Insight into Extracellular Vesicle-Cell Communication: From Cell Recognition to Intracellular Fate.

Extracellular vesicles (EVs) are heterogamous lipid bilayer-enclosed membranous structures secreted by cells. They are comprised of apoptotic bodies, microvesicles, and exosomes, and carry a range of nucleic acids and proteins that are necessary for cell-to-cell communication via interaction on the cells surface. They initiate intracellular signaling pathways or the transference of cargo molecules, which elicit pleiotropic responses in recipient cells in physiological processes, as well as pathological processes, such as cancer. It is therefore important to understand the molecular means by which EVs are taken up into cells. Accordingly, this review summarizes the underlying mechanisms involved in EV targeting and uptake. The primary method of entry by EVs appears to be endocytosis, where clathrin-mediated, caveolae-dependent, macropinocytotic, phagocytotic, and lipid raft-mediated uptake have been variously described as being prevalent. EV uptake mechanisms may depend on proteins and lipids found on the surfaces of both vesicles and target cells. As EVs have been shown to contribute to cancer growth and progression, further exploration and targeting of the gateways utilized by EVs to internalize into tumor cells may assist in the prevention or deceleration of cancer pathogenesis.

The Role of Extracellular Vesicles in Metabolic Reprogramming of the Tumor Microenvironment.

The tumor microenvironment (TME) includes a network of cancerous and non-cancerous cells, together with associated blood vessels, the extracellular matrix, and signaling molecules. The TME contributes to cancer progression during various phases of tumorigenesis, and interactions that take place within the TME have become targets of focus in cancer therapy development. Extracellular vesicles (EVs) are known to be conveyors of genetic material, proteins, and lipids within the TME. One of the hallmarks of cancer is its ability to reprogram metabolism to sustain cell growth and proliferation in a stringent environment. In this review, we provide an overview of TME EV involvement in the metabolic reprogramming of cancer and stromal cells, which favors cancer progression by enhancing angiogenesis, proliferation, metastasis, treatment resistance, and immunoevasion. Targeting the communication mechanisms and systems utilized by TME-EVs is opening a new frontier in cancer therapy.

Extracellular vesicle fusion visualized by cryo-electron microscopy.

Extracellular vesicles (EVs) transfer bioactive molecules between cells in a process reminiscent of enveloped viruses. EV cargo delivery is thought to occur by protein-mediated and pH-dependent membrane fusion of the EV and the cellular membrane. However, there is a lack of methods to identify the fusion proteins and resolve their mechanism. We developed and benchmarked an in vitro biophysical assay to investigate EV membrane fusion. The assay was standardized by directly comparing EV and viral fusion with liposomes. We show that EVs and retroviruses fuse with liposomes mimicking the membrane composition of the late endosome in a pH- and protein-dependent manner. Moreover, we directly visualize the stages of membrane fusion using cryo-electron tomography. We find that, unlike most retroviruses, EVs remain fusogenic after acidification and reneutralization. These results provide novel insights into the EV cargo delivery mechanism and an experimental approach to identify the EV fusion machinery.

The Purification and Characterization of Exosomes from Macrophages.

Macrophages play an essential role in diverse biological processes, from the immune response to inflammatory and neurodegenerative disorders, to various cancers. A macrophage subpopulation, known as tumor-associated macrophages (TAMs), has been shown to promote tumorigenesis, metastasis, and immune escape of cancer cells. Some of the pro-tumorigenic effects of TAMs are mediated via the secretion of nano-vesicles (exosomes) from macrophages to neighboring cells. In this chapter, we describe peritoneal macrophage isolation methods, polarization of TAMs, and purification and characterization of macrophage-derived exosomes.

Exosomes and their role in tumorigenesis and anticancer drug resistance.

Exosomes are a class of extracellular vesicles ranging in size from 40 to 100 nm, which are secreted by both cancer cells and multiple stromal cells in the tumor microenvironment. Following their secretion, exosomes partake in endocrine, paracrine and autocrine signaling. Internalization of exosomes by tumor cells influences several cellular pathways which alter cancer cell physiology. Tumor-derived exosomes secreted by cancer or stromal cells can also confer anticancer drug-resistant traits upon cancer cells. These exosomes promote chemoresistance by transferring their cargo which includes nucleic acids, proteins, and metabolites to cancer cells or act as a decoy for immunotherapeutic targets. Depletion of exosomes can reverse some of the detrimental effects on tumor metabolism and restore drug sensitivity to chemotherapeutic treatment. Herein we discuss various approaches that have been developed to deplete exosomes for therapeutic purposes. The natural composition, low immunogenicity and cytotoxicity of exosomes, along with their ability to specifically target tumor cells, render them an appealing platform for drug delivery. The ability of exosomes to mediate autocrine and paracrine signaling in target cells, along with their natural structure and low immunogenicity render them an attractive vehicle for the delivery of anticancer drugs to tumors.