Natural and Bioengineered Extracellular Vesicles in Diagnosis, Monitoring and Treatment of Cancer

Abstract

Extracellular vesicles (EVs) are cell derived nanovesicles which are implicated in both physiological and pathological intercellular communication, including the initiation, progression, and metastasis of cancer. The exchange of biomolecules between stromal cells and cancer cells via EVs can provide a window to monitor cancer development in real time for better diagnostic and interventional strategies. In addition, the process of secretion and internalization of EVs by stromal and cancer cells in the tumor microenvironment (TME) can be exploited for delivering therapeutics. EVs have the potential to provide a targeted, biocompatible, and efficient delivery platform for the treatment of cancer and other diseases. Natural as well as engineered EVs as nanomedicine have immense potential for disease intervention. Here, we provide an overview of current knowledge of EVs' function in cancer progression, diagnostic and therapeutic applications for EVs in the cancer setting, as well as current EV engineering strategies.

Keywords: Extracellular vesicles (EVs); cancer; cancer immunotherapy; cargo loading methods; diagnosis; drug delivery; immune microenvironment; large scale production; monitoring; tumor microenvironment (TME).

Figure 1.. Subtypes of EVs and their biogenesis.

(A-B) EV subtypes (A) and secretion and biogenesis of ectosomes and exosomes (B). (A) EVs are broadly classified into two categories, ectosomes and exosomes. Other subtypes of EVs such as migrasomes and extracellular secreted particles (ESPs) have been proposed. (B) Plasma membrane budding generates ectosomes or microvesicles ranging in diameter from 50 nm to 1000 nm and include small EVs (40–200 nm), medium EVs (200–500 nm), large EVs/large oncosomes (>500 nm), and apoptotic bodies (derived from apoptotic cells, 500–5000 nm). Exosomes are generated from the endosomal pathway ranging in diameter from 40 nm to 200 nm. Extracellular molecules are endocytosed into cells, forming early sorting endosomes (ESEs), where cargo exchange and membrane fusion with endoplasmic reticulum and Golgi apparatus potentially occurs. ESEs then mature to late sorting endosomes (LSEs), where vesicles are formed in the lumen by a second membrane invagination for the generation of intraluminal vesicles (ILVs). Cargos of ILVs come from endoplasmic reticulum (ER), Golgi apparatus, other cytosolic molecules, as well as extracellular molecules. LSEs give rise to multiple vesicular bodies (MVBs), which can fuse with autophagosomes leading to the degradation of cargos in lysosomes or fuse with lysosomes directly. The digested products can be recycled by the cells. MVBs not fused with autophagosomes or lysosomes can be transferred to and fused with plasma membrane for the secretion of ILVs as exosomes. Created with BioRender.com.

Figure 2.. Multicomponent biomarker analysis employing cancer relevant EVs and the influence of EVs in cancer relevant processes.

EVs from tumors facilitate the 1) metabolism of cancer cells and stromal cells for their survival, proliferation, and other cellular functions; 2) angiogenesis in the stroma to supply oxygen and nutrients for tumor growth; 3) proliferation of cancer cells; 4) reprogramming of immune microenvironment, such as M1 macrophages to M2 macrophages polarization and T cell exhaustion/decreased T cell infiltration; 5) signaling to the tumor microenvironment to impact tumor progression; 6) formation of pre-metastatic niches in distant organs for increased tumor metastasis. Cargos carried by EVs can be DNA, mRNA, miRNA, proteins or peptides, enzymes, lipids, etc. Created with BioRender.com.

Figure 3.. Bioengineered EVs to present cargo on the surface, lumen, or both.

(A-B) Two major engineering strategies for loading therapeutic cargos to EVs: exogenous bioengineered EVs (A) and endogenous bioengineered EVs (B). (A) Exogenous loading method includes 1) passive diffusion; 2) electroporation under an electric field; 3) sonification with ultrasound; 4) hybridization with liposomes; 5) chemical modulation for the conjugation of molecules via chemical groups. (B) Endogenous loading method refers to the modification of parental cells and generation of EVs from there. The modification of parental cells including 1) cellular nanoporation; 2) DNA or 3) mRNA transfection of plasmids into cells; 4) passive diffusion to cells. Cargos loaded into EVs can be DNA, mRNA, proteins or peptides, chemotherapies, etc. Delivery of plasmid DNA to cells results in incorporation of plasmid DNA and the encoded mRNA and protein (either a surface protein or cytoplasmic protein) into EVs. Similarly, mRNA transfection leads to mRNA and the translated protein (either a surface protein or cytoplasmic protein) in EVs. Created with BioRender.com.

Figure 4.. Large-scale EV isolation methods.

Reported methods for large-scale EV isolation include differential ultracentrifugation and density gradient ultracentrifugation, tangential flow filtration, and fast-protein liquid chromatography, consisting of size exclusion, affinity, and ion exchange chromatography. Such methods enable separation of EVs based on their biophysical properties such as size, density, surface markers, and surface charge. Created with BioRender.com.

Figure 5.. Architectural diversity of EVs and potential impacts on EV biodistribution.

EVs demonstrate morphological, charge, and size diversity. Examples of such diversity are included in the figure. Based on what has been reported for synthetic nanoparticles, it is possible that the architecture of EVs can shape their biodistribution. Potential biodistribution based on EV architecture is depicted. Created with BioRender.com.