Emerging role of exosomes as a liquid biopsy tool for diagnosis, prognosis & monitoring treatment response of communicable & non-communicable diseases

Switching from tissue-based traditional biopsies to ‘liquid biopsy’ has taken the diagnostic research a step further as it is painless, rapid, non-invasive and can be consistently undertaken in certain situations providing the ‘real time’ molecular profile and dynamic behaviour of the disease1. Although the free circulating nucleic acids (DNA and RNA) were discovered long back in 1948, their importance as biomarker was first postulated by Leon et al2 in 1977. It is after that when the diagnostic research community found an alternative to solid biopsies in the form of circulating cell-free DNA (ccfDNA) and circulating tumour cells (CTCs) and the technique was called as ‘liquid biopsy’. Liquid biopsy has the notable advantage over traditional tissue biopsies in that it is less stressful to the patients; provides much more comprehensive diagnostic and prognostic information about the disease during, before and after treatment3. It allows the sampling in a minimally invasive or non-invasive manner during the course of the disease, thus avoiding the risks related to tissue sampling and provides the continuous assessment of disease progression and treatment efficacy. Unlike traditional biopsies, liquid biopsies are repeatable and can be performed even in the worst clinical conditions. So far the most common tools of liquid biopsies were CTCs, ccfDNA and circulating cell-free RNA (cfRNA). However, lack of surface markers and scarcity of the desired cell-free nucleic acids are some of the limitations which render these molecules (CTCs, ccfDNAs and cfRNA) less significant to be used as liquid biopsy tools. Recently, the increasing role of ‘exosomes’ in biomedical applications has grabbed the attention of medical researchers.

Exosomes are lipid bilayer membrane vesicles of endosomal origin with 40-150nm diameter4 secreted by almost every cell type of the body and are ubiquitously present in all body fluids5. These are naturally secreted vesicles which are typically defined as a subset of intraluminal vesicles (ILVs) that are secreted upon multivesicular bodies (MVBs) fusion with plasma membrane6. Exosomes were initially supposed to remove the cellular waste from the cells; this thought was further supported by some recent studies, which also indicate the role of exosomes in cellular waste elimination and maintaining cellular homeostasis78. In addition to waste management, exosomes are also involved in cell-cell communication and activating significant biological responses even in distant cells910. Besides being associated with various patho-physiological functions in the cell,10 exosomes have also been shown to play a role as immune modulators1112. Contemplating the unique properties and natural functions of exosomes, researchers have anticipated the deliberate utilization of exosomes in clinical research. The substantial development in the field of exosomes has opened up possibilities for the diagnosis and treatment of many deadly diseases. The researchers are now looking forward to find a profound scope of exosomes in medical interventions like targeted drug delivery and personalized medicines, but till date, the most studied field of exosomes is biomarker analysis. Exosomes being the molecular signature of their originating cells, can provide enough and accurate information about the parent cell. Furthermore, since these are actively and continuously produced, these can be the best source for real-time assessment of disease for prognosis/diagnosis and also for monitoring the treatment response efficacy and relapse1213. The ubiquitous and non-invasive nature of exosomes has encouraged their role in routine diagnosis and treatment response monitoring, thus making them favourable to be used in liquid biopsies. Recently, Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) guidelines14 advocated the use of operational terms such as ‘small extra-cellular vesicles (sEVs)’ instead of ‘exosomes’ unless the origin of the vesicle could be proven yet we have used the term exosome throughout the manuscript to maintain the effective link with published literature and avoid any misunderstanding of exosomes to other sEVs. This review summarizes the existing knowledge of applications, limitations and technical advancements of exosome-based liquid biopsies for the diagnosis, prognosis and treatment response monitoring in communicable and non-communicable diseases and highlighted the future aspects of exosome-based liquid biopsy in clinical interventions.

Biogenesis & cargo loading of exosomes in context of liquid biopsies

The process of exosome biogenesis has been demonstrated to be slightly different from that of other extracellular vesicles. Formation of early endosomes by inward budding of plasma membrane marks the initiation of exosome biogenesis15 and is mediated by the endosomal sorting complex required for transport (ESCRT) like other extracellular vesicles. Early endosomes undergo maturation to transform into late endosomal MVBs, which generate ILVs within their lumen. The formation of ILVs in exosomes has been shown to be either ESCRT-dependent or ESCRT-independent16. ESCRT-independent mechanism involves some trafficking effectors such as tetraspanins or lipids (lysobisphosphatidic acid and ceramides) that bind the cargo to the early endosomal membrane15. This binding leads to the formation of ILVs by the budding of the endosomal membrane into the lumen. Fusion of MVBs with the plasma membrane leads to the secretion of sEVs called ‘exosomes’15.

Exosomes from different origins have their specific cargo (lipids, proteins, DNA, mRNA and miRNA), which constitute the molecular fingerprint of their originating cells517. The enrichment of the cargo into exosomes depends on cell type and cellular homeostasis18. The cargo of the exosomes has been demonstrated to be changed with the changing environment of these cells19. Moreover, exosomes secreted by the same cell line are shown to be heterogeneous20. Cargo loading or cargo sorting in exosomes is a crucial step for determining the fate and function of an exosome. Although there are some studies showing the evidence of cargo loading during extra-cellular vesicle biogenesis212223 there is no certain rule with respect to sorting of cargo like how and when these are loaded into exosomes. On the basis of the mode of loading, cargo sorting may be of two types: ‘active or selective’ cargo sorting and ‘passive or random’ cargo sorting24. Active cargo sorting is a process in which cargo is incorporated selectively into exosomes by accumulating it on the place of nascent exosome biogenesis. The enrichment of miRNAs into exosomes has been demonstrated in several studies compared to their concentration in the cell which indicates that the cargo can be selectively sorted into exosomes2526. The accumulation and incorporation of cargo have been shown to be mediated by some specific adaptor proteins such as ALIX, hnRNPA1 and SYNCRIP272829. These proteins are called trafficking effectors that facilitate the binding of cargo to the endosomal membrane24. While passive or random cargo sorting is a process in which cargo is incorporated randomly into newly forming exosomes and the incorporation of the cargo depends on its concentration (i.e., the rate of its expression in the cell and its surrounding) and also on the rate of extracellular vesicles (EV) production in the cell. However, the same is also applicable in the case of active cargo sorting. Although there are several research studies describing the enrichment of cargo into exosomes, the exact mechanism of the cargo sorting is not known yet. In general,the cargo sorting depends on cell signalling and cell state, so probably the factors that affect the state of the cell also indirectly affect the sorting or selection of cargo in the exosomes. Till date, the identified regulatory factors of cargo sorting are growth factor signalling30 stress signalling31 senescence32 metabolic changes33 and disease-associated changes34 in the cell. The in-depth-knowledge of biogenesis and cargo sorting in exosomes will certainly improve our understanding of the selection of biomarkers in exosomes and, consequently, their applications in the field of liquid biopsies.

Exosomes: as liquid biopsy tool

The need for an advanced, high throughput, rapid and repeatable diagnostic technique has led the researchers to look towards exosomes as a better alternative to be used in liquid biopsies. The liquid biopsy concept relies on a biological source of bioactive substances, i.e. protein, DNA, RNA or metabolite-based biomarkers, which is required for the assessment of the disease and its cure. Exosomes possess some distinct properties which render them favourable to be used in liquid biopsies, showing remarkable supremacy over other liquid biopsy sources, i.e., CTC, cfDNA or cfRNA. Exosomes are ubiquitous in nature and continuously produced and accumulated in body fluids; thus are more abundant than ctcDNAs or cfDNAs. Therefore, exosomes play a crucial role in reflecting better intratumour heterogeneity than CTCs, in cases where only a few CTCs are released like tumours of the central nervous system. Moreover, CTCs, cfDNA and cfRNA are prone to be degraded in biological fluids while exosomes having a lipid bilayer membrane which protect the cargo from degradation and keep it intact35. Furthermore, CTCs and cfDNAs are released during necrosis or apoptosis, while exosomes are actively secreted by living cells and are molecular signatures of their originating cell’s biological complexity, thus allowing for the real-time assessment of the disease. Thus, exosomes are better representative of their parental cells than CTCs/cfDNAs. Several studies have revealed that exosomal DNA/RNA yield higher sensitivity as well as specificity for detecting mutations compared to the total circulating cfDNAs and cfRNAs3637. Collection and enrichment of CTCs and cfDNA are complicated compared to exosomes which are relatively easy to be obtained through classic extraction methods such as ultracentrifugation38. Several other novel extraction methods and commercial isolation kits which require only a small amount of the human body fluid have also been developed which make them feasible for further clinical applications39. Exosomes are more homogeneous in terms of size and shape, which make them suitable and easily distinguishable from other molecules440. Exosomes contain specific surface markers such as CD63, CD9, HSP70 and TS10141 which can be used to discriminate them from other vesicles. Exosomes can be stored for 48-96 h at 4°C at −80°C for a long time and can also be isolated from long-term stored samples42. Storage stability of exosomes makes them significant for further clinical applications as it reduces the sample storage cost and avoids transportation difficulty. Exosomes exhibit specific surface proteins of target cells, a property that can be used in target specific drug deliveries43. The blood–brain barrier which was previously thought to be inaccessible, can now be accessed via exosomes; as these vesicles have the unique property of passing through this barrier44. With exhaustive literature published and clinical trials underway, exosomes have shown great scope and potential to be used in clinical applications for the diagnosis and therapeutics of various diseases (http://pubmed.ncbi.nlm.nih.gov; http://clinicaltrials.gov). The available exosome databases such as ExoCarta45 Vesiclepedia46 urinary exosome protein database47 and EVmiRNA48 are also enriching the field by providing updates about proteins and nucleic acid contents of exosomes, thus helping in planning the novel clinical set ups.

Limitations of exosome-based liquid biopsy

Rapid increase in the involvement of exosomes in clinical applications has drawn attention to the development of rapid and high throughput approaches for the biophysical and biological characterization of exosomes. Analysis of a biofluid-derived specific exosome retaining its biological activity is challenging and demands more careful studies in this field. Some issues that are proving as roadblocks in the path of exosomal application in liquid biopsies are discussed below.

Isolation/purification: The first challenge in exosomal analysis is their isolation and purification. Since exosomes are derived from different cell types and are isolated along with other cellular/molecular components, cellular debris act as contaminants which can hamper subsequent downstream analysis. Hence exosomal fraction needs to be purified before quantification. So far, several methods have been developed for exosomal isolation, including ultrafiltration, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, polymer-based precipitation, size exclusion through membrane, cell sorting, immunoaffinity capture, microfluidics and commercial kits-based isolation. All isolation methods have their own demerits; some have low recovery rate, some are expensive and hectic while some methods are specific but not applicable to large volumes. Even the available commercial kits for exosomal isolation have limitations as they utilize buffers which can hamper the qualitative and quantitative analysis of exosomes. Recent studies have demonstrated that extracellular vesicles isolated through different methods show significant variance in the properties like total miRNA yield, etc127128. Therefore, there is an urgent need for the improvement of extraction/purification devices; moreover, the method of exosome isolation should be selected based on sample type, starting sample volume and subsequent downstream analysis129.

Normalization: Normalization and quantitation of genetic materials isolated from exosomes is also a major challenge. The exosomes isolated from a biofluid do not necessarily contain the genetic materials of targeted tissues. There is always only a fraction of exosomes that are specific to those targeted cells/tissues. Identifying this fraction of exosomes originating from the targeted cells or tissues would enable tracking changes in exosomal levels over time, attributing them to specific source tissues when multiple samples from various targeted tissues need to be collected simultaneously. Instead of theoretical considerations, only empiric determinations will be able to resolve these normalization challenges. The utilization of marker-free and marker-based characterization techniques will help us to ascertain the ratio of exosomes originate from targeted and non-targeted cells/ tissues within a mixed population.

Quantification: There are several methods that can be used to distinguish desired exosomes from other extracellular vesicles or contaminants. Characterization of the exosomes may involve either surface marker-based characterization or shape/size-based characterization. The common methods used for exosome characterization based on exosomal markers are flow cytometry, liquid chromatography, immune-blot analysis, mass spectrometry, western immune blotting and dot blot assay. Although the characterization of exosomes is still a challenge in the exosomal analysis, it is expected to be resolved soon with the use of a combination of micro flow cytometry with droplet based barcoding and sequencing of the RNA content of individual exosomes.

Yield optimization: Although exosomal analysis is being made easy by the introduction and evaluation of novel high throughput technologies for isolation, purification and characterization, it is still not regularly used in clinical investigations due to the requirement of much time and expertise. In order to use exosomes in liquid biopsies, yield optimization is another challenge that is being faced by this field. Yield optimization can be obtained through the development of a single-step device for isolation, purification and characterization of exosomes. Exosome culture may also be a better option to resolve this problem. The existing knowledge about exosomes is the result of the prolonged experimental analysis of cultured exosomes. The yield of exosomes for therapeutic use can also be improved by propagating cell lines of the exosomes derived from a single, well-defined cell type.

Clinical validation/reproducibility: Another challenge for exosomal use in liquid biopsies is clinical validation of exosomes and their comparison to other biomarker platforms. Validation of exosome dosage and potency is difficult due to the heterogeneous nature of the exosomes, derived from cells having different characteristics. Therefore, the exchange of data across various studies and clinical trials also becomes difficult130. Evaluation and standardization of efficacy and dosage of exosomes will help to compare the results across different studies131. For recruiting exosomes in clinical applications, immunogenicity, safety and stability of exosomes have to be investigated at each processing step132.

Disease-associated alterations: Despite technological obstacles, some disease-associated alterations are also posing impediments in this field. Some disease-associated alterations or spontaneous fluctuations in the contents of patient’s biofluid may introduce dynamic noises to the biomarker analysis, which are inevitable and difficult to handle. There must be a strict defining collection protocol so that these introduced dynamic noises can be mitigated to some extent. Despite technical advancements and improvements, there must be a rigorous scrutiny for the selection of these exosome-derived biomarkers before their use as a liquid biopsy tool to increase sensitivity and diagnostic accuracy.

Technical advancements in exosome-based liquid biopsy

The increasing involvement of exosomes in clinical research, entail endeavours to deal with all the associated limitations. In the past few years, the research in exosomal science has witnessed some revolutionary advancement with noticeable increase in the development of numerous highly advanced techniques for isolation, quantification and analysis of exosomes. The rapidly growing field of exosomes requires an automated, fast and user-friendly method that is efficient for further downstream applications. Moreover, MISEV 2018 guidelines strictly recommended (i) determination of quantitative measures of source material along with the quantification of EVs, (ii) characterization of EVs to reassure of having abundant EVs (number of particles, amount of proteins/ lipids), (iii) determination of the presence of associated components, i.e. positive marker (unique for EV subtypes) and (iv) determination of the presence of non-vesicle components, i.e. negative marker for each preparation of EVs14. Therefore, some novel extraction methods or the combinations of methods have been applied in some recent clinical trials with the aim of higher exosome yield, purity and feasibility to downstream applications; like Vn96-peptide-based method133 anion-exchange method134 exosome total isolation chip135 various microfluidic platforms136 and combination of size exclusion chromatography with ultrafiltration.

After isolating exosomes, their characterization is a crucial step for the determination of the quality and quantity of these exosomes, especially when these are to be used in some medical interventions such as liquid biopsies or therapeutic usage. Characterization of exosomes ensure that the particular biomarker or the therapeutic effect is associated with exosomes only and not with any other sources such as cell debris or some other content isolated with these exosomes. General steps of characterization of exosomes include:

Quantification: As per the new recommendations from MISEV, both exosomes and the source of exosomes should be quantitatively measured and mentioned in each experimental preparation14. The source of the exosomes can be indicated as volume/weight/size of the biological sample, mentioning of the type of biological fluid (blood or urine etc.,) is equally important. The most commonly used quantification methods for exosomes are the estimation of total particle number and total protein amount. Particle number can be measured by various light scattering methods such as nanoparticle tracking analysis, high-resolution flow cytometry and dynamic light scattering137.

The total protein can be measured by sodium dodecyl-sulfate polyacrylamide gel electrophoresis and other colorimetric methods such as bicinchoninic acid or bradford method. Despite total protein amount, the total lipid amount can also be determined for quantification of exosomes. For the reliability of quantity, MISEV recommends the reporting of ratios of protein/lipid to total particles along with the global quantification measures14.

Characterization on the basis of protein content: Evaluation of some important protein markers provides a way to characterization of exosomes. According to the MISEV guidelines, at least one protein marker from each following category should be evaluated for the characterization of extra-cellular vesicles: (i) one transmembrane /lipid-bound protein, (ii) one cytosolic protein with membrane or lipid binding ability, (iii) one negative protein marker which is not part of EV but generally co-isolated during EV preparation, (iv)one positive protein marker that distinguishes the EV subtype and (v) one luminal protein with functional activities and the mode of its association with EVs14.

Characterization on basis of single vesicle analysis: The MISEV2018 guidelines recommend reporting of single vesicle analysis results with both close-up as well as and wild field images at high resolution by scanning probe microscopy, atomic force microscopy or super resolution microscopy techniques. MISEV further recommends the analysis using single-particle analyzer such as multi-angle light scattering coupled to asymmetric flow field-flow fractionation (AF4), high-resolution flow cytometry, fluorescence correlation spectroscopy or Raman spectroscopy. Recommendations for the characterization of EVs by MISEV include assessing the topology of EV-associated components, which can be determined by digestion (nuclease and protease), permeabilization and other antibody studies14.

The possibility of the isolation of exosomes specific to diseased cells through specific exosomal surface markers and the application of highly sensitive technologies for the detection and analysis such as BEAMing technology, qNANO GOLD, WES (Whole-exosome sequencing), Tam-seq (Tagged-amplicon deep sequencing), LFIA (Lateral Flow Immuno Assay), ARMS (amplification of refractory mutation system), SPR (surface plasmon resonance) and FFS (fluorescence fluctuation spectroscopy), NGS, digital PCR and PointMan™ DNA enrichment technology will allow the complete assessment of the disease. ‘Lab on a chip’ platforms, in combination with NPs have further improved the efficiency of exosomal analysis in the context of medical intervention.

Commercialization of exosome-based techniques and products

Exosome-based technologies have a promising commercial market lately. In recent years, recognizing the potential of exosomes in disease diagnosis and therapeutics, a large number of commercial companies have emerged and/or begun to invest in the development and production of exosome-based products. These companies are biotechnological/ pharmaceutical firms that are engaged in developing exosome-based techniques and products and launching them in the market. Some of these recently developed exosome-based techniques and products, along with the developing companies are listed in Supplementary Table I.

In general, exosomes used in clinical interventions are regulated as drugs and biological products under the Public Health Service Act and the Federal Food Drug and Cosmetic Act and require FDA approval under ‘Public Safety Notification’ released on December 6, 2019. Aegle Therapeutics Corp. was first to receive FDA approval for its exosome-based product to begin clinical trials in burn victims in April 2018138. Numerous companies have developed their exosome-based diagnostics and therapeutic platforms for further clinical applications. The ExoDx Prostate (IntelliScore) EPI test is the exosome-based diagnostic test for prostate cancer, which was developed by Exosome diagnostics (Exosome Dx) now part of Bio-Techne132. Another biomarker-based test developed by this firm is also an exosome-based detection of EGFR T790M in NSCLC cases which avoids unnecessary tumour biopsies and is more sensitive and specific compared to traditional cfDNA-based tumour biopsies92.Avalon GloboCare Corp. in collaboration with GenExosome Technologies, has also developed diagnostic tests for the detection of saliva-based exosome miRNA biomarker-miR-185139 and for the identification of human angiogenic exosomes140. ExoSTING™ and exoIL™ developed by Codiak Biosciences are the engineered exosomes to be used for therapeutic purposes141142.

Conclusions and future prospects

In the era of evolving minimally invasive diagnostic approaches and precision medicine, the considerable limitations of tissue biopsy have been gradually recognized and liquid biopsy is gaining widespread traction as a non-invasive, rapid and repeatable approach to be used in clinical settings. It is expected that liquid biopsy will drastically revolutionize future diagnostics and patient care and the market of liquid biopsy is likely to increase at the compound annual growth rate of ~19 per cent between 2021 and 2032143. In recent years, exosomes have been demonstrated to have a great potential to serve as novel biomarkers in liquid biopsy and provide new prospects for early diagnosis, prognosis, monitoring of treatment response and progression of communicable and non-communicable diseases. Despite the myriad advantages, the use of exosomes-based liquid biopsy in clinical settings is in its early stage and is still not considered as a standard method for diagnosing different diseases, including cancer. Currently, the clinical applications of exosome-based liquid biopsy are greatly restrained by the lack of effective enrichment technologies for the isolation and categorization of exosomes with high efficiency and purity from different body fluids, highly sensitive tools for the specific detection of exosome content and elevated cost. Although in recent decades, tremendous progress has been made and various methods/technologies have been developed for the isolation of exosomes as well as analysis of the molecular content of the exosome144. However, a standardized protocol for the high-throughput isolation with high-purity, categorization and quantification of exosomes from different body fluids (i.e.plasma, blood, urine, etc.) is not available. Most of the newly developed platforms for the detection of the molecular content of exosomes suffers from limited sensitivity due to the high heterogeneity of different exosome subsets in total exosomes derived from body fluids. The further advancement in technology for the detection and analysis of single exosome may reveal the distinct molecular profile of specific exosomes and can provide accurate information related to particular disease/condition. Therefore, more concerted efforts are required for the development and/or improvement of new cost-effective platforms/techniques with high-sensitivity, high-specificity and high-purity for exosome isolation and detection of molecular content. Since the content of exosomes critically depends on the stimulation and the donor cell environment, the documented disease-specific exosomal biomarkers need to be validated in a different population, including patients with comorbid/coinfection conditions for establishing exosomes as liquid biopsy tool in routine clinical practices.