srm-MIA concentrations were higher in stage IV melanoma patients compared to melanoma free patients and

29

E.Alegre et al./Clinica Chimica Acta454(2016)28–32

healthy controls.S100B and MIA levels in exosomes were also statisti-cally higher in advanced melanoma patients,as compared to both healthy controls and melanoma free-patients (p b 0.01in both cases,Fig.3,Table 1).

To know if there was a change in the secretion of these biomarkers in exosomes,we studied the percentage of S100B or MIA concentrations in exosomes in relation to their serum levels.We observed a signi ?cant decrease in percentage of S100B in exosome from patients in stage IV (median stage IV:17%,Q1–Q3:12%–21%)compared to controls and dis-ease free patients (median control:23%,Q1–Q3:20%–26%;median dis-ease free:24%,Q1–Q3:18%–29%;p b 0.05related to stage IV)(Fig.3).This difference was not observed when MIA was analyzed (median con-trol:18%,Q1–Q3:6%–23%;median disease free:17%,Q1–Q3:14%–26%;median stage IV:17%,Q1–Q3:12%–28%).

Finally,we analyzed the potential utility of TYRP2concentrations in exosomes.There was a signi ?cant correlation in its levels between serum and exosomes (r =0.941;p b 0.01).The percentage of TYRP2in exosomes (median:61%;Q1–Q3:54%–68%;p b 0.01)was signi ?cant-ly higher than the percentage of MIA in exosomes (median:17%;Q1–Q3:11%–26%)or the percentage of S100B in exosomes (median:21%;

Q1–Q3:14%–25%).However,contrary to what occurs with S100B and MIA,we did not detect differences in either serum or exosome levels be-tween control and melanoma patients (Table 1).

These results show that both S100B and MIA concentrations in exosomes from melanoma patients are higher than in healthy controls and disease free patients.

3.3.Diagnostic and prognostic value of S100B and MIA concentrations in exosomes

Next,we analyzed the sensitivity and speci ?city of exo-MIA and exo-S100B to discriminate advanced melanoma patients from healthy con-trols.The ROC analysis demonstrated an AUC of 0.883(95%CI =0.808–0.959;p b 0.01)for exo-MIA,better than srm-MIA (AUC:0.806;95%CI =0.701–0.903;p b 0.01)(Fig.4).With a cut-off value of exo-MIA set at 1.4μg/L,the sensitivity and the speci ?city was 80%.Also,the ROC for exo-S100B had an AUC of 0.840(95%CI =0.745to 0.936;p b 0.01),better than that observed for srm-S100B (AUC:0.806;95%CI =0.709to 0.902;p b 0.01).With a cutoff value of exo-S100B set at 0.015μg/L,the sensitivity and speci ?city was 80%.

Concentrations of MIA in exosomes above the median value were as-sociated with shorter survival (Fig.5).Patients with exo-MIA levels equal or higher than 2.5μg/L showed a median survival of 4months,while patients with lower concentrations presented a median survival of 11months (p b 0.05).Also,patients with exo-S100B concentrations higher than 0.03μg/L showed a median survival of 7months,while pa-tients with lower levels had a median survival of 10months (p =0.296).4.Discussion

We have shown that both S100B and MIA are detected in exosomes obtained from melanoma patients.The method used here to measure S100B employs monoclonal antibodies directed against the β-chain,so it can detect both the AB-and BB-dimers [22].Other proteins of the S100family have been identi ?ed in most proteomic analysis

of

Fig.1.A:Representative experiment (n =4)of size distribution of exosomes obtained from serum and from M8melanoma cell line.B:Representative experiment (n =5)of Western blot incubated with anti-CD63of serum exosomes and of the whole serum.

Table 1

Serum (srm-),exosome (exo-)and exosome-free serum (sn-)levels of MIA,S100B and TYRP2in healthy controls,melanoma-free patient s,and stage IV melanoma patients.Data are expressed as median and interquartile range.P b 0.05.

Controls

Disease free Stage IV

srm-MIA (μg/L)6(5–7) 5.2(3.9–6.6)14(7–45)a ,b exo-MIA (μg/L)1(0.4–1.5)1(0.9–1.7) 2.5(1.5–7.2)a ,b sn-MIA (μg/L) 4.9(4–6)

4.1(3–5)

10(5–33)a ,b

srm-S100B (μg/L)0.06(0.05–0.07)0.04(0.03–0.07)0.22(0.07–0.67)a ,b exo-S100B (μg/L)0.01(0.01–0.02)0.01(0.0–0.02)0.04(0.02–0.09)a ,b sn-S100B (μg/L)0.04(0.04–0.05)0.03(0.02–0.06)0.23(0.06–0.60)a ,b srm-TYRP2(μg/L)13(11–16)12(6–22)9(8–22)exo-TYRP2(μg/L)8(7–9)9(5–11)6(5–11)sn-TYRP2(μg/L)

5(4–7)

3(1–10)

3(2–11)

a Related to control.

b

Related to disease free

patients.

Fig.2.Relationship between MIA (A)and S100B (B)concentrations in exosomes (exo-)and either serum (srm-)or exosome-free supernatant (sn-).R2coef ?cient is indicated for each relationship and p b 0.05was considered signi ?cant.

30 E.Alegre et al./Clinica Chimica Acta 454(2016)28–32

exosomes.S100A1,S100A6and S100A11,which can interact with S100B forming heterodimers [23],have been particularly detected [24].Furthermore,we could also detect the melanoma biomarker S100B by ELISA in exosomes from M8and UMBY melanoma cell lines (data not shown).To our knowledge,this is the ?rst report in which S100B and MIA have been documented in exosomes.However,the pro-portion of S100B in exosomes decreased in stage IV patients which can be due to S100B released by melanoma cells mainly by necrosis and ap-optosis and not due to overexpression or active secretion [25].This was not observed in the case of MIA.MIA is a secreted protein that binds to integrins,particularly α4β1and α5β1[26].These integrins and others are present in exosomes and MIA could be bound to these proteins.Both S100B and MIA,which are well known biomarkers in melanoma [1],can be used to identify exosomes released by melanoma cells.

We have observed that the sensitivity and speci ?city for melanoma of the S100B or MIA assays in exosomes are superior to their correspon-dent analysis in serum.By measuring S100B and MIA in exosomes,in-terferences from other sources can be avoided.With this approach we were able to detect only those proteins selectively delivered,and we ex-cluded those proteins released by cell death,as they are not included in exosomes.Also,apoptotic bodies,debris and other microvesicles that could contain these proteins are usually in the range of 1μm [27],so

they are eliminated in the process of exosome isolation.For this reason,the quanti ?cation of these molecules in exosomes could increase the speci ?city of the biomarker,at the same time that it could permit to identify those exosomes derived from the tumor.Other authors have shown that the analysis of speci ?c tumor proteins in exosomes could improve the sensitivity and speci ?city to detect cancer patients.Peng et al.detected CA125located on the surface of exosomes,which allowed one to identify the exosomes from tumor cells in the ascites of ovarian cancer patients [21].Also,recently,it was shown that Glypican-1in cir-culating serum exosomes can differentiate patients with pancreatic can-cer from healthy subjects or patients with a benign pancreatic disease with absolute speci ?city and sensitivity [28].However,further studies in benign conditions other than melanoma where MIA [29]or S100B [30]are elevated,should be performed [2].As we and others have pre-viously shown in case of serum [1,5,31],it seems that not only does MIA present improved sensitivity and speci ?city in melanoma patients,but also has prognostic value,since advanced stage patients with exo-MIA concentrations higher than 2.5μg/L presented shorter overall survival than patients with lower levels.

Previous authors have shown increased concentrations of the house-keeping exosome proteins CD63and Rab-5b in melanoma patients as compared with healthy controls [32].We have not detected differences in exosome levels between melanoma patients and healthy controls.Probably these discordances could be explained by the different methods used to obtain and measure exosomes.Most other studies an-alyzing proteomic pro ?le in exosomes employ cumbersome technology,such as ultracentrifugation and mass-spectrometry [12,16,17].Impor-tantly,we have performed this study using not very time-consuming technologies available in most clinical laboratories so our methodology can be easily translated into routine [33].Exosomes from clinical sam-ples can be easily obtained using ExoQuick method [18],a technology that captures these microvesicles in “polymer nets ”[34].Peinado et al.described a “melanoma signature ”that included TYRP2in circulating exosomes from subjects with advanced melanoma [17].Although we have observed that serum TYRP2circulates mainly included into exosomes,we have not detected increased concentrations of this pro-tein in the melanoma exosomes,probably due to the different technol-ogies used to isolate exosomes and to analyze this

protein.

Fig.3.Exosome concentrations (A)and ratio between exosome and serum concentrations in percentage (B)for MIA and S100B in stage IV melanoma patients,melanoma free-patients and healthy

controls.

Fig.4.ROC analysis curves for discriminating between healthy controls and advanced melanoma patients.AUC value is indicated for each biomarker.

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In addition to their utility as a diagnostic or prognostic factor,the analysis of exosomes through MIA and S100B measurement could pro-vide additional biological information,as melanoma-derived exosomes are mediators of tumorigenesis.It has been reported that melanoma exosomes increase the metastatic behavior of primary tumors [17]and promote the generation of suppressive myeloid cells [35].In the same way,tumor antigens in exosomes may be useful to induce thera-peutic immune responses.For instance,exosomes obtained from ad-vanced colorectal cancer patients contain CEA and can be used to induce a potent CEA-speci ?c antitumor immune response [19].

In summary,we have shown that S100B and MIA are present in exosomes and that their determination in these microvesicles could be an alternative to their analysis in serum for the diagnosis and prog-nosis of melanoma patients.Further studies will be necessary to charac-terize their value,and to assess their role to differentiate melanoma from other benign conditions.Acknowledgments

Authors declare no con ?ict of interest.This work was supported by a “Fondo de Investigación Sanitaria ”grant [PI14/00274].We like to thank Dra.María Romero for her support in the preparation of the manuscript and Carmen Rodríguez for her technical assistance.References

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Fig.5.Kaplan –Meier plots representing survival for patients with advanced melanoma according to MIA and S100B levels above or below the median value:MIA:2.5μg/L;S100B:0.03μg/L.

32 E.Alegre et al./Clinica Chimica Acta 454(2016)28–32