Secreted primary human malignant mesothelioma exosome signature reflects oncogenic cargo

Abstract

Malignant mesothelioma (MM) is a highly-aggressive heterogeneous malignancy, typically diagnosed at advanced stage. An important area of mesothelioma biology and progression is understanding intercellular communication and the contribution of the secretome. Exosomes are secreted extracellular vesicles shown to shuttle cellular cargo and direct intercellular communication in the tumour microenvironment, facilitate immunoregulation and metastasis. In this study, quantitative proteomics was used to investigate MM-derived exosomes from distinct human models and identify select cargo protein networks associated with angiogenesis, metastasis, and immunoregulation. Utilising bioinformatics pathway/network analyses, and correlation with previous studies on tumour exosomes, we defined a select mesothelioma exosomal signature (mEXOS, 570 proteins) enriched in tumour antigens and various cancer-specific signalling (HPGD/ENO1/OSMR) and secreted modulators (FN1/ITLN1/MAMDC2/PDGFD/GBP1). Notably, such circulating cargo offers unique insights into mesothelioma progression and tumour microenvironment reprogramming. Functionally, we demonstrate that oncogenic exosomes facilitate the migratory capacity of fibroblast/endothelial cells, supporting the systematic model of MM progression associated with vascular remodelling and angiogenesis. We provide biophysical and proteomic characterisation of exosomes, define a unique oncogenic signature (mEXOS), and demonstrate the regulatory capacity of exosomes in cell migration/tube formation assays. These findings contribute to understanding tumour-stromal crosstalk in the context of MM, and potential new diagnostic and therapeutic extracellular targets.

Figure 1. Human malignant mesothelioma cell characterisation.

(a) Phase contrast images of human MM cells JO38, JU77, OLD1612, and LO68 reveal elongated fibroblast-like morphology. (b) Cell viability as determined by the Trypan Blue dye exclusion assay after culture in presence of 5% FCS (foetal calf serum) or 0.5% ITS (insulin-transferrin-selenium) conditions for 24 hr, demonstrating that all cell lines remain >92% viable. Data representative of mean ± SEM of three independent experiments performed in triplicate.

Figure 2. Isolation and characterisation of MM-derived exosomes.

(a) Workflow showing isolation and purification of exosomes from mesothelioma cell lines using serum-free media (SFM) conditions. Exosomes (10 μg) were solubilised in SDS, separated by 1D-SDS-PAGE and fractions (n = 2) subjected to in-gel reduction, alkylation, and tryptic digestion. Extracted peptides were fractionated and identified using mass spectrometry analysis, data processing database searching, informatics and protein annotation. (b) Protein yield (μg/cell dish) for JO38, JU77, OLD1612, and LO68 exosomes is shown (average n = 3). (c) For Western blotting, exosome preparations (10 μg) were separated by 1D-SDS-PAGE, electrotransferred, and probed with exosome markers Alix/PDCD6IP and TSG101. Data representative of three independent experiments. (d) For transmission electron microscopy, exosomes (2 μg) were negatively stained using uranyl acetate and viewed by transmission EM, revealing a relatively homogenous population of round membranous vesicles 30–150 nm in size for all cell types. Scale bar, 100 nm. Representative image from n = 3 and 5 independent fields of view.

Figure 3. Characterisation of mesothelioma-derived exosomes reveals an exosomal-specific signature (mEXOS).

(a) Principal component analysis (PCA). Each symbol represents a biological replicate, and the colour represents the group (model). (b) A four-way Venn diagram of proteins distributed between each MM-derived exosome type is shown, revealing 631 proteins common to each dataset. (c) To determine the classification of proteins in mEXOS we applied a stringent filtering criteria. The total MM-derived exosomal proteins (2,178) were compared with the Vesiclepedia database (comprising 16,085 human entries) and literature searching, of which 506 were unique to this study and not previously reported in the context of extracellular vesicles. Among the 1,672 co-identified proteins, 42 were non-cancer proteins reported in Vesiclepedia. with a further 22 proteins reported shared with mesothelioma and other cancers in Vesiclepedia. Therefore, to determine the unique MM exosome protein signature (mEXOS), we summated the 506, 42, and 22 proteins from these categories to reveal 570 proteins as select exosomal and MM-derived components (Table S3). (d) KEGG pathway analysis of mEXOS, with p-values indicated. (e) Correlation matrix of mEXOS, representing differential abundance based on normalised spectral count (SpC) values between each of the MM models investigated, showing that each individual sample represents clear distribution and similarity with other MM models.

Figure 4. Validation and pathway analysis of mesothelioma-derived exosome cargo.

(a) To validate the relative abundance of proteins identified using GeLC-MS/MS, Western blot analysis was used to compare expression for selected proteins; glypican-1 (HSPG1/GPC1), mesothelin (MSLN), calreticulin (CALR), and other exosome components protocadherin Fat 1 (FAT1), CD81, and CD70. For Western blotting, exosomes (10 μg) were separated by 1D-SDS-PAGE, electrotransferred, and probed with markers as indicated (n = 2). (b) Relative quantitation of label-free spectral counting (SpC) are shown for selected proteins validated using Western immunoblotting (n = 3). (c) Immune system and immune disease related pathways in MM-derived exosomes, with p-values indicated. (d) Cancer cell biology related pathways in MM-derived exosomes, with p-values indicated. (e) Signal transduction related pathways in MM-derived exosomes, with p-values indicated.

Figure 5. Mesothelioma exosomes regulate recipient cells of the tumour microenvironment.

To explore the potential exosome-mediated regulation of cells in the tumour microenvironment, fibroblasts (MEFs, neoHFFs) and endothelial cells (HUVECs) were exposed to MM cell-derived exosomes and recipient cell function analysed. For recipient cell migration, MEF (a), neoHFF (b) and HUVEC (c) cells were investigated using transwell assays over 24 hr in response to exosomes (30 μg/mL) derived from JO38, JU77, OLD1612, and LO68 cells. Vehicle control (DMEM), in addition to exosome-free control (exosome-depleted) were used. Transversing cells were stained with DAPI, imaged, and counted (n = 3; average ± sem; *p < 0.05, **p < 0.01). Tube formation assays using HUVEC cells (7 × 10⁴), supplemented with MM-derived exosomes (30 μg/mL) and controls and seeded onto Matrigel (1 mg/mL). After 24 hr, tube formation was analysed, imaged, and quantified. Scale bar = 50 μm. (representative images from n = 3, average ± sem; *p < 0.05).