Exosomes as Biomarker of Cancer

Aleena Sumrin Shumaila Moazzam Aleena Ahmad Khan Irsa Ramzan Zunaira Batool Sana Kaleem Moazzam Ali Hamid Bashir Muhammad Bilal About the authors

ABSTRACT

Rapid advances in medicine and biotechnology resulted in the development of non-invasive diagnostic and prognostic biomarkers enabling convenient and accurate detection. Exosomes has recently emerged as non-invasive biomarker for a number of diseases including cancer. Exosomes are the small endosome originated membranous vesicles secreted in a number of biological fluids such as serum, saliva, urine, ascites, cerebrospinal fluid, etc. Exosomes contain microRNA proteins and mRNA which can be used as disease specific biomarkers. Here we reviewed recent advancement in the field of exosomes as diagnostic biomarker for cancer along with a brief overview of their biogenesis, function and isolation.

Key words:
Exosome; Biomarker; Cancer biomarker

INTRODUCTION

Exosomes are membrane bound extra cellular vesicles that originate from late endosome, ranging in size from 30 to 150 nano meter. These are released from several types of the cells and can be found circulating in almost all biological fluids. Exosomes were first described with reference to mammalian reticulocytes as circulating vesicles derived from multi vesicular bodies, containing membrane associated proteins11 Pan B-T, Johnstone RM. Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell. 1983;33(3):967-78.. During the last decade a number of studies shaped our understanding regarding composition and function of exosomes. It is known that exosomes carry different molecular components of the cells from which they originate. These include proteins, lipids, microRNA and mRNA22 Théry C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nature Reviews Immunology. 2002;2(8):569-79.. Exosomes were once considered as a mechanism to secrete unwanted substances, but the detection of functional mRNA and microRNA in exosomes has generated enormous interest in studying their role in a variety of human pathologies and development. Exosomes act as a medium of communication between mammalian cells by mediating exchange of genetic material33 Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature cell biology. 2007;9(6):654-9.,44 Bang C, Thum T. Exosomes: New players in cell-cell communication. The international journal of biochemistry & cell biology. 2012;44(11):2060-4..

The lumen of exosomes is filled with cytoplasm, of the cell of their origin; they are a valuable sample of cell’s interior showing enormous diagnostic potential. The main advantages that make exosomes, a promising tool in cancer diagnosis and prognosis include their ability to represent a global landscape of tumour heterogeneity that cannot be appreciated using traditional methods of mutation analysis.

Secondly analysis of circulating exosomes is much safer alternate to currently used invasive biopsies that are very difficult to perform repeatedly. Moreover the personalized nature of exosome based diagnosis like microRNA profiling is highly specific as compared to low specificity of conventional serum biomarkers that imparts marginal advantage in terms of personalized diagnosis if any at all55 An T, Qin S, Xu Y, Tang Y, Huang Y, Situ B, et al. Exosomes serve as tumour markers for personalized diagnostics owing to their important role in cancer metastasis. Journal of extracellular vesicles. 2015;4..

BIOGENESIS OF EXOSOMES

Biogenesis of exosomes starts with the invagination of late endosomal membrane resulting in the formation of smaller vesicles in the lumen of late endosomes /multi vesicular bodies (MVBs). Membrane proteins that are selected for degradation are sorted into intra luminal vesicles of MVBs before fusion with lysosome. Alternatively MVBs fuse with cell membrane and release their luminal vesicles as exosomes (Figure 1). Large vesicles 100 to 1000 nm released directly from cell membrane are called microvesicles66 Booth AM, Fang Y, Fallon JK, Yang J-M, Hildreth JE, Gould SJ. Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. The Journal of cell biology. 2006;172(6):923-35.. The very similar and somewhat overlapping size range of exosomes and microvesicles makes their separation difficult.

Figure 1
Biogenesis and uptake of exosomes. Exosomes biogenesis starts with the formation of intraluminal vesicles in late endosomes following cargo sorting. Both ESCRT dependent and ESCRT independent lipid driven pathways are involved in formation of multi vesicular bodies, MVBs. Exocytic MVB fuse with plasma membrane in a Rab GTPases regulated fashion. Exosome membrane is enriched in sphingomyelin, cholesterol, and ceramide whereas lumen of vesicle is filled with miRNA, mRNA, DNA and proteins. Exosomes released from cancer cell are taken up through endocytosis by neighbouring cells. Once endocytosed by recipient cell exosomes release their cargo, resulting in altered regulation of a variety of biological functions of recipient cell.

Endosomal sorting complex required for transport (ESCRT) is the multi protein complex that regulates formation of MVBs and its components for example Tsg101 is often found associated with exosomes.

Other protein markers found attached with exosomal membrane are also reminiscent of its origin including Rab GTPase, Annexins , SNAREs, Alix and flotillin77 van Niel G, Porto-Carreiro I, Simoes S, Raposo G. Exosomes: a common pathway for a specialized function. Journal of biochemistry. 2006;140(1):13-21..Exosomes isolated by ultracentrifugation appear as cup shaped structures when imaged using electron microscope88 Conde-Vancells J, Rodriguez-Suarez E, Embade N, Gil D, Matthiesen R, Valle M, et al. Characterization and comprehensive proteome profiling of exosomes secreted by hepatocytes. Journal of proteome research.2008;7(12):5157-66..

Exosome content database, ExoCarta shows 9,769 proteins, 1,116 lipids, 3,408 mRNAs, and 2,838 miRNAs that were identified in exosomes from multiple organisms99 Keerthikumar S, Chisanga D, Ariyaratne D, Al Saffar H, Anand S, Zhao K, et al. ExoCarta: a web-based compendium of exosomal cargo. Journal of molecular biology. 2015.. Proteins like Tsg101, tetraspanins,

CD63 and CD81 are commonly found with exosomes and can be used as exosome markers. The lipid content of exosomes includes cholesterol, sphingolipids, phospholipids, and bisphosphates1010 Laulagnier K, Motta C, Hamdi S, Sébastien R, Fauvelle F, Pageaux J-F, et al. Mast cell-and dendritic cellderived exosomes display a specific lipid composition and an unusual membrane organization. Biochemical Journal.2004;380(1):161-71..

Biological function of exosomes depends on their ability to recognise recipient cells. Specificity in target cell recognition is known from studies where B cell exosomes selectively recognize follicular dendritic cells and exosomes from human intestinal epithelial cells targeted dendritic cells1111 Mallegol J, Van Niel G, Lebreton C, Lepelletier Y, Candalh C, Dugave C, et al. T84-intestinal epithelial exosomes bear MHC class II/peptide complexes potentiating antigen presentation by dendritic cells. Gastroenterology. 2007;132(5):1866-76.,1212 Denzer K, van Eijk M, Kleijmeer MJ, Jakobson E, de Groot C, Geuze HJ. Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface. The Journal of Immunology. 2000;165(3):1259-65..

ISOLATION OF EXOSOMES

Different groups investigating exosomal vesicles lack agreement on a universal method for exosome isolation from different body fluids. This is because of exosome size variation, variations in protein/lipid composition or varying percentages of non-specific component aggregation on exosome surface. All these factors affect sedimentation properties of exosomes and can interfere with purification. With the advancement of molecular detection techniques, even minute exosomal components can be quantified. Furthermore co-isolation of contamination other than exosomes creates another level of complexity in the interpretation of exosomal analysis data. The methods used for exosome isolation include ultracentrifugation, ultrafiltration, polymer - based precipitation and immunoaffinity, purification1313 Taylor DD, Shah S. Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods. 2015;87:3-10..

Ultracentrifugation, a “gold standard” method for isolation of exosomes, traditionally employs a centrifugal force in excess of 100,000 x g to a solution of various macromolecules, resulting in sedimentation of high density molecules from the centrifuge axis to less denser components1313 Taylor DD, Shah S. Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods. 2015;87:3-10.. Mostly ultracentrifugation is used along with sucrose density gradient, so the low density exosomes float1414 Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current protocols in cell biology. 2006:3.22. 1-3.. 9.. The method is not fit for high throughput clinical applications due to its labour intensive nature. Ultracentrifugation consumes more time requires expensive laboratory equipments and highly trained personnel1515 Zeringer E, Barta T, Li M, Vlassov AV. Strategies for isolation of exosomes. Cold Spring Harbor protocols. 2015;2015(4):pdb. top074476..

Size based isolation employing ultrafiltration is comparatively less time consuming and requires minimal of specialized equipment, making it a cost effective exosome isolation method1616 Cheruvanky A, Zhou H, Pisitkun T, Kopp JB, Knepper MA, Yuen PS, et al. Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. American Journal of Physiology-Renal Physiology. 2007;292(5):F1657-F61..

Polymer based precipitation methods using polyethylene glycols (PEG) are frequently used for precipitation of viruses and other small particles1717 Yamamoto KR, Alberts BM, Benzinger R, Lawhorne L, Treiber G. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology. 1970;40(3):734-44.

18 Adams A. Concentration of Epstein-Barr virus from cell culture fluids with polyethylene glycol. Journal of General Virology. 1973;20(3):391-4.
-1919 Lewis GD, Metcalf TG. Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples. Applied and environmental microbiology. 1988;54(8):1983-8.. The same technique of precipitation followed by (10,000 to 20,000 x g) centrifugation is being used for isolation of exosomes2020 Lin J, Li J, Huang B, Liu J, Chen X, Chen X-M, et al. Exosomes: novel biomarkers for clinical diagnosis. The Scientific World Journal. 2015;2015.. Commercial products such as Total Exosome Isolation by Life Technologies, ExoSpin by Cell Guidance Systems and ExoQuick by System Biosciences enables fast exosome precipitation from various biological fluids such as milk, blood, urine, amniotic fluid, serum, etc1515 Zeringer E, Barta T, Li M, Vlassov AV. Strategies for isolation of exosomes. Cold Spring Harbor protocols. 2015;2015(4):pdb. top074476.. Various groups have compared commercially available exosome precipitation reagents reporting variation in yield and level of purity that can be achieved for subsequent downstream analysis2121 Rekker K, Saare M, Roost AM, Kubo A-L, Zarovni N, Chiesi A, et al. Comparison of serum exosome isolation methods for microRNA profiling. Clinical biochemistry. 2014;47(1):135-8..

Immunoaffinity capture is another promising new approach for isolating specific exosomes by affinity purification using lectins and antibodies against CD9, CD81, CD63, CD82, EpCAM, Alix and Rab5. For this approach to work, antibodies are immobilized on media like magnetic beads, chromatographic plates, matrices, and filters1414 Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current protocols in cell biology. 2006:3.22. 1-3.. 9.,1515 Zeringer E, Barta T, Li M, Vlassov AV. Strategies for isolation of exosomes. Cold Spring Harbor protocols. 2015;2015(4):pdb. top074476.,2222 Chen C, Skog J, Hsu C-H, Lessard RT, Balaj L, Wurdinger T, et al. Microfluidic isolation and transcriptome analysis of serum microvesicles. Lab on a chip. 2010;10(4):505-11.. Use of specific antibodies gives this method selectivity in isolating subpopulations from circulating exosomes while making it somewhat less desirable method in terms of capturing the true exosome and tumour heterogeneity in clinical samples1313 Taylor DD, Shah S. Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods. 2015;87:3-10..

EXOSOMAL PROTEINS AS DIAGNOSTIC BIOMARKERS

Proteomics is a rapidly emerging field due to advancement in biotechniques and instrumentation. The research and development in proteomics has led to improvements in disease prognosis and diagnosis especially with reference to use of proteins as biomarker. Exosomes also have various proteins either enclosed within the vesicles or present on surface membrane. Latest techniques enabled researchers to detect, quantitate and characterize the proteins of exosomes. Peptide libraries can be prepared from isolated exosomes for comparison of protein profiles. The exosomal proteins have emerged as non-invasive diagnostic and prognostic biomarkers for many types of cancers2323 Simpson RJ, Jensen SS, Lim JW. Proteomic profiling of exosomes: current perspectives. Proteomics. 2008 Oct;8(19):4083-99. PubMed PMID: 18780348.,2424 Choi DS, Kim DK, Kim YK, Gho YS. Proteomics of extracellular vesicles: Exosomes and ectosomes. Mass spectrometry reviews. 2015 Jul-Aug;34(4):474-90. PubMed PMID: 24421117..

Research conducted on exosomes shed in urine during various diseases has led to the development of an entire database of urinary exosome proteins, isolated from healthy human donors. Based on protein mass spectrometric analysis data obtained by NHLBI Epithelial Systems Biology Laboratory, their components, synthesis and functions have been catalogued as well2525 Pisitkun T, Shen RF, Knepper MA. Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A. 2004;101(36):13368-73.,2626 Gonzales PA, Pisitkun T, Hoffert JD, Tchapyjnikov D, Star RA, Kleta R, et al. Large-scale proteomics and phosphoproteomics of urinary exosomes. J Am Soc Nephrol. 2009;20(2):363-79..

Table 1 lists exosomal proteins that can be used as potential biomarkers for various cancers. Some exosomes were derived from body fluids of patients including urine, serum, saliva, plasma, ascites, CSF, etc. while others were isolated from experimental cell lines.

Table 1
Exosomal Proteins in Different Cancers

EXOSOMAL NUCLEIC ACIDS AS BIOMARKER

Exosomes found in body fluids contain significant amount of different RNA species such as mRNA, miRNA, (micro RNA), snRNA (small nuclear RNA) and lncRNA (long non coding RNA) as well as DNA. Recently fragmented ribosomal RNA (rRNA) is discovered as major specie of exosomal RNA4141 Simpson RJ, Kalra H, Mathivanan S. ExoCarta as a resource for exosomal research. Journal of extracellular vesicles. 2012;1.

42 Manterola L, Guruceaga E, Pérez-Larraya JG, González-Huarriz M, Jauregui P, Tejada S, et al. A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool. Neuro-oncology.2014:not218.

43 Ahadi A, Brennan S, Kennedy PJ, Hutvagner G, Tran N. Long non-coding RNAs harboring miRNA seed regions are enriched in prostate cancer exosomes. Scientific reports. 2016;6.

44 Schageman J, Zeringer E, Li M, Barta T, Lea K, Gu J, et al. The complete exosome workflow solution: from isolation to characterization of RNA cargo. BioMed research international. 2013;2013.
-4545 Jenjaroenpun P, Kremenska Y, Nair VM, Kremenskoy M, Joseph B, Kurochkin IV. Characterization of RNA in exosomes secreted by human breast cancer cell lines using next-generation sequencing. PeerJ. 2013;1:e201.. Much of the work conducted on evaluating RNA as biomarker started after Valadi’s discovery of exosomal mRNA and miRNA in 200733 Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature cell biology. 2007;9(6):654-9.. The amount of miRNA is higher in exosomes as compared to their parent cells 46. This is further confirmed by deep sequencing of exosomal RNA species by Huang et al. that concluded miRNA is the most abundant functional RNA specie in exosomes4747 Huang X, Yuan T, Tschannen M, Sun Z, Jacob H, Du M, et al. Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC genomics. 2013;14(1):1.. These discoveries stirred up interest in using miRNA as biomarker of different diseases.

miRNA are short, non-coding single stranded RNA molecules, having length up to 19-23 nucleotides. They regulate gene expression mostly by targeting 3ʹuntranslated regions of mRNAs at post transcriptional level. miRNA plays a vital role in different biological processes that includes apoptosis, cell cycle control and are also associated with disease such as cancers and neurodegenerative disorders4848 Weber JA, Baxter DH, Zhang S, Huang DY, Huang KH, Lee MJ, et al. The microRNA spectrum in 12 body fluids. Clinical chemistry. 2010;56(11):1733-41.,4949 Calin GA, Croce CM. MicroRNA signatures in human cancers. Nature Reviews Cancer. 2006;6(11):857-66.. The composition and concentration of exosomal miRNAs varies among diseased and healthy individuals. This variation shows the potential of using exosome derived miRNAs as non-invasive biomarker. Several studies conducted on different types of cancer have reported cancer specific exosomal miRNAs as biomarker5050 Skog J, Würdinger T, van Rijn S, Meijer DH, Gainche L, Curry WT, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nature cell biology.2008;10(12):1470-6.

51 Silva J, Garcia V, Zaballos A, Provencio M, Lombardia L, Almonacid L, et al. Vesicle-related microRNAs in plasma of nonsmall cell lung cancer patients and correlation with survival. European Respiratory Journal. 2011;37(3):617-23.

52 Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecologic oncology. 2008;110(1):13-21.
-5353 Rabinowits G, Gercel-Taylor C, Day JM, Taylor DD, Kloecker GH. Exosomal microRNA: a diagnostic marker for lung cancer. Clinical lung cancer. 2009;10(1):42-6.. For example, miR-375 and miR-141 are up-regulated in serum of prostate cancer patients as compared to normal individuals5454 Bryant R, Pawlowski T, Catto J, Marsden G, Vessella R, Rhees B, et al. Changes in circulating microRNA levels associated with prostate cancer. British journal of cancer. 2012;106(4):768-74.. Similarly miR-373, miR-200a, miR-200b and miR-200c can be used as diagnostic and prognostic biomarker of ovarian cancer. miR-372 is used as a biomarker of colorectal cancer5656 Yu J, Jin L, Li W, Jiang L, Hu Y, Zhi Q, et al. Serum miR-372 is a diagnostic and prognostic biomarker in patients with early colorectal cancer. Anti-cancer agents in medicinal chemistry. 2015.. Some exosomal miRNAs can be diagnostic or prognostic biomarker of more than one cancer while others are specific for particular cancer. For example, miR-21 is diagnostic biomarker of ovarian, breast, cervical, retinoblastoma, gastric, pancreatic, cervical cancer and laryngeal squamous cell carcinoma (LSCC)5757 Kumar S, Keerthana R, Pazhanimuthu A, Perumal P. Overexpression of circulating miRNA-21 and miRNA-146a in plasma samples of breast cancer patients. Indian journal of biochemistry & biophysics. 2013;50(3):210-4.

58 Liu J, Sun H, Wang X, Yu Q, Li S, Yu X, et al. Increased exosomal microRNA-21 and microRNA-146a levels in the cervicovaginal lavage specimens of patients with cervical cancer. International journal of molecular sciences. 2014;15(1):758-73.

59 Liu SS, Wang YS, Sun YF, Miao LX, Wang J, Li YS, et al. Plasma microRNA-320, microRNA-let-7e and microRNA-21 as novel potential biomarkers for the detection of retinoblastoma. Biomedical reports. 2014;2(3):424-8.

60 Tokuhisa M, Ichikawa Y, Kosaka N, Ochiya T, Yashiro M, Hirakawa K, et al. Exosomal miRNAs from peritoneum lavage fluid as potential prognostic biomarkers of peritoneal metastasis in gastric cancer. PloS one. 2015;10(7):e0130472.

61 Que R, Ding G, Chen J, Cao L. Analysis of serum exosomal microRNAs and clinicopathologic features of patients with pancreatic adenocarcinoma. World journal of surgical oncology. 2013;11(1):1.

62 Ogata-Kawata H, Izumiya M, Kurioka D, Honma Y, Yamada Y, Furuta K, et al. Circulating exosomal microRNAs as biomarkers of colon cancer. PloS one. 2014;9(4):e92921.
-6363 Wang J, Zhou Y, Lu J, Sun Y, Xiao H, Liu M, et al. Combined detection of serum exosomal miR-21 and HOTAIR as diagnostic and prognostic biomarkers for laryngeal squamous cell carcinoma. Medical Oncology. 2014;31(9):1-8..

Besides miRNA exosomes also contains long non-coding RNA (lncRNA) that range in size from several 100-1000 bases. Transcribed in diseased and normal cells, the exact function of lncRNA is not clear, while there are some indications that lncRNA acts as a sponge for miRNA6464 Jacquier A. The complex eukaryotic transcriptome: unexpected pervasive transcription and novel small RNAs. Nature Reviews Genetics. 2009;10(12):833-44.,6565 Johnson JM, Edwards S, Shoemaker D, Schadt EE. Dark matter in the genome: evidence of widespread transcription detected by microarray tiling experiments. TRENDS in Genetics. 2005;21(2):93-102.,6666 Wang Y, Xu Z, Jiang J, Xu C, Kang J, Xiao L, et al. Endogenous miRNA sponge lincRNA-RoR regulates Oct4, Nanog, and Sox2 in human embryonic stem cell self-renewal. Developmental cell. 2013;25(1):69-80.. Prostate cancer antigen 3 (PCA3) was the first identified lncRNA in Prostrate Cancer 6767 Lu W, Zhou D, Glusman G, Utleg AG, White JT, Nelson PS, et al. KLK31P is a novel androgen regulated and transcribed pseudogene of kallikreins that is expressed at lower levels in prostate cancer cells than in normal prostate cells. The Prostate. 2006;66(9):936-44.. Another lncRNA HOTAIR is identified as a serum based diagnostic and prognostic biomarker of LSCC6363 Wang J, Zhou Y, Lu J, Sun Y, Xiao H, Liu M, et al. Combined detection of serum exosomal miR-21 and HOTAIR as diagnostic and prognostic biomarkers for laryngeal squamous cell carcinoma. Medical Oncology. 2014;31(9):1-8.. Enrich motifs identified in exosomal lncRNA align to seed regions of one or more exosomal miRNAs in Prostate cancer. Tumour derived exosomes also contains complete functional mRNAs, proteins and small RNAs that favour tumour growth by changing cell environment. In the presence of fully functional protein machinery mRNA is translated into protein33 Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature cell biology. 2007;9(6):654-9.,5050 Skog J, Würdinger T, van Rijn S, Meijer DH, Gainche L, Curry WT, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nature cell biology.2008;10(12):1470-6.,6868 Sandhu S, Garzon R, editors. Potential applications of microRNAs in cancer diagnosis, prognosis, and treatment. Seminars in oncology; 2011: Elsevier.. Table 2 shows a list of RNA molecules that are up or down regulated in cancers showing their potential as biomarker.

Table 2
Types of RNA as Biomarker in Different Cancers

EXOSOMES FROM OTHER BIOFLUIDS AS BIOMARKER

Exosomes biomarkers have extensively been reported in biological fluid such as blood, plasma and urine. But recently several exosomes biomarkers have been identified in saliva, bronchoalveolar lavage fluid, cerebrospinal fluid, amniotic fluid, breast milk, semen, synovial fluid, bile and malignant ascites8787 Palanisamy V, Sharma S, Deshpande A, Zhou H, Gimzewski J, Wong DT. Nanostructural and transcriptomic analyses of human saliva derived exosomes. PLoS ONE. 2010;5(1):e8577.

88 Keller S, Rupp C, Stoeck A, Runz S, Fogel M, Lugert S, et al. CD24 is a marker of exosomes secreted into urine and amniotic fluid. Kidney international. 2007;72(9):1095-102.
-8989 Aalberts M, van Dissel-Emiliani FM, van Adrichem NP, van Wijnen M, Wauben MH, Stout TA, et al. Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans. Biology of reproduction. 2012;86(3):82.. Several studies demonstrated that exosomal micro RNA from human saliva can be used as diagnostic biomarker. For example, in 2009 Micheal and his co-workers isolated and characterized the miRNA carrying exosomes from saliva. They reported that miRNA in exosomes of Sjogran’s syndrome patients vary from that of healthy persons9090 Michael A, Bajracharya SD, Yuen PS, Zhou H, Star RA, Illei GG, et al. Exosomes from human saliva as a source of microRNA biomarkers. Oral diseases. 2010;16(1):34-8.. These miRNA (hsa-miR-150, hsa-miR-29b, miRPlus-17829, miRPlus-17841, miRPlus-17848, miRPlus-17858) can be used as a diagnostic biomarkers in future. A year later, Palanisamy et al. found that salivary exosomes also contain several protein and mRNA8787 Palanisamy V, Sharma S, Deshpande A, Zhou H, Gimzewski J, Wong DT. Nanostructural and transcriptomic analyses of human saliva derived exosomes. PLoS ONE. 2010;5(1):e8577., which have a potential to be used as biomarkers. Breast cancer exosomes interacts with cells of salivary gland, which in turn change the composition of salivary gland cell derived exosomes both proteomically and transcriptomically9191 Lau CS, Wong DT. Breast cancer exosome-like microvesicles and salivary gland cells interplay alters salivary gland cell-derived exosome-like microvesicles in vitro. PLoS ONE. 2012;7(3):e33037.. These promising discoveries might lead to the development of saliva based biomarkers for breast cancer.Recently it has been establish that salivary exosomes may be used to early detection of pancreatic cancer. Seven genes (Apbb1ip, Aspn, BCO31781, Daf2, FOXP1, Gng2 and Incenp) in saliva derived exosomes after the development of pancreatic cancer. Principe and co-workers highlighted the importance of saliva for early diagnosis of head and neck cancer9393 Principe S, Hui ABY, Bruce J, Sinha A, Liu FF, Kislinger T. Tumor-derived exosomes and microvesicles in head and neck cancer: Implications for tumor biology and biomarker discovery. Proteomics. 2013;13(10-11):1608-23..

A number of exosomal cancer biomarkers were isolated from ascetic fluid. Examples include exosomes of ovarian carcinoma patients that derives from ascities were over-expressing CD24 protein and epithelial cell adhesion molecules (EpCAM)9494 Runz S, Keller S, Rupp C, Stoeck A, Issa Y, Koensgen D, et al. Malignant ascites-derived exosomes of ovarian carcinoma patients contain CD24 and EpCAM. Gynecologic oncology. 2007;107(3):563-71.. Tokuhisa and his co-workers reported that high expression of exosomal miR-21 and miR1225-5p may serve as a promising prognostic biomarker of gastric cancer in malignant ascites samples6060 Tokuhisa M, Ichikawa Y, Kosaka N, Ochiya T, Yashiro M, Hirakawa K, et al. Exosomal miRNAs from peritoneum lavage fluid as potential prognostic biomarkers of peritoneal metastasis in gastric cancer. PloS one. 2015;10(7):e0130472.. Recently in 2015 it has been reported that miRNA contents of CSF derived exosomes can be used as a potential biomarker for therapeutic observation of glioblastoma patients9595 Akers JC, Ramakrishnan V, Kim R, Phillips S, Kaimal V, Mao Y, et al. miRNA contents of cerebrospinal fluid extracellular vesicles in glioblastoma patients. Journal of Neuro-oncology. 2015;123(2):205-16..

Table 3 shows different exosomal cancer biomarkers identified in body fluids other than peripheral blood.

Table 3
Different Types of Exosomal Cancer Biomarker in Body-Fluids.

Further research in this domain will definitely help in the development of new exosomal biomarkers.

CONCLUSION AND FUTURE PROSPECTS

As compared to other biomarkers which are detected in body fluids, exosomal biomarkers give high sensitivity and specificity. Given the name of liquid biopsy, exosomes contain the valuable samples derived from within the cancer cells and stably packaged to survive in blood circulation and other body fluids. Exosomes are secreted by cancer cells during tumour progression and have a great potential to become a routine laboratory practices in future. However their diversity needs to be fully explored before standardised diagnostic procedures can be developed.

REFERENCES

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    Denzer K, van Eijk M, Kleijmeer MJ, Jakobson E, de Groot C, Geuze HJ. Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface. The Journal of Immunology. 2000;165(3):1259-65.
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Publication Dates

  • Publication in this collection
    2018

History

  • Received
    09 Aug 2016
  • Accepted
    23 Oct 2017
Instituto de Tecnologia do Paraná - Tecpar Rua Prof. Algacyr Munhoz Mader, 3775 - CIC, 81350-010 Curitiba PR Brazil, Tel.: +55 41 3316-3052/3054, Fax: +55 41 3346-2872 - Curitiba - PR - Brazil
E-mail: babt@tecpar.br