Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle‐ and exosome‐like populations of KIT‐containing extracellular vesicles

Abstract

Activating mutations in the receptor KIT promote the dysregulated proliferation of human mast cells (huMCs). The resulting neoplastic huMCs secrete extracellular vesicles (EVs) that can transfer oncogenic KIT among other cargo into recipient cells. Despite potential contributions to diseases, KIT‐containing EVs have not been thoroughly investigated. Here, we isolated and characterized KIT‐EV subpopulations released by neoplastic huMCs using an immunocapture approach that selectively isolates EVs containing KIT in its proper topology. Immunocapture of EVs on KIT antibody‐coated electron microscopy (EM) affinity grids allowed to assess the morphology and size of KIT‐EVs. Immunoblot analysis demonstrated KIT‐EVs have a distinct protein profile from KIT‐depleted EVs, contain exosome and microvesicle markers, and are separated into these subtypes by ultracentrifugation. Cell treatment with sphingomyelinase inhibitors shifted the protein content among KIT‐EV subtypes, suggesting different biogenesis routes. Proteomic analysis revealed huMC KIT‐EVs are enriched in proteins involved in signalling, immune responses, and cell migration, suggesting diverse biological functions, and indicated neoplastic huMCs disseminate KIT via shuttling in heterogeneous microvesicle‐ and exosome‐like EVs. Further, selective KIT‐immunocapture will enable the enrichment of specific huMC‐derived EVs from complex human biosamples and facilitate an understanding of their in vivo functions and potential to serve as biomarkers of specific biological pathologies.

Endogenous Alpha-Synuclein is Essential for the Transfer of Pathology by Exosome-Enriched Extracellular Vesicles, Following Inoculation with Preformed Fibrils in vivo

“…2F ). Of note, proteins of organelles that are not related to exosomes and extracellular vesicles in general, such as laminin B1 (LMNB1, nucleus), ribosomal protein S6 and L7 (RSP6, RPL7, ribosome), Golgi matrix protein 130 (GM130, Golgi), carlticulin (CARL, ER) and others [ 49 ], [ 50 ], were not amongst the detected proteins ( Supplementary Table 1 ), highlighting the lack of contaminants and further validating the enrichment of the described brain-derived preparation in exosomes. Moreover, tissue expression analysis pinpointed the hippocampus, brain and brain cortex as the top tissues of origin, further verifying the source-specificity of the isolated ExE-EVs ( Supplementary Fig.…”

A survey of the currently known mast cell mediators with potential relevance for therapy of mast cell-induced symptoms

“…This process is distinct from “piecemeal degranulation” that has additionally been reported (Dvorak 2005 ). MCs can also form synapses for targeted secretion (Table 4 ).With regard to the possibility that, under certain circumstances, almost all molecules that can be produced by a MC might be able to act as mediators, four release options are of particular interest: (1) diffusion of substances into the extracellular space; (2) release of mRNA, microRNA, and proteins expressed in the MC by secretion of exosomes and vesicles (Savage et al 2023 ), some of them containing KIT (Pfeiffer et al 2022 ); (3) formation of nanotubules with exchange of intracellular material which seems to be involved in inducing apoptosis in cancer cells (Ahani et al 2022 ); and (4) formation of MC extracellular traps (Möllerherm et al 2016 ; Table 4). These four mechanisms, by which MCs can use almost any molecule as a mediator, underline the extraordinary role of these cells in our immune system.…”

Extracellular Vesicles as New Players in Drug Delivery: A Focus on Red Blood Cells-Derived EVs

“…Duijvesz and co-workers developed a highly sensitive time-resolved fluorescence immunoassay (TR-FIA) for the capture/detection of EVs using anti-human CD9- or CD63-coated plates [ 98 ]. The high specificity of this approach is generally recognized, but, on the other hand, it is limited by the availability of reliable and cost-effective monoclonal antibodies, or aptamers, specific for EV isolation (often targeting tetraspanin proteins) and also by the necessity of a mild elution method that preserves EV integrity [ 99 , 100 , 101 , 102 ].…”