Melanoma Affects the Composition of Blood Cell-Derived Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) are specifically loaded with nucleic acids, lipids, and proteins from their parental cell. Therefore, the constitution of EVs reflects the type and status of the originating cell and EVs in melanoma patient’s plasma could be indicative for the tumor. Likewise, EVs might influence tumor progression by regulating immune responses. We performed a broad protein characterization of EVs from plasma of melanoma patients and healthy donors as well as from T cells, B cells, natural killer (NK) cells, monocytes, monocyte-derived dendritic cells (moDCs), and platelets using a multiplex bead-based platform. Using this method, we succeeded in analyzing 58 proteins that were differentially displayed on EVs. Hierarchical clustering of protein intensity patterns grouped EVs according to their originating cell type. The analysis of EVs from stimulated B cells and moDCs revealed the transfer of surface proteins to vesicles depending on the cell status. The protein profiles of plasma vesicles resembled the protein profiles of EVs from platelets, antigen-presenting cells and NK cells as shown by platelet markers, co-stimulatory proteins, and a NK cell subpopulation marker. In comparison to healthy plasma vesicles, melanoma plasma vesicles showed altered signals for platelet markers, indicating a changed vesicle secretion or protein loading of EVs by platelets and a lower CD8 signal that might be associated with a diminished activity of NK cells or T cells. As we hardly detected melanoma-derived vesicles in patient’s plasma, we concluded that blood cells induced the observed differences. In summary, our results question a direct effect of melanoma cells on the composition of EVs in melanoma plasma, but rather argue for an indirect influence of melanoma cells on the vesicle secretion or vesicle protein loading by blood cells.

Immunoaffinity‐based isolation of melanoma cell‐derived exosomes from plasma of patients with melanoma

“…Recent studies of EVs in patients with cancer indicate that these vesicles are heterogeneous, encompassing a mixture of EVs derived from normal and tumour cells [8,17]. Only some of EVs populating all body fluids in these patients are tumour-derived and it is likely that the ratios of tumour cell-derived vs normal cell-derived EVs vary from patient to patient.…”

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures

“…Different sample types were subjected to bead-based multiplex EV analysis by flow cytometry (MACSPlex Exosome Kit, human, Miltenyi Biotec) ( 9 , 10 ), with details regarding sample preparation and normalization summarized in Table S2 in Supplementary Material. Unless indicated otherwise, EV-containing samples were processed as follows: Samples were diluted with MACSPlex buffer (MPB) to, or used undiluted at, a final volume of 120 µL and loaded onto wells of a pre-wet and drained MACSPlex 96-well 0.22 µm filter plate before 15 µL of MACSPlex Exosome Capture Beads (containing 39 different antibody-coated bead subsets) were added to each well.…”

Concentration of extracellular vesicles isolated from blood relative to the clinical pathological status of dogs with mast cell tumours

“…In a study on the impact of melanoma on EV formation, most EVs in the isolates originated from PLTs . Helley et al also demonstrated that the number of EVs originating from PLTs also increased in conjunction with advanced malignant tumour stages in human patients.…”