Regulation of cargo selection in exosome biogenesis and its biomedical applications in cancer

Abstract

Extracellular vesicles (EVs), including exosomes, are increasingly recognized as potent mediators of intercellular communication due to their capacity to transport a diverse array of bioactive molecules. They assume vital roles in a wide range of physiological and pathological processes and hold significant promise as emerging disease biomarkers, therapeutic agents, and carriers for drug delivery. Exosomes encompass specific groups of membrane proteins, lipids, nucleic acids, cytosolic proteins, and other signaling molecules within their interior. These cargo molecules dictate targeting specificity and functional roles upon reaching recipient cells. Despite our growing understanding of the significance of exosomes in diverse biological processes, the molecular mechanisms governing the selective sorting and packaging of cargo within exosomes have not been fully elucidated. In this review, we summarize current insights into the molecular mechanisms that regulate the sorting of various molecules into exosomes, the resulting biological functions, and potential clinical applications, with a particular emphasis on their relevance in cancer and other diseases. A comprehensive understanding of the loading processes and mechanisms involved in exosome cargo sorting is essential for uncovering the physiological and pathological roles of exosomes, identifying therapeutic targets, and advancing the clinical development of exosome-based therapeutics.

Fig. 1. Mechanisms of extracellular vesicle (EV) biogenesis and EV components.

EV biogenesis occurs through multiple pathways, including through multivesicular bodies (MVBs) within endosomes, which leads to the secretion of exosomes, and through the plasma membrane, which results in microvesicle (MV) generation. After endocytosis, early endosomes undergo maturation in MVBs, during which they form intraluminal vesicles (ILVs) through inward membrane budding. MVBs either fuse with lysosomes for degradation or dock at the cell periphery for exosome secretion, which is facilitated by the RAB GTPases and SNARE complexes. Other types of EVs include apoptotic bodies, which are released during apoptosis via membrane budding; migrasomes, which originate from retraction fibers and contain internal vesicles; and vesicles, which are formed from amphisomes and result from the fusion of outer autophagosome membranes with late-endosomes. Exosomes carry diverse macromolecules, including signaling proteins, transcriptional regulators, various RNA species, DNA, and lipids.

Fig. 2. Exosome biogenesis mechanisms and cargo sorting pathways.

Multiple molecular mechanisms of intraluminal vesicle (ILV) generation in multivesicular bodies (MVBs) have been revealed. a The classical ESCRT-dependent pathway involves the recognition of ubiquitinated proteins in the endosomal membrane by the ESCRT-0, -I, -II, and -III subcomplexes. The ATPase VPS4 cooperates in a stepwise manner to mediate ILV formation. b In the syndecan-syntenin-ALIX pathway, membrane budding and cargo clustering can occur independently of the early ESCRT machinery, with VPS4 required for the scission step. c Ceramide, which is generated from sphingomyelin by nSMase2, plays a key role in the ESCRT-independent pathway of ILV biogenesis. Ceramide can form lipid raft microdomains and trigger the conversion of ILVs into MVBs. nSMase2 is activated by FAN and can be pharmacologically inhibited by small molecules such as GW4869. The cargoes sorted through this pathway include flotillin, cholesterol, and tetraspanins, which are localized to lipid rafts.

Fig. 3. Exosome engineering strategies for loading cancer therapeutic cargo into exosomes.

Exosomes can be engineered to target internal and modified surface cargoes for cancer therapy. Cargo loading strategies are achieved by incubating therapeutic agents (e.g., pharmacological inhibitors, miRNAs, and recombinant proteins) directly with isolated exosomes (post-loading) or by exposing them to exosome-secreting donor cells, followed by the isolation of loaded exosomes (pre-loading). Post-loading methods require physical treatments to disrupt membrane integrity and allow the cargo to enter the interior of exosomes. Alternatively, exosome-producing cells with genetic expression constructs that encode therapeutic cargoes linked to an exosome sorting domain can be generated. This leads to sorting therapeutic cargo into exosomes. Modified exosomes containing tumor antigens can stimulate antigen-presenting cells and drive antitumor immune responses in the human body. Engineered exosomes can also directly release antitumor cargo to attack cancer cells.