Exosomes were first observed in the early 1980s in the culture media of reticulocytes. They are produced in inward budding multivesicular bodies (MVBs). The resulting intra-luminal vesicles (ILV) are then released through exocytosis into the extracellular space. Based on early observations of this phenomena, the term “exosomes” was coined for such vesicles. Exosomes share similar topology to the plasma membrane and are released by virtually all cell types and have been confirmed in all bodily fluids.

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Protein Exosome Marker

Q: How are Exosomes Identified?

Exosomes can contain a vast array of different proteins depending on their host cell which, and their components are further modulated by cellular state (e.g. stress or activation, or inhibition of specific signaling pathways). Tetraspanins like CD9, CD63 and CD81 are the most common canonical exosome marker proteins, present on the vesicle surface. Surface localization of tetraspanin antigens makes them good candidate targets for immunolabeling and purification of exosomes from biological samples. Components of the endosomal sorting complex required for transport (ESCRT) like TSG101 and Alix, cytoskeletal proteins, integrins and annexins are also enriched on exosomes; these molecules play a pivotal role in exosome targeting and cell adhesion.

Written/Edited by Dr. Stefan Pellenz, PhD

Exosomes are extracellular microvesicles (EMV) excreted by most cells involved in intercellular communication. They are present in bodily fluids and can contain a vast array of different proteins depending on their host cell. Their content is further modulated by the cellular state such as stress or activation, or inhibition of specific signaling pathways. This makes exosomes excellent biomarkers for liquid biopsies e.g. in cancer diagnosis.

exosome biogenesis and protein markers — Exosome Biogenesis begins with the invagination of early endosomes. These mature into late endosomes and then further into multivesicular bodies (MVBs). MVB formation involves the inward budding of the endosomal membrane, leading to the sequestration of cytoplasmic components into intraluminal vesicles (ILVs). ILVs are released through exocytosis into the extracellular space, which earned exosomes their name. Exosomes can contain a wide variety of exosome protein markers depending on their host cell which and the cell’s state, e.g. stress or activation, or inhibition of specific signaling pathways). Tetraspanins like CD9, CD63 and CD81 are the most common canonical exosome marker proteins. Surface localization of tetraspanins makes them particularly well suited for immunolabeling and purification of exosomes from biological samples. Components of the endosomal sorting complex required for transport (ESCRT) like TSG101 and Alix, cytoskeletal proteins, integrins and annexins are also enriched on exosomes. These molecules play a pivotal role in exosome targeting and cell adhesion.

What are Exosomes?
How are Exosomes formed?
How are Exosomes Identified?
Why are Exosomes Important?
How are Exosomes Studied?
Exosome Marker Antibodies
References

What are Exosomes?

Exosomes are small (50-120nm) endosome-derived extracellular microvesicles (EMV) that play a crucial role in intercellular communication by transporting various biomolecules, such as proteins, lipids, and nucleic acids, between cells. They were first observed in the early 1980s in the culture media of reticulocytes. The term exosome is based on the observation that they are released through exocytosis into the extracellular media. Exosomes share similar topology to the plasma membrane and are released by virtually all cell types and have been confirmed in all bodily fluids.

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How are Exosomes formed?

Exosome biogenesis usually begins with the internalization of extracellular material through endocytosis leading to the formation of early endosomes, which are membrane-bound organelles that contain the internalized cargo. These then mature into late endosomes, a process accompanied by changes in membrane composition and cargo sorting. Late endosomes can develop into MVBs, which are characterized by the presence of intraluminal vesicles (ILVs) formed through invagination of the endosomal membrane. In the MVB, proteins, lipids, nucleic acids, and other molecules are sorted into ILVs. This sorting is a critical step in exosome biogenesis and is achieved by the endosomal sorting complexes required for transport (ESCRT). The ESCRT consists of several protein complexes (ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III) that work together to recognize and sequester cargo into budding ILVs. MVBs can then either fuse with lysosomes for degradation, or they can fuse with the plasma membrane, releasing the ILVs into the extracellular space as exosomes.

How are Exosomes Identified?

Exosomes contain a vast array of different proteins depending on their host cell which, and their components are further modulated by cellular state (e.g. stress or activation, or inhibition of specific signaling pathways). Tetraspanins like CD9, CD63 and CD81 are the most common canonical exosome marker proteins, present on the vesicle surface. Surface localization of tetraspanin antigens makes them good candidate targets for immunolabeling and purification of exosomes from biological samples. Components of the endosomal sorting complex required for transport (ESCRT) like TSG101 and Alix, cytoskeletal proteins, integrins and annexins are also enriched on exosomes; these molecules play a pivotal role in exosome targeting and cell adhesion.

Why are Exosomes important?

Secretion of exosomes occurs constitutively though the rate of exosome secretion, and composition of exosomes may be augmented by a variety of intrinsic or extrinsic factors (e.g. cell stress, signaling cascades). Despite their ubiquitous nature, exosomes are considered unconventional secretory pathway components.

Because exosomes are secreted from nearly every cell type, their composition mirrors their host diversity, and depends heavily upon the type of cell from which they originate. Their molecular composition also reflects physiological or pathophysiological changes in their cell or tissue of origin. This makes exosomes excellent biomarkers for liquid biopsies to diagnose and track disease progression. For cancer diagnostics, exosomes have significant advantages over circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) because of their abundance, stability, and the wide variety of contained marker molecules.

Exosomes are also implicated in cell-cell communication. Exosome components may be transferred directly to neighboring cells or may be shuttled across different cells before reaching their end destination via a method known as transcytosis. This way, exosomes can transmit signals across large distances where simple diffusion may be insufficient. Their role in cell-cell communication suggests that exosomes may have a deeper role in many physiological processes; this hypothesis is supported by the observation that exosome signaling plays a direct role in development and patterning, immune response, neuronal communication, and tissue repair.

In some pathologies, exosomes also act as vectors; tumor cell-derived exosomes play an active role in tumor angiogenesis and metastasis. Exosomes shed from stimulated blood cells and the vascular endothelium are involved in neurological disorders such as multiple sclerosis, transient ischemic attacks, and antiphospholipid syndrome. Exosomes may also carry damaged cellular material targeted for destruction and facilitate the spreading of toxic forms of aggregated proteins such as α-synuclein and β-amyloid and contribute to the progression of neurodegenerative diseases. Some research also suggests that exosome transport has been exploited by viral pathogens such as SARS-CoV-2 to travel between host cells and evade immune detection. Because of their small size and simple structure, exosomes may sometimes cross the blood-brain barrier. It has been suggested that exosomes could serve as a delivery system targeting the central nervous system to treat neuropathic diseases without the need for invasive surgery. The use of exosomes to transfer genetic information, or to deliver therapeutic agents is a currently underexplored field that holds vast medicinal potential.

How are Exosomes Studied?

The exosome secretome is vast and diverse, containing many different markers (see http://www.exocarta.org/). The presence of canonical surface markers like those listed above permits purification and in-depth study of exosome secretion and content from different sample types.

antibodies-online offers a range of antibodies and ELISA kits for the detection of know exosome proteins.

Exosome Marker Antibodies

Based on recent literature, the most relevant protein exosome markers include:

Protein	Gene	GeneID	Uniprot	Ref	exocarta Top 100 proteins	TS	EF	LP	TA	CS	AG	MT	AP	HS	EN	RG	CA	II	VI	ND
14-3-3 protein epsilon	YWHAE	7531	P62258	-	22								X
14-3-3 protein beta/alpha	YWHAB	7529	P31946	50								x
14-3-3 protein eta	YWHAH	7533	Q04917	94								x
14-3-3 protein gamma	YWHAG	7532	P61981	54								x
14-3-3 protein theta	YWHAQ	10971	P27348	56								x
14-3-3 protein zeta/delta	YWHAZ	7534	P63104	-	15								X
78 kDa glucose-regulated protein	HSPA5	3309	P11021	(1)	35									X
Actin, cytoplasmic 1	ACTB	60	P60709	-	5					X
ADAM10	ADAM10	102	O14672	(2)	-										X	X	X			X
Alix	PDCD6IP	10015	Q8WUM4	(3)	2		X
Alpha-Enolase	ENO1	2023	P06733	(4)	9										X
Alpha-Synclein	SNCA	6622	P37840	(32)	-								X							X
Aminopeptidase N	ANPEP	290	P15144	-	-						X							X
Beta-amyloid	APP	351	P05067	(5)	-															X
Annexin A1	ANXA1	301	P04083	53				x
Annexin A11	ANXA11	311	P50995	68				x
Annexin A4	ANXA4	307	P09525	72				x
Annexin A6	ANXA6	309	P08133	67				x
Annexin A5	ANXA5	308	P08758	(6)	20				X									X
Annexin A2	ANXA2	302	P07355	(7)	6				X
AP-1	JUN	3725	P05412	-	-											X	X
ATP citrate lyase	ACLY	47	P53396	-	72										X
ATPase	ATP1A1	476	P05023	-	39										X
Basigin	BSG	682	P35613	(8)	-											X	X
Caveolin-1	CAV1	857	Q03135	(9), (10)	-			X									X
CD9	CD9	928	P21926	(11)	1	X
CD11a	ITGAL	3683	P20701	(12)	-				X								X
CD11b	ITGAX	3687	P11215	(12)	-				X								X
CD11c	ITGAM	3684	P20702	(12)	-				X								X
CD29	ITGB1	3688	P05556	(12)	34				X								X
CD37	CD37	951	P11049	(11)	-	X
CD44	CD44	960	P16070	(13)	-				X								X
CD49f	ITGA6	3655	P23229	(12)	89				X								X
CD53	CD53	963	P19397	-	x
CD63	CD63	967	P08962	(10), (11)	7	X											X
CD81	CD81	975	P60033	(14)	24	X												X
CD82	CD82	3732	P27701	(11)	-	X
CD142	TF	2152	P13726	(15)	-											X	X
CD146	MCAM	4162	P43121	(15)	-				X								X
CD163	CD163	9332	Q86VB7	(15)	-											X	X	X
Clathrin heavy chain 1	CLTC	1213	Q00610	-	23					X
Claudin-1	CLDN1	9076	O95832	(8)	-												X
Cofilin-1	CFL1	1072	P23528	-	25					X
		-	-	(16)	-														X
		-	-	(16)	-														X
EF-1-alpha-1	EEF1A1	1915	P68104	(4)	14											X
EF2	EEF2	1938	P13639	-	17											X
EGFR	EGFR	1956	P00533	(15)	-												X
Ep-CAM	EPCAM	4072	P16422	(17), (18)	-				X								X
Fatty acid synthase	FASN	2194	P49327	(3)	21										X		X
Fibronectin	FN1	2335	P02751	93
Flotillin-1	FLOT1	10211	O75955	(18), (19)	41			X									X
Flotillin-2	FLOT2	2319	Q14254	(19)	-			X
Fructose-bisphosphate aldolase A	ALDOA	226	P04075	-	18										X
Gelsolin	GSN	2934	P06396	92					x							x
Glyceraldehyde-3-phosphate dehydrogenase	GAPDH	2597	P04406	-	4										X
HCV core protein		-	-	(20)	-														X
Heat shock 70 kDa protein 1A	HSPA1A	3303	P0DMV8	51									x
Heat shock protein HSP 90-alpha	HSP90AA1	3320	P07900	(1)	10									X				X
Heat shock protein HSP 90-beta	HSP90AB1	3326	P08238	(1)	19									X
Heparanase	HPSE	10855	Q9Y251	(21)	-										X		X
		-	-	(20)	-														X
		-	-	(20)	-														X
HLA-DRA	HLA-DRA	3122	P01903	(22)	-						X						X	X
	HLA-G	3135	P17693	(23)	-						X						X	X
Hsc70	HSPA8	3312	P11142	(1)	3									X
		-	-	(16)	-														X
	Tax	-	-	(20)	-														X
Huntingtin	HTT	3064	P42858	(5)	-		X													X
ICAM-1	ICAM1	3383	P05362	(24)	-				X
Leucine-rich receptor kinase 2	LRRK2	120892	Q5S007	(5)	-															X
L-lactate dehydrogenase A chain	LDHA	3939	P00338	-	13										X
Lysosome-associated membrane glycoprotein 1	LAMP1	3916	P11279	(25)	-						X						X
Lysosome-associated membrane glycoprotein 2	LAMP2	3920	P13473	(23)	88						X						X
MHCI		-	-	(26)	-						X							X
MHCII		-	-	(26)	-						X							X
MUC1	MUC1	4582	P15941	(15)	-				X								X
N-cadherin	CDH2	1000	P19022	(15)	-				X								X
Phosphoglycerate kinase 1	PGK1	5230	P00558	(4)	16										X
Placental Alkaline Phosphatase	ALPP	250	P05187	(15)	-										X		X
Prion proteins		-	-	(5)	-															X
Prostate-specific antigen	KLK3	354	P07288	(27)	-										X		X
Pyruvate kinase PKM	PKM	5315	P14618	(28)	12										X		X
Rab-14	RAB14	51552	P61106	-	75							X
Rab-5a	RAB5A	5868	P20339	-	80							X
Rab-5b	RAB5B	5869	P61020	-	86							X
Rab-5c	RAB5C	5878	P51148	-	64							X
Rab-7	RAB7A	7879	P51149	-	61							X
Rap 1B	RAP1B	5908	P61224	-	33							X
Superoxide dismutase	SOD1	6647	P00441	-										x					x
Syndecan-1	SDC1	6382	P18827	(29)	-		X
Syndecan-4	SDC4	6385	P31431	(29)	-		X
Syntenin-1	SDCBP	6386	O00560	(30)	8		X
TARDBP	TDP-43	23435	Q13148	-															x
Transitional endoplasmic reticulum ATPase	VCP	7415	P55072	26
Triosephosphate isomerase	TPI1	7167	P60174	27										x
Tumor-Associated Glycoprotein	TAG-72	-	-	(15)	-												X
Tetraspanin-8	Tspan8	7103	P19075	(15)	-	X											X
Tsg101	TSG101	7251	Q99816	(31)	11		X
Tubulin alpha-1C chain	TUBA1C	84790	Q9BQE3	-					x
Tubulin alpha-4A chain	TUBA4A	7277	P68366	-					x
Tubulin beta-2B chain	TUBB2B	347733	Q9BVA1	-					x
Tubulin beta-4B chain	TUBB4B	10383	P68371	-					x
Vacuolar-sorting protein 35	VPS35	55737	Q96QK1	(5)	-		X													X