The Analysis of Inflammation-Related Proteins in a Cargo of Exosomes Derived from the Serum of Uveal Melanoma Patients Reveals Potential Biomarkers of Disease Progression

Abstract

Background: Uveal melanoma (UM) is the most common intraocular tumour in adults with a poor prognosis and extremely high mortality rate due to the development of metastatic disease. However, despite relatively good knowledge about the histological and genetic risk factors for metastasis development, there is no specific biomarker that would allow early detection of UM progression. Recently, exosomes and their molecular cargo have been widely studied in the search for potential biomarkers in several cancers. The purpose of this study was to analyze the inflammation-related protein cargo of exosomes derived from the serum of primary and metastatic UM patients and healthy donors.

Methods: The exosomes were isolated from the serum of primary and metastatic UM patients and healthy donors. Using multiplex immunoassay technology, we analyzed the concentration of 37 inflammation-related proteins in obtained exosomes.

Results: The analysis of protein cargo showed several molecules related to inflammation, such as interferon-gamma, interleukin 2, 22 and 12(p40), Pentraxin-3, TNFSF13B and TNFSF8 which were significantly enriched in metastatic UM exosomes. We showed a significant correlation between the disease stage and the concentration of these inflammation-related proteins from exosomal cargo.

Conclusions: Based on the obtained results, we propose the panel of exosomal proteins for early detection of uveal melanoma progression into metastatic disease.

Keywords: biomarkers; exosomes; inflammation-related proteins; uveal melanoma.

Figure 1

The characteristics of exosomes isolated from the serum of uveal melanoma patients with primary and metastatic disease (UM-exo primary and UM-exo metastatic) and healthy donors (HD-exo). The scanning electron microscopy (SEM) showed the proper shape and size (exosomes marked with white arrows) (A). The isolated exosomes indicate high levels of acetylcholinesterase activity. Data represent the mean ± SD from three technical replicates of absorbance readout at 405 nm from the pooled serum (B). The expression of proteins characteristic for small extracellular vesicles—endosomal origin (LAMP1, Alix, CD63), their protein cargo (TGFβ1), the tumour-origin (MLANA), and absence of organelle-specific proteins (Calnexin) was confirmed by Western blot. The band marked with * were obtained from the short-time exposure of the same membrane, due to differences in the protein expression level between the cell line lysate and exosomes (C). The uncropped Western Blot images can be found in Figure S1.

Figure 2

The general comparison of immunomodulatory protein in exosomal cargo in healthy donors and UM patients. All the concentrations are displayed in (pg/mL), only the proteins effectively detected are shown. (A) Heatmap representing mean concentrations of all the analyzed cytokines in HD-exo (n = 20) and UM-exo (n = 20) groups; (B) table showing mean concentrations and p-values indicating the statistical significance; (C) for better visualization of the differences in cytokine concentrations, the heatmap was divided into three panels with a distinct concentration range: 13,000–2000 pg/mL, 1400–100 pg/mL, 100–0 pg/mL. The p-value was calculated with the unpaired t-test or Mann-Whitney test.

Figure 3

Immunomodulatory protein cargo of exosomes from healthy donors and UM patients. All the concentrations are displayed in (pg/mL), only the proteins effectively detected are shown. (A) Heatmap representing mean concentrations of all the analyzed cytokines in HD-exo, primary UM-exo and metastatic-exo groups; (B) table showing mean concentrations and p-values indicating the statistical significance; (C) to better visualize the differences in cytokine concentrations, the heatmap was divided into three panels with concentration range: 13,000–2000 pg/mL, 1400–100 pg/mL, 100–0 pg/mL. The p-value was calculated based on one-way ANOVA (data with normal distribution) or Kruskal–Wallis test (samples without normal distribution).

Figure 4

Box and whiskers plots present the concentration of interleukins and interferons detected in exosomes derived from healthy donors, primary and metastatic UM patients. Results are displayed as a median concentration in picograms per milliliter. The box and whiskers were calculated using the Tukey method. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p > 0.0001: Based on one-way ANOVA or Kruskal–Wallis test with Dunn’s post hoc multiple comparison test.

Figure 5

Box and whiskers plots presenting concentration of TNF superfamily, extracellular matrix and its modifying proteins in exosomes derived from healthy donors, primary and metastatic UM patients. Results are displayed as a median concentration in picograms per milliliter. Box and whiskers were calculated using the Tukey method. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p > 0.0001: based on One-way ANOVA or Kruskal–Wallis test with Dunn’s post hoc multiple comparison test.