Circulating melanoma exosomes as diagnostic and prognosis biomarkers

doi:10.1016/j.cca.2015.12.031

Clinica Chimica Acta

Volume 454, 15 February 2016, Pages 28-32

https://doi.org/10.1016/j.cca.2015.12.031 Get rights and content

Highlights

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MIA and S100B can be easily detected in exosomes using routine techniques.
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Their levels in exosomes and serum are higher in stage IV melanoma patients.
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MIA and S100B analysis in exosomes have high sensitivity and specificity.
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Higher MIA levels in exosomes were associated with shorter survival.

Abstract

Background

Malignant melanoma is an aggressive cancer with an increasing incidence. Exosomes are actively secreted microvesicles, whose characteristics reflect 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 protein 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 quantified in serum, exosomes, and exosome-free serum.

Results

Both MIA and S100B were detected in exosomes and correlated significantly with serum concentrations (S100B: r = 0.968; MIA: r = 0.799; p < 0.001). MIA and S100B concentrations in exosomes were significantly higher in melanoma patients than in healthy controls and disease-free patients. However, TYRP2 concentrations in exosomes did not differ between these three groups. ROC curves analysis rendered AUCs for MIA of 0.883 (p < 0.01) and of 0.840 for S100B (p < 0.01). Patients with exosome MIA concentration higher than 2.5 μg/L showed shorter median survival related to those with lower level (4 versus 11 months; p < 0.05).

Conclusions

MIA and S100B can be detected in exosomes from melanoma patients and their quantification presents diagnostic and prognostic utility.

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 advanced melanoma [1], [2]. MIA is a small soluble protein of 11 kDa secreted by malignant melanoma cells. S100 is a 21 kDa dimeric protein composed of 2 subunits, α or β, being the αβ heterodimer expressed by melanoma cells [3]. LDH concentrations higher than reference range can classify patients with metastatic melanoma in a more advanced stage (M1c) [4]. Both MIA and S100B serum concentrations are elevated in advanced melanoma and their measurement can be useful as prognostic factors and to monitor the disease in stages III and IV [1], [5]. However they show some limitations of specificity 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 inhibitors [8], [9]. Exosomes are actively secreted microvesicles derived from the cellular endosomal membrane with sizes ranging from 30 to 200 nm [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 result, exosomes are now considered to play a pivotal role in tumor development and progression [17].

The composition of nucleic acids and proteins cargo of exosomes reflects the cells they originate from [18]. In fact, Peinado et al. [17] found that exosomes isolated from stage IV melanoma patients characteristically contain the proteins tyrosinase-related protein 2 (dopachrome tautomerase, TYRP2), Very Late Antigen 4, Heat Shock Protein 70, HSP90 isoform, and MET oncoprotein [17]. In addition, melanoma exosomes seem to play a role in the metastasis to lymph nodes. Particularly, TYRP2 expression in exosomes has been associated with metastatic progression in advanced melanoma [17].

Tumor-derived exosomes have been detected in several biological fluids and can carry tumor specific antigens, such as carcinoembryogenic antigen in ascitic exosomes from colon carcinoma [19], prostate specific antigen in urinary exosomes from prostate cancer [20], or CA125 in ascitic exosomes from ovarian carcinoma [21]. The expression of tumor-specific antigens by exosomes can render these particles useful for cancer diagnosis 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.

Section snippets

Sample collection

Peripheral blood samples were collected after obtaining informed consent from 53 advanced melanoma patients (mean age: 55 years; males: 54%), 18 melanoma disease-free patients (mean age: 54 years; males: 43%) and from 25 healthy volunteers (mean age: 41 years; males: 29%) used as age and sex matched control groups. Blood samples were centrifuged and serum was isolated and kept at − 80 °C until analysis. The study was approved by our institution's Ethical Review Board.

M8 and UMBY melanoma cell lines

Identification of serum exosomes

The mean size of the microvesicles obtained by precipitation with ExoQuick and by ultracentrifugation was lower than 200 nm, similar to that described for exosomes [10] (Fig. 1A). Exosomes isolated from plasma were characterized by Western blot using anti-CD63 and, as expected, we observed the predicted band at 53 kDa (Fig. 1B). In conclusion, we could identify these microvesicles obtained by precipitation as exosomes.

The concentration of exosomes isolated using the ExoQuick reagent was estimated

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 S100 family have been identified in most proteomic analysis of exosomes. S100A1, S100A6 and S100A11, which can interact with S100B forming heterodimers [23], have been particularly detected [24]. Furthermore, we could also detect the

Acknowledgments

Authors declare no conflict 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.

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View full text

Circulating melanoma exosomes as diagnostic and prognosis biomarkers

Highlights

Abstract

Background

Methods

Results

Conclusions

Introduction

Section snippets

Sample collection

Identification of serum exosomes

Discussion

Acknowledgments

Adv. Clin. Chem.

Mol. Ther.

J. Biol. Chem.

Clin. Chim. Acta

Clin. Biochem.

Evaluation of multiple serum markers in advanced melanoma

Tumour Biol.

Protein and non-protein biomarkers in melanoma: a critical update

Amino Acids

S100-Beta, melanoma-inhibiting activity, and lactate dehydrogenase discriminate progressive from nonprogressive American Joint Committee on Cancer stage IV melanoma

J. Clin. Oncol.

S-100β and MIA in advanced melanoma in relation to prognostic factors

Anticancer Res.

Prospective monitoring of adjuvant treatment in high-risk melanoma patients: lactate dehydrogenase and protein S-100B as indicators of relapse

Melanoma Res.

S-100B: a stronger prognostic biomarker than LDH in stage IIIB–C melanoma

Ann. Surg. Oncol.

Quantitative cell-free circulating BRAFV600E mutation analysis by use of droplet digital PCR in the follow-up of patients with melanoma being treated with BRAF inhibitors

Clin. Chem.

BRAF mutation analysis in circulating free tumor DNA of melanoma patients treated with BRAF inhibitors

Melanoma Res.

Membrane vesicles as conveyors of immune responses

Nat. Rev. Immunol.

Caveolin-1 tumor-promoting role in human melanoma

Int. J. Cancer

Identifying mRNA, microRNA and protein profiles of melanoma exosomes

PLoS ONE

Tumor-released microvesicles as vehicles of immunosuppression

Cancer Res.