Routes and mechanisms of extracellular vesicle uptake

Figures & data

Table I. Compounds, chemicals and peptides used to inhibit EV uptake

Pathways blocked	Inhibitor	Target	Treatment recipient
Endocytosis	Heparin	Heparan sulphate proteoglycans	Glioblastoma multiforme primary tumour cells (25); SW-780 bladder cancer cells (32);
Endocytosis	α-difluoromethylornithine (DFMO)	Heparan sulphate proteoglycans	Glioblastoma multiforme primary tumour cells (25);
Endocytosis	Asialofetuin	Galectin-5	Macrophages (24);
Endocytosis	Human receptor–associated protein (RAP)	CD91	Dendritic cells (79);
Endocytosis	RGD (Arg-Gly-Asp) peptide	Fibronectin	Macrophages (23); Dendritic cells (30);
Endocytosis	Ethylenediaminetetra acetic acid (EDTA)	Calcium	Macrophages (24); Dendritic cells (30, 44); bone marrow–derived dendritic cells (22);
Endocytosis	Cytochalasin D	Actin	Human macrophages (23); SKOV-3 ovarian cancer cells (41); RAW-264.7 macrophages (27); Microglia (28); Dendritic cells (30, 44); Bone marrow–derived dendritic cells (22); human A549 alveolar epithelial cells (77); Human umbilical cord endothelial cells (26); HeLa cells (26);
Endocytosis	Cytochalasin B	Actin	Macrophages (24);
Endocytosis	Latrunculin A	Actin	Human umbilical cord endothelial cells (26); HeLa cells (26);
Endocytosis	Latrunculin B	Actin	RAW-264.7 macrophages (27);
Clathrin- and caveolin-dependent endocytosis	NSC23766	Dynamin	Microglia (28);
Clathrin- and caveolin-dependent endocytosis	Dynasore	Dynamin-2	Macrophages (24); Microglia (28);
Clathrin-dependent endocytosis	Chlorpromazine	Dopamine receptors, serotonin receptors, histamine receptors, α1- and α2-adrenergic receptors and M1 and M2 muscarinic acetylcholine receptors	SKOV-3 ovarian cancer cells (41); RAW-264.7 macrophages (27);
Macropinocytosis	5-(N-Ethyl-N-isopropyl)amiloride (EIPA)	Sodium/proton exchanger	SKOV-3 ovarian cancer cells (41); RAW-264.7 macrophages (27);
Macropinocytosis	Amiloride	Sodium/proton exchanger	Microglia (28);
Macropinocytosis	Bafilomycin A Monensin and Chloroquine	H(+)-ATPase activity (increase pH)	Microglia (28);
Phagocytosis and macropinocytosis	Annexin-V	Phosphatidylserine	Microglia (28, 31); Ovarian cancer patient ascites-derived EVs (10); Neuro-2A mouse neuroblastoma cells (31);
Phagocytosis	Wortmannin	Phosphoinositide 3-kinases (PI3Ks)	RAW-264.7 macrophages (27);
Phagocytosis	LY294002	Phosphoinositide 3-kinases (PI3Ks)	RAW-264.7 macrophages (27);
Lipid raft–mediated endocytosis	Methyl-β-cyclodextrin (MβCD)	Cholesterol	SKOV-3 ovarian cancer cells (41); RAW-264.7 macrophages (27); BT-549 breast cancer cells (47); Human umbilical cord endothelial cells (26); U87-MG glioblastoma cells (26);
Lipid raft–mediated endocytosis	Filipin	Cholesterol	Bone marrow–derived dendritic cells (22); Melanoma cells (34); Human umbilical cord endothelial cells (26);
Lipid raft–mediated endocytosis	Simvastatin	Cholesterol	Human umbilical cord endothelial cells (26);
Lipid raft–mediated endocytosis	Fumonisin B1 and N-butyldeoxynojirimycin hydrochloride	Glycosphingolipid	Pre-treatment of EV-producing Jurkat cells (80); HEK-293T kidney cells (80);
Lipid raft–mediated endocytosis	U0126	ERK1/2	Human umbilical cord endothelial cells (26), HeLa cells (26); Mouse embryonic fibroblasts (26);
Membrane fusion	Proton pump inhibitor (AstraZeneca)	Sodium reabsorption (decrease pH)	Melanoma cells (34);

Fig. 1.  Pathways shown to participate in EV uptake by target cells. EVs transport signals between cells. EVs have been shown to be internalized by cells through phagocytosis, clathrin- and caveolin-mediated endocytosis. There is also evidence to support their interaction with lipid rafts resulting in EV uptake. Lipid rafts are involved in both clathrin- and caveolin-mediated endocytosis. EVs can be internalized by macropinocytosis where membrane protrusions or blebs extend from the cell, fold backwards around the EVs and enclose them into the lumen of a macropinosome; alternatively EVs are macropinocytosed after becoming caught in membrane ruffles. EVs may also deliver their protein, mRNA and miRNA cargo by fusion with the plasma membrane. Alternatively, intraluminal EVs may fuse with the endosomal limiting membrane following endocytosis to enable their EV contents to elicit a phenotypic response.

Table II. Antibodies used to block EV uptake

Target	Treatment recipient
ICAM-1	Dendritic cells (30); lymph node cells and spleen cells (38);
LFA-1	Dendritic cells (43, 44); CD8+ ConA T cells (45); T cells (70);
TIM-4	RAW-264.7 macrophages (27); BaF3 bone marrow pro-B cells (79);
MFG-E8	Dendritic cells (30);
DC-SIGN	Monocyte-derived dendritic cells (29);
DEC205	Dendritic cells (44);
H-2Kb	Dendritic cells (43);
Tspan8	Rat aortic endothelial cells (35);
CD9	Dendritic cells (30); Lung fibroblasts (35); rat pancreatic adenocarcinoma BSp73ASML (ASML) cell-derived EVs (38);
CD11a	Dendritic cells (30);
CD11b	Lymph node cells and spleen cells (38);
CD11c	Lymph node cells and spleen cells (38);
CD44	Lymph node cells and spleen cells (38);
CD49c	Lung fibroblasts (35);
CD49d	Rat aortic endothelial cells (35); lymph node cells and spleen cells (38);
αv (CD51)	Dendritic cells (30);
β3 (CD61)	Dendritic cells (30);
CD62L	Lymph node cells and spleen cells (38);
CD81	Dendritic cells (30); rat pancreatic adenocarcinoma BSp73ASML (ASML) cell-derived EVs (38);
CD91	Dendritic cells (79);
CD106	Rat aortic endothelial cells (35);
CD151	Lung fibroblasts (35);

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