EXOSOMES-DISCOVERY-SEPARATION AND FUTUR USE IN DIAGNOSIS AND THERAPY

This presentation illustrates the discovery of exosomes-methods of separation ,and future perspectives in disease diagnosis and therapy .

1. Exosomes : Magic Players in Molecular Diagnosis Ekbal Mohamed Abo- hashem (MD) Professor of Clinical Pathology Mansoura University-Egypt

2. AGENDA: The evolution of exosome application . Exosomes as liquid biopsy markers . Exosomes extraction methods . Commercial development of exosomes . Exosome therapy and drug delivery . Challenges for the use of exosomes in clinical practice .

3. It was over 50 years ago that researchers first observed minute particulates in human plasma. They found that this material — which they termed “platelet dust” — was lipid-rich and likely to be involved in platelet activation. Yet it was not until the 1980s that these 30-150nm extracellular vesicles were first defined and the name ‘exosomes’ was coined. The evolution of exosome applications

4. Like liposomes, exosomes are comprised of a lipid membrane and an inner aqueous medium. However, exosomes were found to be more complex in structure, containing a large array of proteins and lipids. Exosomes are subsequently released from the endosomal compartment into the extracellular space by fusion with the plasma membrane.

5. Upon release from the secreting cell, they transmit messages to recipient cells through several mechanisms, including surface receptor interaction, membrane fusion, as well as receptor-mediated endocytosis, phagocytosis, and/or micropinocytosis .

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8. Exosomes play critical roles in various physiological and pathological processes, including cancer, pregnancy disorders, cardiovascular diseases, and immune responses. Due to the considerable limitations of solid biopsy , It is imperative to introduce liquid biopsy to clinical practice to reduce invasive operations and promote more precise medical intervention.

9. Exosomes as Liquid Biopsy Markers : Exosomes show superiority over conventional serum-based biomarkers, particularly in regard to diagnostic sensitivity and accuracy : First, exosomes are secreted by living cells and contain biological information from the parental cells and are more representative than cell-free DNA (cfDNA), which is secreted during necrosis or apoptosis.

10. Second, exosome identification is clear and simple. Exosomes express specific proteins such as CD63, ALIX, TS101, and HSP70, which can be used as markers to effectively distinguish exosomes from other vesicles, and their specific cup-shaped characteristic makes them identifiable by electron microscopy.

11. Third, They are also innately stable, with a lipid bilayer that enables them to withstand degradation even within harsh microenvironments. In terms of practicality, exosomes can be easily and non-invasively isolated from biological fluids such as urine, blood, and even tears. Once extracted, they can be stored by freezing, freeze-drying, or spray-drying.

12. Exosomes prefer to be isolated in pH 7 solution rather than in acidic environments. The high biological stability can reduce the cost of sample short-term storage and the difficulty of transportation, which greatly enhances the clinical applicability of exosomes.

13. Fourth, unlike many conventional serum biomarkers, exosomes can pass through the blood brain barrier, a highly selective boundary providing information about brain cells that would be otherwise difficult to obtain. This may also provide a means to deliver therapeutic cargo to the brain, helping to treat cancer and traumatic brain injuries.

14. Fifth, compared with circulating tumor cells (CTCs), exosomes are relatively convenient to obtain from almost any body fluids. Generally, despite the slightly more complicated DNA extraction procedures, exosomal DNA possesses more abundant biological information and higher accuracy for prognosis prediction than cfDNA.

15. Exosomes represent an excellent and promising liquid biopsy marker and have their application as a potential complement to personalized medicine in some common malignant tumors, pregnancy disorders, cardiovascular diseases, and organ transplantation . Owing to the great prospects of exosomes in clinical applications, a commercial chain of exosome research-related technologies has been formed and is still under development

16. Before exosomes can be utilized in large-scale medical practice, it is crucial that these nano-sized particles are precisely distinguished from a wide spectrum of cellular debris and interfering components. There is no single standardized approach to exosome separation and analysis, with each approach providing unique strengths and limitations. EXOSOME EXTRACTION METHODS :

17. Exosome extraction methods are mainly based on their physical characteristics and components. Importantly, exosome quality will be greatly affected by different exosome extraction procedure. Currently, methods for exosome extraction can be commonly divided into : Differential ultracentrifugation, Density gradient/cushion centrifugation, Size exclusion chromatography (SEC), Precipitation, (Immuno-) affinity capture, Microfluidic approaches, etc EXOSOME EXTRACTION METHODS ( cont.)

18. In addition ,multiple recent extraction and detection technologies have been developed such as : The microfluidic chip Integrated extraction and quantitative analysis of exosomal nucleic acids and proteins, with high specificity and intact yield of exosomes, small required sample volume and simple, time-saving operation. Moreover, a novel urine-based EV extraction and enrichment method was established .This new technology takes only 30 min to enrich EVs from 4 ml urine.

19. A combination of several of these methods has been suggested as a promising strategy for the improvement of the isolation outcome. This is to provide exosome subsets with high purity with respect to size, morphology, concentration, the presence of exosome-enriched markers, and the lack of contaminants

20. Various test kits are now available ,aiming at developing novel and precise exosome technology as liquid biopsy tool for diagnosis of multiple cancers, including lung and prostate cancer. An example is The ExoDx Prostate® ( IntelliScore ) (EPI) test ,a star production of exosome diagnostics. This is a urine-based and completely noninvasive test designed to assist physicians in assessing whether an individual patient over 50 years with 2–10 ng/ml prostate-specific antigen, presenting for a needle biopsy, is at greater risk for high-grade prostate cancer; therefore, the patient can avoid unnecessary biopsy and, instead, continue to follow up. COMMERCIAL DEVELOPMENT OF EXOSOMES:

21. Exosome-based strategies for the diagnosis of tumors Recently, an exosome-based tumor diagnostic assay called ExoTEST ™ ( HansaBioMed , Estonia) was developed. ExoTEST is a sandwich ELISA developed to capture and quantify exosomes in human plasma and other body fluids ,and in cell culture supernatants. In this method, proteins of exosomes such as CD63 and Rab5b, and a tumor-associated marker (e.g., CAV1) are exploited for immunoisolation of nanovesicles . The accuracy of the novel method was established by western blot and flow cytometry analysis.

22. ExoTEST provides the possibility of screening multiple markers on one sample, defining conditions with similar clinical presentation and overlap of molecular pathology.

23. Establishment and development of the first salivary-based exosome miRNA biomarker-miR-185 as a dual target for the diagnosis and treatment of oral cancer. ( Genexosome Technologies ). Preliminary studies have shown that salivary exosome protein and miRNA content can be potentially useful to detect disease conditions, such as Sjogren’s syndrome , and exosomes from amniotic fluids could provide a potent tool for prenatal diagnosis . Salivary exosomes:

24. Cells located towards the drainage system, such as kidney tubular cells, provide exosomal markers that are not detectable in whole urine . Urine exosomal preparations from prostate cancer patients contain mRNA of PCA3 and TMPRSS2, a product resulting from an ERG fusion chromosomal rearrangement . Similarly, in urine exosomes, diagnostic and prognostic markers of renal ischemia/reperfusion injury or antidiuretic hormone action, such as aquaporin -1 and -2, have been found . All these molecules are not easily detected in whole urine. Urinary Exosomes:

25. Exosomes: novel biomarkers for neurodegenerative diseases? Most of the misfolded proteins associated with neurodegeneration are shuttled via exosomes. Superoxide dismutase, a-synuclein, amyloid and tau are involved in amyotrophic lateral sclerosis, Parkinson’s disease, Alzheimer’s disease (AD) and tauopathies, respectively, are all processed via the MVB pathway and secreted in association with exosomes by cultured cells . Exosome-associated tau is also present in human cerebrospinal fluid samples of early AD patients.

26. Exosomes not only contain precious biomarkers but their profiling can be used to identify specific altered pathways: Patient urine exosomes and PC-3 cell culture-derived vesicles both also have an increased level of d-catenin In some cases of bladder cancer, eight upregulated exosomal proteins were identified. Interestingly, it was found that they largely relate to the EGF receptor signaling mechanism

27. Once exosomes have been extracted and purified, how do we then turn them into effective drug delivery systems? Luckily exosomes are made for this role, combining the benefits of both synthetic nanocarriers and cell-mediated drug delivery systems, while avoiding their limitations. The first step to harnessing these properties is ‘cargo loading’, the process of packing exosomes with therapeutic materials. Exosome :a rising star in therapy and drug delivery:

28. Several cargo loading methods have been employed for this purpose, each with their own advantages and disadvantages. Though the potential applications of exosome therapy are wide-ranging, by far the most common area of exosomal research is cancer, followed by inflammation and infection. Directing exosomes to express and deliver therapeutic drug candidates , was developed by ( Codiak BioSciences - engEx ™ Platform ) .

29. ExoSTING is a major and promising immune therapeutic candidate targeting cancer . It is highly potent with minimal toxic potential. It is rarely affected by serum systemic cytokines and preserves the vitality of effector T cells and antigen presenting cells in tumors to maintain sustained immune protection. Recently, exoSTING is being developed for solid tumor therapy that activates the “STING” receptor in immune cells.

30. Oral administration of antisense oligonucleotides with PureTech’s milk exosome-based technology to transform conventional intravenous injection therapy for improved efficacy and reduced toxicity ( PureTech Health in collaboration with Roche).

31. Challenges for the use of exosomes in clinical practice : A standardization of the classification and extraction method of exosomes for different body liquids is urgently needed. More efficient methods with a low biofluid volume requirement and high purity and yield are the foundation of subsequent applications. The identification of specific subtypes of EVs is urgently needed, as different vesicles may exert various biological effects.

32. It is necessary to standardize the protocols and identification methods when attempting to use exosomes widely in clinical testing. Additionally, more reliable biomarkers should be confirmed.

33. It may be a better direction to validate documented biomarkers on a larger scale to create new panels for multiple fields As potential therapeutic cargo, the biological safety, targeted efficacy, and adverse effects of exosomes must be confirmed before clinical use.