**5. Exosomes as biomarkers and therapeutic targets**

All cells in the body secrete ExMVs, a heterogeneous population of bilayer vesicles with membrane that transport and deliver loads of proteins and nucleic acids to the recipient cells, allowing cell-cell communication. The exosomes, of endosomal origin, regulate normal and pathological processes. Healthy subjects and patients with different diseases release exosomes with different RNA and protein contents into the circulation, which can serve as biomarkers [45]. Compared to conventional biomarkers in serum or urine samples, exosomal biomarkers have greater sensitivity and specificity due to their high stability. They are present in almost all body fluids that harbor molecular components, exosomal proteins, and miRNA, and they are carriers of genetic information, which can be used for diagnosis. Although most RNAs found in exosomes are nucleotide fragments of degraded RNA with a length of <200 nm, some full-length RNA may be present. For example, circulating exosomal miRNAs are equivalent to those of the cancer cells of origin [45].

The power of nanovesicles as biomarkers depends on the enrichment of the exosomal classification markers, which otherwise only represent a very small proportion (<0.01%) of the total proteome of body fluids. The enrichment of the exosomal biomarkers of diagnosis will help in the discovery of new biomarkers to provide more precise information related to the origin of each exosome. A proteomic analysis and characterization of the plasma exosomes is essential, and gel permeation chromatography has been used to purify TNFR1 exosomal-like vesicles from the low-density lipoprotein (LDL) fraction. With this multistage purification scheme, it was possible to identify the 66 proteins of the circulating healthy exosomes, in the plasma, including proteins, both cytosolic and membrane-associated, as extracellular secreted proteins and associated with the cells identified with vesicular trafficking. The advantage of this analysis is that it allows the separation of different populations of vesicles according to their size. This reduces complexity in the identification of proteins in the plasma sample that may contain more than 1 million different intermixed proteins, and the discovery of peroxisome proliferator-activated receptor gamma (PPARγ) as a component of plasma exosomes will allow identifying a new pathway for the paracrine transfer of nuclear receptors specific in each pathology [46].

### **5.1. Exosomal proteins as diagnostic biomarkers**

The molecular content of exosomes is the fingerprint of the cell type that released it and its current status; most viable cells release extracellular environment, protein secretions of exosomes, and when fused with the plasma membrane, appear in the blood and urine, so they are easily accessible, and can be used as biomarkers, for the diagnosis and prognosis of malignant tumors and other pathologies [47, 48].

Due to their cellular origin, exosomes express protein markers specific to the endosomal pathway, such as tetraspanins (CD63, CD9 and CD81), heat shock proteins (Hsp70), proteins of the Rab family, Tsg101 and Alix, which are not found in other vesicles of similar size. Their function is to eliminate damaged or aged cellular molecules, to protect cells from the accumulation of waste or drugs, participate in physiological and pathological processes, and have a wide variety of clinical applications, ranging from biomarkers to cancer therapy [49]. Proteomic and biochemical analysis of the purified exosomes revealed that the bilayer membrane of phospholipids is embedded with various proteins and lipids originating in the parental cells. These can serve as surface markers for the characterization and differentiation of exosomes from other types of microvesicles [50]. Exosomes contain various proteins, which express specific cellular functions, so they can serve as biomarkers for the diagnosis of liver, kidney, and cancer diseases [51]. Proteins in urinary exosomes are easily available through nontoxic means, are invasive, and are useful in diagnosis, especially for diseases of the urinary tract [52].

## **5.2. Exosomal nucleic acids as diagnostic biomarkers**

A new generation of drug delivery systems can be mediated by exosomes and ExMVs; because they have high administration efficiency and low immunogenicity, new therapies can be implemented, and standardization is achieved with isolation techniques, with high functional efficiency, solid performance, scalable production, adequate storage, and efficient loading methods, which do not damage its molecular integrity and the movement of the elements *in vivo* as novel "nano-vehicles" [43]. The elements derived from cells generate an endogenous mechanism for intercellular communication; they are vehicles with the capacity to transfer biological information and the potential use can be as means of drug delivery [44].

All cells in the body secrete ExMVs, a heterogeneous population of bilayer vesicles with membrane that transport and deliver loads of proteins and nucleic acids to the recipient cells, allowing cell-cell communication. The exosomes, of endosomal origin, regulate normal and pathological processes. Healthy subjects and patients with different diseases release exosomes with different RNA and protein contents into the circulation, which can serve as biomarkers [45]. Compared to conventional biomarkers in serum or urine samples, exosomal biomarkers have greater sensitivity and specificity due to their high stability. They are present in almost all body fluids that harbor molecular components, exosomal proteins, and miRNA, and they are carriers of genetic information, which can be used for diagnosis. Although most RNAs found in exosomes are nucleotide fragments of degraded RNA with a length of <200 nm, some full-length RNA may be present. For example, circulating exosomal miRNAs are equivalent

The power of nanovesicles as biomarkers depends on the enrichment of the exosomal classification markers, which otherwise only represent a very small proportion (<0.01%) of the total proteome of body fluids. The enrichment of the exosomal biomarkers of diagnosis will help in the discovery of new biomarkers to provide more precise information related to the origin of each exosome. A proteomic analysis and characterization of the plasma exosomes is essential, and gel permeation chromatography has been used to purify TNFR1 exosomal-like vesicles from the low-density lipoprotein (LDL) fraction. With this multistage purification scheme, it was possible to identify the 66 proteins of the circulating healthy exosomes, in the plasma, including proteins, both cytosolic and membrane-associated, as extracellular secreted proteins and associated with the cells identified with vesicular trafficking. The advantage of this analysis is that it allows the separation of different populations of vesicles according to their size. This reduces complexity in the identification of proteins in the plasma sample that may contain more than 1 million different intermixed proteins, and the discovery of peroxisome proliferator-activated receptor gamma (PPARγ) as a component of plasma exosomes will allow identifying a new

pathway for the paracrine transfer of nuclear receptors specific in each pathology [46].

The molecular content of exosomes is the fingerprint of the cell type that released it and its current status; most viable cells release extracellular environment, protein secretions of

**5. Exosomes as biomarkers and therapeutic targets**

194 Stromal Cells - Structure, Function, and Therapeutic Implications

to those of the cancer cells of origin [45].

**5.1. Exosomal proteins as diagnostic biomarkers**

Exosomes contain exosomal RNAs, especially miRNAs that function as diagnostic biomarkers, are protected from RNase-dependent degradation, are detected in circulating plasma, and serve for the diagnosis of ovarian cancer [53, 54]. Nanostructure analysis and study of the transcriptome of exosomes that transport RNA are diagnostic and found in breast milk, saliva, blood, urine, malignant ascites, amniotic fluid, bronchoalveolar secretion, and synovial fluid [55, 56].

Urinary extracellular vesicles (uEV) are released in the nephron of the kidney and urinary tract. Specific proteomic and transcriptomic markers provide information on the cell of origin and are a reservoir for the discovery of biomarkers in kidney diseases. The uEV are a new means of cell signaling, renal tubular cells, and can provide exosomal markers not detectable in urine. Renal biopsy is an invasive technique with complications such as infection and hemorrhage. The analysis of proteomic and transcriptomic changes of uEV in different disease states as a biomarker can be a noninvasive alternative to biopsy [57].

In conclusion, the most important biomedical utility of exosomes is their application as biomarkers in clinical diagnosis. Compared with those detected in conventional samples, such as serum or urine, exosomal biomarkers provide comparable or superior sensitivity and specificity, attributed to their excellent stability, and the exosomal biomarkers of biofluids can be easily obtained. Recent technical advances in the isolation of exosomes will make diagnostics more beneficial.
