En bloc release of MVB‐like small extracellular vesicle clusters by colorectal carcinoma cells

Abstract

Small extracellular vesicles (EVs) are membrane enclosed structures that are usually released from cells upon exocytosis of multivesicular bodies (MVBs) as a collection of separate, free EVs. In this study, we analysed paraffin embedded sections of archived human colorectal cancer samples. We studied 3D reconstructions of confocal microscopic images complemented by HyVolution and STED imaging. Unexpectedly, we found evidence that large, MVB-like aggregates of ALIX/CD63 positive EV clusters were released en bloc by migrating tumour cells. These structures were often captured with partial or complete extra-cytoplasmic localization at the interface of the plasma membrane of the tumour cell and the stroma. Their diameter ranged between 0.62 and 1.94 μm (mean±S.D.: 1.17 ± 0.34 μm). Highresolution 3D reconstruction showed that these extracellular MVB-like EV clusters were composed of distinguishable internal particles of small EV size (mean±S.D.: 128.96 ± 16.73 nm). In vitro, HT29 colorectal cancer cells also showed the release of similar structures as confirmed by immunohistochemistry and immune electron microscopy. Our results provide evidence for an en bloc transmission of MVB-like EV clusters through the plasma membrane. Immunofluorescent-based detection of the MVB like small EV clusters in archived pathological samples may represent a novel and unique opportunity which enables analysis of EV release in situ in human tissues.

S-Adenosylmethionine Treatment of Colorectal Cancer Cell Lines Alters DNA Methylation, DNA Repair and Tumor Progression-Related Gene Expression

“…For immunocytochemistry, HT-29 and SW480 cells were grown in glass coverslips, placed into 6-well plates as described before by Valcz et al [ 27 ]. Following SAM treatment, cells were fixed with 4% paraformaldehyde for 10 min at 4 °C.…”

Exosomes: Their Role in Pathogenesis, Diagnosis and Treatment of Diseases

“…The release of small EVs or exosomes differs from migracytosis, which involves the translocation of cytoplasmic material to migrasomes, followed by their release when the retraction fibers break [ 12 ]. In addition, the uptake of small EVs may have an effect on recipient cells different from that of multivesicular body (MVB)-like EVs, whose release may lead to a relatively delayed effect on the microenvironment [ 13 ]. Molecular composition of exosomes: exosomes are surrounded by a phospholipid bilayer and contain numerous molecules, including proteins, lipids, DNA and several types of RNA.…”

Metabolic wastes are extracellularly disposed by excretosomes, nanotubes and exophers in mouse HT22 cells through an autophagic vesicle clustering mechanism

“…Our study indicated that there is a constitutive microtubule-directed autophagic vesicle clustering process to degrade and to excrete metabolic waste materials in wild type HT22 cells. The extracellular disposal of membrane enclosed MVB-like small vesicle clusters has recently described by a number of studies 33,34 . One of these studies with colorectal carcinoma cells provided evidence 30 that the clustered small vesicle release relates to LC3 and RAB7 signaling 34 .…”

“…The extracellular disposal of membrane enclosed MVB-like small vesicle clusters has recently described by a number of studies 33,34 . One of these studies with colorectal carcinoma cells provided evidence 30 that the clustered small vesicle release relates to LC3 and RAB7 signaling 34 . Cell migration and nanotube-like retraction fiber breakage was proposed as a releasing mechanism for migrasomes, a type of multivesicular extracellular vesicles 33 , which might help to explain the phenomenon of excretosome release at cellular nanotube terminals.…”