Quantitative proteomics of extracellular vesicles derived from human primary and metastatic colorectal cancer cells

Figures & data

Fig. 1.  Purification and characterization of SW480 EVs and SW620 EVs. (a) Cells were incubated with media containing 10% FBS or serum-free media for 24 h. Serum-free medium did not induce apoptosis under our experimental conditions, as shown by an assay for cleaved PARP. As a positive control for apoptosis, cells were treated with 10 µM of staurosporine (STA) in serum-free media for 24 h 68. (b) Fractions of OptiPrep density gradients were analysed by Western blotting. CD63 and CD81, marker proteins of EVs 1, were detected in fraction 3. (c) The size distribution of EVs was measured by NTA indicating an average diameter of 159.1±11.1 nm (ranged from 23.0 to 636.3 nm) and 165.5±8.6 nm (ranged from 26.7 to 574.7 nm) for SW480 EVs and SW620 EVs, respectively. (d) Electron microscopy revealed that purified EVs were small closed vesicles lacking apoptotic bodies, cellular debris or protein aggregates. Scale bar = 200 nm.

Fig. 2.  Quantitative proteomic analysis of SW480 EVs and SW620 EVs. (a) Schematic workflow of the quantitative proteomics of EVs indicates the number of proteins in each step. (b) The Venn diagram shows the overlapped proteins in SW480 EVs and SW620 EVs. The GO analyses for cellular components (c) and biological processes (d) of SW480 EVs and SW620 EVs. Note that proteins normally have several GO annotations.

Fig. 3.  Comparison of EV proteomes with secretomes and other CRC cell-derived EV proteomes. The number of identified proteins and GO cellular component annotation were compared between EVs and secretomes 11 derived from SW480 (a and b) and SW620 (c and d). Note that proteins normally have several GO annotations. p<10⁻⁵ is denoted by *. The Venn diagrams show the overlap of proteins identified by SW480 EVs and SW620 EVs with HT29 EVs and LIM1215 EVs (e) and CRC ascites-derived EVs (f).

Fig. 4.  Comparative proteomic analysis of SW480 EVs and SW620 EVs. (a) Using the APEX tool 13, the relative abundances of vesicular proteins were calculated and their abundances were given in arbitrary units. Black dots show the 302 common EV proteins with expression changes of less than 1.5-fold. Blue dots indicate the 368 SW480 EV-enriched proteins with more than a 1.5-fold upregulation in protein abundance compared to SW620 EVs or only identified in SW480 EVs. Red dots denote the 359 SW620 EV-enriched proteins with more than a 1.5-fold upregulation in protein abundance over SW480 EVs or only identified in SW620 EVs. (b) Predicted fold-changes based on quantitative proteomics were validated by Western blotting of whole cell lysates (50 µg) and EVs (2 µg). Results are representative of 2 experiments. (c) Proteomes of common EV proteins, SW480 EV-enriched proteins and SW620 EV-enriched proteins were analysed by GO biological process annotations. Note that proteins normally have several GO annotations.

Table I. Cell adhesion-associated EV proteins enriched in SW480 EVs compared to SW620 EVs

Protein	UniProt accession number	Gene symbol	Estimated abundance of vesicular protein		Fold change (SW620 EVs/SW480 EVs)
			SW480 EVs	SW620 EVs
Integrin-associated protein
Calcium and integrin-binding protein 1	Q99828	CIB1		68.0
Galectin-3-binding protein	Q08380	LGALS3BP		91.0
Integrin alpha-1	P56199	ITGA1	14.7
Integrin alpha-2	P17301	ITGA2	70.0	53.8	0.8
Integrin alpha-3	P26006	ITGA3	105.4	19.7	0.2
Integrin alpha-5	P08648	ITGA5	21.1
Integrin alpha-6	P23229	ITGA6	223.4	179.9	0.8
Integrin alpha-V	P06756	ITGAV	163.4	15.9	0.1
Integrin beta-1	P05556	ITGB1	196.3	110.1	0.6
Integrin beta-4	P16144	ITGB4	210.7	144.9	0.7
Integrin beta-5	P18084	ITGB5	151.3	37.7	0.2
Integrin-linked protein kinase	Q13418	ILK	32.0
Lactadherin	Q08431	MFGE8	335.0	271.8	0.8
LIM and senescent cell antigen-like-containing domain protein 1	P48059	LIMS1	90.0
Vitronectin	P04004	VTN		32.4
Other adhesion-related protein
40S ribosomal protein SA	P08865	RPSA	66.4	141.7	2.1
ADP-ribosylation factor 6	P62330	ARF6	306.8	290.3	0.9
Alpha-parvin	Q9NVD7	PARVA	40.3
Amyloid beta A4 protein	P05067	APP	26.1	24.6	0.9
Basement membrane-specific heparan sulfate proteoglycan core protein	P98160	HSPG2		7.9
CD2-associated protein	Q9Y5K6	CD2AP	41.4	45.5	1.1
CD9 antigen	P21926	CD9	935.7	964.9	1.0
CD44 antigen	P16070	CD44	281.9	62.4	0.2
CD97 antigen	P48960	CD97	72.5	58.8	0.8
CD151 antigen	P48509	CD151	102.6	78.6	0.8
Cell surface glycoprotein MUC18	P43121	MCAM	126.7
Coagulation factor V	P12259	F5	11.0
Coronin-1A	P31146	CORO1A	27.6
Discoidin, CUB and LCCL domain-containing protein 2	Q96PD2	DCBLD2	18.7	23.8	1.3
Disintegrin and metalloproteinase domain-containing protein 15	Q13444	ADAM15	31.6
Ephrin-B1	P98172	EFNB1	151.7
Epidermal growth factor receptor	P00533	EGFR	98.8
Ezrin	P15311	EZR	243.4	166.8	0.7
Fermitin family homolog 2	Q96AC1	FERMT2	14.7
Flotillin-2	Q14254	FLOT2	17.6	16.9	1.0
G-protein coupled receptor 56	Q9Y653	GPR56		85.0
GDP-mannose 4,6 dehydratase	O60547	GMDS	28.8	60.2	2.1
Intercellular adhesion molecule 1	P05362	ICAM1	381.5	67.9	0.2
LIM domain only protein 7	Q8WWI1	LMO7		9.1
Liprin-beta-1	Q86W92	PPFIBP1	91.5	14.5	0.2
Lymphocyte function-associated antigen 3	P19256	CD58	126.9
Moesin	P26038	MSN	317.3	236.1	0.7
Myelin protein zero-like protein 2	O60487	MPZL2	75.4
Myosin-9	P35579	MYH9	110.4	155.9	1.4
Neural cell adhesion molecule L1	P32004	L1CAM	69.9	11.8	0.2
Neuropilin-1	O14786	NRP1	22.6
Neuroplastin	Q9Y639	NPTN	40.2	34.6	0.9
Nucleoside diphosphate kinase A	P15531	NME1	542.4	349.2	0.6
Peripheral plasma membrane protein CASK	O14936	CASK	20.4	11.2	0.5
Poliovirus receptor-related protein 2	Q92692	PVRL2	53.4
Protein LAP2	Q96RT1	ERBB2IP	16.7	7.2	0.4
Protein scribble homolog	Q14160	SCRIB	57.9	18.7	0.3
Protein sidekick-1	Q7Z5N4	SDK1	6.7
Rho-related GTP-binding protein RhoB	P62745	RHOB	44.2
Sorbin and SH3 domain-containing protein 1	Q9BX66	SORBS1	22.1
Syntaxin-binding protein 1	P61764	STXBP1	68.2
Syntaxin-binding protein 3	O00186	STXBP3	105.6	62.7	0.6
Transforming growth factor-beta-induced protein ig-h3	Q15582	TGFBI	56.8	23.0	0.4
Transforming protein RhoA	P61586	RHOA	427.8	432.8	1.0
Trophoblast glycoprotein	Q13641	TPBG	228.0	110.0	0.5
Tyrosine-protein kinase SYK	P43405	SYK	66.7	20.8	0.3
Tyrosine-protein kinase transmembrane receptor ROR2	Q01974	ROR2	19.5
Tyrosine-protein kinase-like 7	Q13308	PTK7	60.8	12.6	0.2
Junction protein
Afadin	P55196	MLLT4	15.3	8.4	0.6
Cadherin-13	P55290	CDH13	33.8	37.5	1.1
Catenin alpha-1	P35221	CTNNA1	146.2	176.1	1.2
Catenin beta-1	P35222	CTNNB1	181.1	204.9	1.1
Catenin delta-1	O60716	CTNND1	144.9	153.9	1.1
Desmoglein-2	Q14126	DSG2		10.7
Disks large homolog 1	Q12959	DLG1	157.9	82.7	0.5
Junction plakoglobin	P14923	JUP	78.6	66.3	0.8
Junctional adhesion molecule A	Q9Y624	F11R	183.0	131.1	0.7
Plakophilin-2	Q99959	PKP2	158.3	101.9	0.6
Plakophilin-3	Q9Y446	PKP3	84.2	89.4	1.1
Plakophilin-4	Q99569	PKP4	31.4	12.2	0.4
Vinculin	P18206	VCL	47.3	112.7	2.4
Zyxin	Q15942	ZYX		38.4

Table II. CRC progression- and metastasis-related proteins enriched in SW620 EVs compared to SW480 EVs

Protein	UniProt accession number	Gene symbol	Estimated abundance of vesicular protein		Fold change (SW620 EVs/SW480 EVs)	SW480 secretome	SW620 secretome	HT29 EVs	LIM1215 EVs	CRC asictes EVs	Reference
			SW480 EVs	SW620 EVs
Cancer progression
CD276 antigen	Q5ZPR3	CD276	34.1	90.8	2.7					O	39
Fascin	Q16658	FSCN1	108.1	176.3	1.6	O	O			O	33
Myristoylated alanine-rich C-kinase substrate	P29966	MARCKS	144.9	298.9	2.1			O		O	34
Myosin-10	P35580	MYH10	31.2	73.6	2.4			O	O	O	36
Myosin-Ib	O43795	MYO1B	98.9	154.6	1.6					O	37
Serine/threonine-protein kinase PAK 4	O96013	PAK4		42.7							35
Prominin-1	O43490	PROM1		107.3						O	40
Serine-threonine kinase receptor-associated protein	Q9Y3F4	STRAP	43.0	78.3	1.8						41
Vinculin	P18206	VCL	47.3	112.7	2.4	O	O			O	38
Nuclease-sensitive element-binding protein 1	P67809	YBX1		88.9							42
Diagnostic indicator of metastatic cancer with poor prognosis
Annexin A1	P04083	ANXA1	35.4	148.9	4.2		O	O		O	43
Annexin A11	P50995	ANXA11	86.6	161.8	1.9			O	O	O	43
High mobility group protein HMG-I/HMG-Y	P17096	HMGA		254.6							44
Overexpressed protein in CRC
Dipeptidase 1	P16444	DPEP1		118.1					O		45
Formin-like protein 1	O95466	FMNL1		10.2							46,47
Formin-like protein 2	Q96PY5	FMNL2		18.1							46,47
Lysosome-associated membrane glycoprotein 1	P11279	LAMP1	40.8	96.2	2.4			O		O	48,49
Tyrosine-protein kinase Lck	P06239	LCK		289.1						O	50
Plastin-2	P13796	LCP1		166.9						O	51
Metastasis-associated in colon cancer protein 1	Q6ZN28	MACC1		28.0							52
Solute carrier family 12 member 2	P55011	SLC12A2	29.5	56.2	1.9			O	O	O	53,54
Transgelin-2	P37802	TAGLN2	189.0	436.6	2.3	O	O	O	O	O	24
Vimentin	P08670	VIM	36.9	177.8	4.8	O	O				55
Overexpressed protein in other metastatic cancer
Adenylate kinase 2, mitochondrial	P54819	AK2		121.5							56
Stress-70 protein, mitochondrial	P38646	HSPA9		32.8				O			57
Galectin-3-binding protein	Q08380	LGALS3BP		91.0					O		58
Protein S100-A14	Q9HCY8	S100A14		177.9				O	O	O	61
Protein S100-A4	P26447	S100A4		250.1						O	59–61
Protein S100-A6	P06703	S100A6		139.8				O		O	59,60
Protein S100-A9	P06702	S100A9		90.6						O	59,60
Syntenin-1	O00560	SDCBP	435.7	755.0	1.7	O	O	O	O	O	62
Syntenin-2	Q9H190	SDCBP2		118.4					O	O	62
Multidrug resistance
Multidrug resistance-associated protein 4	O15439	ABCC4	28.0	45.2	1.6						64
Calcyclin-binding protein	Q9HB71	CACYBP		158.1							12,66
Glutathione S-transferase P	P09211	GSTP1	295.9	596.3	2.0	O	O	O	O	O	67

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