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Review

Exosomes: A Source for New and Old Biomarkers in Cancer

by
Mariantonia Logozzi
,
Davide Mizzoni
,
Rossella Di Raimo
and
Stefano Fais
*
Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
*
Author to whom correspondence should be addressed.
Cancers 2020, 12(9), 2566; https://0-doi-org.brum.beds.ac.uk/10.3390/cancers12092566
Submission received: 28 July 2020 / Revised: 4 September 2020 / Accepted: 7 September 2020 / Published: 9 September 2020
Versions Notes

Abstract

:

Simple Summary

The follow-up of patients with tumors needs new or at least more reliable biomarkers to avoid invasive approaches. Unfortunately, the existing biomarkers too often have generated more problems than having proven to be sufficiently helpful for the clinical oncologists. Very recently, translational research has focused on extracellular vesicles, with size ranging between micro to nano as potential sources of new tumor biomarkers. In particular, nanovesicles (called exosomes) that are variably released from virtually all cells, have shown to be a potential source of new tumor biomarkers but also a preferential delivery system for well-known biomarkers, such as PSA and CEA. The clinical data supporting this new research area are, unfortunately, very few, but the existing reports are very encouraging. We review and discuss the existing literature supporting a key role of exosomes as a source of tumor biomarkers, together with some unexpected discoveries.

Abstract

Clinical oncology needs reliable tumor biomarkers to allow a follow-up of tumor patients who do not necessarily need invasive approaches. To date, the existing biomarkers are not sufficiently reliable, and many of them have generated more problems than facilitating the commitment of clinical oncologists. Over the last decades, a broad family of extracellular vesicles, with size ranging between micro to nano, has been raised as a new hope for potential sources of new tumor biomarkers. However, while knowledge in the field is increasing, we do not currently have definitive information allowing a clinical use of extracellular vesicles in cancer clinics. Recent evidence provides new perspective in clinical oncology, based on data showing that circulating nanovesicles called exosomes may represent a valuable source of tumor biomarkers. In this review, we discuss the existing clinical data supporting a key role of exosomes as a source of tumor biomarkers, including proteins and miRNAs, but also discuss the importance of the expression of known tumor biomarkers when expressed on exosomes.

1. What Exosomes Are

Exosomes are 40–180 nm extracellular vesicles that are released from virtually all cells under normal and pathological conditions [1,2,3,4,5]. They form by membrane invagination of late endosomes and are released in the extracellular microenvironment after multivesicular bodies (MVBs) fusion with the plasma membrane [1,2,6,7]. Thus, the exosomes proteins’ make up derives from a cascade of internal vesicle fusion, MVB formation, and their fusion with the plasma membrane makes. Exosomes express specific markers of the endosomal pathway, such as tetraspanins (CD63, CD9, and CD81), but also heat shock proteins (HSP70) and proteins from the Rab family, Tsg101 and Alix, which are not detectable in other types of vesicles of similar size [1,2,8]. For similar reasons, exosomes may express markers acquired during the process of internal vesicles’ fusion with the plasma membrane, thus witnessing the cellular source [1,2]. We know that hematopoietic cells (B cells, T cells, dendritic cells, mast cells, and platelets), intestinal epithelial cells, Schwann cells, neuronal cells, adipocytes, fibroblasts (NIH3T3), and tumor cells release vesicles extracellularly [8,9]. However, exosomes have a characteristic lipid bilayer composition of their membrane, and they contain a cargo of nucleic acids, including DNAs, messenger RNAs (mRNAs), and microRNAs (miRNAs) [1,2]. Recently, it has been shown that exosomes deliver proteins exerting a functional activity. From a functional point of view, extracellular vesicles (EVs) were originally suggested to be involved in the removal of unnecessary molecules that are poorly degraded by the intracellular lysosomal system [10]. Indeed, exosomes are not only cell ‘cleaners’, but crucial actors in cell-to-cell communication [1,2,3,5,11,12,13,14,15]. They are detectable in virtually all biological fluids, including plasma, urine, cerebrospinal fluid, epididymal fluid, amniotic fluid, malignant and pleural effusions of ascites, bronchoalveolar lavage fluid, synovial fluid, and breast milk, suggesting a that EVs may have a critical role in connecting tissues, organs and compartments of our body [1,9,16,17,18], but also transferring infectious agents including viruses and pathological prion proteins [13,19].
Knowledge of EVs is rapidly increasing and what we are learning is extremely challenging. In fact, the available data support a key role of this broad family of vesicles in both the homeostasis of our body and the pathophysiology of the vast majority of human diseases, including cancer, thus representing a valuable source of disease biomarkers [1,2,20,21,22,23,24,25,26,27,28,29,30].
Presently, the protein cargo of EVs and exosomes that have been purified from human body fluids have shown some level of variability, due to both the broad spectrum of cellular sources and the inter-individual variability [20,31,32,33]. The choice of the exosome source is pivotal to obtain reliable and reproducible results, considering the different biophysical and chemical properties of each biological fluid. It is, in fact, mandatory that, despite the possible lipoproteins contamination, plasma and serum are by far the body fluids witnessing a systemic situation; while, for instance, urine and stool may represent a source of exosomes from the urinary tract and the gut, respectively [1,2,34]. Moreover, the plasma is considered the most suitable source of exosomes being deprived of fibrinogen, thus allowing the best exosome recovery in terms of number and cargo (proteins and RNA) [1,9,22,34].
Thanks to a general improvement of the technical equipment to analyze EVs, as represented by nanoparticle tracking analysis (NTA), immune captured based technologies, and nanoscale flow cytometry (NFC), we can now obtain reliable quantitative and qualitative information potentially useful for future clinical application [16,20,35,36].

2. Isolation Methods of Plasma-Derived Exosomes

A consensus agreement on a standard method for isolation of plasma-derived exosomes still lacks actually, while many approaches have been proposed in the case of cell culture supernatants. However, ultracentrifugation is currently the widely used technique for exosomes isolation; it is based on the differential sedimentation rate of particles according to their size, shape, and density. Although this technique is time-consuming and raising some problems on the vesicle integrity, there is a general agreement supporting its use is the most versatile between the available methods, and probably the one providing the most complete extracellular vesicle yield independently from the source [2,33,37,38]. Another method that is used is an iodixanol/sucrose gradient or sucrose density cushion protocol (commonly defined as the density gradient ultracentrifugation). However, while it allows better purification of the EVs population, this technique leads to a great loss of nanovesicles during fractionation; and in plasma samples, it does not allow for the satisfactory separations of vesicles from apolipoproteins and blood HDL [39]. Size exclusion chromatography (SEC) has become a popular technique to purify exosomes from body fluids, in particular from plasma. It is a customizable procedure using different types of matrices/resins commercially available to enhance pick resolution of particles (increase in column length) of the following spectroscopic analysis, or concentrate samples from a large volume (increase in column diameter) [38]. SEC isolation excludes albumin from the exosome separations, but on the other side, it, together of being cost and time expensive, has shown to be not reliable as a technique to be translated into the clinical use, in as much as it provided variable results on the purity of samples between different laboratories [39].
Moreover, SEC leads to the formation of aggregates that affects the quality of the product, as it has been shown with all the methodologies based on polymeric precipitation mixtures [39]. Exosomes can be isolated from human body fluids by immunocapture or immunoaffinity, as well. These methods provide a methodology aimed at immunocapturing exosomes on antibodies attached to either plastic wells or immunomagnetic beads or chromatographic matrices or microfluidic platforms, all based on the specific recognition of exosomal membrane marker by monoclonal antibodies [33,37]. There is a general consensus on the reliability of this approach with a high potentiality for clinical application [37,38]. It currently has some limitations, mostly due to the low exosome yield and some problems in eluting the complexes exosomes-antibodies and some level of non-specific binding of other micro/nanocorpes to the substrates [33,37,38]. A further method that appears promising is the exosome purification, based on microfluidics allowing a better purity of exosomes isolation, in a very short time [33]. However, it did not show an ideal performance in terms of the number of EVs/exosomes obtained, and presently lacks an acceptable level of standardization [33]. In summary, while it is not perfect, the best approach is to use ultracentrifugation with implementation with immunocapture-based techniques. Of course, the goal of future techniques should be to set up methodologies that need to be validated—and are, hopefully, adaptable to all the clinical laboratories worldwide.

3. A Role of Exosomes in Cancer Progression and Metastasis

Malignant cancers have some phenotypes in common, including hypoxia, low nutrient supply, and extracellular acidosis [40,41,42,43]. Recently, EVs (especially exosomes) have emerged as an alternative mediator of cell-to-cell communication within the tumor microenvironment (TME) and cancer metastasis [41,42,43]. A marked increase in the exosomes, released under acidic pH (6.5) has been shown as compared to a physiological pH (7.4) [4]. This occurred independently from the tumor histotype, meaning that prostate cancer, melanoma, osteosarcoma, breast, and colorectal carcinoma-derived cell lines all showed an increased exosomes, released when cultured at acidic condition [4]. Moreover, the exosomes released at low pH showed always a smaller size as compared to those released at pH 7.4, which showed a more heterogeneous size [4]. The increase in the exosome release at in vitro acidic condition was comparable to the increased plasmatic exosome levels measurable in prostate cancer patients as compared to different controls, including inflammatory conditions, such as patients with benign prostate hypertrophy or healthy individuals [35]. However, there is increasing evidence that argues tumor microenvironmental acidity may actively participate in determining an increased release of exosomes in cancer conditions [5]. In fact, exosomes play a key role in tumor growth and metastasis through the formation of tumor niches in target organs together with inducing malignant transformation in resident mesenchymal stem cells [3,14,44,45].
The ensemble of these data shows that exosomes have a prominent role in favoring both the growth of primary tumors and their metastatic spread [44,45]. However, some data support a role of exosomes as primary actors in a recent Darwinian-like theory on tumor formation, hypothesizing that tumor progression passes through a microenvironment-determined progressive selection of cells with very high adaptive features—in turn, allowing tumors to live and grow in a very hostile microenvironment [46,47,48,49]. In fact, the experimental evidence supports a key role of the acidic tumor milieu in boosting exosome release from malignant cells [5]. It appears, therefore, conceivable that the exosome hyperproduction by tumor cells may be induced by the toxic microenvironment that probably selects cells that use an extracellular release of vesicles to eliminate toxic molecules to prevent their intracellular accumulation. Recent evidence suggests that exosomes released in acidic condition express ions transporters, such as Carbonic Anhydrase IX (CA IX), that on exosomes exerts a full enzymatic function [50]. A further investigation has shown that increased CA-related enzymatic function is measurable in plasmatic exosomes from cancer patients [51], suggesting that CA-IX expression and activity in plasmatic exosomes preparation may represent a new valuable cancer biomarker in the near future [20,52].
The versatile cargo of EVs, on the one hand, allows them to play a multitude of roles within the tumor microenvironment; on the other hand, it renders EVs a very promising source of tumor biomarkers [22,53], including protein, lipids, and a series of nucleic acids (i.e., DNAs, mRNAs, and miRNA) [17,21]. The harsh microenvironmental conditions of hypoxia, acidity, and low nutrient supply are responsible for the increased exosome release by tumor cells, as well as the hyperexpression of known tumor markers, such as PSA [35], and proteins related to ion/proton transport (e.g., V-ATPase, CA-IX) [50].
An issue that deserves some discussion is the increase in the number of circulating exosomes, that while in vitro related to the acidic pH, has been shown into the plasma of tumor patients as well [35]. This was hypothesized in 2017 [22] on the basis of clinical data obtained using an immunocapture-based technique [16] and confirmed with different techniques in tumor patients and independently from tumor histologies [4,35,54]. Moreover, pre-clinical in vivo experiments, reported a direct correlation between the tumor mass and the levels of plasmatic exosomes [16]. This was supported by some clinical reports showing that the surgical removal of the primary tumor has led to a dramatic drop down of the plasmatic exosome levels [55,56]. This suggests that the amount of circulating exosome levels may represent a way to monitor the effectiveness of both medical and surgical therapies, and also extremely useful in the patients’ follow-up. On the other hand, it appears conceivable that this huge amount of circulating tumor exosomes may represent a real danger for cancer patients, given the role of exosomes in tumor metastasis [3,14], also suggesting that therapies aimed at reducing exosome production by tumors may represent a goal of future anti-tumor treatments.

4. The Clinical Relevance of Exosomes as Biomarkers of Cancer

Research on exosomes as a source of tumor markers, covering several cancer types, is increasing (Table 1); nevertheless, only a few exosome-based diagnostic assays are currently available for clinical use.
An immunocapture-based ELISA (IC-ELISA) test pioneered an attempt to quantify and characterize plasmatic exosomes [16]. With this approach, it has been shown that CD63+ and CAV1+ plasma exosomes levels were significantly higher in melanoma patients as compared to healthy donors [16]. On the basis of the early described test, the IC-ELISA has been recently modified and compared with other emerging technologies, such as NTA and NFC [35]. The ensemble of these three techniques confirmed that under acidic condition, cancer cells released an increased number of exosomes [35] and that this was a common feature of virtually all cancers [4]. In the same study, both IC-ELISA and NFC showed that in acidic conditions, human prostate cancer cells released increased amounts of exosomes expressing PSA [35]. However, this approach was exploited in a prospective clinical study in prostate cancer patients, as compared to both patients with benign prostate hypertrophy (BPH) and healthy controls [36]. The results showed that the levels of plasmatic exosomes expressing PSA were significantly increased in patients with prostate cancer, with significantly higher sensitivity and specificity as compared to the standard serum PSA in the same patients [36]. A careful statistical analysis, comparing the serum PSA to the exosome PSA levels (as determined by either IC-ELISA or NFC), showed that the exosome-related measures are significantly correlated, thus reflecting the same biological phenomenon, but also that serum PSA and exosome PSA are independent values [36]. This confirmed the impossibility of serum PSA to discriminate Pca from BPH patients, while the plasmatic levels of exosomes expressing PSA clearly distinguished not only cancer patients from healthy individuals but cancer patients from patients with a non-tumor condition, such as BPH [36]. This is a result of paramount importance, inasmuch as serum PSA determination, while it is currently used worldwide for Pca early diagnosis and clinical follow-up, has sadly shown a high number of false positives and false negatives with many unwanted consequences [85].
These data highly support the use of IC-based technologies for both characterization and quantification of circulating exosomes, providing potential new sources of clinical biomarkers for screening tests, diagnosis, and follow-up of cancer patients, and possibly also patients with other diseases. IC-ELISA should deserve very much attention for several reasons including: (i) It is noninvasive; (ii) it is rapid, affordable, specific, quantitative, and versatile (easily extendable to other markers/conditions); it requires small amounts of sample and has multiple readouts; (iv) it allows both screenings and follow-up applications; and lastly (v) it can be translated with reasonable costs to all the research and clinical laboratories worldwide. Most of all, IC-ELISA provides the possibility of screening multiple markers within the same sample, and therefore, the possibility to explore in the same plasma sample the expression on exosomes of know tumor biomarkers, surrogate tumor biomarkers, and hopefully new tumor biomarkers.

5. Clinical Data on the Plasmatic Exosome Levels and the Hyperexpression of Known Tumor Biomarkers

As specified above, a current limitation of exosome diagnostic applications is the lack of standardization for methods concerning both exosome isolation and further analysis [33,37,38]. However, despite a number of commercially available assays for exosome isolation providing fast protocols, the approach allowing the best yield still requires ultracentrifugation protocols of large volumes [2,33,37,38]. Another important concern derives from the evidence is that the vast majority of exosome purification from human body fluids contain both the heavy and light chains of the immunoglobulins—this is intriguing information, but at the same time represents a great limit to perform reliable qualitative approaches, such as Western blot analysis [2,33,37].
By using an immunocapture-based assay, it was shown that patients with advanced melanoma patients (stage III and IV) had higher plasmatic exosomes levels as compared to healthy donors, with the best results with exosomes expressing caveolin-1 [16]. These data suggested the ability of exosomes-associated caveolin-1 to detect advanced melanoma more effectively than the Lactate dehydrogenase (LDH) serum levels commonly used in the follow-up of melanoma patients [16]. These findings are supported by recent studies showing that in melanoma patients, MIA and S100B-positive exosomes were significantly higher than in healthy controls [58] and that the amount of serum exosomes from patients with different hematological malignancies is significantly higher than in healthy individuals [84]. This last study identified markers on circulating nanovesicles reflecting their cellular source, including CD19 in B cell neoplasm, CD38 in multiple myeloma, CD13 in myeloid tumors, and CD30 in Hodgkin’s lymphoma [84].
A clinical study has shown that patients with Glioblastoma (GBM) have higher EVs plasma levels and that the EVs levels significantly decrease after surgical resection and raise again at recurrence [54], with very promising implication for therapy response, and monitoring.
According to these findings, a study has shown an increase of exosome levels in the urine of prostate cancer (PCA) patients using a time-resolved fluorescence immunoassay [86]. A clinical study performed in patients with colorectal cancer showed that the levels of plasmatic exosomes were statistically higher than in healthy controls [75]. A study, published in 2005, has shown that plasmatic exosomes from colon cancer patients expressed carcinoembryonic antigen (CEA), together with death receptors ligands [77]. In prostate cancer patients, the number of plasmatic exosomes, measured by NTA, was higher than in the plasma of healthy controls [60]. Recently, a milestone study has shown that glypican-1 (GPC1) positive exosomes were detectable in the serum of patients with pancreatic cancer with a high level of specificity and sensitivity, as compared to both healthy subjects and patients with a benign pancreatic disease [69]. However, the same study showed that high levels of GPC1 on exosomes can be detected in breast cancer patients as well [69], suggesting that exosome GPC1 is not a tumor-specific marker.
The ensemble of these clinical reports suggests the use of plasmatic exosomes quantification as a new and suitable method for the clinical follow-up of cancer patients. In further supporting this hypothesis, it has been shown that the effect of the treatment with Imatinib in patients with gastrointestinal stromal tumor (GIST) may be monitored by measuring the plasmatic levels of EVs before and after treatment [87]. Thus, suggesting that the effectiveness of a therapy may be evaluated by measuring the EVs levels in cancer patients. A recent clinical study performed in patients with Oral Squamous Cell Carcinoma has shown that surgical treatment induced a dramatic reduction of the plasmatic levels of exosomes expressing CD63 as early as one week after resection, as assessed by IC-ELISA [56]. A pre-clinical study has shown that treatment with proton pump inhibitors of a xenograft model of human melanoma induced a significant reduction of plasmatic EV levels consistent with a reduction of the tumor size [88]. This approach may be used in evaluating the effectiveness of radiotherapy as well [89].
Probably the most convincing pre-clinical evidence in this sense is that showing a direct correlation between exosome levels and the tumor size [16], suggesting that circulating exosomes in cancer patients may represent roughly an esteem of the tumor mass, including both the primary tumor and the metastasis.
A crucial issue is that the assessment of EVs levels in cancer patients may be highly improved if implemented with highly specific markers. To this purpose, it is mandatory to carry on with studies looking at new EVs-related tumor biomarkers, including proteins, lipids, and nucleic acids. Some clinical studies have shown that tumor patients’ exosomes express higher levels of some known tumor markers such as CEA in colon cancer patients [77], but also many other markers, while not always with completely reliable data (reviewed in [21]). Two recent studies on PSA are a milestone example on the importance of the reappraisal of exosomal expression of known tumor biomarkers. The first one showed the increased expression of PSA in exosomes that are purified from both prostate cancer cells under acidic culture conditions and plasma of prostate cancer patients [35]. More importantly, a prospective clinical study has shown that the plasmatic levels of exosome expressing PSA was significantly higher in patients with prostate cancer as compared to both patients with BPH and healthy individual patients [36]. These data were obtained by implementing two different techniques, both using monoclonal antibodies against exosome antigens and PSA, i.e., IC-ELISA and NFC. The prospective clinical study was performed in 240 individuals, including 80 PC patients, 80 BPH patients, and 80 healthy CTR [36]. The statistical analysis showed that pooling together the two analysis, the PSA exosome determination got to 96% of sensitivity and 100% specificity in distinguishing PC from BPH patients, while the IC-ELISA alone showed 98.57% of sensitivity and 80.28% of specificity in distinguishing PC patients from BPH patients, and 100% of both sensitivity and specificity in distinguishing PC patients from healthy individuals [36]. This last result has suggested that IC-ELISA may be exploited in future screening tests performed in young men to the purpose of getting to a reliable early diagnosis of prostate cancer with a non-invasive technique.
In addition to plasma, urine is another body fluid that can be easily exploited in the clinical management of cancer patients, particularly in patients with genitourinary tract malignancies. Interestingly, proteomic analysis of urinary EVs may represent a valuable diagnostic tool in certain kidney pathologies [90]. The authors showed that polycystin-1 and polycystin-2 expression, together with that of other Ca2+-binding proteins (annexin A1, annexin A2, protein S100-A9, protein S100-A8, and retinoic acid-induced protein 3) expressed in EVs preparations from patients’ urines are significantly altered in kidney diseases, including tumors [90].
Exosomes of the prostate epithelium that are released into semen or urine (prosteasome) also contain important molecular information related to certain types of tumors [91,92]. For example, analysis of urine-derived exosomes of prostate cancer patients identified tumor biomarkers, such as prostate-specific antigen and PCA3 [86,92,93,94,95]. Increased level of d-catenin has shown in exosomes preparations obtained from both prostate cancer patients’ urine and PC-3 cell culture supernatants [96]. Some potential exosome-associated biomarkers related to the EGF receptor signaling mechanism were shown in bladder cancer as well [97,98].
An entirely new and exciting area is the identification of tumor-associated nucleic acids within the exosome preparation obtained from patients’ body fluids. The reports suggesting that RNAs are pretty detectable in exosome samples are increasing, also suggesting that within exosomes the RNAs are protected from degradation by ribonucleases enriching the human body fluids [99].

6. Circulating Exosomal miRNAs as Tumor Biomarkers

Besides the protein cargo, the exosomes isolated from different body fluids contain a significant amount of nucleic acids, such as mRNA, miRNA, long non-coding RNA (lncRNA), as well as DNA. Starting from Valadi’s discovery of exosomal mRNAs and miRNAs [15], exosomal miRNAs have been extensively studied as they are the most abundant in exosomes and EVs [100]. Important differences have been shown in exosomal miRNAs composition and amount between cancer patients and healthy individuals, supporting their use as potential non-invasive clinical biomarkers [101] (summarized in Table 2).
However, the use of exosomal miRNA in clinical oncology has been to date limited by technical and analytical biases that affect the yield, integrity, and purity of the exosomal miRNA [2,102]. Indeed, the miRNAs in biological fluids are either packaged in the vesicles or associated with RNA-binding protein (e.g., Argonaute 2) or with lipoprotein complexes (mostly HDL and LDL) [2,102,103]. During ultracentrifugation for exosomes isolation, circulating non-exosomal RNA are pelleted together with exosomal preparations with subsequent RNA contamination in EVs preparations [103]. One possible approach to solve this drawback could be to immunocapture exosome/EVs before carrying on with miRNA detection. In fact, through this technique, exosomes may be attached to plastic plates covered with antibodies recognizing specific exosomal markers (or even a specific tumor marker if available) and perform miRNAs characterization on the attached exosome/EVs population. It appears a feasible technical approach that will make a more reliable RNA characterization on EVs purification from patients’ samples.
Table
Table 2. Detection of exosomal microRNAs as tumor biomarkers in serum and plasma.
Table 2. Detection of exosomal microRNAs as tumor biomarkers in serum and plasma.
PathologyBiomarkerSourceIsolation MethodPotential UseReference
Prostate cancermiR-141, miR-375SerumFiltration-based capture of exosomesDiagnosis/Stage Determination[104]
miR-1290, miR-375PlasmaPolymeric precipitationPrognosis[105]
miR-141SerumPolymeric precipitationDiagnosis[106]
Colorectal cancerlet-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, miR-23aSerumUCEarly Diagnosis[107]
miR-19SerumUC, Polymeric precipitationPrognosis[108]
miR-4772-3pSerumPolymeric precipitationPrognosis for recurrent stage II, III[109]
miR-21PlasmaUCPrognosis[110]
miR-221PlasmaUCPrognosis[111]
Ovarian cancermiR-21, miR-214, miR-200a, miR-200b, miR-200c, miR-203, miR-205, miR-141SerumModified magnetic activated cell sorting (MACS) procedureEarly Diagnosis/Prognosis[112]
miR-373, miR-200a, miR-200b, miR-200cSerumPolymeric precipitationDiagnosis/Prognosis[113]
miR-21, miR-100, miR-200b, miR-320PlasmaPolymeric precipitationDiagnosis[114]
Breast cancermiR-101, miR-372,
miR-373		SerumPolymeric precipitationDiagnosis[115]
miR-1246, miR-21PlasmaUC, Polymeric precipitationDiagnosis[116]
Lung cancermiR-151a-5p, miR-30a-3p, miR-200b-5p, miR-629, miR-100, miR-154-3pPlasmaPolymeric precipitationEarly Diagnosis[117]
Esophageal Squamous Cell CarcinomamiR-21PlasmaPolymeric precipitationEarly Diagnosis/Therapy[118]
Laryngeal Squamous Cell CarcinomamiR-21and HOTAIR (lncRNA)SerumPolymeric precipitationDiagnosis/Prognosis[119]
Pancreatic cancermiR-17-5p, miR-21SerumUCDiagnosis/Prognosis[120]
miR-1246, miR-4644, miR-3976, miR-4306SerumUC, Density gradient centrifugationDiagnosis[74]
miR-10b, miR-21, miR-30c, miR-181a, miR-let7aPlasmaUCDiagnosis[121]
miR-191, miR-21,
miR-451a		SerumPolymeric precipitationDiagnosis[122]
miR-451aPlasmaUCPrognosis[123]
Gastric cancermiR-423-5pSerumPolymeric precipitationDiagnosis/Prognosis[124]
Hepatocellular carcinomamiR-18a, miR-221,
miR-222, miR-224		SerumPolymeric precipitationDiagnosis[125]
miR-718SerumUCDiagnosis/Prognosis/Recurrence[126]
UC, ultracentrifugation.

7. The Strategic Role of Immunocapture-Based Approach in the Development of Exosome-Based Diagnostic Approach in Cancer

When our group described for the first time the immunocapture-based approach in exosome-based cancer diagnostic [16] (patent Exo-test, PCT/EE2009/000001), it received many criticisms in the ISEV community, mostly based on the use of anti-Rab5 antibodies to capture the EVs. However, in the last five years, many groups across the world have reported both pre-clinical and clinical data obtained with the same approach, and thus, supporting an agreed use of this methodology in cancer diagnostics [127,128]. We had the chance to compare a modified IC-ELISA to both NFC and NTA in their potential clinical relevance. IC-ELISA showed many advantages as compared to both the other techniques inasmuch it allows at the same time to quantify and characterize the clinical sample, and to include broader populations of EVs in terms of size, from nano to micro [35,36].
Moreover, differently to NTA, IC-ELISA evaluated the expression of a broad spectrum of biomarkers, including both those EVs specific and those that may represent either specific or surrogate tumor biomarkers [16,35,36,56] (Table 3), and most of all, in a considerable amount of clinical sample at the same time.
The IC-ELISA has the key advantage to be exportable in the majority of the clinical laboratories worldwide, needing to be implemented with the use of an ultracentrifuge that presently is the most accepted technical approach to obtain EVs and exosomes from either pre-clinical or clinical samples [2]. The immunocapture-base technique may also represent a valuable new tool for differentiating EVs through their protein expression, and thus, allowing them to perform nucleic acid analyses in different EVs subpopulation, and this could be of paramount importance for clinical research based on micro-RNAs studies [99].

8. Conclusions

Incorporating IC-ELISA technology into a platform for exosome-associated mRNA analysis is expected to enable detection and quantification of plasma RNAs of well-defined tumor origin, providing highly sensitive and specific assessment while avoiding some of the ‘noise’ that hampers quantitative real-time PCR and microarray analysis of whole-body fluids. Besides, exosomes can be enriched in mRNAs and miRNAs, which are hardly detectable in the parent tissue where their signal is covered by the presence of a higher number of molecules. Thus, exosomes can well represent the source for the identification of novel disease-associated markers.
Altogether these data suggest that quantification and characterization of EVs in human body fluid may be highly helpful as a new non-invasive diagnostic tool for the clinical management of cancer patients. The different expression of tumor biomarkers in circulating exosomes may lead to early diagnosis, to improve the tumor staging, as well as to evaluate the progression of the disease. In fact, the clinical study showing the relevance of exosomes expressing PSA in prostate cancer diagnosis suggests that future clinical investigation should also be aimed at verifying a potential new role of the old tumor biomarkers when expressed on exosomes. However, further clinical studies are needed to validate the use of either plasmatic or other body-fluids derived exosomes in the clinical practice, with considerable advantages both for patients, avoiding or limiting unnecessary invasive procedures, and hopefully significantly reducing the costs of the public health.

Author Contributions

Conceptualization: S.F., M.L.; Data curation: S.F., M.L., D.M., R.D.R.; Investigation: S.F., M.L.; Project administration: S.F.; Supervision: S.F.; Visualization: S.F., M.L., D.M., R.D.R.; Writing—original draft: S.F., M.L.; Writing—review & editing: S.F., M.L., D.M., R.D.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Yáñez-Mó, M.; Siljander, P.R.-M.; Andreu, Z.; Bedina Zavec, A.; Borràs, F.E.; Buzas, E.I.; Buzas, K.; Casal, E.; Cappello, F.; Carvalho, J.; et al. Biological properties of extracellular vesicles and their physiological functions. J. Extracell. Vesicles 2015, 4, 27066. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Théry, C.; Witwer, K.W.; Aikawa, E.; Alcaraz, M.J.; Anderson, J.D.; Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G.K.; et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 2018, 7, 1535750. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Spugnini, E.; Logozzi, M.; Di Raimo, R.; Mizzoni, D.; Fais, S. A Role of Tumor-Released Exosomes in Paracrine Dissemination and Metastasis. Int. J. Mol. Sci. 2018, 19, 3968. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Logozzi, M.; Mizzoni, D.; Angelini, D.; Di Raimo, R.; Falchi, M.; Battistini, L.; Fais, S. Microenvironmental pH and Exosome Levels Interplay in Human Cancer Cell Lines of Different Histotypes. Cancers 2018, 10, 370. [Google Scholar] [CrossRef] [Green Version]
  5. Logozzi, M.; Spugnini, E.; Mizzoni, D.; Di Raimo, R.; Fais, S. Extracellular acidity and increased exosome release as key phenotypes of malignant tumors. Cancer Metastasis Rev. 2019, 38, 93–101. [Google Scholar] [CrossRef]
  6. Cocucci, E.; Meldolesi, J. Ectosomes and exosomes: Shedding the confusion between extracellular vesicles. Trends Cell Biol. 2015, 25, 364–372. [Google Scholar] [CrossRef]
  7. Zhang, Y.; Liu, Y.; Liu, H.; Tang, W.H. Exosomes: Biogenesis, biologic function and clinical potential. Cell Biosci. 2019, 9, 19. [Google Scholar] [CrossRef]
  8. van der Pol, E.; Böing, A.N.; Harrison, P.; Sturk, A.; Nieuwland, R. Classification, Functions, and Clinical Relevance of Extracellular Vesicles. Pharmacol. Rev. 2012, 64, 676–705. [Google Scholar] [CrossRef] [Green Version]
  9. Properzi, F.; Logozzi, M.; Fais, S. Exosomes: The future of biomarkers in medicine. Biomark. Med. 2013, 7, 769–778. [Google Scholar] [CrossRef]
  10. Johnstone, R.M.; Adam, M.; Hammond, J.R.; Orr, L.; Turbide, C. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J. Biol. Chem. 1987, 262, 9412–9420. [Google Scholar]
  11. Andreola, G.; Rivoltini, L.; Castelli, C.; Huber, V.; Perego, P.; Deho, P.; Squarcina, P.; Accornero, P.; Lozupone, F.; Lugini, L.; et al. Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles. J. Exp. Med. 2002, 195, 1303–1316. [Google Scholar] [CrossRef] [PubMed]
  12. Camussi, G.; Deregibus, M.-C.; Bruno, S.; Grange, C.; Fonsato, V.; Tetta, C. Exosome/microvesicle-mediated epigenetic reprogramming of cells. Am. J. Cancer Res. 2011, 1, 98–110. [Google Scholar] [PubMed]
  13. Canitano, A.; Venturi, G.; Borghi, M.; Ammendolia, M.G.; Fais, S. Exosomes released in vitro from Epstein–Barr virus (EBV)-infected cells contain EBV-encoded latent phase mRNAs. Cancer Lett. 2013, 337, 193–199. [Google Scholar] [CrossRef]
  14. Zhao, H.; Achreja, A.; Iessi, E.; Logozzi, M.; Mizzoni, D.; Di Raimo, R.; Nagrath, D.; Fais, S. The key role of extracellular vesicles in the metastatic process. Biochim. Biophys. Acta Rev. Cancer 2018, 1869, 64–77. [Google Scholar] [CrossRef] [PubMed]
  15. Valadi, H.; Ekström, K.; Bossios, A.; Sjöstrand, M.; Lee, J.J.; Lötvall, J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 2007, 9, 654–659. [Google Scholar] [CrossRef] [Green Version]
  16. Logozzi, M.; De Milito, A.; Lugini, L.; Borghi, M.; Calabrò, L.; Spada, M.; Perdicchio, M.; Marino, M.L.; Federici, C.; Iessi, E.; et al. High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients. PLoS ONE 2009, 4, e5219. [Google Scholar] [CrossRef] [Green Version]
  17. Fais, S.; Logozzi, M.; Lugini, L.; Federici, C.; Azzarito, T.; Zarovni, N.; Chiesi, A. Exosomes: The ideal nanovectors for biodelivery. J. Biol. Chem. 2013, 394, 1–15. [Google Scholar] [CrossRef]
  18. Alvarez-Erviti, L.; Seow, Y.; Yin, H.; Betts, C.; Lakhal, S.; Wood, M.J.A. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 2011, 29, 341–345. [Google Scholar] [CrossRef]
  19. Properzi, F.; Logozzi, M.; Abdel-Haq, H.; Federici, C.; Lugini, L.; Azzarito, T.; Cristofaro, I.; di Sevo, D.; Ferroni, E.; Cardone, F.; et al. Detection of exosomal prions in blood by immunochemistry techniques. J. Gen. Virol. 2015, 96, 1969–1974. [Google Scholar] [CrossRef]
  20. Fais, S.; O’Driscoll, L.; Borras, F.E.; Buzas, E.; Camussi, G.; Cappello, F.; Carvalho, J.; Cordeiro da Silva, A.; Del Portillo, H.; El Andaloussi, S.; et al. Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine. ACS Nano 2016, 10, 3886–3899. [Google Scholar] [CrossRef]
  21. Zocco, D.; Ferruzzi, P.; Cappello, F.; Kuo, W.P.; Fais, S. Extracellular Vesicles as Shuttles of Tumor Biomarkers and Anti-Tumor Drugs. Front. Oncol. 2014, 4. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Cappello, F.; Logozzi, M.; Campanella, C.; Bavisotto, C.C.; Marcilla, A.; Properzi, F.; Fais, S. Exosome levels in human body fluids: A tumor marker by themselves? Eur. J. Pharm. Sci. 2017, 96, 93–98. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Li, A.; Zhang, T.; Zheng, M.; Liu, Y.; Chen, Z. Exosomal proteins as potential markers of tumor diagnosis. J. Hematol. Oncol. 2017, 10, 175. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Soung, Y.; Ford, S.; Zhang, V.; Chung, J. Exosomes in Cancer Diagnostics. Cancers 2017, 9, 8. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Sumrin, A.; Moazzam, S.; Khan, A.A.; Ramzan, I.; Batool, Z.; Kaleem, S.; Ali, M.; Bashir, H.; Bilal, M. Exosomes as Biomarker of Cancer. Braz. Arch. Biol. Technol. 2018, 61. [Google Scholar] [CrossRef]
  26. Huang, T.; Deng, C.-X. Current Progresses of Exosomes as Cancer Diagnostic and Prognostic Biomarkers. Int. J. Biol. Sci. 2019, 15, 1–11. [Google Scholar] [CrossRef]
  27. Vafaei, S.; Fattahi, F.; Ebrahimi, M.; Janani, L.; Shariftabrizi, A.; Madjd, Z. Common molecular markers between circulating tumor cells and blood exosomes in colorectal cancer: A systematic and analytical review. Cancer Manag. Res. 2019, 11, 8669–8698. [Google Scholar] [CrossRef] [Green Version]
  28. Meng, Y.; Sun, J.; Wang, X.; Hu, T.; Ma, Y.; Kong, C.; Piao, H.; Yu, T.; Zhang, G. Exosomes: A Promising Avenue for the Diagnosis of Breast Cancer. Technol. Cancer Res. Treat. 2019, 18, 153303381882142. [Google Scholar] [CrossRef] [Green Version]
  29. Wong, C.-H.; Chen, Y.-C. Clinical significance of exosomes as potential biomarkers in cancer. World J. Clin. Cases 2019, 7, 171–190. [Google Scholar] [CrossRef]
  30. Campanella, C.; Caruso Bavisotto, C.; Logozzi, M.; Marino Gammazza, A.; Mizzoni, D.; Cappello, F.; Fais, S. On the Choice of the Extracellular Vesicles for Therapeutic Purposes. Int. J. Mol. Sci. 2019, 20, 236. [Google Scholar] [CrossRef] [Green Version]
  31. Boukouris, S.; Mathivanan, S. Exosomes in bodily fluids are a highly stable resource of disease biomarkers. Proteom. Clin. Appl. 2015, 9, 358–367. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Ludwig, N.; Whiteside, T.L.; Reichert, T.E. Challenges in Exosome Isolation and Analysis in Health and Disease. Int. J. Mol. Sci. 2019, 20, 4684. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Li, X.; Corbett, A.L.; Taatizadeh, E.; Tasnim, N.; Little, J.P.; Garnis, C.; Daugaard, M.; Guns, E.; Hoorfar, M.; Li, I.T.S. Challenges and opportunities in exosome research—Perspectives from biology, engineering, and cancer therapy. APL Bioeng. 2019, 3, 011503. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Witwer, K.W.; Buzás, E.I.; Bemis, L.T.; Bora, A.; Lässer, C.; Lötvall, J.; Nolte-‘t Hoen, E.N.; Piper, M.G.; Sivaraman, S.; Skog, J.; et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J. Extracell. Vesicles 2013, 2, 20360. [Google Scholar] [CrossRef]
  35. Logozzi, M.; Angelini, D.F.; Iessi, E.; Mizzoni, D.; Di Raimo, R.; Federici, C.; Lugini, L.; Borsellino, G.; Gentilucci, A.; Pierella, F.; et al. Increased PSA expression on prostate cancer exosomes in in vitro condition and in cancer patients. Cancer Lett. 2017, 403, 318–329. [Google Scholar] [CrossRef]
  36. Logozzi, M.; Angelini, D.F.; Giuliani, A.; Mizzoni, D.; Di Raimo, R.; Maggi, M.; Gentilucci, A.; Marzio, V.; Salciccia, S.; Borsellino, G.; et al. Increased Plasmatic Levels of PSA-Expressing Exosomes Distinguish Prostate Cancer Patients from Benign Prostatic Hyperplasia: A Prospective Study. Cancers 2019, 11, 1449. [Google Scholar] [CrossRef] [Green Version]
  37. Yu, L.-L.; Zhu, J.; Liu, J.-X.; Jiang, F.; Ni, W.-K.; Qu, L.-S.; Ni, R.-Z.; Lu, C.-H.; Xiao, M.-B. A Comparison of Traditional and Novel Methods for the Separation of Exosomes from Human Samples. BioMed Res. Int. 2018, 2018, 1–9. [Google Scholar] [CrossRef] [Green Version]
  38. Konoshenko, M.Y.; Lekchnov, E.A.; Vlassov, A.V.; Laktionov, P.P. Isolation of Extracellular Vesicles: General Methodologies and Latest Trends. BioMed Res. Int. 2018, 2018, 1–27. [Google Scholar] [CrossRef]
  39. Villa, F.; Quarto, R.; Tasso, R. Extracellular Vesicles as Natural, Safe and Efficient Drug Delivery Systems. Pharmaceutics 2019, 11, 557. [Google Scholar] [CrossRef] [Green Version]
  40. Fais, S.; Overholtzer, M. Cell-in-cell phenomena in cancer. Nat. Rev. Cancer 2018, 18, 758–766. [Google Scholar] [CrossRef]
  41. Pillai, S.R.; Damaghi, M.; Marunaka, Y.; Spugnini, E.P.; Fais, S.; Gillies, R.J. Causes, consequences, and therapy of tumors acidosis. Cancer Metastasis Rev. 2019, 38, 205–222. [Google Scholar] [CrossRef] [PubMed]
  42. Fais, S.; Venturi, G.; Gatenby, B. Microenvironmental acidosis in carcinogenesis and metastases: New strategies in prevention and therapy. Cancer Metastasis Rev. 2014, 33, 1095–1108. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Gillies, R.J.; Pilot, C.; Marunaka, Y.; Fais, S. Targeting acidity in cancer and diabetes. Biochim. Biophys. Acta Rev. Cancer 2019, 1871, 273–280. [Google Scholar] [CrossRef]
  44. Peinado, H.; Zhang, H.; Matei, I.R.; Costa-Silva, B.; Hoshino, A.; Rodrigues, G.; Psaila, B.; Kaplan, R.N.; Bromberg, J.F.; Kang, Y.; et al. Pre-metastatic niches: Organ-specific homes for metastases. Nat. Rev. Cancer 2017, 17, 302–317. [Google Scholar] [CrossRef] [PubMed]
  45. Lugini, L.; Valtieri, M.; Federici, C.; Cecchetti, S.; Meschini, S.; Condello, M.; Signore, M.; Fais, S. Exosomes from human colorectal cancer induce a tumor-like behavior in colonic mesenchymal stromal cells. Oncotarget 2016, 7. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Ardaševa, A.; Gatenby, R.A.; Anderson, A.R.A.; Byrne, H.M.; Maini, P.K.; Lorenzi, T. Evolutionary dynamics of competing phenotype-structured populations in periodically fluctuating environments. J. Math. Biol. 2019. [Google Scholar] [CrossRef] [Green Version]
  47. Gatenby, R.A.; Zhang, J.; Brown, J.S. First Strike–Second Strike Strategies in Metastatic Cancer: Lessons from the Evolutionary Dynamics of Extinction. Cancer Res. 2019, 79, 3174–3177. [Google Scholar] [CrossRef] [Green Version]
  48. Gillies, R.J.; Brown, J.S.; Anderson, A.R.A.; Gatenby, R.A. Eco-evolutionary causes and consequences of temporal changes in intratumoural blood flow. Nat. Rev. Cancer 2018, 18, 576–585. [Google Scholar] [CrossRef]
  49. Mazzocca, A.; Ferraro, G.; Misciagna, G.; Fais, S. Moving the systemic evolutionary approach to cancer forward: Therapeutic implications. Med. Hypotheses 2018, 121, 80–87. [Google Scholar] [CrossRef]
  50. Logozzi, M.; Capasso, C.; Di Raimo, R.; Del Prete, S.; Mizzoni, D.; Falchi, M.; Supuran, C.T.; Fais, S. Prostate cancer cells and exosomes in acidic condition show increased carbonic anhydrase IX expression and activity. J. Enzyme Inhib. Med. Chem. 2019, 34, 272–278. [Google Scholar] [CrossRef] [Green Version]
  51. Logozzi, M.; Mizzoni, D.; Capasso, C.; Del Prete, S.; Di Raimo, R.; Falchi, M.; Angelini, D.F.; Sciarra, A.; Maggi, M.; Supuran, C.T.; et al. Plasmatic exosomes from prostate cancer patients show increased carbonic anhydrase IX expression and activity and low pH. J. Enzyme Inhib. Med. Chem. 2020, 35, 280–288. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. Lener, T.; Gimona, M.; Aigner, L.; Börger, V.; Buzas, E.; Camussi, G.; Chaput, N.; Chatterjee, D.; Court, F.A.; del Portillo, H.A.; et al. Applying extracellular vesicles based therapeutics in clinical trials–an ISEV position paper. J. Extracell. Vesicles 2015, 4, 30087. [Google Scholar] [CrossRef] [PubMed]
  53. O’Driscoll, L.; Stoorvogel, W.; Théry, C.; Buzas, E.; Nazarenko, I.; Siljander, P.; Yáñez-Mó, M.; Fais, S.; Giebel, B.; Yliperttula, M. European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD). Eur. J. Pharm. Sci. 2017, 98, 1–3. [Google Scholar] [CrossRef]
  54. Osti, D.; Del Bene, M.; Rappa, G.; Santos, M.; Matafora, V.; Richichi, C.; Faletti, S.; Beznoussenko, G.V.; Mironov, A.; Bachi, A.; et al. Clinical Significance of Extracellular Vesicles in Plasma from Glioblastoma Patients. Clin. Cancer Res. 2019, 25, 266–276. [Google Scholar] [CrossRef] [Green Version]
  55. Campanella, C.; Rappa, F.; Sciumè, C.; Marino Gammazza, A.; Barone, R.; Bucchieri, F.; David, S.; Curcurù, G.; Caruso Bavisotto, C.; Pitruzzella, A.; et al. Heat shock protein 60 levels in tissue and circulating exosomes in human large bowel cancer before and after ablative surgery: Exosomal Hsp60 in Large Bowel Cancer. Cancer 2015, 121, 3230–3239. [Google Scholar] [CrossRef] [PubMed]
  56. Rodríguez Zorrilla, S.; Pérez-Sayans, M.; Fais, S.; Logozzi, M.; Gallas Torreira, M.; García García, A. A Pilot Clinical Study on the Prognostic Relevance of Plasmatic Exosomes Levels in Oral Squamous Cell Carcinoma Patients. Cancers 2019, 11, 429. [Google Scholar] [CrossRef] [Green Version]
  57. Peinado, H.; Alečković, M.; Lavotshkin, S.; Matei, I.; Costa-Silva, B.; Moreno-Bueno, G.; Hergueta-Redondo, M.; Williams, C.; García-Santos, G.; Ghajar, C.M.; et al. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat. Med. 2012, 18, 883–891. [Google Scholar] [CrossRef] [Green Version]
  58. Alegre, E.; Zubiri, L.; Perez-Gracia, J.L.; González-Cao, M.; Soria, L.; Martín-Algarra, S.; González, A. Circulating melanoma exosomes as diagnostic and prognosis biomarkers. Clin. Chim. Acta 2016, 454, 28–32. [Google Scholar] [CrossRef]
  59. Khan, S.; Jutzy, J.M.S.; Valenzuela, M.M.A.; Turay, D.; Aspe, J.R.; Ashok, A.; Mirshahidi, S.; Mercola, D.; Lilly, M.B.; Wall, N.R. Plasma-Derived Exosomal Survivin, a Plausible Biomarker for Early Detection of Prostate Cancer. PLoS ONE 2012, 7, e46737. [Google Scholar] [CrossRef] [Green Version]
  60. Nawaz, M.; Camussi, G.; Valadi, H.; Nazarenko, I.; Ekström, K.; Wang, X.; Principe, S.; Shah, N.; Ashraf, N.M.; Fatima, F.; et al. The emerging role of extracellular vesicles as biomarkers for urogenital cancers. Nat. Rev. Urol. 2014, 11, 688–701. [Google Scholar] [CrossRef]
  61. Gabriel, K.; Ingram, A.; Austin, R.; Kapoor, A.; Tang, D.; Majeed, F.; Qureshi, T.; Al-Nedawi, K. Regulation of the Tumor Suppressor PTEN through Exosomes: A Diagnostic Potential for Prostate Cancer. PLoS ONE 2013, 8, e70047. [Google Scholar] [CrossRef]
  62. Beach, A.; Zhang, H.-G.; Ratajczak, M.Z.; Kakar, S.S. Exosomes: An overview of biogenesis, composition and role in ovarian cancer. J. Ovarian Res. 2014, 7, 14. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. An, T.; Qin, S.; Xu, Y.; Tang, Y.; Huang, Y.; Situ, B.; Inal, J.M.; Zheng, L. Exosomes serve as tumour markers for personalized diagnostics owing to their important role in cancer metastasis. J. Extracell. Vesicles 2015, 4, 27522. [Google Scholar] [CrossRef] [PubMed]
  64. Zhao, Z.; Yang, Y.; Zeng, Y.; He, M. A microfluidic ExoSearch chip for multiplexed exosome detection towards blood-based ovarian cancer diagnosis. Lab Chip 2016, 16, 489–496. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Szajnik, M.; Derbis, M.; Lach, M.; Patalas, P.; Michalak, M.; Drzewiecka, H.; Szpurek, D.; Nowakowski, A.; Spaczynski, M.; Baranowski, W.; et al. Exosomes in Plasma of Patients with Ovarian Carcinoma: Potential Biomarkers of Tumor Progression and Response to Therapy. Gynecol. Obstet. 2013, 4, 3. [Google Scholar] [CrossRef] [Green Version]
  66. Chen, Y.; Wang, L.; Zhu, Y.; Chen, Z.; Qi, X.; Jin, L.; Jin, J.; Hua, D.; Ma, X. Breast cancer resistance protein (BCRP)-containing circulating microvesicles contribute to chemoresistance in breast cancer. Oncol. Lett. 2015, 10, 3742–3748. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  67. Fang, S.; Tian, H.; Li, X.; Jin, D.; Li, X.; Kong, J.; Yang, C.; Yang, X.; Lu, Y.; Luo, Y.; et al. Clinical application of a microfluidic chip for immunocapture and quantification of circulating exosomes to assist breast cancer diagnosis and molecular classification. PLoS ONE 2017, 12, e0175050. [Google Scholar] [CrossRef] [Green Version]
  68. Ciravolo, V.; Huber, V.; Ghedini, G.C.; Venturelli, E.; Bianchi, F.; Campiglio, M.; Morelli, D.; Villa, A.; Mina, P.D.; Menard, S.; et al. Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy. J. Cell. Physiol. 2012, 227, 658–667. [Google Scholar] [CrossRef]
  69. Melo, S.A.; Luecke, L.B.; Kahlert, C.; Fernandez, A.F.; Gammon, S.T.; Kaye, J.; LeBleu, V.S.; Mittendorf, E.A.; Weitz, J.; Rahbari, N.; et al. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 2015, 523, 177–182. [Google Scholar] [CrossRef] [Green Version]
  70. Moon, P.-G.; Lee, J.-E.; Cho, Y.-E.; Lee, S.J.; Chae, Y.S.; Jung, J.H.; Kim, I.-S.; Park, H.Y.; Baek, M.-C. Fibronectin on circulating extracellular vesicles as a liquid biopsy to detect breast cancer. Oncotarget 2016, 7, 40189–40199. [Google Scholar] [CrossRef] [Green Version]
  71. Vardaki, I.; Ceder, S.; Rutishauser, D.; Baltatzis, G.; Foukakis, T.; Panaretakis, T. Periostin is identified as a putative metastatic marker in breast cancer-derived exosomes. Oncotarget 2016, 7. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  72. Moon, P.-G.; Lee, J.-E.; Cho, Y.-E.; Lee, S.J.; Jung, J.H.; Chae, Y.S.; Bae, H.-I.; Kim, Y.-B.; Kim, I.-S.; Park, H.Y.; et al. Identification of Developmental Endothelial Locus-1 on Circulating Extracellular Vesicles as a Novel Biomarker for Early Breast Cancer Detection. Clin. Cancer Res. 2016, 22, 1757–1766. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Lee, S.J.; Lee, J.; Jung, J.H.; Park, H.Y.; Lee, C.-H.; Moon, P.-G.; Baek, M.-C.; Jeong, J.-H.; Park, J.; Chae, Y.S. Exosomal Del-1 as a potent diagnostic marker for breast cancer: A prospective cohort study. J. Clin. Oncol. 2017, 35, 11548. [Google Scholar] [CrossRef]
  74. Madhavan, B.; Yue, S.; Galli, U.; Rana, S.; Gross, W.; Müller, M.; Giese, N.A.; Kalthoff, H.; Becker, T.; Büchler, M.W.; et al. Combined evaluation of a panel of protein and miRNA serum-exosome biomarkers for pancreatic cancer diagnosis increases sensitivity and specificity. Int. J. Cancer 2015, 136, 2616–2627. [Google Scholar] [CrossRef]
  75. Silva, J.; Garcia, V.; Rodriguez, M.; Compte, M.; Cisneros, E.; Veguillas, P.; Garcia, J.M.; Dominguez, G.; Campos-Martin, Y.; Cuevas, J.; et al. Analysis of exosome release and its prognostic value in human colorectal cancer. Genes. Chromosomes Cancer 2012, 51, 409–418. [Google Scholar] [CrossRef]
  76. Li, J.; Chen, Y.; Guo, X.; Zhou, L.; Jia, Z.; Peng, Z.; Tang, Y.; Liu, W.; Zhu, B.; Wang, L.; et al. GPC1 exosome and its regulatory miRNAs are specific markers for the detection and target therapy of colorectal cancer. J. Cell. Mol. Med. 2017, 21, 838–847. [Google Scholar] [CrossRef]
  77. Huber, V.; Fais, S.; Iero, M.; Lugini, L.; Canese, P.; Squarcina, P.; Zaccheddu, A.; Colone, M.; Arancia, G.; Gentile, M.; et al. Human colorectal cancer cells induce T-cell death through release of proapoptotic microvesicles: Role in immune escape. Gastroenterology 2005, 128, 1796–1804. [Google Scholar] [CrossRef]
  78. Yokoyama, S.; Takeuchi, A.; Yamaguchi, S.; Mitani, Y.; Watanabe, T.; Matsuda, K.; Hotta, T.; Shively, J.E.; Yamaue, H. Clinical implications of carcinoembryonic antigen distribution in serum exosomal fraction-Measurement by ELISA. PLoS ONE 2017, 12, e0183337. [Google Scholar] [CrossRef]
  79. Yoshioka, Y.; Kosaka, N.; Konishi, Y.; Ohta, H.; Okamoto, H.; Sonoda, H.; Nonaka, R.; Yamamoto, H.; Ishii, H.; Mori, M.; et al. Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen. Nat. Commun. 2014, 5, 3591. [Google Scholar] [CrossRef]
  80. Tian, Y.; Ma, L.; Gong, M.; Su, G.; Zhu, S.; Zhang, W.; Wang, S.; Li, Z.; Chen, C.; Li, L.; et al. Protein Profiling and Sizing of Extracellular Vesicles from Colorectal Cancer Patients via Flow Cytometry. ACS Nano 2018, 12, 671–680. [Google Scholar] [CrossRef]
  81. Yoon, J.H.; Ham, I.-H.; Kim, O.; Ashktorab, H.; Smoot, D.T.; Nam, S.W.; Lee, J.Y.; Hur, H.; Park, W.S. Gastrokine 1 protein is a potential theragnostic target for gastric cancer. Gastric Cancer 2018, 21, 956–967. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  82. Yen, E.-Y.; Miaw, S.-C.; Yu, J.-S.; Lai, I.-R. Exosomal TGF-β1 is correlated with lymphatic metastasis of gastric cancers. Am. J. Cancer Res. 2017, 7, 2199–2208. [Google Scholar] [PubMed]
  83. Sandfeld-Paulsen, B.; Aggerholm-Pedersen, N.; Baek, R.; Jakobsen, K.R.; Meldgaard, P.; Folkersen, B.H.; Rasmussen, T.R.; Varming, K.; Jørgensen, M.M.; Sorensen, B.S. Exosomal proteins as prognostic biomarkers in non-small cell lung cancer. Mol. Oncol. 2016, 10, 1595–1602. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  84. Caivano, A.; Laurenzana, I.; De Luca, L.; La Rocca, F.; Simeon, V.; Trino, S.; D’Auria, F.; Traficante, A.; Maietti, M.; Izzo, T.; et al. High serum levels of extracellular vesicles expressing malignancy-related markers are released in patients with various types of hematological neoplastic disorders. Tumor Biol. 2015, 36, 9739–9752. [Google Scholar] [CrossRef] [PubMed]
  85. Hoffman, R.M. Screening for Prostate Cancer. N. Engl. J. Med. 2011, 365, 2013–2019. [Google Scholar] [CrossRef]
  86. Duijvesz, D.; Versluis, C.Y.L.; van der Fels, C.A.M.; Vredenbregt-van den Berg, M.S.; Leivo, J.; Peltola, M.T.; Bangma, C.H.; Pettersson, K.S.I.; Jenster, G. Immuno-based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer: Extracellular vesicles as marker for PCa. Int. J. Cancer 2015, 137, 2869–2878. [Google Scholar] [CrossRef]
  87. Ogorevc, E.; Štukelj, R.; Bedina-Zavec, A.; Šuštar, V.; Šimundić, M.; Kralj-Iglič, V.; Janša, R. A 32-month follow-up study of nanovesicle concentrations in blood from 12 patients with gastrointestinal stromal tumour treated with imatinib. Biochem. Soc. Trans. 2013, 41, 303–308. [Google Scholar] [CrossRef]
  88. Federici, C.; Petrucci, F.; Caimi, S.; Cesolini, A.; Logozzi, M.; Borghi, M.; D’Ilio, S.; Lugini, L.; Violante, N.; Azzarito, T.; et al. Exosome Release and Low pH Belong to a Framework of Resistance of Human Melanoma Cells to Cisplatin. PLoS ONE 2014, 9, e88193. [Google Scholar] [CrossRef] [Green Version]
  89. Mutschelknaus, L.; Peters, C.; Winkler, K.; Yentrapalli, R.; Heider, T.; Atkinson, M.J.; Moertl, S. Exosomes Derived from Squamous Head and Neck Cancer Promote Cell Survival after Ionizing Radiation. PLoS ONE 2016, 11, e0152213. [Google Scholar] [CrossRef]
  90. Pocsfalvi, G.; Raj, D.A.A.; Fiume, I.; Vilasi, A.; Trepiccione, F.; Capasso, G. Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease. Proteom. Clin. Appl. 2015, 9, 552–567. [Google Scholar] [CrossRef]
  91. Ronquist, G.; Nilsson, B.O. The Janus-faced nature of prostasomes: Their pluripotency favours the normal reproductive process and malignant prostate growth. Prostate Cancer Prostatic Dis. 2004, 7, 21–31. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  92. Nilsson, J.; Skog, J.; Nordstrand, A.; Baranov, V.; Mincheva-Nilsson, L.; Breakefield, X.O.; Widmark, A. Prostate cancer-derived urine exosomes: A novel approach to biomarkers for prostate cancer. Br. J. Cancer 2009, 100, 1603–1607. [Google Scholar] [CrossRef] [PubMed]
  93. Mitchell, P.J.; Welton, J.; Staffurth, J.; Court, J.; Mason, M.D.; Tabi, Z.; Clayton, A. Can urinary exosomes act as treatment response markers in prostate cancer? J. Transl. Med. 2009, 7, 4. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  94. Øverbye, A.; Skotland, T.; Koehler, C.J.; Thiede, B.; Seierstad, T.; Berge, V.; Sandvig, K.; Llorente, A. Identification of prostate cancer biomarkers in urinary exosomes. Oncotarget 2015, 6. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  95. Fujita, K.; Nonomura, N. Urinary biomarkers of prostate cancer. Int. J. Urol. 2018, 25, 770–779. [Google Scholar] [CrossRef] [Green Version]
  96. Lu, Q.; Zhang, J.; Allison, R.; Gay, H.; Yang, W.-X.; Bhowmick, N.A.; Frelix, G.; Shappell, S.; Chen, Y.-H. Identification of extracellular δ-catenin accumulation for prostate cancer detection. Prostate 2009, 69, 411–418. [Google Scholar] [CrossRef] [Green Version]
  97. Smalley, D.M.; Sheman, N.E.; Nelson, K.; Theodorescu, D. Isolation and Identification of Potential Urinary Microparticle Biomarkers of Bladder Cancer. J. Proteome Res. 2008, 7, 2088–2096. [Google Scholar] [CrossRef]
  98. Lee, J.; McKinney, K.Q.; Pavlopoulos, A.J.; Niu, M.; Kang, J.W.; Oh, J.W.; Kim, K.P.; Hwang, S. Altered Proteome of Extracellular Vesicles Derived from Bladder Cancer Patients Urine. Mol. Cells 2018, 41, 179–187. [Google Scholar] [CrossRef]
  99. Keller, S.; Ridinger, J.; Rupp, A.-K.; Janssen, J.W.; Altevogt, P. Body fluid derived exosomes as a novel template for clinical diagnostics. J. Transl. Med. 2011, 9, 86. [Google Scholar] [CrossRef] [Green Version]
  100. Huang, X.; Yuan, T.; Tschannen, M.; Sun, Z.; Jacob, H.; Du, M.; Liang, M.; Dittmar, R.L.; Liu, Y.; Liang, M.; et al. Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genom. 2013, 14, 319. [Google Scholar] [CrossRef] [Green Version]
  101. Lapitz, A.; Arbelaiz, A.; O’Rourke, C.J.; Lavin, J.L.; Casta, A.L.; Ibarra, C.; Jimeno, J.P.; Santos-Laso, A.; Izquierdo-Sanchez, L.; Krawczyk, M.; et al. Patients with Cholangiocarcinoma Present Specific RNA Profiles in Serum and Urine Extracellular Vesicles Mirroring the Tumor Expression: Novel Liquid Biopsy Biomarkers for Disease Diagnosis. Cells 2020, 9, 721. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  102. Mateescu, B.; Kowal, E.J.K.; van Balkom, B.W.M.; Bartel, S.; Bhattacharyya, S.N.; Buzás, E.I.; Buck, A.H.; de Candia, P.; Chow, F.W.N.; Das, S.; et al. Obstacles and opportunities in the functional analysis of extracellular vesicle RNA-an ISEV position paper. J. Extracell. Vesicles 2017, 6, 1286095. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  103. Arroyo, J.D.; Chevillet, J.R.; Kroh, E.M.; Ruf, I.K.; Pritchard, C.C.; Gibson, D.F.; Mitchell, P.S.; Bennett, C.F.; Pogosova-Agadjanyan, E.L.; Stirewalt, D.L.; et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc. Natl. Acad. Sci. USA 2011, 108, 5003–5008. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  104. Bryant, R.J.; Pawlowski, T.; Catto, J.W.F.; Marsden, G.; Vessella, R.L.; Rhees, B.; Kuslich, C.; Visakorpi, T.; Hamdy, F.C. Changes in circulating microRNA levels associated with prostate cancer. Br. J. Cancer 2012, 106, 768–774. [Google Scholar] [CrossRef] [Green Version]
  105. Huang, X.; Yuan, T.; Liang, M.; Du, M.; Xia, S.; Dittmar, R.; Wang, D.; See, W.; Costello, B.A.; Quevedo, F.; et al. Exosomal miR-1290 and miR-375 as Prognostic Markers in Castration-resistant Prostate Cancer. Eur. Urol. 2015, 67, 33–41. [Google Scholar] [CrossRef] [Green Version]
  106. Li, Z.; Ma, Y.-Y.; Wang, J.; Zeng, X.-F.; Li, R.; Kang, W.; Hao, X.-K. Exosomal microRNA-141 is upregulated in the serum of prostate cancer patients. OncoTargets Ther. 2016, 9, 139–148. [Google Scholar] [CrossRef] [Green Version]
  107. Ogata-Kawata, H.; Izumiya, M.; Kurioka, D.; Honma, Y.; Yamada, Y.; Furuta, K.; Gunji, T.; Ohta, H.; Okamoto, H.; Sonoda, H.; et al. Circulating Exosomal microRNAs as Biomarkers of Colon Cancer. PLoS ONE 2014, 9, e92921. [Google Scholar] [CrossRef]
  108. Matsumura, T.; Sugimachi, K.; Iinuma, H.; Takahashi, Y.; Kurashige, J.; Sawada, G.; Ueda, M.; Uchi, R.; Ueo, H.; Takano, Y.; et al. Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer. Br. J. Cancer 2015, 113, 275–281. [Google Scholar] [CrossRef]
  109. Liu, C.; Eng, C.; Shen, J.; Lu, Y.; Takata, Y.; Mehdizadeh, A.; Chang, G.J.; Rodriguez-Bigas, M.A.; Li, Y.; Chang, P.; et al. Serum exosomal miR-4772-3p is a predictor of tumor recurrence in stage II and III colon cancer. Oncotarget 2016, 7, 76250–76260. [Google Scholar] [CrossRef] [Green Version]
  110. Tsukamoto, M.; Iinuma, H.; Yagi, T.; Matsuda, K.; Hashiguchi, Y. Circulating Exosomal MicroRNA-21 as a Biomarker in Each Tumor Stage of Colorectal Cancer. Oncology 2017, 92, 360–370. [Google Scholar] [CrossRef]
  111. Liu, L.; Meng, T.; Yang, X.-H.; Sayim, P.; Lei, C.; Jin, B.; Ge, L.; Wang, H.-J. Prognostic and predictive value of long non-coding RNA GAS5 and mircoRNA-221 in colorectal cancer and their effects on colorectal cancer cell proliferation, migration and invasion. Cancer Biomark. 2018, 22, 283–299. [Google Scholar] [CrossRef] [PubMed]
  112. Taylor, D.D.; Gercel-Taylor, C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol. Oncol. 2008, 110, 13–21. [Google Scholar] [CrossRef] [PubMed]
  113. Meng, X.; Müller, V.; Milde-Langosch, K.; Trillsch, F.; Pantel, K.; Schwarzenbach, H. Diagnostic and prognostic relevance of circulating exosomal miR-373, miR-200a, miR-200b and miR-200c in patients with epithelial ovarian cancer. Oncotarget 2016, 7. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  114. Pan, C.; Stevic, I.; Müller, V.; Ni, Q.; Oliveira-Ferrer, L.; Pantel, K.; Schwarzenbach, H. Exosomal microRNAs as tumor markers in epithelial ovarian cancer. Mol. Oncol. 2018, 12, 1935–1948. [Google Scholar] [CrossRef] [Green Version]
  115. Eichelser, C.; Stückrath, I.; Müller, V.; Milde-Langosch, K.; Wikman, H.; Pantel, K.; Schwarzenbach, H. Increased serum levels of circulating exosomal microRNA-373 in receptor-negative breast cancer patients. Oncotarget 2014, 5, 9650–9663. [Google Scholar] [CrossRef] [Green Version]
  116. Hannafon, B.N.; Trigoso, Y.D.; Calloway, C.L.; Zhao, Y.D.; Lum, D.H.; Welm, A.L.; Zhao, Z.J.; Blick, K.E.; Dooley, W.C.; Ding, W.Q. Plasma exosome microRNAs are indicative of breast cancer. Breast Cancer Res. 2016, 18, 90. [Google Scholar] [CrossRef] [Green Version]
  117. Cazzoli, R.; Buttitta, F.; Di Nicola, M.; Malatesta, S.; Marchetti, A.; Rom, W.N.; Pass, H.I. microRNAs Derived from Circulating Exosomes as Noninvasive Biomarkers for Screening and Diagnosing Lung Cancer. J. Thorac. Oncol. 2013, 8, 1156–1162. [Google Scholar] [CrossRef] [Green Version]
  118. Tanaka, Y.; Kamohara, H.; Kinoshita, K.; Kurashige, J.; Ishimoto, T.; Iwatsuki, M.; Watanabe, M.; Baba, H. Clinical impact of serum exosomal microRNA-21 as a clinical biomarker in human esophageal squamous cell carcinoma: Exosomal MicroRNA-21 Expression in ESCC. Cancer 2013, 119, 1159–1167. [Google Scholar] [CrossRef]
  119. Wang, J.; Zhou, Y.; Lu, J.; Sun, Y.; Xiao, H.; Liu, M.; Tian, L. Combined detection of serum exosomal miR-21 and HOTAIR as diagnostic and prognostic biomarkers for laryngeal squamous cell carcinoma. Med. Oncol. 2014, 31, 148. [Google Scholar] [CrossRef]
  120. Que, R.; Ding, G.; Chen, J.; Cao, L. Analysis of serum exosomal microRNAs and clinicopathologic features of patients with pancreatic adenocarcinoma. World J. Surg. Oncol. 2013, 11, 219. [Google Scholar] [CrossRef] [Green Version]
  121. Lai, X.; Wang, M.; McElyea, S.D.; Sherman, S.; House, M.; Korc, M. A microRNA signature in circulating exosomes is superior to exosomal glypican-1 levels for diagnosing pancreatic cancer. Cancer Lett. 2017, 393, 86–93. [Google Scholar] [CrossRef] [Green Version]
  122. Goto, T.; Fujiya, M.; Konishi, H.; Sasajima, J.; Fujibayashi, S.; Hayashi, A.; Utsumi, T.; Sato, H.; Iwama, T.; Ijiri, M.; et al. An elevated expression of serum exosomal microRNA-191, −21, −451a of pancreatic neoplasm is considered to be efficient diagnostic marker. BMC Cancer 2018, 18, 116. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  123. Takahasi, K.; Iinuma, H.; Wada, K.; Minezaki, S.; Kawamura, S.; Kainuma, M.; Ikeda, Y.; Shibuya, M.; Miura, F.; Sano, K. Usefulness of exosome-encapsulated microRNA-451a as a minimally invasive biomarker for prediction of recurrence and prognosis in pancreatic ductal adenocarcinoma. J. Hepato-Bil-Pan. Sci. 2018, 25, 155–161. [Google Scholar] [CrossRef] [PubMed]
  124. Yang, H.; Fu, H.; Wang, B.; Zhang, X.; Mao, J.; Li, X.; Wang, M.; Sun, Z.; Qian, H.; Xu, W. Exosomal miR-423-5p targets SUFU to promote cancer growth and metastasis and serves as a novel marker for gastric cancer. Mol. Carcinog. 2018, 57, 1223–1236. [Google Scholar] [CrossRef] [PubMed]
  125. Sohn, W.; Kim, J.; Kang, S.H.; Yang, S.R.; Cho, J.-Y.; Cho, H.C.; Shim, S.G.; Paik, Y.-H. Serum exosomal microRNAs as novel biomarkers for hepatocellular carcinoma. Exp. Mol. Med. 2015, 47, e184. [Google Scholar] [CrossRef] [PubMed]
  126. Sugimachi, K.; Matsumura, T.; Hirata, H.; Uchi, R.; Ueda, M.; Ueo, H.; Shinden, Y.; Iguchi, T.; Eguchi, H.; Shirabe, K.; et al. Identification of a bona fide microRNA biomarker in serum exosomes that predicts hepatocellular carcinoma recurrence after liver transplantation. Br. J. Cancer 2015, 112, 532–538. [Google Scholar] [CrossRef]
  127. Jang, S.C.; Crescitelli, R.; Cvjetkovic, A.; Belgrano, V.; Olofsson Bagge, R.; Sundfeldt, K.; Ochiya, T.; Kalluri, R.; Lötvall, J. Mitochondrial protein enriched extracellular vesicles discovered in human melanoma tissues can be detected in patient plasma. J. Extracell. Vesicles 2019, 8, 1635420. [Google Scholar] [CrossRef] [Green Version]
  128. Lázaro-Ibáñez, E.; Lässer, C.; Shelke, G.V.; Crescitelli, R.; Jang, S.C.; Cvjetkovic, A.; García-Rodríguez, A.; Lötvall, J. DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology. J. Extracell. Vesicles 2019, 8, 1656993. [Google Scholar] [CrossRef] [Green Version]
  129. Li, S.; Zhao, Y.; Chen, W.; Yin, L.; Zhu, J.; Zhang, H.; Cai, C.; Li, P.; Huang, L.; Ma, P. Exosomal ephrinA2 derived from serum as a potential biomarker for prostate cancer. J. Cancer 2018, 9, 2659–2665. [Google Scholar] [CrossRef]
  130. Chen, C.-L.; Lai, Y.-F.; Tang, P.; Chien, K.-Y.; Yu, J.-S.; Tsai, C.-H.; Chen, H.-W.; Wu, C.-C.; Chung, T.; Hsu, C.-W.; et al. Comparative and Targeted Proteomic Analyses of Urinary Microparticles from Bladder Cancer and Hernia Patients. J. Proteome Res. 2012, 11, 5611–5629. [Google Scholar] [CrossRef]
  131. Sun, B.; Li, Y.; Zhou, Y.; Ng, T.K.; Zhao, C.; Gan, Q.; Gu, X.; Xiang, J. Circulating exosomal CPNE3 as a diagnostic and prognostic biomarker for colorectal cancer. J. Cell. Physiol. 2019, 234, 1416–1425. [Google Scholar] [CrossRef] [PubMed]
  132. Sun, N.; Sun, S.G.; Lu, Z.L.; He, J. [Diagnostic value of protein markers in plasma exosomes of lung squamous cell carcinoma]. Zhonghua Zhong Liu Za Zhi 2018, 40, 418–421. [Google Scholar] [CrossRef] [PubMed]
Table
Table 1. Protein tumor markers on plasmatic exosomes.
Table 1. Protein tumor markers on plasmatic exosomes.
PathologyExosomal Proteins (Biomarkers)Potential UseSourceIsolation MethodReferences
MelanomaCaveolin-1DiagnosisPlasmaUC[16]
HSP70, HSP90PrognosisPlasmaUC, Density gradient centrifugation[57]
MIA, S100BDiagnosis/PrognosisSerumPolymeric precipitation[58]
Prostate Cancer (Pca)PSAScreening/Early DiagnosisPlasmaUC[35,36]
CA IXDiagnosisPlasmaUC[51]
SurvivinEarly DiagnosisPlasmaUC[59]
Exosomes levelsDiagnosis/Prognosis/Disease surveillancePlasmaUC[60]
PTENDiagnosisPlasmaUC[61]
Ovarian CancerEpCAM,
CD24,
CA-125		DiagnosisPlasmaMicrofluidic chip[62,63,64]
TGF-beta1,
MAGE3/6,		Diagnosis/Prognosis/Therapy monitoringPlasmaUC[65]
Breast cancerBreast cancer resistance protein (BCRP)Prognostic biomarkerPlasmaUC[66]
Her2Diagnosis/Molecular classificationPlasmaMicrofluidic chip[67]
Trastuzumab resistance/Tumor aggressivenessPlasmaUC[68]
Glypican-1Screening/DiagnosisSerumUC[69]
FibronectinEarly DiagnosisPlasmaELISA[70]
PeriostinDiagnosisPlasmaUC[71]
Del-1Early Diagnosis/PrognosisPlasmaUC[72,73]
Pancreatic cancerCD44v6, Tspan 8, EpCAM, CD104Diagnosis/PrognosisSerumUC[74]
Glypican-1Screening/DiagnosisSerumUC[69]
Colorectal cancerHsp60Diagnosis/TherapyPlasmaUC[55]
TSAP6/CEADiagnosis/PrognosisPlasmaUC[75]
Glypican-1Diagnosis/Therapy target (treatment)PlasmaImmunocapture assays[76]
CEADiagnosisSerumUC/Polymeric precipitation[77,78]
CD147DiagnosisSerumUC[79]
PlasmaUC[80]
Gastric cancerGKN1Diagnosis/PrognosisSerumPolymeric precipitation[81]
TGF-β1DiagnosisPlasmaUC[82]
Lung cancerNY-ESO-1PrognosisPlasmaExtracellular Vesicle Array[83]
Oral Squamous Cell CarcinomaCAV-1Follow upPlasmaUC[56]
Hematological tumorsCD9, CD13, CD19, CD30, CD38, CD63DiagnosisSerumUC[84]
UC, ultracentrifugation.
Table
Table 3. Detection of exosomal tumor biomarkers in body fluids by immunocapture-based ELISA (IC-ELISA).
Table 3. Detection of exosomal tumor biomarkers in body fluids by immunocapture-based ELISA (IC-ELISA).
PathologyBiomarkerApproach UsedSourcePotential UseClinical Study Size (N)Reference
MelanomaCaveolin-1IC-ELISAPlasmaEarly DiagnosisControl N = 58; Disease N = 90[16]
MIA, S100BIC-ELISASerumDiagnosis/PrognosisControl N = 25; Disease free N = 18; Disease (stage IV) N = 53[58]
MT-CO2, COX6cIC-ELISAPlasmaDiagnosisControl N = 6; Disease N = 21[127]
Prostate cancerPSAIC-ELISA, NSFCPlasmaScreening/Early
diagnosis		Control N = 15; BPH N = 15; Disease N = 15[35]
Control N = 80; BPH N = 80; Disease N = 80[36]
SurvivinIC-ELISAPlasmaEarly diagnosisControl N = 8; BPH N = 20; Disease N = 39[59]
ephrinA2IC-ELISASerumDiagnosisControl N = 20; BPH N = 21; Disease N = 50[129]
Bladder cancerTACSTD2Flow cytometry, LC-MS/MS, LC-MRM/MS, IC-ELISAUrineDiagnosisControl N = 81; Disease N = 140[130]
Colorectal cancerHsp60IHC, IC-ELISA, IEMPlasmaDiagnosis/TherapyControl N = 40; Disease N = 57[55]
CEAIC-ELISASerumDiagnosisControl N = 8; Disease N = 116[78]
CPNE3IC-ELISAPlasmaDiagnosis/PrognosisControl N = 32; Disease N = 92[131]
Ovarian cancerMT-CO2, COX6cIC-ELISAPlasmaDiagnosisControl N = 6; Disease N = 62[127]
Breast cancerFibronectinIC-ELISAPlasmaEarly DiagnosisControl N = 70;
After surgery N = 40; Benign disease N = 55; Noncancerous diseases N = 80; Disease N = 240		[70]
Del-1IC-ELISAPlasmaDiagnosis/Monitoring/TreatmentControl N = 81;
After surgery N = 50; Benign disease N = 64; Noncancerous diseases N = 98; Disease N = 269		[72]
Control N = 22; Disease N = 114[73]
MT-CO2, COX6cIC-ELISAPlasmaDiagnosisControl N = 6; Disease N = 13[127]
Lung squamous cell carcinoma14-3-3ζIC-ELISAPlasmaDiagnosisControl N = 17; Disease N = 17[132]
Oral Squamous Cell CarcinomaCAV-1IC-ELISAPlasmaFollow upDisease N = 10 (before and after surgery)[56]
BPH, benign prostatic hyperplasia; IC-ELISA, immunocapture based-enzyme-linked immunosorbent assay; IEM, immunoelectron microscopy; IHC, immunohistochemistry; LC-MS/MS, liquid chromatography–tandem mass spectrometry; LC-MRM/MS, liquid chromatography–multiple reaction monitoring-mass spectrometry; NSFC, nanoscale flow-cytometry.

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Logozzi, M.; Mizzoni, D.; Di Raimo, R.; Fais, S. Exosomes: A Source for New and Old Biomarkers in Cancer. Cancers 2020, 12, 2566. https://0-doi-org.brum.beds.ac.uk/10.3390/cancers12092566

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Logozzi M, Mizzoni D, Di Raimo R, Fais S. Exosomes: A Source for New and Old Biomarkers in Cancer. Cancers. 2020; 12(9):2566. https://0-doi-org.brum.beds.ac.uk/10.3390/cancers12092566

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Logozzi, Mariantonia, Davide Mizzoni, Rossella Di Raimo, and Stefano Fais. 2020. "Exosomes: A Source for New and Old Biomarkers in Cancer" Cancers 12, no. 9: 2566. https://0-doi-org.brum.beds.ac.uk/10.3390/cancers12092566

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