Exosomes as Biomarker of Cancer

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 proteins¹. 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 mRNA². 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 material^3,4.

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 all⁵.

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 microvesicles⁶. 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 flotillin⁷.Exosomes isolated by ultracentrifugation appear as cup shaped structures when imaged using electron microscope⁸.

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 organisms⁹. 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 bisphosphates¹⁰.

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 cells^11,12.

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, purification¹³.

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 components¹³. Mostly ultracentrifugation is used along with sucrose density gradient, so the low density exosomes float¹⁴. 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 personnel¹⁵.

Size based isolation employing ultrafiltration is comparatively less time consuming and requires minimal of specialized equipment, making it a cost effective exosome isolation method¹⁶.

Polymer based precipitation methods using polyethylene glycols (PEG) are frequently used for precipitation of viruses and other small particles^17-19. The same technique of precipitation followed by (10,000 to 20,000 x g) centrifugation is being used for isolation of exosomes²⁰. 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, etc¹⁵. Various groups have compared commercially available exosome precipitation reagents reporting variation in yield and level of purity that can be achieved for subsequent downstream analysis²¹.

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 filters^14,15,22. 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 samples¹³.

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 cancers^23,24.

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 well^25,26.

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

Diseases	Exosomal Proteins	Level/Expression	Potencial Use	Methodology	Source	Ref.
Melanoma and other malignant cancers	CD63 and Caveolin 1 enriched exosomes.	Elevated	Diagnosis	In-house sandwich ELISA	Plasma	27
	β1 integrin, α6 integrin, basigin, CD 73.	Elevated surface expression	Prognosis	Mass spectrometry, Western blot, Flow cytometry		28
Non-small cell lung carcinoma (NSCLC)	CD9, CD81	Elevated signal	Diagnosis	Extracellular Vesicle Array (EV Array)	Plasma	29
	CD 63	Co-variation in signal as compared to control
	FAM3C	Over expression	Prognosis	LTQ-FT mass spectrometry	NSCLC cell lines	30
Prostate Cancer (PCa)	FASN, XPO1 and PDCD6IP proteins	Elevated expression	Diagnosis	LCFTMS/ Western blot/ Immuno-histochemistry	Immortalized primary prostate epithelial cells	31
	ENO-1	Decreased expression
Ovarian Cancer	Epithelial cell adhesion molecule (EpCAM) and CD24	Over expression	Diagnosis	Magnetic activated cell sorting procedure (MACS)	Plasma/ Ovarian tumor derived exosomes	32 33
	TGF-beta1, MAGE3/6, L1CAM, ADAM10, EMMPRIN, Claudin-4	Over expression	Diagnosis/ Prognosis	Fluorescent microscopy and cytofluorographic analysis.	Ascites/Blood	34
	TSG101 and Alix	Over expression	Prognosis	Protein profiling	Ovarian cancer cell lines
Hepatitus C induced HCC	CD81	Over expression	Diagnosis	Immunoblotting and densitometry.	Exosomal serum fraction	20
Bladder cancer	MUC1, β1 integrin, α6 integrin, CD44, CD10, CD 73	Increased surface expression	Diagnosis/ Prognosis	Mass spectrometry/ Western blot/ Flow cytometry	HT1376 bladder cancer cells	28
	HBA, HBB and TACSTD2	Upregulated expression	Prognosis	Mass spectrometry	Urine
Breast cancer	Survivin (Inhibitor of apoptosis), MUC 1, β1 integrin, CD73	Elevated level	Diagnosis/ Prognosis	SDS PAGE, Protein staining, Western Blot	Serum exosomes/ Saliva	35
	Breast cancer resistance protein (BCRP)	Elevated level	Prognostic biomarker	Immuno fluorescence	Plasma	36
Pancreatic cancer	CD44v6, Tspan 8, EpCAM, CD104	Elevated level	Diagnosis/ Prognosis	Flow cytometry	Serum exosomes	37
	Glypician 1 (Proteoglycan) circulating exosomes (GPC1+crExos)	Upregulated surface expression	Diagnosis	Mass spectrometry/ Flow cytometry	Serum/ Mice with pancreatic tumors	38
Colorectal cancer	CD81, CD63, CD9	Elevated	Diagnosis/ Prognosis	SDS PAGE, Protein staining, Western Blot	Colorectal cancer cell lines./Ascites	39
	Claudin 3	High level of detection	Diagnosis	Nano-LC-MS/MS analysis and 1-Dimensional SDS-PAGE	CRC-ascites	33
Gastric cancer	MAGE-1 and Her-2/neu+, CRC6	High expression	Prediction/ Prognosis Little diagnostic Value	Protein profiling	Platelet Depleted Plasma	40
	CxCR4	Down regulated expression

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 RNA^41-45. Much of the work conducted on evaluating RNA as biomarker started after Valadi’s discovery of exosomal mRNA and miRNA in 2007³. 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 exosomes⁴⁷. 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 disorders^48,49. 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 biomarker^50-53. For example, miR-375 and miR-141 are up-regulated in serum of prostate cancer patients as compared to normal individuals⁵⁴. 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 cancer⁵⁶. 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)^57-63.

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 miRNA^64,65,66. Prostate cancer antigen 3 (PCA3) was the first identified lncRNA in Prostrate Cancer ⁶⁷. Another lncRNA HOTAIR is identified as a serum based diagnostic and prognostic biomarker of LSCC⁶³. 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 protein^3,50,68. 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

Pathology	Biomarker	Level	Source	Study type	Potential	Ref.
Prostate Cancer (PC)	miR-375 and miR-141	Up-regulation	Serum	Cohort Study	Diagnosis and Stage Determination	54
	miR-107 and miR-574-3p	Up-regulation	Urine
	miR-34a	Down-regulation	Cell line conditioned medium (CM)	Cell Line Models	Predictive Biomarker for Response to Docetaxel in PCa Progression	69
	miR-1290 and miR-375	Higher	Plasma	Cohort Study	Prognostic Biomarker in Castration-Resistant PCa	70
	hsa-41 miR375, hsa-miR21 and hsa-miR574	Higher	Serum	Non Cohort Study	Discriminating Biomarker	71
	miR-141	Up-regulation	Serum	Cohort Study	Diagnosis	72
	miR-574-3p, miR-141-5p and miR-21-5p	Up-regulation	Urine	Non Cohort Study and Cell Line Model	Diagnosis	73
Ovarian Cancer	miR-21, miR-214, miR-200a, miR-200b, miR-200c, miR-203, miR-205 and miR-141	Elevated	Serum	Non Cohort Study	Diagnosis	52
	Let-7	High expression in SKOV-3	Cell Culture Media	Cell Line Models (SKOV-3 and OVCAR-3)	Diagnosis	74
	miR-200	Expressed only in OVCAR-3
	miR-21	Over Expression	-	Non Study Cohort	Diagnosis	75
	miR-30a-5p	Up-regulation	Urine	Non Study Cohort and Cell Line Model	Diagnostic and Therapeutic target of Ovarian Serous Carcinoma	76
	miR-373, miR-200a, miR-200b and miR- 200c	Elevated	Serum	Cohort Study	Diagnosis and Prognosis	55
Breast Cancer	miR-21 and miR-146ª	Over expression	Plasma	Non Cohort Study	Diagnosis	62
	miR-106a	Up-regulated	Plasma	Non Cohort Study	Biomarker for metastatic Breast Cancer	77
Colorectal Cancer (CRC)	let-7a, miR-1246, miR-1229, miR-23a, miR-223, miR-21, and miR-150	Higher	Serum	Non Cohort Study	Diagnosis	62
	miR-19a	Over expression	Serum	Non Cohort Study	Prognostic Biomarker	78
	miR-372	Up-regulation	Serum	Non Cohort Study	Diagnosis and Prognosis	56
Lung Cancer	miR-125a-5p, miR-145 and miR-146a*	Over expression	Serum	Non Cohort Study	Diagnosis	79
	miR-29a-3p and miR-150-5p	Down-regulation	Plasma	Cohort Study	Predict response To Radiation Therapy	80
	miR-21 and miR-155	Up-regulation	Serum and Conditioned media	Non Study Cohort (Cell lines and recurrent nude mouse xenograft)	Diagnosis	81
Gastric Cancer (GC)	miR-214, miR-221 and miR-222	Up-regulation	Serum	Non Cohort Study	Diagnosis and Prognosis	82
	miR-21 and miR-1225-5p	Higher	Malignant ascites and Peritoneal lavage fluid	Non Cohort Study	Diagnosis and Prognosis of Peritoneum Dissection of Gastric Cancer	60
Cervical Cancer (CC)	miR-21 and miR-146a	Up-regulation	Cervicovaginal lavage Fluid	Non Cohort Study	Non-invasive CC screening	58
Retinoblastoma	miR-320, miR-let-7e, and miR-21	Down-regulation	Plasma	Non Cohort Study	Diagnosis	59
Osophageal squamous cell carcinoma (ESCC)	miR-1246	Up-regulation	Serum	Non Cohort Study	Diagnosis and Prognosis	83
Hepatocellular Carcinoma (HCC)	miR-18a, miR-221, miR-222, and miR-224	Up-regulation	Serum	Non Cohort Study	Diagnosis	84
	miR-101, miR-106b, miR-122, and miR-195	Down- Regulation
Pancreatic Cancer	miR-17-5p and miR-21	Higher	Serum	Non Cohort Study	Diagnosis	61
Laryngeal Squamous Cell Carcinoma	miR-21and HOTAIR (lncRNA)	Higher	Serum	Non Cohort Study	Diagnosis and Prognosis	63
Papillary Thyroid Cancer (PTC)	miR-146b and miR-222	Over expression	-	Cell Line Model	Biomarker of PTC recurrence	85
Melanoma	miR-125b	Down-regulation	Serum	Non Cohort Study	Prognosis	86
Glioblastoma	miR-320 and miR-574-3p	-	Serum	Cohort Study	Diagnosis	42
	RNU6-1 (snRNA)	Up-regulation

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 ascites^87-89. 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 persons⁹⁰. 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 mRNA⁸⁷, 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 transcriptomically⁹¹. 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 cancer⁹³.

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)⁹⁴. 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 samples⁶⁰. 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 patients⁹⁵.

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.

Bio Fluid	Disease	Biomarker (Protein/RNA)	Ref.
Saliva	Breast cancer		91
	Pancreatic cancer	mRNA	87 , 92
	Sjogren’s syndrome	miRNA	90
	Head and neck cancer	mRNA, miRNA	93
Ascities	Ovarian cancer	Protein (CD24, EpCAM)	94
		Protein (MMP2, MMP9, uPA)	96
	Gastric cancer	miRNA	60
	Colorectal cancer	Protein (claudin-3)	97
CSF	Glioblastoma	miRNA (miR-21)	95
Milk /ductal fluid	Breast cancer	miRNA (miR-16, 1246, 451 and miR-720)	98
Bile	Cholangiocarcinoma	miRNA	99

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.

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Publication Dates

• Publication in this collection
  2018

History

• Received
  09 Aug 2016
• Accepted
  23 Oct 2017