Exosomes, the Key Players in Cell-To-Cell Communication as the Universal Nano-Sized Disease Sensors of the Future

Exosomes are nanovesicles shed by cells into the extracellular space. From an engineering point of view, exosomes can be assimilated to a complex system, that is, an arrangement of parts that together exhibit a behavior that the individual constituents do not. The authors argue that the unique composition of exosomes as well as their involvement in cell-to-cell communication and monitoring a multitude of diseases, justify entirely looking at exosomes, in the future, as universal sensors of diseases. In this paper, after the short background information on exosomes, the authors show how they are involved in the progress of different diseases.

Exosomes, a class of extracellular (EV) vesicles, are unique nano-sized cargo-bearing biological vesicles, secreted by almost all normal and cancer cells into the extracellular space. Exosomes are the smallest of the extracellular vesicles with their size in the range of 30-150 nm, they are highly heterogeneous and are present in all body fluids. It has to be mentioned that, because of the heterogeneity of exosomes and the overlap of physicochemical properties of different extracellular vesicles, the separation and purification of exosomes are still difficult.
In 1983, Johnstone et al. 1,2 studied the maturation process of erythrocytes from sheep reticulocytes and discovered a 100 nm vesicle, having a lipid bilayer structure, and named the vesicle "exosome" in 1987. Transferrin receptors on reticulocyte plasma membranes were also studied by the group of Stahl. 3 The structure of exosomes and the encapsulated substances were studied in the upcoming years and it was found that similar to cells, these tiny vesicles have an extraordinarily complex structure and composition. The membrane components consist of lipids and proteins and depend on the type of cell producing the exosome (Fig. 1a). The molecular content of exosomes mirrors that of the releasing cell type.
As shown in Fig. 1a, the proteins in the exosome membrane are tetraspanins (CD9, CD63, CD81, etc., used as exosome markers), integrin, etc. Proteins and nucleic acids such as RNA and DNA have been found encapsulated in exosomes (double-stranded DNA, micro-RNAs, and messenger RNA).
One of the most significant realizations in the field was the discovery that exosomes shed by a cell have the capacity to affect the functioning of other cells, and also as tools of communication between cells. 4 Exosomes are capable to convey biological information across different cells and cell types and can also serve as carriers of therapeutic molecules across biological barriers. At this point, after discovering the role of exosomes in cell-to-cell communication, suddenly, exosomes arrived at the front stage. The field of exosomes literally exploded and the EV research proliferated around the world. Thousands of papers have been published, new journals, dedicated exclusively to the study of exosomes appeared both in Europe and North America, and the first international meeting for EVs (called exosomes at the time) was organized by Rose Johnstone and held in Montreal in 2005. The International Society for Extracellular Vesicles (ISEV) was founded in 2011.
The interaction of exosomes with the nearby or distant recipient cells may happen, either by binding with the surface receptors on the target cell, activating the cell by generating a downstream signaling cascade, or the exosomes can be uptaken by fusion with the plasma membrane and release their cargo into the cytosol, changing the pathophysiology of many diseases, including that of cancer (Figs. 1b, 1c).
Exosomes can be captured from body fluids and cultured media with best results by using immunoaffinity approaches that mostly rely on antibodies directed against exosome surface markers. However, there are critical challenges as no standard methods are available to isolate, detect and characterize exosomes. For a comprehensive review on their isolation and detection as well as their benefits for future: clinical assays, Ref. 5 is recommended.
Many studies have shown that the cancer cell-derived exosomes contain specific nucleic acids and proteins that reflect the type of cancer cells. Therefore, exosomes can be utilized as biomarkers for early cancer diagnosis.

Role of Exosomes in Disease Progression and Therapeutic Applications
Cancer studies.-Cancer progression.-Generally, exosomes have been described in the literature as promoters of cancer progression and their antitumor functions are not yet well known. It has been found that exosomes facilitate tumorigenesis by regulating angiogenesis, immunity, and metastasis. The regulation of cancer progression by exosomes is shown in Fig. 2.
Briefly, it is thought that the first step to metastasis is the invasion of cancer cells. It was reported that plasminogen is activated by Heat Shock Proteins (HSP90) existing on the surface of cancer cellderived exosomes. Plasmin, formed by the activation of plasminogen, is believed to enhance the invasion of cancer cells. It has been reported that angiogenesis, supplying oxygen and nutrients to cancer cells, contributes to the enlargement of the lesions.
In spite of promoting tumorigenesis, it was demonstrated that exosomes derived from tumors contained tetraspanins and other tumor antigens, important for the stimulation of immune cells. The role of exosomes in different types of cancer as well as in neurological disorders will be briefly discussed in the following part.
Cancer types.-Hepatocellular carcinoma One of the most lethal forms of cancer with less than 10% survival rates over 5 years is hepatocellular carcinoma (HCC). Scientists found that exosomes are promoting proliferation, growth, invasion, and metastasis of HCC cells. 6 They are involved in the diagnosis, prognosis, and treatment of HCC. Furthermore, recent research on the isolation, purification, characterization, and analysis z E-mail: pmuthu@alcor.concordia.ca of HCC-derived exosomes has reported the possibility of encapsulating chemotherapeutic drugs into exosomes that can be then injected into the body in order to treat or kill cancer cells. In addition, by intravenous or intratumor injection of exosomes, the toxicity, resulting from drugs' free diffusion can be prevented. Exosomes can also be labeled with certain antigens to trigger an immune response and antitumor effects.
Pancreatic cancer The exosomes' function and their molecular mechanisms in the progression of pancreatic cancer and their role in the progress of metastasis have been the subject of many studies. They pointed to several conditions, including diabetes, inflammation, and viral infections, which may cause pancreas precancerous diseases. 7,8 Removing the cause of the disease, and targeting relevant exosomes, may help prevent the development of pancreatic cancer and provide early diagnostic methods.
Prostate cancer Research has been carried out by different groups, regarding the use of exosomes' biomarkers and their capacity to be therapeutic agents for increasing the survival rate of prostate cancer (PCa) which is the second most common cancer-related death among men. 9 The blood prostate-specific antigen (PSA) method cannot detect aggressive prostate cancer, instead, the liquid biopsy technique may be a suitable option to track tumor progression over time. 10 Lung cancer Lung cancer is also one of the causes of death worldwide that can be diagnosed only at a late stage, or after cancer has spread to other parts of the body. Many studies have focused on cancer-derived exosome isolation, purification, and characterization as well as their effect on lung cancer diagnostic and therapy. 7,11 Breast cancer Different research groups have worked on the role of exosomes in breast cancer therapy resistance and cancer progression as well as the outline for potential clinical use of exosomes. 12 Moreover, Brain cancer The secretion of exosomes by malignant cells may result in a signaling process that leads to abnormal cell growth and may contribute to the hypoxic environment, typical of tumors. At the same time, it has been reported that in glioblastoma, and medulloblastoma, exosomes showed immune properties, 13 by transporting heat shock proteins that show an antitumor immunity. On the other hand, exosomal miRNAs may influence neuroblastoma resistance to chemotherapy or may affect brain metastasis.
Neurodegenerative diseases.-Neurodegenerative diseases like Alzheimer's and Parkinson's can be propagated by exosomes via exporting misfolded proteins. It has been reported that identifying the levels of exosomes that contain a specific cargo may result in predicting Alzheimer's disease development up to ten years in advance. 14 Exosomes show ambiguity, they are either, neuroprotective, or neurodegenerative. In Alzheimer's disease, the extracellular vesicles have been proven to participate in the dispersing of Aβ, which is a protein fragment that accumulates in the brain and is associated with Alzheimer's disease, thus, disseminating Aβ pathology.
Other chronic diseases.-Diabetes mellitus and related complications.-Exosomes can be used as a biomarker or therapeutic carrier for diabetes mellitus metabolic disorder treatment and its complications such as eye disorder, diabetic wound healing, cardiovascular diseases, neuropathy, and nephropathy. Also, there are studies that have investigated the use of exosomes for therapy, their effects on enhancing glucose and lipid metabolisms, and their dual role in stifling/stimulating insulin resistance.
Spinal cord injuries.-Worldwide, a number of people are living with disabilities following a spinal cord injury that can result in neural loss and axon/myelin necrosis as well. In response to spinal cord injury, exosomes of bone marrow stem cells can be used as nanocarriers, promote axonal regeneration and angiogenesis, improve structural components and electrophysiology, and reduce inflammation.
Vaccination.-Therapeutic strategies like vaccination can also be developed using EV delivery systems. Recent studies show that exosomes are composed of immunomodulatory features which can lead to EV-based vaccines that can be used against various kinds of diseases such as cancer, virus-related cancers (like HPV and HBV), infectious diseases, and coronavirus.
Research in the field of exosomes has become multidisciplinary and scientists from fields such as engineering, physics, and artificial intelligence bring more and more their contributions. Not only looking at the problems from different angles, using new techniques and tools but also creating a new mindset, will help unveil the mysteries of exosomes and their functionalities.
One of the fields that provided much information on exosomes, by developing methods for their isolation and detection, is microfluidics. Mostly based on immunoaffinity, microfluidic separation, and detection methods have been developed and evolved over the years. Our group developed recently a magnetic particle-based device by using streptavidin-coated magnetic particles (magnetic immunocapture), including a 3D mixer and a sedimentation unit. In this device, the exosomes are isolated faster with a high isolation efficiency. (Fig. 3a).
Advancements in exosome isolation and detection techniques.-Research in the field of exosomes has become multidisciplinary and scientists from fields such as engineering, physics, and artificial intelligence bring more and more their contributions. Not only looking at the problems from different angles, using new techniques and tools but also creating a new mindset, will help unveil the mysteries of exosomes and their functionalities.
One of the fields that provided much information on exosomes, by developing methods for their isolation and detection, is microfluidics. Mostly based on immunoaffinity, microfluidic separation, and detection methods have been developed and evolved over the years.
How exosomes could serve as biosensors for cancer detection?.-As mentioned, exosomes can be released by both cancerous and non-cancerous cells, but their concentration increases substantially as the tumor progresses, as compared to other biomarkers such as tumor antigen, CTC, and ctDNA. For example, in a study of a glioblastoma patient's plasma, the concentration of exosomes was found to be approximately 50 times higher than that of a healthy individual. Exosomes are highly stable and are capable of safeguarding nucleic acids and proteins that play a crucial role in cancer development. Therefore, EVs hold immense potential as they can be used in cancer therapy, prognosis, and serve as promising biomarkers for the early detection of cancer. Several studies have shown that in many types of cancer, the concentration of total cancer cells and their exosome-bound HSPs are elevated and play various roles in cancer biology. Figures 3b and 3c demonstrate the relationship between the plasmonic shift and exosome concentration during breast cancer progression. The tested range of concentrations in this study, covers a wide range from early-stage, non-cancerous diseases to fully developed cancerous conditions. It can be inferred that the LSPR shift is proportional to the concentration of exosomes and can effectively detect cancer from early to advanced stages. 16 Researchers have also investigated the detection of glioma-derived exosomes with a biotinylated antibody-functionalized titanium nitride plasmonic biosensor. They showed that the biosensor exhibits a high degree of sensitivity and specificity in identifying glioma-derived exosomes, which makes it a potential diagnostic and therapeutic instrument for glioma and other cancer types. The biosensor's ability to accurately and precisely detect exosomes implies that it may have practical applications in clinical practice. 17 In another work, a biosensor based on localized surface plasmon resonance (LSPR) was developed to simultaneously detect two types of extracellular vesicles (EVs), derived from different cancer cells. The biosensor exhibited high sensitivity and specificity in detecting tumor-derived EVs and was able to distinguish between EVs from different types of cancer cells. 18

Future Needs and Prospects
Until now, exosomes have generated considerable interest as diagnostic biomarkers and therapeutic cargo carriers.
As shown in this short paper, because of the complexity of exosomes' composition, their effect on most types of cancer is ambivalent. Two different components of the same cargo may influence the progress of cancer in completely opposite ways; one may help to disseminate cancer, promoting cell death while another may drive cell survival, stimulating immunomodulation. Some of the mechanisms involved in these processes are already known, and others will be brought to light, probably, during the next decades. For now, even if exosomal markers are associated with several cancer types, only a few diagnostic assays are available for clinical use but this situation will change rapidly in the future as exosomes have an immense capacity to "feel" the evolution of a multitude of diseases, such as infectious diseases like HIV and viral hepatitis, autoimmune diseases like multiple sclerosis and lupus, cardiovascular diseases, including heart failure and myocardial infarction, traumatic injuries and various types of cancers. The development of exosomes as disease sensors will depend on an extended collaboration between the engineers who are the builders of the most sophisticated devices to separate and detect exosomes and the biologists who are using them to gain an extended knowledge, regarding the evolution of various diseases. The authors of this paper speculate that, depending on the success of this interdisciplinary work, exosomes will be able the fulfill, sooner or later, their promise to become disease sensors.

Conclusions
The importance of exosomes in future medicine is beyond any doubt but,presently, the lack of efficient isolation and detection methods hinders their clinical applications.
In this paper, the authors advanced the idea of sensors that are more complex than those that simply measure a parameter or a property and, based on a value, detect the concentration of a compound. In this enlarged concept of sensor, exosomes, biological nanoparticles of an extreme complexity, will be able to detect a disease, including cancer, at the very beginning. The number of exosomes as well as the changes in their composition will follow the progress of the disease and will be involved in the therapy as well.
In summary, it is no longer in the realm of science fiction to look at exosomes as universal disease sensors.  16