Mass Spectrometry-Based Proteomic Characterization of Cutaneous Melanoma Ectosomes Reveals the Presence of Cancer-Related Molecules

Abstract

Cutaneous melanoma (CM) is an aggressive type of skin cancer for which effective biomarkers are still needed. Recently, the protein content of extracellular vesicles (ectosomes and exosomes) became increasingly investigated in terms of its functional role in CM and as a source of novel biomarkers; however, the data concerning the proteome of CM-derived ectosomes is very limited. We used the shotgun nanoLC–MS/MS approach to the profile protein content of ectosomes from primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cell lines. Additionally, the effect exerted by CM ectosomes on recipient cells was assessed in terms of cell proliferation (Alamar Blue assay) and migratory properties (wound healing assay). All cell lines secreted heterogeneous populations of ectosomes enriched in the common set of proteins. A total of 1507 unique proteins were identified, with many of them involved in cancer cell proliferation, migration, escape from apoptosis, epithelial–mesenchymal transition and angiogenesis. Isolated ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of different cancer-promoting molecules. Taken together, these results confirm the significant role of ectosomes in several biological processes leading to CM development and progression, and might be used as a starting point for further studies exploring their diagnostic and prognostic potential.

uPAR-expressing melanoma exosomes promote angiogenesis by VE-Cadherin, EGFR and uPAR overexpression and rise of ERK1,2 signaling in endothelial cells

“…1c) showed several components with different dimension. The mean size of this distribution was 291.5 ± 4.3 nm according to the previous observations [31]. TEM analysis revealed, as evident in Fig.…”

Proteomic Profiling of Ectosomes Derived from Paired Urothelial Bladder Cancer and Normal Cells Reveals the Presence of Biologically-Relevant Molecules

“…So far, similar studies have not been conducted on the latter population of EVs. Nevertheless, ectosomes can also contain a specific protein profile enriched in cancer biomarkers; this was demonstrated in our previous studies for melanoma [19,20] as well as other studies on breast [21], head and neck [22], and ovarian cancer [23]. In the present study we used a liquid chromatography coupled with tandem mass spectrometry-based (nanoLC-MS/MS) proteomic approach to investigate the protein content of ectosomes released in vitro by T-24 UBC and HCV-29 normal ureter epithelial cells.…”

Low-Vacuum Filtration as an Alternative Extracellular Vesicle Concentration Method: A Comparison with Ultracentrifugation and Differential Centrifugation

“…We found that LVF samples had the highest intensity of Hsp70 and CD63 bands, while the Arf-6 and CD81 bands had similar low intensity in the samples isolated by all tested methods. Ectosome samples are usually enriched in Arf-6, the protein involved directly in the shedding of plasma membrane-derived EVs, but not in exosome biogenesis [ 24 ]. In our study, relatively low expression of Arf-6 in comparison to the exosome marker CD63 was observed, showing that exosome proteins dominated in all samples.…”

“…Differential centrifugation was performed according to the previously described protocol [ 24 ]. After the three preparatory centrifugations, samples were transferred to 50 (mL) polycarbonate centrifuge tubes and centrifuged for 20 (min) at 18,000 (g) at 4 (°C) (Sorvall LYNX 6000 Superspeed Centrifuge, Thermo Fisher Scientific, Waltham, MA, USA).…”