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.

Keywords: biomarkers; cutaneous melanoma; ectosomes; extracellular vesicles; invasion; metastasis; proteomics.

Figure 1

Analysis of ectosome sample purity. (A) TEM analysis of cutaneous melanoma (CM)-derived ectosomes. Size distributions are presented on histograms. Mean diameter ± standard deviation was calculated for all observed vesicles (n) from a given sample. (B) Nanoparticle Tracking Analysis (NTA) analysis of CM-derived ectosomes. Results from five independent measurements for each CM cell line are presented on graphs. The shaded area depicts standard deviation. (C) Western blot analysis of extracellular vesicle (EV) markers. Fifty μg of proteins from whole-cell protein extracts (lines C) and ectosome samples (lines E) separated by 10% SDS-PAGE and transferred into PVDF membrane were probed with anti-CD63 (1:2000), anti-HSP70 (1:2000) and anti-ARF6 (1:500) as primary antibodies and anti-mouse IgG-HRP (1:400) as a secondary antibody. WM115 (primary) and WM266-4 (metastatic), cell lines originating from the same individual, radial/vertical growth phase and lymph node metastasis, respectively; primary WM793 cell line, representing the vertical growth phase; WM1205Lu, a metastatic variant of WM793 cells obtained from lung metastasis.

Figure 2

(A) Number of proteins identified in two biological replicates of each CM ectosome sample by at least two peptides. (B) Venn diagram illustrating the number of proteins shared between given ectosome samples. (C) Percentage of proteins shared between given ectosome samples. Venn diagrams illustrating the number of proteins shared between ectosomes released by isogenic (D) and primary or metastatic CM cells (E). Venn diagram illustrating protein overlap between CM ectosomes and Vesiclepedia database as a reference (F). Ectosomes were isolated from WM115 (primary) and WM266-4 (metastatic) cell lines originating from the same individual, radial/vertical growth phase and lymph node metastasis, respectively; primary WM793 cell line, representing vertical growth phase; and WM1205Lu, a metastatic variant of WM793 cells obtained from lung metastasis.

Figure 3

Gene ontology (GO) analysis of CM ectosomal proteins from four different cell lines performed with the use of FunRich 2.0 software with UniProt (release 2019_11) database as a reference. For each GO term (“Cellular compartment” (A), “Molecular function” (B), “Biological process” (C)), six categories with the highest statistical significance of protein enrichment within the specific category (p < 0.001) were presented on graphs. Ectosomes were isolated from WM115 (primary) and WM266-4 (metastatic) cell lines originating from the same individual, radial/vertical growth phase and lymph node metastasis, respectively; primary WM793 cell line, representing vertical growth phase; and WM1205Lu, a metastatic variant of WM793 cells obtained from lung metastasis.

Figure 4

Gene ontology annotations by the biological process for each CM ectosome sample performed with the use of FunRich 2.0 software via UniProt (release 2019_11) database. Numbers of proteins within the chosen cancer-related categories (with statistical significance of protein enrichment within the given category calculated as −log10(p value)) are shown on the heat map. Ectosomes were isolated from WM115 (primary) and WM266-4 (metastatic) cell lines originating from the same individual, radial/vertical growth phase and lymph node metastasis, respectively; primary WM793 cell line, representing vertical growth phase; and WM1205Lu, a metastatic variant of WM793 cells obtained from lung metastasis.

Figure 5

Gene ontology annotations by the biological process for each CM ectosome sample performed via UniProt (release 2019_11) database. Percentages of proteins within the chosen cancer-related categories for ectosomes from all or metastatic cell lines are presented on the graph. Ectosomes were isolated from WM115 (primary) and WM266-4 (metastatic) cell lines originating from the same individual, radial/vertical growth phase and lymph node metastasis, respectively; primary WM793 cell line, representing vertical growth phase; and WM1205Lu, a metastatic variant of WM793 cells obtained from lung metastasis.

Figure 6

Effect exerted by self-derived ectosomes and ectosomes released by their isogenic metastatic cells, on the motility of primary WM115 and WM793 cells. Wound healing assay was performed after 18 h of incubation with ectosomes. (A) Representative images were taken at 0 h and at 18 h. (B) Graphs presenting relative velocity of wound closure calculated from three repetitions. “*” denotes statistically significant differences (Tukey’s post-hoc test, p value < 0.05).

Figure 7

Effect of incubation with self-derived ectosomes and ectosomes released by their isogenic metastatic cells, on proliferation of primary WM115 and WM793 cells. Alamar Blue assay was carried out after 18 h of incubation with ectosomes. All experiments were conducted in triplicate. “*” denotes statistically significant differences (Tukey’s post-hoc test, p value < 0.05).