Introductory Chapter: Role of Extracellular Vesicles in Human Diseases and Therapy

1. Introduction

Extracellular vesicles (EVs) are nanoscale vesicles secreted by cells that mediate horizontal cargo transport from donor to recipient cell, thereby establishing cell-cell communication and signaling [1]. EVs are lipid bilayer-delimited particles released spontaneously by almost all types of cells. The EVs contain cargo, including proteins, nucleic acids, lipids, metabolites, and even organelles, representing the parent cell’s physiological state (Figure 1) [1, 2]. There are three primary subtypes of EVs identified by their size and biological processes; exosomes (~30–150 nm), microvesicles (~100–1000 nm), and apoptotic bodies (~1000–5000 nm). The exosome biogenesis process is quite intriguing where multivesicular bodies (MVBs) are specialized endosomal compartments containing multiple intraluminal vesicles (ILVs). ILVs are generated by the inward budding of endosomal membranes within MVBs and followed by MVBs fusion with the plasma membrane and extracellular exocytosis-based release as exosomes (Figure 2) [3]. Microvesicles are generated by the budding outward of the healthy cell’s plasma membrane. In comparison, apoptotic bodies are plasma membrane blebs of cells that originate during apoptosis. Several EV subgroups have been hypothesized, including ectosomes, microparticles, oncosomes, and others, in addition to the three primary forms, but the dearth of established biomarkers and the lack of standardized isolation techniques have resulted in misconceptions in classifying EV subgroups [4]. This book presents a comprehensive overview of EVs and has three sections: the biology of extracellular vesicles; the second is the role of extracellular vesicles in human diseases, and third is extracellular vesicles and cancer.

Figure 1.

Centre: showing exosome composition, including proteins, lipids, carbohydrates, nucleic acids, mRNA, miRNA, non-coding RNA, and DNA. Proteins in the exosome include heat shock proteins (HSP), cytoskeletal proteins (ESCRT components), membrane transporters, fusion proteins, growth factors and cytokines, Tetraspanins, Flotillin, ligands like TRAIL (TNF-related apoptosis-inducing ligand), FasL (Fas ligand) and receptors like TfR (transferrin receptor). The different methods commonly used for exosome isolation is shown in the yellow ring. Four corners: shows the different sources of exosomes, including bodily fluid, somatic cells, stem cells and diseased.

Figure 2.

Exosome biogenesis: exosome biogenesis initiates with the intraluminal vesicles (ILVs) formation within the multi-vesicular body (MVB), followed by the fusion of MVB with the plasma membrane. After fusion, these ILVs are secreted as exosomes, although some are chosen for destruction by the lysosome. Exosomes carry lipids, RNA, DNA, proteins, adhesion molecules, receptors, and other functional.

2. Biology of extracellular vesicles

The mechanism of EV internalization in the target cell may be mediated by multiple mechanisms, EV-surface contact molecule interaction to boost juxtracrine downstream signaling, fuse with the membrane to deposit payloads into the cytosol, or are taken up by phagocytosis, macropinocytosis, or receptor-mediated endocytosis is unclear [1, 5]. EVs play a crucial role in facilitating the cell-cell environment under normal and pathological conditions and in the pathogenesis of many diseases [3]. This section reviews the relationship between EV composition and interactions with biological membranes before delivering EV cargo to the target cells and stimuli-induced EV release. This section presents EVs biogenesis, different cargo loading mechanisms of EVs, their release, and the role of G Proteins. This book also discusses the scientific advances that have made it feasible to address the existing bottlenecks associated with the isolation and characterization of EV subsets from body fluids. The roadmap for effective immunocapture and molecular characterization is presented along with the review on immunoaffinity-based techniques for separating specific EV subsets from plasma and biofluids.

Exosomes play an important role in cell-cell communication, signal transduction, immune response in normal and disease backgrounds. Their possible use as diagnostic and prognostic biomarkers and the leverage to use them as therapeutic carrier vehicles have sparked tremendous clinical interest. The main constituents of exosomes are proteins, nucleic acids, and metabolites, as summarized in Figure 1. Among the highly enriched proteins in exosomes, the tetraspanins (CD9, CD63, CD81, CD82) help exosome-cell fusion, while the Heat shock proteins (HSP70, HSP90) are involved in stress response and antigen-binding and presentation, and other proteins (Alix, TSG101) are involved in exosome release. Some of these proteins are involved in exosome biosynthesis (Alix, flotillin, and TSG101), whereas others are considered exosomal biomarkers (e.g., TSG101, HSP70, CD81, and CD63) (Figure 1).

An intriguing question is how the cellular cargo is selectively sorted in the exosomes? This section of the book addresses the EV biogenesis, cargo loading mechanisms, their release, and the role of G Proteins (Figure 2). Another critical aspect of EV biology is how they are taken up by recipient cells? Whether EVs naturally cross biological barriers or need genetic modifications? This section highlights several critical areas, including the interplay of EVs with biological membranes, EV target cell internalization mechanism, the relationship between EV composition and interactions with biological membranes, and stimuli-induced EV release. It is also essential to understand the biophysical aspect of cellular vesicles’ morphology and formation mechanisms discussed in this section. Figure 1 elaborates exosome’s hallmarks and shows different isolation techniques, including size exclusion chromatography, polymeric precipitation, electrokinetic microarray chip, ultracentrifugation-based, immunoaffinity capture, and sucrose density gradient.

This book also specially emphasizes the bottlenecks associated with the isolation and characterization of EV subsets from plasma, thereby limiting a better understanding of their biological significance. A chapter reviews the immunoaffinity-based techniques for separating specific EV subsets from plasma and presents a roadmap for effective immunocapture and molecular characterization. This section also discusses other popular techniques of exosome production in vitro and suggests the challenges of in vivo physiological or pathological characterization of exosomes. The usual sources of exosomes are bodily fluids (plasma, saliva, urine, etc.), somatic cells (cardiomyocytes, fibroblast, pneumocyte, etc.), stem cells or pluripotent cells, and tumor or diseased cells (Figure 1). Red blood cell (RBC) contamination presents a significant challenge in EV isolation from urine for the non-invasive source of disease biomarkers. This section presents an Innovative method describing the removal of RBCs contamination from the urine fraction. This section elaborates the scientific advances that have made it feasible to characterize and engineer EVs, leading to their use as tools in biomarker discovery and disease diagnosis, prognosis, therapeutic application, and theranostics [6]. The potential of liquid biopsy is significant and can be essential for both diagnosis and therapy monitoring [7]. Blood and saliva EVs may assist achieve this without needing tissue samples.

3. Role of extracellular vesicles in human diseases

This section emphasizes emerging data confirming the role of EVs in the pathogenesis of diseases. The role of EVs in relation to inflammation, stress protection, and vascular integrity is also presented and may help better understand tissue resolution and vascular restoration. Another exciting area that has been discussed in this section is the role of EV-transmitted cargos in embryonic development and reproduction-related diseases and clinical translation. This fascinating chapter covers this critical subject and may aid fundamental understanding and clinical translation. Potential applications from circulating biomarkers for early illness detection to future therapeutic carriers for halting disease progression and regenerating damaged tissue/organs for potential regenerative medicine-based applications are also discussed. An emerging area is the studies related to the role of EVs in influencing HIV-1 pathogenesis, how HIV-1 factors target EVs, EVs as an antiretroviral therapy option, and their potential use as diagnostics prognostics, and theranostics in relation to H1V patient management [8]. Another fascinating area is the role of protozoan parasite-derived EVs in mediating host immunomodulation, pathogenesis, and parasite disease development, especially in the context of Leishmania, Toxoplasma, Plasmodium, and Trypanosoma [9]. This section deals with studies investigating new immunotherapeutic models based on protozoan parasite immunomodulation approaches and discusses many aspects of protozoan EV-based strategies to create innovative immunotherapeutic approaches.

4. Extracellular vesicles and cancer

This section presents recent studies on EVs’ pathological and translational potential in malignancies. Cancer-derived EV payloads preserve their molecular features, and cancer cells actively discharge EVs into easily accessible body fluids [10]. The transport of cancer-associated biomolecules by EVs from cancer cells promotes cancer development and reflects changes in cancer status during treatment. EVs bearing tumor antigens are also studied as cancer vaccines to induce tumor-specific anti-tumor immunity. Tumor cell-derived EVs stimulate immunosuppression, angiogenesis, metastasis, metabolic reprogramming, and other processes in the tumor microenvironment. Tumor-derived exosomes (TEX’s) ability to inhibit or boost the immune system is thrilling and intriguing. The occurrence or absence of immunological recipient cells in the TME may affect the outcome of TEX-driven interactions [11]. Transducers that create juxtracrine or paracrine signals may modify immunological recipient cell suppressive pathways, resulting in accelerated tumor development. Due to TEX-based antibody sequestration, immunotherapies may not work fully. TEX-induced immunostimulatory signals may alter the TME to promote immune activation rather than tumor development. TEXs are excellent diagnostic and prognostic biomarkers [12].

The diversity, cargo composition, and molecular mechanism of phenotypic transfer of TEX to recipient cells and vice versa is a critical question. Cancer-derived EV payloads preserve their molecular features, and cancer cells actively discharge EVs into easily accessible body fluids [13]. The transport of cancer-associated biochemicals by EVs from cancer cells promotes cancer development and reflects changes in cancer status during treatment. Moreover, this section discusses the role of EVs in resistance to treatment and diagnostics and being attractive indicators for assessing therapeutic response. EVs produced by disseminated tumor cells chemotactically attract circulating tumor cells (CTCs) and stimulate nearby stromal cells to produce extracellular matrix components like integrins, collagens, and laminin proteins to promote metastatic cell-extracellular matrix remodeling by modulating neighboring tumor cells and stromal cells, promoting tumor invasion and metastasis. Tumor-derived EVs carry molecular signatures specific to the tumor’s genetic complexity and may be used as minimally invasive cancer immunotherapy biomarkers. Through secretory factors and miRNAs, tumor exosomes have been demonstrated to facilitate distant cell-cell contact, resulting in the creation of pro-tumorigenic microenvironments favorable to metastatic spread. EV-induced fibroblast activation, ECM synthesis, angiogenesis, and immunological regulation are essential for metastatic dissemination. This section presents many aspects of the EV-based mechanism involved in metastasis.

The role of EVs in resistance to cancer treatment and diagnostics and being attractive indicators for assessing therapeutic response. Radiation is now often coupled with immunotherapy [13, 14]. EVs may also reduce chemoresistance by carrying RNA forms, and therefore activity regulation of EVs may overcome immunotherapy resistance. Also discussed many aspects of EVs/exosomes and their potential in targeting chemoresistance, radio-resistance, and cancer management. Tumor-derived EVs serve as excellent diagnostic and prognostic biomarkers. The critical bioactivities of tumor-derived exosomes using examples of their cargo molecules are also presented. EVs are immune cell evaders and are currently being investigated as potential diagnostic biomarkers and drug delivery vehicles. Exosomal immune checkpoint regulators may serve as clinical predictors for treatment response or recurrence in a variety of different malignancies. It may be possible that exosome-based paracrine mediators will be necessary for tailoring immune-based therapies to different tumors.

This section also reviews the role of EVs and the potential to use them in the management of difficult to diagnose and treat cancers, like ovarian cancer and breast cancer. Oncologic malignancies such as ovarian cancer are difficult to diagnose, with dismal results, and critically need new treatments. This section describes EVs’ role as a critical player in the spread of ovarian cancer, and EVs may help us learn more about ovarian cancer proliferation and metastasis while also revealing potential new therapeutics. Breast cancer is the most frequent cancer among women, and understanding the role of EVs in facilitating intercellular communication between cancer and stromal cells and its therapeutic possibilities for breast cancer therapy is critical. This book also discussed the information gaps for clinical translation of EVs and pointed out the current research projects on developing EVs as biomarkers or therapeutic delivery systems. The solutions to improve EVs’ efficacy as cancer treatments are also presented. Moreover, the direct and indirect cell surface modification is discussed, emphasizing ongoing and finished clinical studies utilizing naturally generated EVs to treat breast cancer. This book also presents the loopholes for clinical translation of EVs and points out potential future research directions for therapeutic translation and cancer therapy. This anthology of chapters is presented with a broad audience in mind and will serve as a valuable must-have resource to basic biologists, translational scientists, and clinicians.