Evaluation of exosomal non-coding RNAs in cancer using high-throughput sequencing

The gold standard for solid tumors' diagnosis is surgical tissue biopsies usually done after a series of imaging scans [1‐3]. The time, cost, repeatability, accessibility, and invasiveness of tissue biopsy-based diagnostics make them less desirable, leading to a late-stage diagnosis [4‐7]. For cancer patients to achieve the best possible treatment outcome, it is imperative to develop non-invasive and cost-efficient diagnostic methods that have the potential for early tumor detection and monitoring while the patient is undergoing treatment [3]. Liquid biopsy and molecular profiling of biofluids have gained increasing attention in recent decades due to their less invasive approaches, real-time tumor status insights, low cost, and ability to address tumor heterogeneity [8, 9]. The presence of biological materials in biofluids enables researchers to detect tumors occurring in distant tissues that are not symptomatic enough to be detected by conventional imaging techniques [10]. In fact, cancer care has been revolutionized through liquid biopsy, which has enabled early detection, improved diagnosis, predicted and determined therapeutic outcomes, and directed precision medicine [9]. Based on the source of tumor-derived constituents in biofluids, liquid biopsies for cancer are categorized into: (i) extracellular vehicles (EVs) and tumor-derived exosomes, (ii) circulating tumor cells (CTCs), and (iii) circulating tumor DNA (ctDNA) [8, 9]. Exosomes with sizes between 30 and 150 nm are small extracellular vesicles comprised of a lipid bilayer membrane and are responsible for intercellular communication. Stemming from the endosomal pathway within their parental cells, exosomes enclose a wide range of molecules including proteins, lipids, deoxyribonucleic acid (DNA), and different kinds of ribonucleic acids (RNAs) [10]. These contents are important for intercellular communication, both in normal physiological processes as well as in pathological conditions such as immune responses, pregnancy disorders, and cancer [11]. The presence and the stability of exosomes in most body fluids and their contents' similarity to parental cells make them promising candidates for developing new approaches for cancer diagnosis [12, 13]. Using high-throughput sequencing technologies, researchers have found that exosomes comprise different RNA populations including messenger RNAs (mRNAs), transferred RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), small nuclear RNAs (snRNAs), circular RNAs (circRNAs), long non-coding RNAs (lncRNAs), and P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) [14‐16]. The current review discusses the history and biogenesis of exosomes, NGS technology, and the potential ncRNAs biomarkers in different types of cancer detected by NGS. Furthermore, a brief overview of the advantages and challenges of exosome-based diagnostic techniques is provided.

About four decades ago, Pan, Stahl, and Johnstone discovered EVs while studying the loss of transferrin during reticulocyte maturation in blood. They believed that these vesicles are sprouted from different parts of the plasma membrane of cultured cells [17‐20]. Another group of researchers found that EVs result from germination into the intracellular endosome, which eventually forms multivesicular bodies (MVBs). MVBs produce intraluminal vesicles (ILVs) called exosomes. After a while, it turned out that exosomes are involved in intercellular communication [21, 22].

Exosomes with endocytic origin are morphologically very small vesicles whose structure is rich in macromolecules such as proteins and nucleic acids [10, 23]. Their biogenesis involves several stages of a complex biological process that have not yet been fully and accurately determined. The first processing stage begins with the lipid raft domains of the plasma membrane. The primary intracellular endosomes are obtained by budding into the membrane and then turning into the final endosomes via the Golgi apparatus. During this process, intraluminal vesicles are formed through the ESCRT-dependent mechanism (endosomal sorting complexes required for transmission) or non-ESCRT-dependent mechanisms within the final endosomes (Fig. 1). During the budding process of the endosomal membrane, biomolecules are accumulated inside them and form MVBs [24, 25]. As mentioned, ESCRT mediates the process of biogenesis [17] and contains a total of 20 proteins that are generally divided into four categories of ESCRT-0,-I,-II,-III [26, 27]. ESCRT-0 binds to ubiquitinated proteins in the endosomal membrane [28]. ESCRT-I and -II are responsible for membrane deformation in the form of buds with consecutive cargo [27], and ESCRT-III is responsible for the vesicular incision [27, 29]. Under physiological and pathological conditions, exosomes can be secreted from a variety of cancer cells [30, 31], platelets [32], mast cells [30], dendritic cells [33], astrocytes [34], B and T cells [35], etc. Numerous studies have shown that in addition to cells and tissues, exosomes can be contained in fluids such as blood [36], serum [37], amniotic fluid [38], saliva [37], and breast milk [39]. Depending on the cell or tissue type, the secretion of exosomes is regulated by two mechanisms: constitutive mechanism that employs Rab GTPases protein family [40‐43] and/or inducible mechanism regulated by several activating factors such as heat shock, hypoxia, DNA damage, increasing of intracellular calcium, and thrombin [40, 44, 45]. Since exosomes have an important role in intercellular communication, they can target a wide variety of cell types at both close and distant target sites to transfer cargo molecules, such as proteins, lipids, and nucleic acids [46]. The proteins that exosomes carry can be MHC I, II [30], tetraspanins such as CD9, CD63, CD81, CD82, CD54, and CD11b [24, 47], HSPs (60, 70, 90) [24, 48], Rab and annexins [49], Clathrin, Alix, and so on [24]. The main biomolecules in the exosome besides proteins are RNAs. To be more specific, in addition to three major RNAs (mRNA, tRNA, and rRNA) involved in protein synthesis, other non-coding RNAs such as miRNAs, small interfering RNAs (siRNAs), circRNA [50], lncRNA [51], small nucleolar RNAs (snoRNAs), and snRNA (all have a vital role in gene expression regulation at both transcriptional and post-transcriptional levels) [52‐55] are the main RNAs molecules in the exosomes. Furthermore, piRNAs are also another small non-coding RNA molecules found in exosomes and are resistant to chemical reactions and temperature changes. In animal cells, piRNAs are expressed and assemble into the protein-DNA complex through the Argonaute proteins [56‐59]. Many researchers have also found single-stranded [60] and double-stranded DNA within exosomes [61]. Batagov et al. provided evidences that exosomes can transport the 3-UTR fragments of a mRNA, suggesting that these fragments can have a regulatory effect or can be translated into proteins [62].

The following section provides an overview of noncoding RNA nomenclature to help readers understand the terms used in our article. Generally, the homology and function, location, and other factors related to non-coding RNAs contribute to their classification [63]. For example, the name microRNA is indicative of the small size of active RNA molecules, or the name snoRNA, which is an abbreviation for small nucleolar RNA, refers to RNA molecules inside a cell’s nucleolus [64]. Regarding miRNAs, nomenclature can be as follows: a symbol for the human miRNA gene that has been approved by the Human Genome Organization (HUGO) Gene Nomenclature Committee (HGNC) is MIR# format. For example, MIR17 represents a miRNA gene; mir-17 represents a miRNA stem-loop, and miR-17 represents a mature miRNA. Furthermore, each species’ name commences with three letters specific to it. When the letter “has” is used, it refers to humans (Homo sapiens), “hsa-mir-220” as an example, or it refers to rats (Rattus norvegicus) when “rno” is used such as “rno-mir-1”. To identify the members of the same family, the letter is added to the suffix (e.g. hsa-mir-465a and hsa-mir-465b). The 3p’ or 5p’ suffix can also be added if the data are insufficient to determine which sequence is dominant. It is also worth noting that let-7 and lin-4 are notable historical exceptions [64‐67].

On the other hand, lncRNA genes consist of a dispersed set of loci related only by their size, which exceed 200 bases, do not have conserved sequence homology, and perform a variety of functions. Generally, it is a known function of the product that determines the naming of lncRNAs. It would be preferable if genes were named based on their normal function rather than mutant phenotypes. Names for genes must be concise and do not try to include all known information about them. As an example, in lncRNA XIST, 'XIST' stands for 'X (inactive)-specific transcript', which is responsible for transcriptionally silencing one of the X chromosomes pairs. No known functions for lncRNA genes are determined by their genomic context [63, 64].

Circulating RNA is generated by the back-splicing of exons from a pre-mRNA, which results in a 3′,5′-phosphodiester bond and creates a circular RNA linked to itself. A nomenclature scheme for cirRNAs has been proposed: “cir[gene symbol]-n”, where the “gene” symbolizes the unspliced host gene, and “n” corresponds to an iterative five-digit number; for example, circPARN-00001 is the first circRNA that has been named for the host gene Poly(A)-specific ribonuclease (PARN) [64].

Due to the precise diagnostic and prognostic results provided by exosomes, they have become an interesting research topic in the field of medicine [68‐70]. Because exosomes carry biomolecular markers in many types of diseases and are also used as agents to deliver a variety of therapeutic molecules, they can be employed as an efficient tool to detect and treat a wide variety of diseases [71‐74]. Besides, there are other advantages of exosomes, including their ease of use, cost-effectiveness, and pain reduction, which allow clinics to consider using exosomes to diagnose many types of diseases, especially cancer, as an alternative to classical surgical methods [75]. Studies that analyze RNAs in exosomes for identifying various types of cancer have been frequently carried out in recent years. For example, Skog et al. examined the serum of patients with glioblastoma and isolated exosomes containing mRNAs and miRNAs. They found that tumor-derived microvesicles are used as a means of transmitting genetic information to the target cells [76]. Takeshita et al. reported that the miR-1246 expression could be used as a prognostic and a diagnostic tool for esophageal squamous cell carcinoma as exosomes could protect miRNA from degradation by RNase [77].

In the 1990s, many methods were developed for sequencing DNA molecules, and after 2000 the commercialization of NGS platforms was initiated quickly by the companies [78]. This method is a high-throughput sequencing that can perform millions of sequencing reactions simultaneously. NGS is one of the most widely used and advanced tools to identify diseases such as cancer [79, 80]. Using the NGS method, exome, and genome sequencing, chromatin immunoprecipitation sequencing (ChIP-Seq), transcript profiling, and epigenome characterization can be performed [81, 82]. Based on NGS, DNA from cancer samples can be sequenced from a few thousand nucleotides (targeted panel sequencing) to 40–50 million nucleotides (whole-exome sequencing (WES)) and up to 3.3 billion bases (whole-genome sequencing (WGS)).[83]. Currently, targeted gene panels are used in clinics to search for genomic variation in a wide variety of disease-related genes, and whole-exome sequencing is used to identify variation throughout the exome [84]. Over the past few years, NGS has been shown to provide useful information about various cancers and mutations associated with the various types of cancer [85, 86]. Data from the TCGA (The Cancer Genome Atlas), which utilized large-scale genome sequencing to identify markers involved in the 33 cancer types, have contributed to an improvement in cancer diagnosis, treatment, and prevention [87, 88]. Additionally, RNA-sequencing (RNA-seq) has revealed that all forms of RNA, from coding RNA to non-coding RNA, can be detected in exosomes which can be useful for cancer diagnosis [26, 98‐103]. Figure 2 illustrates the steps involved in identifying biomarkers of cancer using NGS.

The following section discusses exosome biomarkers related to various cancer types. Since NGS has become a routine technology in cancer research in recent years and it can provide valuable information about various cancers, the present study attempted to narrow its focus on studies that utilize NGS to analyze exosome noncoding RNAs in cancers. Furthermore, the selections were based on cancers having similar supporting studies.

Exosomes offer numerous advantages over other diagnostic methods. Firstly, they are secreted by virtually all living cells, exist in various body fluids, and their overall levels are commonly elevated in most diseases while retaining biological information from the parent cells. These features indicate their importance in reflecting the status of parental cells and the convenience of their extractions [9, 143‐149]. For instance, obtaining exosomes from body fluids is more convenient than isolating CTCs as these cells are minimal in the blood [150, 151]. With many known classic extraction approaches and a remarkable number of new methods under development, exosomes are more qualified for clinical application than CTCs [150, 152].

Second, exosomes with lipid bilayers could stabilize and protect biomacromolecules against proteinases, RNases, and other enzymatic activity existing in the biofluids, allowing them to be stored for an extended period unlike various biomarker assays requiring fresh biofluids [153‐157]. The storage of exosomes at 4 °C for 24 h followed by freezing at − 80 °C will not change markedly the quality of exosomal markers when compared with immediate storage at − 80 °C and fresh urine samples [158]. Indeed, the high biological stability of exosomes could greatly increase their clinical applications while reducing the cost of sample short-term storage and comforting their transportation [159].

Third, exosomes greatly enhance sensitivity and specificity by reducing the complexity of the biological matrix and reducing noises in the assay, thereby enabling the detection of low abundance biomacromolecules. [160‐162]. The amplified sensitivity of exosome-based biomarkers versus total serum and urine biomarkers has been shown by several studies [163‐165]. For example, in patients with colorectal cancers, exosomal miRNAs extracted from sera showed higher sensitivity (90%) in comparison to carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) serum biomarkers with 30.7% and 16% sensitivity, respectively [165]. In addition, it is challenging to distinguish the noises of normal cells from the signal of cancerous cells while transcriptome analysis of RNA is conducted [166]. Additionally, when RNA is directly isolated from plasma, it is even more challenging to analyze due to the overwhelming megakaryocyte RNA background caused by platelet-derived RNA. [167]. The fact that exosomes contain tumor-specific surface proteins makes them uniquely able to eliminate normal cells noises [166].

Fourth, as far as DNA is concerned, it is more valuable than other sources of DNA, for example, exosomes contain more cfDNA and mitochondrial DNA copies than plasma, which makes them more effective for detecting cancer [168, 169]. Moreover, exosomal DNA mutation frequency has higher detection sensitivity and specificity and greater prognostic value compared with cfDNA [170‐173].

Fifth, NGS-based studies have shown that exosomes contain a variety of RNA biotypes such as circRNAs [9] lncRNA [155, 174], mRNAs [76, 175‐177], miRNAs [178], mRNA fragments [62], piwi-interacting RNA, and fragments of numerous noncoding RNAs including transfer RNA, ribosomal RNA, Y RNAs, and vault RNA [16, 155, 179‐183]. Currently, mRNAs are the most typical biomarkers with clinical applications even though, microRNA studies have dominated the investigations on clinical biomarkers as they are plentiful RNA biotypes with well-known regulatory capabilities (Table 1). The longer RNA biotypes particularly mRNAs with well-known mutations have been known as ‘low hanging fruit’ [9]. Long RNAs not only make the assessment of gene expression levels and the recognition of tumor-specific somatic modifications possible but also make it possible to study the processes indicating disease state or progression [62, 155, 179]. The detection of circRNAs specific to diseases provides effective insights that cannot be obtained by employing small RNA, DNA, or protein assays [9].

Sixth, exosomes can improve the sensitivity of mutation detection. Compared with ctDNA alone, circulating nucleic acids especially exosomal RNAs could increase the whole number of mutant copies accessible for sampling [9, 171]. Combined analysis of ctDNA and exosomal RNA showed that the epidermal growth factor receptor (EGFR) gene activating mutations had almost 10 times more copies. It suggests that exosomal RNA may increase the potential for detection of mutations in blood samples, particularly when very few copies of ctDNA are available at the beginning of diseases [9, 171]. Moreover, the follow-up of EGFR, Kirsten rat sarcoma virus (KRAS) and, v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) genes mutations in ctDNA and exosomes over time revealed that the combined analysis of tumor materials from exosomes with ctDNA could markedly improve the achievement of a liquid biopsy test [171].

Some obstacles are limiting the clinical application of exosomes as tumor markers. The first limitation is the lack of a rapid, efficient, and cost-effective exosome isolation method, which is essential for reproducible analytical results [157]. Ultracentrifugation, ultrafiltration, density-gradient ultracentrifugation, immunoprecipitation, and size-exclusion chromatography are some of the more common methods used for exosome isolation [184, 185]. Unfortunately, there have been reports that these methods are extremely costly, time-consuming, cumbersome, and they cannot remove impurities [186]. The gold standard for the isolation and purity of exosomes is ultracentrifugation; however, it is the most time-consuming method [3, 187‐189]. The second challenge is the identification and quantification of exosomes. Flow cytometry, enzyme-linked immunosorbent, nanoparticle tracking analysis (NTA) are commonly used methods for the quantification of exosomes, each with its own advantages and disadvantages [190]. For example, flow cytometry can detect and sort numerous exosomes by species. However, it requires expensive equipment and yields inconsistent results [3]. Enzyme-linked immunoassay (ELISA) is used for more specific capturing of tumor-derived exosomes by applying specific antibodies against tumor markers such as Glypican-1 (GPC-1), Caveolin-1 (CAV1), or heat shock protein 90 (HSP90) [3]. However, the biological noise or contamination caused by other biomolecules in the sample endangers the qualification of ELISA for exosome identification [3]. NTA could identify the type of exosome, but it is time-consuming as well and requires expensive equipment [3]. An ideal method should address these limitations in terms of purity, cost, equipment, and time to be clinically applicable [3]. Third, the great heterogeneity of extracellular vesicles adds an extra level of difficulty in their purification. There have been many studies developing exosome-based biomarkers, but they have employed different methods for purifying the exosomes, resulting in difficulties with normalizing the results and making comparisons difficult [186]. Despite the argument that exosomes should be classified as blood cells, it has been shown that exosomes have obvious heterogeneity due to their contents as well as their surface characterization [186].

In recent decades, the failure of conventional solid biopsy in addressing cancer detection has highlighted the importance of liquid biopsy as a minimally invasive and alternative approach for prognosis, diagnosis, and monitoring of cancer patients. A liquid biopsy is acquired from various body fluids and consists of abundant biological information from the originated cells, such as CTC, exosomes, ctDNA, or cell-free DNA, as well as circulating RNAs. The advances in NGS make CTCs and ctDNAs more cost-effective, but they suffer from several limitations, including the limited number of CTCs in the blood or the degradation of ctDNA soon after release. On the other hand, exosomes widely exist in all body fluids, carry about 90% of circulating tumor DNAs, and can protect different biomolecules, such as nucleic acids, proteins, and lipids, against degradation. More importantly, exosomes carrying different biotypes of nucleic acids, reflecting the earlier stages of pathological states, provide a promising platform for cancer prognosis, diagnosis, and the follow-up of treatment in precision and personalized medicine.

Unfortunately, the clinical application of exosomes is currently limited by several factors, including the lack of an ideal isolation and characterization method and the inability to classify exosomes secreted by normal cells and tumor cells. Hence, the need for addressing these limitations to use the unique potential of exosomes for capturing the dynamic complexity of cancer is currently highly demanded.