text:
IL‐2 modulates Th2 cell responses to glucocorticosteroid: A cause of persistent type 2 inflammation?

Abstract Background Glucocorticosteroids (GCs) are the main treatment for asthma as they reduce type 2 cytokine expression and induce apoptosis. Asthma severity is associated with type 2 inflammation, circulating Th2 cells and higher GC requirements. Objective The aim of this study was to assess whether ex vivo production of interleukin 2 (IL‐2), a T‐cell survival factor, associated with clinical features of asthma severity, the proportion of blood Th2 cells and Th2 cell responses to GC. Methods Peripheral blood from asthma patients (n = 18) was obtained and the proportion of Th2 cells determined by flow cytometry. Peripheral blood cells were activated with mitogen (24 hours) and supernatant levels of IL‐2 and IL‐13 measured by enzyme‐linked immunosorbent assay. In vitro differentiated Th2 cells were treated with dexamethasone (DEX) and IL‐2 and assessed for apoptosis by flow cytometry (annexin V). Level of messenger RNA (mRNA) for antiapoptotic (BCL‐2) and proapoptotic (BIM) genes, IL‐13, GC receptor (GR) and FKBP5 were determined by quantitative real‐time polymerase chain reaction. GR binding was assessed by chromatin immunoprecipitation. Results IL‐2 produced by activated peripheral blood cells correlated negatively with lung function and positively with a daily dose of inhaled GC. When patients were stratified based on IL‐2 level, high IL‐2 producers made more IL‐13 and had a higher proportion of circulating Th2 cells. In vitro, increasing the level of IL‐2 in the culture media was associated with resistance to DEX‐induced apoptosis, with more BCL‐2/less BIM mRNA. Th2 cells cultured in high IL‐2 had more IL‐13, less GR mRNA, showed reduced binding of the GR to FKBP5, a known GC‐induced gene, and required higher concentrations of DEX for cytokine suppression. Conclusions and Clinical Relevance IL‐2 downregulates Th2 cell responses to GC, supporting both their survival and pro‐inflammatory capacity. These results suggest that a patient's potential to produce IL‐2 may be a determinant in asthma severity.

K E Y W O R D S apoptosis, asthma, IL-13, IL-2, steroid, Th2 cells

| INTRODUCTION
Asthma is a syndrome characterized by symptoms of diverse pathogenesis. 1,2 Type 2 cytokines promote many of the processes responsible for the development of asthma and symptom manifestation. Interleukin 4 (IL-4) is essential for helper T cell (Th2) cell differentiation and drives B-cell isotype switching to immunoglobulin E (IgE); IL-5 is a differentiation factor for eosinophils and mediates their egress from the bone marrow during allergic responses; IL-13 mediates airway hyperresponsiveness through inflammatory cell infiltration, smooth muscle contraction, and epithelial secretions 3 . These cytokines are produced by both type 2 CD4 + Th2 cells 4,5 and group 2 innate lymphoid cells (ILC2s). 6 Th2 cells are allergen-specific memory T cells that circulate between the lymph nodes and periphery and as such can rapidly infiltrate tissues upon allergen exposure. [7][8][9] While both cell types contribute to overall type 2 cytokine levels, the immunologic memory provided by Th2 cells is considered to mediate the persistent nature of allergic inflammation. 8,10 Glucocorticoids (GCs) are the main treatment for asthma. 11 Their efficacy is considered, in large part, to be due to their ability to suppress type 2 cytokines, in vivo, 12 ex vivo 13 and in vitro. 14 In most cases, inhaled GC therapy is sufficient to achieve asthma control, though the amount required varies amongst patients. 15 In severe asthma, even high doses of inhaled GC fail to control symptoms and adequately improve poor lung function. 15 Studies show that moderate/severe asthmatics, despite taking high dose inhaled steroid, experienced improved exacerbation rates, lung function, and eosinophilia after using anti-Th2 cytokine therapies, [16][17][18][19] revealing that persistent type 2 inflammation was a factor in their disease severity.
Another anti-inflammatory effect of GC is their ability to induce apoptosis. 20 This has been well demonstrated for primary human eosinophils, 21,22 though for T cells the effects vary based on subset examined. For instance, GCs effectively induce apoptosis of thymocytes, 23 while memory T cells are less sensitive. 24,25 Recently, we and others reported higher levels of Th2 cells in the blood 26 and bronchoalveolar lavage (BAL) of severe compared to nonsevere asthmatics. 27 As such, asthma severity may be related not only to persistent type 2 cytokine expression but also to the inability of GC therapy to eliminate Th2 cells.
IL-2 is a T cell growth and survival factor that promotes differentiation of the memory T cell phenotype. 28 In T-cell lines, IL-2 was shown to interfere with the GC receptor (GR) nuclear translocation, reducing signaling 29 and to inhibit GC-induced apoptosis. 30 Single nucleotide polymorphisms in IL-2 and the IL-2R are associated with asthma severity, 31 suggesting the strength of the IL-2 pathway may regulate GC responsiveness. Despite this work, studies linking IL-2 levels in patients with GC requirements, immune cell profiles and mechanism of action within human Th2 cells have not been examined. Here, we measured IL-2 production from peripheral blood cells and assessed its relationship with clinical features of asthma, type 2 inflammation and the effect of this cytokine on GC responses of human Th2 cells.

| Subjects
This study was approved by the University of Alberta Human Ethics Review Board (approval number PRO1784). All subjects gave informed consent. Subjects were recruited from the tertiary care Asthma Clinic at the University of Alberta during their initial clinic visit. A history of respiratory health was taken and lung function testing performed to assess forced expiratory volume in 1 second (FEV 1 ) and forced vital capacity (FVC). Blood was obtained for complete blood count (CBC), IgE measurement, cellular function, and Th2 cell profiling. Severe asthma was defined as patients on high dose inhaled corticosteroids (ICS, ≥1000 µg/day fluticasone equivalent), a second line controller (long-acting β agonist, leukotriene modifier/or theophylline) and/or oral corticosteroid (OCS) therapy for more than or equal to 50% of the previous year who remain uncontrolled despite this therapy. 15 2.2 | Peripheral blood cell activation and IL-2 measurement Minneapolis, MN) and IL-13 (851.630.005; Diaclone, Besançon, France) enzyme-linked immunosorbent assay. All samples were run in duplicate, had low variability between replicates and were all within the standard curve of the assay. The lower limit of detection for IL-2 was 1.7 pg/mL and IL-13 was 3.1 pg/mL.
2.3 | Fluorescence-activated cell sorting 2.3.1 | Profiling of peripheral blood cells The proportion of helper T cells and Th2 cells in peripheral blood were identified by whole blood staining as in Ref. 26 In brief, antibodies against CD4 (Clone 1F6; Serotec, Oxford, UK) and CRTh2 (clone BM16; Miltenyi Biotech, San Diego, CA) were used to determine the proportion of cells exhibiting positive staining for these markers, as assessed by flow cytometry. The proportion of helper T cells was identified by low side scatter (SSC low ) and high CD4 (high) . Th2 cells were (SSC low ), CD4 (high) and CRTh2 positive and reported as a proportion of total white blood cells (WBC). Flow cytometry data were collected on BD LSR Fortessa using FACSDiva software (Becton, Dickinson and Company, Franklin Lakes, NJ). Gates were set in accordance with the profiles of the isotype control and/or negative control beads.

| Chromatin immunoprecipitation
Primary Th2 cells were fixed (1% formaldehyde, 10 minutes) followed by the addition of glycine (0. DNA was purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Quantitative polymerase chain reaction was performed using SsoFast EvaGreen Supermix (Bio-Rad). Primers used were for FKBP5 (F-TAACCACAT CAAGCGAGCTG; R-GCATGGTTTAGGGGTTCTTG). Negative control for ChIP was IP using mouse IgG. Data were analyzed using ΔΔC t method. ΔC t for each sample was calculated as C t input −C t pulldown and the 2 C −ΔΔ t was the ΔC t of IgG subtracted from ΔC t of target antibody. Data are presented relative to vehicle control. The GEO SuperSeries accession number GSE36890 38 was retrieved for evaluation of Signal transducer and activator of transcription 5 (STAT5) binding to BCL-2 and IL-13 loci. STAT5A binding in wild-type control and IL-2 treated splenocytes was retrieved as.bed.gz file and converted to BigWig for viewing in the UCSC Genome Browser.

| RNA sequencing
Total RNA was isolated from using a Qiagen RNeasy Plus Kit, eluted in 30 μL RNAse-free water and concentration determined (Nanodrop Quantification). Bioanalysis of RNA was conducted using an Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). rRNA-depleted (HMR) stranded libraries were produced using the services of Genome Quebec. Libraries were multiplexed and sequenced on an Illumina HiSeq 4000 (Illumina, San Diego, CA) in PE100 mode. Paired-end FASTQ files were evaluated for overall quality using FastQC Read Quality reports (Galaxy Version 0.72 www.usegalaxy.org). Sequencing adapters were removed from paired-end files using IlluminaClip in trimmomatic. 39 Maximum mismatch count = 2 and sliding window trimming Nbases = 4, AVGQual = 20. Trimmed reads were aligned to Hg38 using HiSat2 (Version 2.1.0) in fr mode. No-mixed/no-discordant behavior disabled. Gene expression in RNA-Seq experiments was determined using featureCounts 40 (Version 1.6.2) in stranded (forward) mode, using Gene-ID features from Gencode.v29.annotation GFF feature type filterexon, disabled multimapping, minimum mapping quality-10. Gene expression in RNA-Seq experiments was determined using featureCounts 40 (Version 1.6.2) in stranded (forward) mode, using Gene-ID features from Gencode.-v29.annotation GFF feature type filter-exon, disabled multimapping, minimum mapping quality-10. RNAseq data are available upon request.

| Statistical analysis
Patients were stratified by median IL-2 (42 600 pg/mL) as in Refs. 34,41 Receiver operator curve (ROC) analyses were also used as in Ref. 42 using FEV 1 less than 80% predicted or taking more than or equal to 1000 μg/day inhaled steroid and gave a similar cut-point (42 993 pg/mL; FEV 1 : Sen = 0.77, Spec = 0.77; Inhaled Steroid: Sen = 0.8, Spec = 0.62). Stratified data were analyzed for statistical significance using Pearson's the χ 2 test for categorical data and independent t test for continuous variables. Correlations were determined using Pearson's or Spearman's correlation, depending on the normality of the data. For cell culture experiments, statistical significance for apoptosis and gene expression were determined by analysis of variance with post hoc analysis (Student-Newman-Keuls method) or Student t test. Data were analyzed using SigmaPlot Version 12.5 and considered significant with P < 0.05.

| Ethical approval
This study was approved by the University of Alberta Human Ethics Review Board (approval number PRO1784) and the Western University Health Science Research Ethics Board (approval number 106770). All subjects gave informed consent.

IL-2 associates with asthma severity
Patients were recruited (n = 18) from new referrals to our Asthma Center and clinically characterized to assess asthma severity. The amount of IL-2 produced by activated peripheral blood cells was variable, ranging from 14 to 102 933 pg/mL. Patients stratified based on median IL-2 production (42 600 pg/mL) showed no difference in age, body mass index, IgE levels or smoking status. However, those with high IL-2 (>median) did have lower FEV 1 and were taking a higher daily dose of inhaled corticosteroid (Table 1). Analyzing the whole population together, IL-2 production was inversely correlated with FEV 1 (r = −0.558, P = 0.0162; Figure 1A) and positively correlated with total daily dose of inhaled corticosteroid (r = 0.561, P = 0.0155; Figure 1B), suggesting its association with asthma severity. While this study population consisted of only four severe asthmatics, our analysis does suggest this group is characterized by higher IL-2 production (56 380 pg/mL) compared to nonsevere asthmatics (32 133 pg/mL, P = 0.07). Further, when patients were stratified based on the daily dose of inhaled steroid, those taking more than or equal to 1000 μg/day had higher IL-2 production ( Table 2).

IL-2 associates with type 2 inflammation
The propensity for high IL-2 production was also related to the degree of type 2 inflammation. Supernatants from patients with high IL-2 following activation of their peripheral blood cells contained 1.9-fold more IL-13 (584.1 vs 306.8; Table 1) and flow cytometry staining of whole blood showed these patients had higher proportions of CD4 + T cells (7.99 vs 4.55) and Th2 cells (0.35 vs 0.17; CD4 + CRTh2 + T cells as a proportion of total white blood cells; Table 1). IL-2 production correlated with the proportion of both CD4 + T cells ( Figure 1C, r s = 0.490, P = 0.038) and Th2 cells ( Figure 1D, r s = 0.511, P = 0.030). Th2 cells also correlated with total daily dose of inhaled corticosteroid ( Figure 1E, r = 0.583, P = 0.011). cell response to DEX (10 −6 M, 48 hours). Interestingly, we failed to see any increase in annexin V + cells (Figure 2A), indicating there was no effect on apoptosis and/or cell death. This was surprising since this level of DEX was able to induce apoptosis in an immortalized T cell line (CCRF CEM, Figure 2B). A major difference between these two experiments was that the primary Th2 cells were cultured in IL-2 (5 ng/mL). Considering that patients with high IL-2 production had more Th2 cells (Table 1), we next assessed whether titrating the level of this growth factor would alter Th2 cell sensitivity to GC-induced apoptosis. When Th2 cells were cultured with DEX and lower concentrations of IL-2 a significant increase in annexin V + cells was observed ( Figure 2C). Indeed, Th2 cells showed sensitivity to 10-fold less DEX (10 −7 M) when cultured with low IL-2 (1.25 ng/mL; Figure 2D) compared to high IL-2 (5 ng/mL; Figure 2C). Apoptosis is regulated by the expression of proapoptotic and antiapoptotic genes of the BCL-2 family, with the balance between these referred to as the BCL-2 rheostat. 43,44 Since IL-2 induces the antiapoptotic gene BCL-2 45 and GC induces the proapoptotic gene BIM, 46 we assessed the expression of these factors. Th2 cells cultured in high IL-2 had more BCL-2 and less BIM mRNA compared to Th2  | 117 cells cultured in low IL-2 ( Figure 3A). This is likely a transcriptional effect since we found (using GSE36890 38 ) that IL-2 treatment of murine splenocytes mediates STAT5 binding the BCL-2 locus ( Figure 3B). The ratio of BCL-2 to BIM (BCL-2:BIM) was significantly higher in Th2 cells cultured in high vs low IL-2 ( Figure 3C) and remained higher following DEX treatment ( Figure 3D). Indeed, when the data for all three DEX concentrations were combined (10 −8 -10 −6 M), the BCL-2:BIM ratio of Th2 cells cultured with high vs low IL-2 was significantly higher ( Figure 3E), suggesting that it regulates Th2 cell susceptibility to GCinduced apoptosis.

| The IL-2-GC axis regulates the suppression of type 2 cytokines
Since patients exhibiting high IL-2 production from peripheral blood cells also produced more IL-13 (Table 1), we assessed whether IL-2 directly regulates IL-13 expression. We found that Th2 cells cultured with high IL-2 (24 hours) expressed significantly more IL-13 mRNA compared to those cultured with low IL-2 ( Figure 4A). ChIP-sequencing analysis showed (GSE36890 38 ) that IL-2 treatment of murine splenocytes mediated STAT5 binding to the Th2 locus control region ( Figure 4B), a known enhancer of IL-13 expression. 47 Since it did not influence Th2 cell numbers ( Figure 4C), this would suggest the IL-2 effect was due to heightened activation of IL-13 transcription rather than Th2 cell proliferation.
To assess the effect of IL-2 on the ability of GC to suppress type 2 cytokines, we cultured Th2 cells with varying concentrations of IL-2 and DEX. When Th2 cells were cultured in high IL-2 the ability of DEX to suppress IL-13 mRNA was significantly less than when cells were cultured with low IL-2 ( Figure 4D). This did not appear to be due to IL-2 influencing a shift toward Th17 or Treg phenotypes, as mRNA levels for IL-17 family members and IL-10 were low (Table 3). To assess the durability of GC suppression following activation, Th2 cells were pretreated with DEX (24 hours), washed and activated with mitogen in the presence or absence of DEX (24 hours) and high IL-2. Th2 cells receiving DEX as both a pretreatment and during activation exhibited almost complete suppression of IL-13 mRNA (88% and 96%, respectively), while cells receiving DEX only as a pretreatment had substantially more IL-13 mRNA following activation ( Figure 4E).

| IL-2 inhibits GR expression and signaling
To examine the mechanism underlying our observations that IL-2 dampens the ability of GC to induce apoptosis and suppress IL-13, we assessed expression of the GR, total levels as well as GR beta (β), a dominant negative isoform associated with reduced GC sensitivity. 48 The data are presented relative to control GR level (vehicle, low IL-2) and show that total GR mRNA was fairly abundant (~C t 27; Figure 5A), while there were extremely low-to-no levels of the GRβ isoform (C t >37 cycles to not detected; Figure 5B). This result indicates that induction of the dominant negative GRβ isoform is likely not the mechanism underlying our finding of IL-2 dampening the effects of GC. However, Th2 cells cultured in high IL-2 had lower levels of total GR mRNA than those cultured in low IL-2 ( Figure 5A). As such, we next assessed if this reduction in GR mRNA was sufficient to influence GR signaling. To do this, we measured FKBP5 expression, a gene known to be induced by GC. 49 We found that Th2 cells cultured in high IL-2 had significantly less FKBP5 mRNA (in response to all DEX concentrations) compared to those cultured in low IL-2 ( Figure 5C). We developed a ChIP assay for GR, which demonstrated that Th2 cells cultured in high IL-2 exhibited significantly less GR binding to a regulatory element within the FKBP5 locus than those cultured in low IL-2 ( Figure 5D). Furthermore, the level of GR mRNA was inversely correlated with IL-13 mRNA (r s = −0.66, P = 0.000002; Figure 5E). Collectively, these data suggest that IL-2 dampening GR signaling resulted in the higher levels of IL-13 mRNA ( Figure 4D).

| DISCUSSION
IL-2 is elevated in the airways of symptomatic vs asymptomatic asthmatics 50,51 and in steroid-resistant vs sensitive asthmatics. 52 Our study extends these earlier findings by assessing the relationship between peripheral IL-2, clinical indices of asthma severity and immune cell profile. We found that peripheral blood cell production of IL-2 correlates with poor lung function, increased GC requirements and heightened type 2 inflammation. In vitro human Th2 cells maintained in high IL-2 exhibited lower expression of the GR and were less responsive to GC-induced apoptosis and cytokine suppression. Though our study was not designed to examine steroid-resistant asthma per se, which involves assessment presteroid/ poststeroid treatment as in Ref. 52 we did observe that patients with higher inhaled steroid requirements produced more IL-2. Collectively, our results suggest that an environment of high IL-2 may mediate asthma severity by dampening GC responsiveness, resulting in persistence of Th2 cells and production of type 2 cytokines.
Since IL-2 is a growth factor known to mediate proliferation of CD4 + T cells, 53 the higher proportion of Th2 cells in those exhibiting high IL-2 production could be due to increasing CD4 + T cell proliferation and/or Th2 cell development. 54 However, our in vitro data showed that Th2 cells cultured in high IL-2 exhibited a BCL-2:BIM ratio above 1 (ie, more BCL-2 than BIM). Though IL-2 induction of BCL-2 is well documented, 45,55 to our knowledge this is the first report of IL-2 suppressing BIM expression. Further, our integrated analysis of the BCL-2:BIM ratio suggests IL-2 modulates both sides of this rheostat within human Th2 expression, similar to a previous report with antigen-specific Th clones. 56 Both mechanisms may be transcriptional, as we found that IL-2 induces STAT5 binding to BCL-2 and IL-13 loci.
IL-2 dampened the capacity of GC to induce apoptosis. This may be due to its reducing GR expression and signaling, further shifting the BCL-2:BIM ratio, as GR binding to the BIM locus, has been shown. 57 In the case of IL-13, we found an inverse correlation between GR and IL-13 mRNA levels, suggesting that the IL-2 effect on F I G U R E 4 IL-2 dampens GC-mediated suppression of IL-13. A, Th2 cells cultured with high IL-2 (5 ng/mL) had significantly more IL-13 mRNA compared to those cultured in low IL-2 (1.25 ng/mL; n = 6). B, Genome browser tracks for STAT5 ChIP under control and IL-2 replete conditions within the Th2 locus control region, an enhancer of IL-13. C, Th2 cells cultured in high or low IL-2 showed no difference in cell number (n = 6). D, The ability of DEX treatment to suppress IL-13 mRNA was significantly less when Th2 cells were cultured in high IL-2 vs low IL-2 (n = 6). E, Continual exposure to DEX was required for suppression of IL-13, since Th2 cells pretreated with DEX (24 hours) and then activated (ACT) with PMA/ionomycin (24 hours) in the absence of DEX (DEX/ACT) had significantly more IL-13 mRNA than Th2 cells receiving DEX both as a pretreatment and during activation (DEX/ACT + DEX; n = 3). Data are presented as mean ± standard error. *P < 0.05 comparison of DEX vs vehicle for each IL-2 concentration; # P < 0.05, comparison of each DEX concentration high vs low IL-2. ChIP, chromatin immunoprecipitation; DEX, dexamethasone; IL-2, interleukin 2; mRNA, messenger RNA; STAT5, signal transducer and activator of transcription 5; Th2, T-helper cell GC-induced cytokine suppression was also due to reduced GR signaling. Indeed, GR has been shown to interfere with many factors that mediate IL-13 transcription such as STAT5, 29,54 proteins within the AP-1 proteins complex 58 and nuclear factor-κB. 59-61 While our results do not rule out other mechanisms influencing GC sensitivity in vivo, such as level of GRβ 48 and posttranslational modifications that reduce GR nuclear translocation, 62 they do suggest that IL-2 has a transcriptional effect independent of these pathways. Our data also show that while increasing GC concentration resulted in more suppression there was always more IL-13 in high vs low IL-2 conditions, suggesting that simply increasing GC dose in patients with high IL-2 may not be effective. Indeed, McKeever et al 63   compared to 52% in those that maintained usual GC dose. Our findings indicate this approach may be more effective if focused on patients who are "low IL-2 producers," that is, those with a potentially better chance of responding to increased GC.
The strong, super-physiologic activation with PMA and ionomycin resulted in high production of IL-2 from the patients' peripheral blood cells in 1 mL of blood. This method has been published by others studying functional human immune responses, [32][33][34] and showed a similar degree of variability in IL-2 and tumor necrosis factor α production, whether cytokines levels were corrected for total cell numbers or not. 34 In our in vitro system, we empirically developed a model of high and low IL-2 based on typical concentrations used for Th2 cell cultures 10,37 as well as functional readouts of resistance/susceptibility to GC-induced apoptosis. Concentrations were lower than observed after ex vivo mitogenic activation of patients' cells, but similar to levels found to be produced from in vivo antigen-activated T cells 64 and BAL lymphocytes activated using beads coated with αCD2/CD3/CD28 13 and so may be more physiologically relevant. The source of IL-2 within whole blood could be naïve and/or memory CD4 + T cells, 64 Th1 cells, 4 CD8 + T cells 65 or even dendritic cells previously exposure to Gram-negative bacteria. 66 Dendritic cell production would suggest prior infections may influence one's propensity to produce IL-2 and could even occur in utero, as IL-2 promoter methylation at birth was associated with asthma severity in childhood. 67 IL-2 genetic variants are also associated with asthma 68,69 as well as inflammatory bowel disease, type 1 diabetes and multiple sclerosis. 68,70,71 Together, these data suggest that control of IL-2, whether genetic or epigenetic, may be fundamental to the development of immune disease.
Our finding that suppression of IL-13 required continual exposure to GC, since mRNA levels were double if DEX was received only as a pretreatment and not also during activation. These data suggest that unless (a) the dose of GC is sufficient to induce Th2 cell apoptosis and (b) exposure to GC is continual, suppression of IL-13 is temporary. This could be important clinically since asthma phenotyping efforts recently proposed a classification based on type 2 cytokine expression. 72 However, if a patient is taking a GC dose sufficient to suppress cytokines, but insufficient to eliminate Th2 cells, this could result in misclassification of type 2-low asthma. Using a cell-based approach, such as eosinophils and/or Th2 cells to classify type 2-high asthma may improve identification of this phenotype. Further, it may help identify responders to new therapies that directly target type-2 cells and their development, such agents blocking CRTh2 and TSLP. 2,73,74 In summary, our study shows for the first time that IL-2 reduces GC responsiveness of human Th2 cells by downregulating GR expression, supporting both their survival and pro-inflammatory capacity in the face of GC treatment. These results suggest that a patient's potential to produce IL-2 may be a determinant in asthma severity and that measuring peripheral levels of IL-2 could help identify those in need of increased ICS dose and/or alternative therapies.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
JMJD2A facilitates growth and inhibits apoptosis of cervical cancer cells by downregulating tumor suppressor miR-491-5p

Cervical cancer remains the second most common malignancy for women worldwide. Jumonji domain containing 2A (JMJD2A), a member of the JmjC domain-containing family of JMJD2 proteins, is capable of regulating cancer-associated genes, including genes involved in the cell cycle, proliferation, apoptosis, invasion and metastasis. However, its role in human cervical cancer has yet to be elucidated. microRNA (miR)-491-5p, a mature form of miR-491, has been shown to function as a tumor suppressor gene in vitro by inducing apoptosis and inhibiting proliferation and invasion in various types of cancer. However, the underlying mechanism remains to be elucidated. In the present study it was observed that JMJD2A expression was significantly upregulated in human cervical cancer cell lines and cervical epithelial carcinoma tissues. A high JMJD2A level predicted poor overall and disease-free survival rate and may serve as an independent prognostic factor for adverse outcome. JMJD2A increased cervical cancer cell and colony numbers in vitro, increased the tumor weight in a mouse xenograft model, and decreased the apoptotic rate by downregulating the pro-apoptotic proteins Bax, p21 and active caspase-3, and upregulating the anti-apoptotic protein Bcl-2. Transfection experiments indicated that the role of JMJD2A in cervical cancer was mediated, at least in part, by the repression of miR-491-5p. In summary, JMJD2A was identified as an oncogenic protein in human cervical cancer that significantly affected cell and colony numbers, tumor weight and apoptosis via the downregulation of miR-491-5p, which acts as a tumor suppressor in cervical cancer. Therefore, JMJD2A may serve as a prognostic factor and potential target for intervention in cervical cancer.

Introduction
Cervical cancer is a preventable disease, and reductions in its incidence and mortality have been achieved (1); however, it remains the second common malignancy in women worldwide (2). Research into the molecular mechanisms associated with the pathogenesis of cervical cancer is important for its treatment and prevention (3). microRNAs (miRNAs) are a class of short, non-coding RNAs ~22 nucleotides long that regulate gene expression by binding to the 3' untranslated regions (3'-UTRs) of target mRNAs in a sequence-specific manner, resulting in translational repression and/or gene silencing (4,5). A number of studies have demonstrated that miRNAs, functioning as either onco-miRNAs or tumor suppressors, perform important roles during cancer progression (6)(7)(8). At present, >2,500 miRNAs have been identified in humans (miRBase database version 20.0) (9). However, few studies have investigated the association between miRNA and the tumorigenesis of cervical cancer (10). miRNA (miR)-491-5p, which is a mature form of miR-491, functions as a tumor suppressor gene in vitro, as it induces the apoptosis and inhibits the proliferation of ovarian (11), colorectal (12), pancreatic (13) and breast (14) cancer cells. In addition, miR-491-5p inhibits the invasion of glioma (15), breast (16) and oral squamous (17) cancer cells. However, the role of miR-491 in cervical cancer cells remains unknown.
Jumonji domain containing 2A (JMJD2A), a member of the JmjC domain-containing family of JMJD2 proteins (JMJD2A-JMJD2D), recognizes di-and tri-methylated histone H3 lysine 9 (H3K9) and H3K36, and trimethylated H1.4K26 as substrates (18). This leads to the promotion of an open chromatin state and contributes to transcriptional activation and the regulation of cancer-associated genes, including those involved in the cell cycle, cell proliferation, apoptosis, invasion and metastasis (19). JMJD2A is involved in several types of cancer, including ductal carcinoma, lung, breast, ovarian, bladder and colon cancer, renal adenocarcinoma, and head and neck squamous cell carcinoma (20)(21)(22)(23)(24). It has been demonstrated that miR-491-5p exerts inhibitory effects on breast cancer cell growth by directly targeting the 3'UTR of JMJD2B mRNA and blocking the estrogen receptor (ER) α-mediated signaling pathway (25). Additionally, a previous study indicated that JMJD2A may contribute to breast tumor formation by stimulating ERα activity (26). These results suggest that JMJD2A may exhibit its oncogenic function by regulating the expression of miR-491-5p.
Thus, the aim of the present study was to investigate the function of JMJD2A in human cervical cancer and determine whether its role is dependent on miR-491-5p. The expression of JMJD2A in human cervical cancer cell lines was evaluated to determine whether it is an oncogenic protein. Additionally, the association of JMJD2A levels with overall and disease-free survival rates and the potential of JMJD2A as an independent prognostic factor for adverse outcomes were investigated. Furthermore, the effects of JMJD2A on cervical cancer cell growth and apoptosis were evaluated.

Materials and methods
Human specimens. A total of 38 primary cervical epithelial carcinoma tissues were collected from patients (n=38; mean age of 50±0.7 years old). Normal cervical tissues were collected from patients who underwent hysterectomy as a result of benign gynecological diseases (n=20; mean age of 46±0.9 years old). Specimens were obtained from patients admitted to the Banan People's Hospital of Chongqing (Chongqing, China) between November 2014 and July 2016. Informed consent was obtained, and the present study was approved by the Ethics Committee of the Banan People's Hospital of Chongqing. Tissue samples were immediately snap-frozen in liquid nitrogen and stored at -80˚C prior to use. The mean JMJD2A level of the cervical cancer tissues was evaluated using western blotting as later detailed. Tissues exhibiting lower JMJD2A expression compared with the mean were classified as the JMJD2A low group, while tissues exhibiting higher JMJD2A expression compared with the mean were classified as the JMJD2A high group. The mean miR-491-5p level of the cervical cancer tissues was evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) as later detailed. Tissues in which miR-491-5p levels were lower than the mean level were classified as the miR-491-5p low group, while tissues with higher miR-491-5p levels compared with the mean level were classified as the miR-491-5p high group.
Cell culture and oligonucleotide transfection. The human cervical cancer cell lines HeLa, CaSki, C-4-I, SiHa and C-33 A were obtained from the Cell Bank of Shanghai, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human cervical cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) under an atmosphere of 5% CO 2 at 37˚C. Normal human cervical cells were grown in a 100-mm plastic dish and cultured under the aforementioned conditions. All procedures involving clinical specimens were approved by the Banan People's Hospital of Chongqing.
Cell viability assay. The viability of cervical cancer cells was determined using an MTS kit (CellTiter 96 AQ; Promega Corporation, Madison, WI, USA). Briefly, cells (5x10 3 cells/well) were seeded in 96-well plates. After 12 h, a fresh mixture of MTS and phenazine methosulfate was added and the cells were incubated for 2-4 h at 37˚C. An MR7000 microplate reader (Dynatech International, LLC, Melville, NY, USA) was used to measure the absorbance at 490 nm.
RNA isolation and RT-qPCR. Post-transfection, total RNA (tRNA) was extracted from human cervical cancer cells using TRIzol ® (Thermo Fisher Scientific, Inc.), following the manufacturer's protocol. Following spectrophotometric measurement (260 nm) of tRNA concentration using an UV spectrophotometer and subsequent analysis of the quality of the tRNA using 2% agarose gels stained with ethidium bromide at room temperature for 30 min, 3 mg tRNA was subjected to reverse transcription (RT) using M-MLV reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) at 25˚C for 10 min, 37˚C for 100 min and 90˚C for 5 sec and 4˚C for 5 min.

Mouse xenograft experiment.
Mouse xenograft experiments were performed as described previously (30). A total of 40 female athymic nude (nu/nu) mice (3-4 weeks old; weighing 14-16 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The mice were bred and maintained under specific pathogen-free conditions. Animals were housed under a controlled temperature (25±1˚C) and humidity (50%), with a 12/12 h light/dark cycle and free access to food and water. HeLa, c-4-1 or SiHa cells (5x10 6 ) transfected with Ad-JMJD2A, Sh-JMJD2A or the corresponding controls (Ad-NC and Sh-NC) were inoculated subcutaneously into the left and right flanks of the mice (n=10/group). The mice were sacrificed and the tumors were weighed 3 weeks after inoculation. The weight of the animals at the time of sacrifice was 12-18 g and there was no significant change in the weight of each animal over the experimental period. Tumor volumes were calculated using the following formula: π/6 x a 2 x b, where a is the short axis and b is the long axis. The largest subcutaneous tumor detected in the present study had a diameter of 1.9 cm. No mouse exhibited multiple subcutaneous tumors. Notably, discomfort was considered a humane endpoints and euthanasia by isoflurane inhalation was applied to immediately sacrifice those animals exhibiting such symptoms. The protocol was approved by the Ethics Committee of Banan People's Hospital of Chongqing.
Statistical analysis. Statistical analysis was performed using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard error of the mean. One-way analysis of variance followed by Dunnett's post hoc test was used to analyze differences among multiple groups. The association between JMJD2A levels and miR-491-5p was tested using Spearman rank correlation. Survival rate analysis was performed using the Kaplan-Meier method, and differences between the groups were tested using the log-rank test.

JMJD2A is highly expressed in cervical cancer cells and strongly associated with survival rate.
To investigate the role of JMJD2A in cervical cancer, the expression of JMJD2A in five cervical cancer cell lines (HeLa, CaSki, C-4-I, SiHa and C-33 A) was examined. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results (Fig. 1A) and western blot analysis (Fig. 1B) revealed that JMJD2A mRNA and protein levels were clearly higher in cervical cancer cell lines compared with normal cervical cells, suggesting that JMJD2A is highly expressed in human cervical cancer.
To further investigate the association of JMJD2A with cervical cancer, the 60-month overall survival rates and disease-free survival rates of patients with low (lower than the mean level) and high (higher than the mean level) JMJD2A expression levels were analyzed. The results demonstrated that a low JMJD2A level was associated with high overall and disease-free survival rates, while a high JMJD2A level was associated with poor overall and disease-free survival rates ( Fig. 1C and D). These observations indicate that JMJD2A has an association with survival rate and may potentially serve as an independent prognostic factor.

JMJD2A overexpression induces the growth of cervical cancer cells.
To explore whether JMJD2A participates in the growth and apoptosis of cervical cancer cells, JMJD2A was knocked down or overexpressed in the human cervical cancer cell lines HeLa, C-4-I, and SiHa. The knockdown and overexpression efficiencies were confirmed by RT-qPCR ( Fig. 2A). Next, the cell viability at different time-points after transfection (0, 24, 48 and 72 h) was determined. The results demonstrated that JMJD2A knockdown (sh-JMJD2A) significantly reduced the number of surviving cells, and the inhibition of cell growth appeared to be time-dependent. In addition, JMJD2A knockdown significantly decreased the number of colonies in vitro. In the mouse xenograft model, tumor weights for mice implanted  with the sh-JMJD2A HeLa, C-4-I and SiHa cells were significantly reduced compared with those in the respective sh-NC group (Fig. 2B-F). Conversely, JMJD2A overexpression (Ad-JMJD2A) increased the proliferation and colony formation of cervical cancer cells in vitro, and increased the weight of the xenograft tumors compared with those in the respective control group (Ad-NC). These findings indicate that JMJD2A regulates cervical cancer growth in vitro. (Fig. 2G) revealed that JMJD2A knockdown induced cellular apoptosis in HeLa, C-4-I and SiHa cells, whereas JMJD2A overexpression significantly attenuated cellular apoptosis in these cell lines, resulting in a markedly low apoptotic rate. The results of western blot analysis (Fig. 3A) demonstrated that JMJD2A knockdown upregulated the expression of pro-apoptotic proteins (Bax, p21 and active caspase-3) and downregulated the level of the anti-apoptotic protein Bcl-2. However, it exhibited no clear effect on the expression of the apoptosis-related protein pro caspase-3.

JMJD2A overexpression inhibits apoptosis of cervical cancer cells. Flow cytometric analysis
JMJD2A knockdown induces miR-491-5p expression in cervical cancer cells. miR-491-5p has been reported to act as a tumor suppressor and is downregulated in a number of cancer cell lines (31)(32)(33). The results of the present study demonstrated that miR-491-5p was downregulated in the human cervical cancer lines HeLa, CaSki, C-4-I, SiHa and C-33 A (Fig. 3B). To explore the correlation between JMJD2A and miR-491-5p levels in the cervical cancer tissues, linear regression analysis was performed. The data demonstrated that the miR-491-5p and JMJD2A levels were significantly and negatively correlated (Fig. 3C). In addition, when JMJD2A was knocked down or overexpressed in cervical cancer cells, significant inductive and suppressive effects on miR-491-5p expression were observed, respectively (Fig. 3D). The miR-491-5p mRNA levels of the cervical cancer tissues were used to divide the patients into miR-491-5p low and miR-491-5p high expression groups. Kaplan-Meier curves were plotted for analysis of the overall survival rates (Fig. 3E) and indicated that a low miR-491-5p level predicted a poor overall survival rate. Together, these results suggest that miR-491-5p expression is downregulated in cervical cancer and a low miR-491-5p expression level is predictive of a poor survival rate.
miR-491-5p knockdown reverses the effects of sh-JMJD2A on cervical cancer growth. In order to examine the potential regulative mechanism, LNA-miR-491-5p and LNA-NC were respectively transfected into sh-JMJD2A and sh-NC cells. The miR-491-5p levels of the transfected were validated using RT-qPCR (Fig. 3F).  (Fig. 4) demonstrated that following the knockdown of miR-491-5p (sh-NC + LNA-miR-491-5p), the cell and colony numbers were significantly higher, and the apoptotic rate was significantly lower than those in the control (sh-NC + LNA-NC). In addition, JMJD2A knockdown (sh-JMJD2A + LNA-NC) significantly decreased the cell and colony numbers, and induced cell apoptosis in comparison with the control (sh-NC + LNA-NC). In the cells with knockdown of miR-491-5p and JMJD2A (sh-JMJD2A + LNA-miR-491-5p), the effects of JMJD2A knockdown were significantly attenuated, as evidenced by the increase of cell and colony numbers and the reduction of cell apoptotic rate compared with those in the sh-JMJD2A + LNA-NC group, and the results were comparable to those in the control group (sh-NC + LNA-NC).

Discussion
Although the functions of JMJD2A in various types of cancer have been demonstrated previously, its role in human cervical cancer remains unclear. The present study identified that JMJD2A is overexpressed in human cervical cancer, which is consistent with a previous study reporting that JMJD2A is overexpressed in human breast, lung, head and neck, uterine, endometrial and ovarian cancer, and stomach and renal adenocarcinoma (34). In addition, log-rank analysis in the present study indicated that high JMJD2A expression predicts poor overall and disease-free survival rates. These results suggest that JMJD2A is overexpressed in human cervical cancer and may be an independent prognostic factor.
The potential mechanisms of JMJD2A in various types of cancer have been investigated in previous studies. In one study, the overexpression of JMJD2A was detected in human breast cancer cells, and it was observed that JMJD2A forms a complex with estrogen receptor (ER)α in vivo, and the downregulation of JMJD2A decreased the expression of cyclin D1, a prominent ERα target gene and cell cycle regulator (26). Another study demonstrated that JMJD2A promotes cellular transformation by blocking cellular senescence through transcriptional repression of the tumor suppressor CHD5 (23). Furthermore, JMJD2A has been reported to be involved in human carcinogenesis through regulation of the G1/S transition in human bladder and lung cancers (22). Another study suggested that the JMJD2A level correlates with the level of the pro-apoptotic microRNA miR-34a in gastric cancer tissues and JMJD2A represses the expression of miR-34a by decreasing its promoter activity (35).
The potential mechanism underlying the roles of JMJD2A in human cervical cancer was investigated in the present study. JMJD2A was found to regulate cervical cancer cell growth and apoptosis; more specifically, JMJD2A deficiency induced cervical cancer cell apoptosis by upregulating pro-apoptotic proteins (Bax, p21 and active caspase-3) and downregulating the anti-apoptotic protein Bcl-2. In addition, miR-491-5p, which is reported to act as tumor suppressor in a number of different cancers, was demonstrated to be negatively correlated with the expression of JMJD2A in cervical cancer. The present study also revealed that JMJD2A knockdown significantly upregulated the expression of miR-491-5p. The significant inhibition of cell growth and increased apoptosis of cervical cancer cells induced by sh-JMJD2A were markedly reversed by the suppression of miR-491-5p. Together, these results indicate that JMJD2A participates in the proliferation and apoptosis of human cervical cancer cells and its role is partly mediated via the regulation of miR-491-5p. However, the mechanism underlying the effects on the regulation of miR-491-5p expression requires further exploration.
The mechanism by which miR-491-5p acts in tumor suppression was not fully investigated in the present study. Previous studies found that miR-491-5p suppressed the growth of oral squamous cell carcinoma, pancreatic cancer and glioblastoma by targeting GIT1, EGFR and CDK6 genes (14,17,36). Another study indicated that miR-491-5p targeting TP53 and Bcl-X L induced cell apoptosis in SW1990 pancreatic cancer cells through a mitochondria-mediated pathway (13). However, the genes that miR-491-5p targets in human cervical cancer have not yet been identified.
In summary, JMJD2A was identified as an oncogenic protein in human cervical cancer. Its effects on cell and colony numbers, tumor weight and apoptosis may be mediated by the downregulation of miR-491-5p, which serves a role as an anti-onco-miRNA in cervical cancer. Therefore, JMJD2A could serve as an independent prognostic factor and potential target for intervention in cervical cancer.


Melanoma-Derived Extracellular Vesicles: Focus on Their Proteome

Malignant melanoma is one of the most aggressive types of cancer, and its incidence is increasing rapidly each year. Despite the extensive research into improved diagnostic and treatment methods, early detection and disease constraint still present significant challenges. As successful isolation protocols have been developed, extracellular vesicles (EVs) have become the subject of extensive investigation in terms of their role in cancer progression and as a possible source of disease biomarkers. Besides functional studies, quantitative and qualitative proteomics have recently emerged as promising tools for the advancement of melanoma biomarkers. Nevertheless, the amount of data concerning the proteome of melanoma-derived EVs is still very limited. In this review we cover the current knowledge on protein content of melanoma-derived EVs, with a focus on their potential role in the development and progression of melanomas.

Introduction
Malignant melanoma is one of the most aggressive types of cancer, caused by neoplastic transformation of melanocytes (pigment-producing cells). Despite the prevalence of their cutaneous localization (>91%), primary melanoma tumors can also develop within the uveal tract or mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts [1]. Global melanoma incidence is constantly rising, with approximately 350,000 new cases each year. Although melanoma incidence is 10-fold lower than the incidence of other skin cancers, its capacity to rapidly metastasize contributes to one of the highest mortality rates among all cancers, at about 60,000 deaths per year [2].
Despite the extensive research into improved diagnostic and treatment methods, early detection and disease constraint still present significant challenges. Melanoma markers routinely used in clinical practice for staging, prognostics, treatment selection, and follow-up include lactate dehydrogenase (LDH), S100B protein, melanoma inhibitory activity (MIA) protein, and BRAFV 600E mutation [3]. These classic blood markers are constantly being revisited due to their insufficient diagnostic sensitivity and specificity. On the other hand, novel potential circulating biomarkers, such as cell-free DNA [4], microRNA [5], and circulating tumor cells [6], or a combination of autoantibodies [7], are being considered.
As successful protocols for the isolation of the extracellular vesicles (EVs) have been developed, they have become the subject of extensive investigation in terms of their role in cancer progression, and as a possible source of disease biomarkers. Viable cells predominantly release two types of EVs: ectosomes (also called microvesicles), which are directly shed from the cell surface, and exosomes, which are released after the fusion of multivesicular bodies (MVBs) with the plasma membrane. In the case Specific contents of EVs and their action towards the recipient cells depend on their molecular composition, which is determined by their origin cells. Proteins, lipids, and nucleic acids that are specific for a given cell type can be detected in respective EV populations, thus contributing to their prognostic and diagnostic value for individual health conditions, including different types of cancer [9][10][11]. Therefore, quantitative and qualitative proteomics of melanoma-derived EVs have recently emerged as promising tools for the advancement of melanoma biomarkers, but the amount of relevant data is still very limited. Nevertheless, the extensive proteomic analyses that have been carried out so far have confirmed the presence of melanoma-specific antigens and multiple oncogenic proteins in cargoes of melanoma-derived EVs. In this review we cover the current knowledge on protein content of melanoma-derived EVs, with a focus on their potential role in the development and progression of melanoma. Specific contents of EVs and their action towards the recipient cells depend on their molecular composition, which is determined by their origin cells. Proteins, lipids, and nucleic acids that are specific for a given cell type can be detected in respective EV populations, thus contributing to their prognostic and diagnostic value for individual health conditions, including different types of cancer [9][10][11]. Therefore, quantitative and qualitative proteomics of melanoma-derived EVs have recently emerged as promising tools for the advancement of melanoma biomarkers, but the amount of relevant data is still very limited. Nevertheless, the extensive proteomic analyses that have been carried out so far have confirmed the presence of melanoma-specific antigens and multiple oncogenic proteins in cargoes of melanoma-derived EVs. In this review we cover the current knowledge on protein content of melanoma-derived EVs, with a focus on their potential role in the development and progression of melanoma.

The Role of EVs in Melanoma Biology
All populations of EVs can be released by normal, as well as transformed, cancer cells. Upon their delivery to the recipient cells, EVs modulate a number of biological processes, including intercellular signaling, protein transport, cell proliferation, and cancer development. Melanoma-derived exosomes were shown to stimulate proliferation of cancer cells, to mediate activation of the epithelial-mesenchymal transition (EMT), and to induce pre-metastatic niche formation [12]. They also promote degradation of the extracellular matrix (ECM) by metalloproteinases (MMPs) [13], activate integrin signaling, and provide ligands for epidermal growth factor receptor (EGFR) and Notch receptor [12]. Furthermore, melanoma-derived exosomes modulate multiple functions of the immune system, most often by counteracting the anti-melanoma immune response [14]. They have also been shown to influence bone marrow progenitor cells toward a pro-metastatic phenotype through receptor tyrosine kinase MET [15] to regulate differentiation and maturation of antigen-presenting cells [16], to control survival and apoptosis of effector T-cells, and to control cytokine production and cytotoxicity of natural killer (NK) cells [12].
Although the vast majority of studies on melanoma-derived EVs have focused only on the exosome population, ectosomes may also facilitate disease development and progression. In the 1980s, different melanoma cell lines were shown to release ectosomes, which displayed the ability to boost metastatic potential when internalized by less invasive tumor cells [17]. More recent studies reported a two-fold higher number of ectosomes released in vitro by melanoma cells in comparison to normal melanocytes [18]. In addition, melanoma-derived ectosomes showed higher levels of tissue factor (TF), the main clotting initiator associated with cancer-related procoagulant state [18]. Melanoma-derived ectosomes may also promote tumor invasion and metastasis by transferring matrix-degrading metalloproteinases or their endogenous activators [19][20][21]. Finally, ectosomes may participate in tumor-stroma or tumor-immune cell interactions. Ectosome-mediated transformation of normal fibroblasts to tumor-associated fibroblasts has already been described in melanoma [22], as well as suppression of immune response by vesicle-associated Fas ligand (FasL) [23].
Even less is known about the role of apoptotic bodies in melanoma. However, in a sole study, apoptotic bodies released by mouse B16-F1 melanoma cells were isolated and revealed the highest procoagulant activity in in vitro coagulation assays in comparison to exosomes and ectosomes [24]. The procoagulant activity was attributed to the abundance of TF and phosphatidylserine in the apoptotic bodies under analysis. Additionally, mice inoculated with B16-F1 apoptotic bodies prior to inoculation with tumorigenic B16-F1 cells were protected from tumor development for up to 60 days, while a shorter tumor-free period was observed in mice immunized with B16-F1 exosomes or ectosomes. These results clearly demonstrated the involvement of melanoma-derived apoptotic bodies in procoagulant and immunogenic functions and their contribution to the prothrombotic state, as well as to anti-cancer immunity [24].

Isolation of Melanoma-Derived EVs for Proteomic Analysis
The credibility of proteomic data concerning any population of EVs depends primarily on the sample purity. Every single protocol for vesicle preparation and isolation should ideally lead to the obtainment of uncontaminated fractions of interest, i.e., within the predefined size and density range, free of cells, cellular debris, organelles, microorganisms, or aggregates, and characterized by a presence or absence of specific protein markers. Despite several decades of research and technological advancement, isolation of EVs still poses a significant challenge. Since the chosen isolation method may influence further analytical steps due to different chemical or physical factors used during the procedure, it should be decided based on the source of EVs (cell cultures or body fluids) and the specific downstream applications.
Regarding the sources of melanoma-derived EVs for proteomic studies, only conditioned culture media have been used so far. Except for one study using primary cell cultures established from biopsies of uveal melanoma tumors [25], other research works were performed using commercially available cutaneous melanoma cell lines representing various stages of the disease [24,[26][27][28][29][30]. For maximum vesicle recovery, conditioned media should be collected from subconfluent (60-80%) melanoma cells [24,[26][27][28][29][30]. Another good practice at the stage of melanoma cell culture is the use of fetal bovine (or calf) serum previously depleted of EVs [24,[26][27][28][29][30], although bovine-specific protein sequences can be also eliminated during analysis of MS data. Alternatively, melanoma cells can be cultured for 24-48 h [25] in serum-free media, which is also thought to increase the amounts of released EVs. Regarding vesicle yield, expanded surface roller bottles for a maximal cell:supernatant ratio were recently used in proteomic studies of melanoma-derived exosomes [27].
The further preparation of EV samples for MS-based proteomic analysis requires a careful methodical approach, however the majority of known isolation methods can be successfully adapted. The most commonly used method for melanoma EV isolation is differential centrifugation, where centrifugal force determines the size of isolated particles. Besides centrifugal force (g-force), each protocol based solely or partially on centrifugation should include other factors that may to some degree influence EV sedimentation, i.e. rotor type, tube size, sample volume and viscosity, and temperature. Also, when changing the centrifuge, in many protocols centrifugation time is often adjusted by K-factor. However, Livshts et al. [31] demonstrated that such practice may be erroneous in the case of fixed-angle rotors. Instead they provided an equation to calculate the proportion of pelleted EVs of a given size based on applied centrifugation force, centrifugation time, and the sedimentation path lengths of a given fixed-angle or swinging bucket rotor. Using this method the centrifugation time required for complete sedimentation of the EVs of a given size can be easily calculated, and individual differential centrifugation protocols adequate for the centrifuge available can be developed.
Regardless of the differences at later stages, for the first centrifugation step low centrifugal forces, ranging from 300× g [25] up to 3,200× g [24,32], were routinely used to remove remaining cells and cell debris. Higher centrifugal forces were used to pellet larger vesicles (ectosomes and apoptotic bodies), however, their overlapping sizes (0.1-1 µm for ectosomes, 0.1-5 µm for apoptotic bodies) and densities makes their complete separation difficult. Muhsin-Sharafaldine et al. [24] used 25,000× g centrifugation to isolate both ectosomes and apoptotic bodies from in vitro culture of mouse B16-F1 melanoma cells. The only difference between isolation procedures was that supernatant for isolation of apoptotic bodies came from the cell culture treated with doxorubicin (pro-apoptotic agent), while vesicles from the untreated culture were considered as an ectosome population [24]. This approach can be justified by the fact that viable melanoma cells in a culture should not release apoptotic bodies. However, their presence in small amounts cannot be ruled out and should be analyzed with the use of exemplary apoptotic markers, such as histones or gp96 protein [33]. In addition, ectosomes and apoptotic bodies isolated according to the aforementioned protocol showed only a slight shift in terms of their diameter distribution, with modal values of approximately 250 nm for ectosomes and 350 nm for apoptotic bodies [24].
In contrast to high 25,000× g centrifugal force, another proteomic study on melanoma-derived EVs used centrifugation at only 10,000× g to pellet ectosomes released by mouse B16-F10 melanoma cells [29]. Such low centrifugal forces increase the risk of contamination with apoptotic bodies, but almost completely exclude the contamination with exosomes. In fact, quantitative analysis of proteomic data revealed a significant depletion of exosomal markers in the obtained melanoma ectosomes [29]. Taken together, these results suggest the need to carefully distinguish between melanoma-derived ectosomes and apoptotic bodies. Centrifugation steps of up to 16,000× g allow the separation of the population of larger apoptotic bodies, however, the use of 16,000-25,000× g will most likely produce a mixed population of ectosomes and smaller apoptotic bodies. The presence of the latter can be efficiently reduced if apoptotic processes in a cell culture are not over-induced.
For isolation of exosomes, intermediate steps up to 30,000× g can be used to remove cells, cell debris, and larger vesicles, followed by ultracentrifugation at 100,000-150,000× g to pellet exosomes. In proteomic studies on melanoma-derived exosomes, ultracentrifugation was often proceeded by ultrafiltration and concentration of the previously obtained supernatant with the use of 0.22 µm filters [24] or 100-500 kDa molecular weight cut-off membranes [27,29]. The ultracentrifugation step can be further modified by the addition of a density gradient, in which vesicles are separated according to their size and density. In this method, vesicles move in the gradient medium to the point where their density is equal to the density of the medium. Regarding commercially available kits, OptiPrep was used to isolate exosomes released by melanoma Mel501 cells on a 10-40% iodixanol gradient [30].
In another proteomic study on melanoma-derived EVs, the discontinuous step-gradient of 0.4-2.5 M sucrose in pH 7.4 HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer allowed for separation of two distinct exosome subpopulations. When a pre-cleaned and concentrated sample was loaded on top of a gradient, one exosome population was recovered, while loading the medium on the bottom of the tube and covering it with a sucrose gradient (the so-called bottom loading or upward displacement) resulted in separation of low density exosomes (1.12-1.19 g/mL) and high density exosomes (1.26-1.29 g/mL) [29]. Since ultracentrifugation can potentially affect exosome integrity, size-exclusion chromatography (SEC), which preserves all biophysical properties of EVs, was simultaneously used to prove that subpopulations of low-and high-density exosomes were not artifacts created during ultracentrifugation. In SEC, porous beads are used as a stationary phase. Large particles are eluted earlier from the column (they do not enter the pores), while smaller particles remain in the filtration column for longer (they are trapped in pores of the stationary phase). In the aforementioned study, melanoma-derived exosomes were isolated with the use of a Sephacryl column and then again successfully separated into two populations on a sucrose gradient [29]. Also, a gradient of low osmotic solution of Nycodenz was similarly used to separate exosomes. Another successful separation of low-and high-density exosomes ruled out the possibility that their different mobility within the sucrose gradient resulted solely from the damage to the exosomes caused by the hyperosmotic nature of the sucrose gradient [29]. In addition, after the use of different gradients for exosome isolation, it is necessary to remove any additional reagents from the sample before proceeding with the LC-MS protocol. For instance, melanoma-derived exosomes isolated on a sucrose gradient were dialyzed (10 kDa cut-off) against phosphate buffered saline (PBS) in order to remove sucrose, and later purified by the FASP (filter-aided sample preparation) method using ultrafiltration units with a 3 kDa cut-off [24].
Furthermore, isolation of exosomes does not necessarily require the ultracentrifugation step. As was shown by Xiao et al. [27], melanoma-derived exosomes can be isolated for proteomic studies with the use of additional precipitation agents. Cell culture media (previously depleted of cells, cell debris, and larger vesicles) were incubated with the commercially available Exoquick-TC polymeri c precipitation buffer, and exosomes were then pelleted by centrifugation at 10,000× g only [27]. Unlike other isolation methods, Exoquick-TC and similar kits are dedicated to smaller vesicles (approximately 60-180 nm in diameter), and thus still not applicable for isolation of ectosomes or apoptotic bodies. Furthermore, co-precipitation of protein aggregates is also commonly observed after the use of polymeric precipitation agents, and this can limit the identification of surface antigens in various analytical methods, for instance in flow cytometry.
To study the functional impact of melanoma-derived EVs present in patients' body fluids on disease progression, isolation of EVs exclusively of melanoma origin from the total EV pool is necessary. Recently, Sharma et al. [34] developed an immunoaffinity-based method for exosome capture from the plasma of melanoma patients. Using mouse 763.74 antibody specific for the chondroitin sulfate proteoglycan 4 precursor (CSPG4) epitope uniquely expressed on melanoma cells, they successfully separated melanoma from non-melanoma exosomes confirmed by thorough analysis of their protein cargo. However, this method requires morphologically intact, non-aggregated exosomes, which carry the epitope of interest. Additionally, plasma-derived EVs are often coated by plasma proteins, which limit the access to surface antigens, thus the initial removal of plasma contaminants by SEC was proposed [34].
Finally, purity of an EV sample obtained with the use of a given isolation protocol should be analyzed in terms of vesicle size and the presence or absence of marker molecules. Methods that have been used for size characterization of melanoma-derived EVs include transmission electron microscopy (TEM) [24,[27][28][29]32], nanoparticle tracking analysis (NTA) [28,29], and dynamic light scattering (DLS) [24], all of which also provide information on vesicle concentration. Phenotyping of melanoma EVs may be performed using flow cytometry [24] or western blot [27][28][29][30]. The commonly used markers for exosomes were tetraspanins (CD9, CD81), tumor susceptibility gene protein 101 (Tsg101), Alix, and HSP70. Other populations of melanoma-derived EVs were characterized by depletion or lower expression of exosomal markers, however, for apoptotic bodies histones or gp96 protein can be used [33], while ectosomes typically show enrichment in β1 integrins, CD40, TyA antigen, peptidase C1a, or Ezrin [35]. Such an approach follows the guidelines from the Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) [36], which does not propose molecular markers that could specifically characterize a single EV population; their presence should rather be analyzed in terms of their relative depletion or enrichment. Considering mechanisms of EV biogenesis, for each population membrane-or endosome-associated protein, markers should be provided, alongside exemplary cytosolic or secreted proteins that are expected to be recovered in isolated EVs. As a negative control the absence of nuclear, mitochondrial, or cytoskeletal proteins can be demonstrated for exosomes and ectosomes, while apoptotic bodies formed during cell fragmentation should contain such proteins. In terms of contamination, the presence of major components of non-EV co-isolated structures (e.g., aggregates of serum albumins or apolipoproteins, aggregates of uromodulin in urine, ribosomal proteins, etc.) should also be excluded.

Proteomic Techniques in Studies on Melanoma-Derived EVs
High throughput, specificity, and sensitivity have caused mass spectrometry to become the major tool used for identifying and characterizing molecular contents of EVs (mainly proteins), but also lipid and metabolite composition. A number of EV-oriented studies using qualitative and quantitative proteomics are constantly increasing, creating alternative analytical approaches, such as gel-based or shotgun proteomics. Novel, state-of-the-art methods allow identification of over 1,000 proteins in one EV sample, regardless of its source, i.e., cell cultures or body fluids [37].
Most of the latest research on EV proteome (summarized in Table 1) utilizes the common proteomic approach based on tryptic digestion of proteins, followed by separation of peptides by nano HPLC and their analysis by tandem mass spectrometry (MS/MS). In some cases the complexity of the EV sample is reduced prior to tryptic digestion by 1D or 2D SDS-PAGE (one/two-dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) separation of proteins. Lazar et al. [28] used 1D SDS-PAGE to separate exosomal proteins from 7 melanoma cell lines. The gels were routinely stained with Coomassie Brilliant G-250 Blue and each protein band was analyzed by mass spectrometry. Boussiadia et al. [30] also applied 1D SDS-PAGE to separate proteins from exosomes released by Mel501 melanoma cells, but the entire gel lines were cut into 25 slices, avoiding the loss of proteins (e.g., those located on the band margins, or not stained), which is inevitable when cutting out only stained bands.
In comparison to 1D SDS-PAGE, 2D SDS-PAGE includes an isoelectric focusing (IEF) step prior to separation of proteins by their molecular mass. It allows for separation and visualization of a much higher number of proteins from the complex samples, and for their more adequate quantitative analysis. Pardo et al. [38] used 2D SDS-PAGE before LC-MS/MS analysis of the conditioned media from the culture primary uveal melanoma UM-A cell line. Although no particular population of EVs was isolated, the study identified 133 proteins (the majority of them involved in melanoma biology) in excised gel spots. Analogous analyses of sera from uveal melanoma patients and healthy control revealed, based on different spot sizes, a differential expression of gp100 and cathepsin D that were later investigated as possible melanoma biomarkers [38]. MeWo and SK-MEL-28 human melanoma cell lines exosomes 0.1 µm filtration followed by ultracentrifugation at 100,000× g 553 proteins common to all populations procoagulant proteins were more abundant in ectosomes and apoptotic bodies than in exosomes, with tissue factor (and lipid-phosphatidylserine) critical for procoagulant activity [24] B16F10 mouse melanoma cell line exosomes, ectosomes 10,000× g to pellet ectosomes; exosomes were then pelleted from the remaining supernatant at 110,000× g; sucrose density and Nycodenz gradients were also applied to further separate exosomes; independent isolation of exosomes was performed using size exclusion chromatography (SEC) nanoLC-MS/MS a total of 4421 proteins; 1540 proteins common to all populations, and 533, 354, and 110 proteins were identified exclusively in ectosomes and lowand high-density exosomes, respectively bottom-loading (instead of top-loading) of exosomes on sucrose density and Nycodenz gradients resulted in separation of high-and low-density exosomes displaying distinct protein profiles [29] melanoma A375 and normal melanocytic HEMa-LP cell lines exosomes 500 kDa cut-off ultrafiltration followed by ultracentrifugation at 100,000× g, Exoquick-TC precipitation 2D DIGE-LC-MS/MS total number not provided, 114 protein spots detected differential expression of annexin A1, annexin A2, syntenin-1, and hyaluronian and proteoglycan link protein 1 between melanocytes and melanoma exosomes [27] primary cell cultures established from 14 original tumor specimens of uveal melanoma patients not isolated, entire secretome was analyzed -label-free nanoLC-MS/MS total of 1843 proteins (758 with at least 3 unique peptides) a subsets of 83 up-regulated and 80 down-regulated proteins in uveal melanoma secretome [38] sera of uveal melanoma patients and healthy controls not isolated, entire secretome was analyzed -2D SDS-PAGE-LS-MS (CapLC system coupled to a Q-TOF spectrometer) 133 (on average) cathepsin D and gp100 protein levels increased in sera of uveal melanoma patients [25] To demonstrate differential expression of a protein within EV cargo, 2D DIGE (differential in-gel electrophoresis) can also be used. This method has an obvious advantage over traditional 2D SDS-PAGE, because it allows separation of up to three samples, stained differentially by fluorescent dyes (Cy3, Cy5, and Cy2) on one gel, thus overcoming in-gel differences. Xiao et al. [27] used 2D DIGE to compare protein profiles of exosomes released by normal HEMa-LP melanocytes and metastatic A375 melanoma cells. Finally, the major downfall of 2D techniques is that hydrophobic membrane proteins precipitate during IEF. Considering the membrane origin of EVs, a significant part of the EV proteome may not be observed and identified by MS if IEF is applied beforehand.
All of the aforementioned methods allowed for identification of up to 300 proteins in a single melanoma-derived EV sample. These numbers can be maximized with the use of a shotgun proteomics workflow, which has become a major tool, especially in large-scale proteomic profiling. The standard protocol starts with digestion of an intact (unseparated) protein mixture, thus the analysis is not limited by the modest separation efficiency of SDS-PAGE. Particularly, low-abundance proteins, aberrant proteins, as well as membrane proteins, can be more successfully identified. In studies based on shotgun proteomics, the number of proteins identified in melanoma-derived EV samples ranged from 539 [25] to even 2893 [29], proving the advantage of this approach.
Finally, shotgun proteomics can also be applied for quantitative analysis. Although no common methods of labeling, such as iTRAQ (isobaric tags for relative and absolute quantitation) or SILAC (stable isotope labeling in cell culture), have been applied to profile proteomes of melanoma-derived EVs so far, they proved useful in proteomic analysis of EVs from other types of cancer cells. For instance, Jeppesen et al. [39] used iTRAQ to identify proteins with differential expression in EVs from non-metastatic and metastatic bladder cancer cell lines, while Inder et al. [40,41] applied SILAC to compare proteome of EVs and of entire secretomes of prostate cancer PC3 cells. Alternatively, label-free quantitation can be performed based on spectral counting (SC), or spectral peak intensities (total ion current (TIC))-based approach. Boussadia et al. [30] used spectral counting quantification-exponentially-modified protein abundance index (emPAI) to compare proteomes of exosomes released be melanoma Mel501 cells cultured under normal and acidic pH.

Promising Results From In Vitro Studies on Melanoma-Derived EVs
The first study demonstrating the potential of global proteomic profiling of melanoma-derived EVs was performed by Mears et al. [26]. Protein profiles of exosomes purified from cell supernatants of two melanoma cell lines, MeWo and SK-MEL-28, were obtained using 1D PAGE alone and 2D PAGE, followed by MS analysis. Protein profiles obtained by these two different methods were highly congruent between exosomes but differed from the corresponding profiles of the total cell lysates. The 2D gels obtained for exosomes showed approximately 25% less protein spots in comparison to the total cell lysates, and only 45-50% of proteins were shared between corresponding lysates and exosomes, according to subsequent MS analysis. Forty-one exosomal proteins identified by MS included heat shock proteins, enzymes, and structural proteins with a variety of functions (according to Gene Ontology (GO) analysis), such as guanosine triphosphate (GTP)-binding, tetraspanin binding or apoptosis-related action. Interestingly, proteins present in whole-cell lysates that were absent in exosomes were mostly of mitochondrial, ER, Golgi, and lysosomal origin, which supports the endosomal origin of exosomes. This study was also the first to demonstrate the presence of several proteins in exosomes, namely ezrin, radixin, prostaglandin regulatory-like protein (PGRL), p120 catenin, syntaxin-binding proteins 1 and 2, Septin 2 (Nedd5), and tryptophan/aspartic acid (WD) repeat-containing protein 1 [26].
It would be useful clinically if proteomic profiling of melanoma-derived EVs allowed one to distinguish them from EVs released by non-transformed cells, and to indicate any differentially expressed proteins. For exosomes released by the highly metastatic A375 melanoma cell line and HEMa-LP normal melanocytes, 2D DIGE-MS analysis identified 11 differentially expressed proteins, including annexin A1, annexin A2, syntenin-1, and hyaluronan and proteoglycan link protein 1 (HAPLN1), all involved in angiogenesis, invasion, migration, and metastasis of melanoma cells [27].
In subsequent functional studies, normal melanocytes acquired invasive properties after incubation with melanoma-derived exosomes, suggesting that the specific protein cargo of tumor-derived exosomes may influence the process of carcinogenesis. Additionally, differential expression of 8 out of 11 exosomal proteins identified by MS correlated with expression levels of corresponding mRNA and upstream miRNAs that were also detected in melanoma-and melanocyte-derived exosomes [27].
Furthermore, the molecular content of EVs reflects the current state their origin cells. In that aspect proteomic profiling of melanoma-derived EVs may be used to indicate the proteins with differential expression, depending on the disease stage. Possibility of such application was preliminarily explored in studies using exosomes derived from 7 melanoma cell lines (3 non-tumorigenic, 2 tumorigenic, and 2 metastatic) [28]. Twenty-five percent of the proteins were shared between exosomes of all 7 origins, while the remaining 75% were present in exosomes released only by one or several cell lines. This suggested some degree of specificity and the dependence of the protein signature on the aggressiveness of a given cell line. Interestingly, according to gene ontology (GO) analysis categories of biological functions, such as cell motility, regulation of apoptosis, and angiogenesis, were significantly enriched only in the case of exosomes derived from metastatic melanoma cells [28].
It is generally accepted that besides the aforementioned exosomes, cells release at least two other types of EVs, i.e., ectosomes and apoptotic bodies. Due to their different biogenesis, these three populations have distinctive molecular contents and exert specific actions towards recipient cells. As the recent studies have shown, the proteomic approach can be applied alongside other methods to analyze phenotypic and functional differences between vesicle types originating from melanoma cells. MS-based profiling of exosomes, ectosomes, and apoptotic bodies derived from mouse B16-F1 melanoma in GO analysis revealed the enrichment of histones (H2A, H2B, H3.1, and H4) and heat shock proteins (HSPA5 and HSC71) in exosomes compared to ectosomes and apoptotic bodies. Additionally, based on ion scores, exosomes were enriched in tetraspanins CD9 and CD81, which belong to main exosomal markers [24]. Although the aforementioned enrichment of exosomes with histones should be considered as a possible contamination, Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) guidelines state that individual components from the endoplasmic reticulum, Golgi, mitochondrial, or nuclear compartments may be sorted into exosomes, especially in pathologic conditions that could affect protein incorporation [36].
Other studies considered the existence of subpopulations of exosomes that were obtained from mouse B16-F10 melanoma conditioned cell culture media using ultracentrifugation with sucrose density gradient [29]. Low-and high-density exosomes, alongside ectosome fraction for comparison, were subjected to MS analysis that allowed identification of 4,421 proteins in total. As many as 1,540 proteins were shared by all 3 populations, while 533, 354, and 110 proteins were identified exclusively in ectosomes, low-density exosomes, and high-density exosomes, respectively. Although exosomal markers (ALIX, CD9, CD63, and CD81) were detected in all subpopulations, their relative abundance was higher in both exosome subpopulations, as compared to ectosomes. The potential functional differences between the proteins identified across EV populations were revealed by GO analysis. Categories such as "oxidoreductases" and "dehydrogenases" were enriched in ectosomes and low-density exosomes, but not in high-density exosomes, suggesting the presence of mitochondrial components in ectosomes and low-density exosomes. In contrast, high-density exosomes showed higher enrichment within the categories of "translation", "ribosomal proteins", and "ribonucleoproteins". Considering proteins identified in only one of EV subpopulations, low-density exosomes, were enriched in "small GTPase regulatory activity"-and "vesicle mediated transport"-related proteins, while high-density exosomes did not show the enrichment in any of GO categories, this suggests that low-and high-density exosomes represent two distinct subpopulations with unique protein compositions [29]. Despite the enrichment of high-density exosomes in ribonucleoproteins, questions may be raised as to whether this subpopulation originates from the remnants of ribosomal aggregates, e.g., from ruptured cells. Since ribosomes and mitochondria have similar densities to exosomes, they can be co-isolated, therefore the presence of exemplary mitochondrial or ribosomal markers should be analyzed in similar studies.
Finally, in vivo studies using cell culture-derived EVs come with certain limitations in terms of providing precise cellular conditions that would mimic the actual tumor microenvironment. For instance, the extracellular pH in the central region of tumors decreases because of lactate accumulation, while regular medium change in cell culture prevents the environment from getting acidic. Boussadia et al. [30] performed quantitative proteomic analysis of Mel501-derived exosomes obtained from cell cultures grown in standard conditions and in pH artificially lowered to pH 6.0. After 1D SDS-PAGE and in-gel trypsinization of the obtained bands 186 and 157, proteins were identified in control and pH 6.0 exosomes, respectively. Based on emPAI indices, the list of up-and down-regulated proteins in pH 6.0 exosomes was generated and subjected to functional annotation analysis using the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database. Exosomes released under acidic conditions showed a significant enrichment within categories such as "proteoglycans", "focal adhesion", and "protein processing in endoplasmic reticulum", while proteins involved in melanogenesis were down-regulated, suggesting the increased aggressiveness and reduced melanin content of Mel501 cells caused by pH 6.0. Subsequent GO analysis confirmed pH 6.0-associated abundance of proteins closely related to melanoma cell migration and with ability to influence melanoma malignancy. Enriched categories included cell-cell adhesion, leukocyte migration, regulation of cell shape, small GTPase mediated signal transduction, and epidermal growth factor receptor (EGFR) signaling pathways [30].

Clinical Significance of Proteomic Studies on Melanoma-Derived EVs
Despite the extensive research, the number of protein biomarkers with adequate clinical values in melanoma cells is still very limited. Development of quantitative proteomic studies on EVs isolated from different body fluids of melanoma patients holds promise for identification and validation of novel cancer cell specific biomarker candidates. Nevertheless, so far only a few comprehensive proteomic studies on melanoma secretomes have used patients' samples. Firstly, the levels of established melanoma biomarkers, namely MIA, S100B, and tyrosinase-related protein 2 (TYRP2), were compared between exosomes isolated from the sera and total serum samples of stage IV melanoma patients and healthy controls. Higher concentrations of MIA and S100B were revealed in exosomes obtained from cancer-affected individuals, suggesting their potential diagnostic and prognostic utility [42]. Other studies showed higher levels of cathepsin D and gp100 in sera of patients with uveal melanoma compared to healthy volunteers' sera [38]. Also, in a more recent study four melanocytic and fourteen primary uveal melanoma cell cultures were established from either cadaver (normal ocular melanocytes) or patient specimens (melanoma). The 2D SDS-PAGE LC-MS comparative analyses of their respective secretomes revealed 163 proteins differentially expressed between secretomes of melanoma and non-melanoma origin, with the levels of MIA and GDF15 (growth/differentiation factor 15 precursor protein) showing the most significant increase [25]. Although no particular population of EVs from sera was isolated in the last two studies mentioned above, it would be worth exploring whether these levels are reflected in sera-derived EVs. On the other hand, proteins that do not show significant enrichment or depletion in the entire sera or cell culture secretome may still be differentially expressed in particular populations of EVs isolated therefrom, and further evaluated as potential biomarkers.
Because of such limited data of clinical origin, the previously mentioned findings from the in vivo studies should be thoroughly reviewed. Future research could focus on biomarker candidates that were revealed in EVs released by melanoma cells, which could be used to direct the studies using patients' samples in order to verify whether their differentiating levels are reflected at the organism level and are indeed clinically relevant. In that respect, apoptotic bodies released by mouse B16-F1 melanoma cells showed enrichment of the melanoma tumor-associated premelanosome protein (PMEL) [24]. Another proteomic study identified a set of proteins within exosome released by Mel501 cells, i.e., proto-oncogenes (HRAS, NRAS), tissue inhibitor of metalloproteinase 3 (TIMP3), heath shock protein isoforms (HSP90AB1, HSP90B1, HSPAIL, HSPA5), glucosidase II α subunit (GANAB), and actin-binding proteins (gelsolin, cofilin), all of which were previously found up-regulated in melanoma patients and associated with poor prognosis [30].
EVs are also carriers of established melanoma type-specific antigens. Using LC-MS, Lazar et al. [28] demonstrated the selective presence of melanoma-associated antigen 4, melanoma-associated antigen B2, and melanoma antigen recognized by T-cells in exosomes released by 7 different melanoma cell lines, suggesting that chosen exosomal proteins may prove useful in distinguishing between different melanoma subtypes. The same study also revealed the presence of proteins belonging to melanoma oncogenic pathways, including proto-oncogenes (NRAS, tyrosine-protein kinases Src, c-Met, and c-Kit), epidermal growth factor receptor (EGFR), and melanoma cell adhesion molecule (MCAM), which may all be involved in early melanoma development. Also, melanocyte-specific premelanosome protein (PMEL) was found by proteomic studies in exosomes derived from several melanoma cancer cell lines [43]. As access to deeper blood vessels is not achieved by non-transformed melanocytes unless vertical tumor growth occurs, circulating (plasma) melanocyte-specific exosomal protein markers could serve as an early indication of invasive melanoma growth. Finally, melanoma-derived exosomes were shown to contain a number of immunosuppressive proteins, such as galectins (LGALS1 and LGALS3) and 5 -nucleotidase (NT5E). Therefore, they may favor tumor escape from immune surveillance, which may contribute to, and explain, the ineffectiveness of some immunotherapies [28].
Unfortunately, so far only one clinical study (clinicaltrials.gov identifier: NCT02439008), exploring melanoma-derived exosomes as a source of potential protein biomarkers of radio-and immunotherapy response has been registered. This is in contrast to numerous proteomic studies in clinical settings, which follow different types of cancer. For instance, proteomic analysis of urinary EVs from prostate cancer patients revealed several markers for different cancer stages based on Gleason scores [44], while other studies identified changes in expression of 14 proteins correlated with glioblastoma invasiveness [45]. The diagnostic and prognostic potential of melanoma-derived EVs has also been demonstrated by a proteomic study [15]. A specific highly-expressed cargo (including Met oncoprotein) was identified in exosomes from metastatic B16-F10 melanoma cells in comparison to exosomes from less invasive B16-F1 cells. Functional in vivo studies show a direct contribution of exosomal Met in the education of bone marrow progenitor cells in establishing pre-metastatic niches in the lung and bone. Following this finding, exosomes from patients' sera were also subjected to proteomic analysis and revealed a melanoma-specific exosomal protein signature that may potentially be used in disease staging and prognostics. With regard to breast and pancreatic cancer, proteomic analysis revealed specific protein profiles for exosomes derived from organotropic metastatic cancer cells [46]. When applied to melanoma EVs, such an approach may allow prediction of organ-specific sites of melanoma metastasis based on EV proteomic profiles.
Finally, EVs are also widely used as drug delivery systems (DDSs) [47]. Recently, attempts have been made to create artificial vesicles that would mimic the properties of natural EVs. The proteomic approach was recently used to compare protein cargo of natural neuroblastoma exosomes and exosome-mimetic vesicles derived from neuroblastoma cells by simple extrusion [48]. Proteomic data revealed a subset of proteins that were abundant in artificial vesicles in comparison to natural exosomes. Artificial vesicles contained proteins representing the whole parental cell proteome, whereas the exosomal protein set reflected their endosomal origin. This property of extrusion-made vesicles may alleviate the need for endosomal sorting of endogenous therapeutic proteins into exosomes, potentially also in melanoma.

Post-Transitional Modifications of Melanoma-Derived EVs: A Future Perspective
Proteomic techniques may also be applied to study post-translational modifications (PTMs) of proteins from melanoma-derived EVs. PTMs may influence several aspects of EV biology, including their biogenesis, protein sorting and localization, physical stability, and pathophysiologal action towards recipient cells. Glycosylation is the most common PMT and specific changes in glycosylation are considered the major hallmarks of cancer. Studies using Western Blot, flow cytometry, and lectin microarrays revealed the enrichment of melanoma-derived exosomes in high mannose, polylactosamine, α-2,6 sialic acid, and complex N-linked glycans [49,50], with the latter being directly involved in exosomal protein sorting [50]. Additionally, ectosomes were shown to be enriched mainly with fucose and complex type N-glycans with bisecting N-acetylglucosamine residues, which was demonstrated in our recent lectinomic study [32].
Proteomic techniques have not been applied to study PTMs in melanoma-derived EVs so far. However, glycosylation of EVs released by ovarian cancer cells has been analyzed by MS, which revealed the enrichment of heavily glycosylated tumor-associated galectin-3-binding protein G (LGALS3BP) in EVs [51]. Another MS-based study described the shift from high-mannose-type N-glycans to complex-type N-glycans in urinary EVs of patients with galactosemia [52]. Such changes in glycosylation with regards to particular disease states underline the importance of their analysis in the context of melanoma-derived EVs. In contrast to Western Blot, flow cytometry, or lectin microarray, which only confirm the presence of a given glycoepitope, MS-based analysis allows matching of specific glycan structures to proteins that possess them. Therefore, identification of particular proteins with aberrant glycosylation within EVs by MS should become a considerable direction in melanoma biomarker discovery.
Besides glycosylation, the presence of another PTMs in melanoma-derived EVs should be investigated. For instance, phosphorylation plays an important role in intercellular signaling and has already been addressed in the context of EVs by MS-based study. Gonzales et al. [53] identified 14 phosphoproteins in urinary exosomes from healthy donors, and multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter protein. In an actual clinical context, label-free quantitative proteomic studies recently revealed 144 phosphoproteins that were differentially expressed in EVs from sera of breast cancer patients and healthy donors [54], indicating the need for similar studies on EVs derived from other types of cancer, including melanoma. Also, single proteomic studies reported the presence of ubiquitinated, sumoylated, oxidated, and myristoylated proteins in different types of EVs. The presence of these modifications may also affect protein sorting, EV aggregation, or their binding to the recipient cells, and has not been evaluated in melanoma-derived EVs so far [55].

Conclusions
A significant amount of data obtained from proteomic studies on melanoma-derived EVs provides a valuable background for identification of novel melanoma biomarkers and for their adjustment towards clinical use. Comprehensive proteomic characterization could be also used as a starting point for further studies on the molecular mechanisms leading to melanoma development and progression, and on the identification of the molecular pathways altered in melanoma subtypes, which could explain the various responses to the existing therapeutic treatments for this cancer. Nevertheless, the studies based on the proteomic analyses of melanoma-derived EVs only create the premise for the future prospective, large, independent, and multicenter trials that are necessary for the validation of any potential biomarker of cancer occurrence, tumor progression. or metastasis. However, the results of the already published studies are promising and suggest that these investigations should be continued in the future for melanoma, a disease for which effective biomarkers are lacking. Unfortunately, unlike well-explored methods for EV isolation and size characterization, the protocols for proteomic studies on melanoma-derived EVs are in the early stages of development. The few related studies that have been published so far used significantly different approaches, therefore, basic procedures for such experiments are still in need of normalization and verification. Funding: Writing of this review was supported by the Jagiellonian University (K/ZDS/008062 and K/DSC/005541).

Conflicts of Interest:
The authors declare no conflict of interest.