Liposome co-incubation with cancer cells secreted exosomes (extracellular vesicles) with different proteins expressions and different uptake pathways.

We recently showed that in vitro incubation of cells with liposomes of varying compositions can increase exosome secretion and increase the yield of harvested exosomes (extracellular vesicles, EVs). This might foster their potential therapeutic implementations. In the current study, we investigated the surface proteins and the uptake of the harvested exosomes (EVs) to see if the incubation of cells with liposomes would change the biological properties of these exosomes (EVs). Interestingly, exosomes (EVs) induced by solid cationic liposomes lacked some major exosome marker proteins such as CD9, flotillin-1, annexin-A2 and EGF, and subsequently had lower levels of cellular uptake upon re-incubation with donor cancer cells. However, exosomes (EVs) induced under normal condition and by fluid cationic liposomes, displayed the entire spectrum of proteins, and exhibited higher uptake by the donor cancer cells. Although endocytosis was the major uptake pathway of exosomes (EVs) by tumor cells, endocytosis could occur via more than one mechanism. Higher exosome uptake was observed in donor B16BL6 cells than in allogeneic C26 cells, indicating that donor cells might interact specifically with their exosomes (EVs) and avidly internalize them. Taken together, these results suggest a technique for controlling the characteristics of secreted exosomes (EVs) by incubating donor cancer cells with liposomes of varying physiochemical properties.

Introduction

Extracellular vesicles, EVs (exosomes) are nano-sized biological vesicles that are secreted by various cell types such as tumor cells, B cells and dendritic cells. They can be isolated from both extracellular biological fluid and conditioned culture medium[1]. Recent observations suggest that these natural vesicles mediate cell-cell communication in many biological processes[2,3].

Since exosomes (EVs) have an innate ability to carry macromolecules such as proteins, DNA, mRNA and miRNAs, they have the potential to function as carriers to deliver payloads to target cells for therapeutic and diagnostic purposes[1,4]. Indeed, exosomes (EVs) have shown promising therapeutic results in the treatment of cancer, Parkinson’s disease and inflammatory disorders[5-9]. Hence, a number of clinical trials have been designed to study exosomes (EVs) as drug delivery tools, particularly to tumors[10,11]. However, therapeutic applications have been restricted by low exosome (EV) yields and by low uptake by the target cells; these hurdles have to be overcome before they can realize their potential as drug carriers[12].

We recently reported that the incubation of cancer cells with liposome formulations of different physiochemical properties enhanced exosome (EV) secretion and increased exosome (EV) yield by conventional separation methods[13]. Fluid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)-based cationic liposomes were more effective than solid HSPC (hydrogenated soy phosphatidylcholine)-based liposome in increasing yield. In addition, the collected exosomes (EVs) showed different uptake propensities depending on the properties of the liposome preparations used for incubation. Uninduced exosomes (EVs) and exosomes (EVs) induced by fluid cationic liposomes had higher uptake than exosomes (EVs) induced by solid cationic liposomes. Further experiments are needed to understand the mechanisms behind these observations.

The mechanisms of interaction of exosomes (EVs) with cells and how this influences their uptake by recipient cells is not well understood, even the basic question of whether exosome (EV) uptake occurs through endocytosis or direct membrane fusion. Clarifying the mechanism of exosome (EV) uptake is the key to their development as drug delivery system. Many reports have demonstrated that exosome (EV) uptake by target cells is driven heterogeneously via various mechanisms, depending on the nature of the exosome (EV) surface membrane proteins available to interact with the membrane receptors of target cells[14-19]. One class of exosome (EV) surface proteins is the tetraspanins, which are thought to be exosome (EV) markers with a role in the adhesion of exosomes (EVs) to recipient cells, facilitating exosome (EV) uptake[20]. For instance, CD9 and CD81 participate in attachment and uptake of exosomes (EVs) by dendritic cells[15]. Flotillin-1, a plasma membrane microdomain, is another exosomal surface protein that controls the clathrin independent endocytosis pathway in cells[16,21]. In addition, EGF (Epidermal growth factor) is another exosomal surface protein with a predominant role in the uptake process via EGFR (EGF receptor)-mediated endocytosis[22]. Similarly, Annexin-A2 mediates endocytic entry to cells[23]. Other plausible mechanism for exosome (EV) uptake may be clathrin-dependent endocytosis, lipid raft-mediated endocytosis, phagocytosis and/or macropinocytosis[15,17-19].

In this study, we expanded our previous research to reveal the importance and role of exosome (EV) proteins in the uptake of exosomes (EVs) induced by liposomes with varying physiochemical properties. In addition, we have studied the uptake mechanisms of exosomes (EVs) by different cancer cell lines, induced by changing liposome properties. The results indicate that the induced exosomes (EVs) display different expression of surface proteins and different endocytosis pathways, which might reflect the amount and selectivity of exosome (EV) uptake.

Results

Cellular uptake study

The cellular uptake of exosomes (EVs) collected after 48 h incubations was studied under either normal conditions (exo-N), or after stimulation with 1 mM HSPC-based liposomes (exo-S1) and 0.05 mM DOPE-based liposomes (exo-S2). The uptake of exo-S1 was lower than that of exo-N and exo-S2 (Fig. 1), in agreement with our previous observations[13].

Figure 1

Uptake of exosomes (EVs) by cancer cells. The percentage of B16BL6 cells taking up exosomes (EVs) was evaluated by flow cytometry after incubation of PKH67-labeled exosomes (EVs) with donor cancer cells (B16BL6). Data represents the mean ± SD (n = 3) after subtracting the background. A one way ANOVA test (Tukey’s test) was applied. *p < 0.05 and **p < 0.01.

Analysis of exosomal proteins

Many previous studies have reported the contribution of exosomal surface proteins to their cellular uptake, and have suggested their potential as drug delivery vehicles[1,4,14]. Accordingly, protein analysis of the collected exosomes (EVs) was conducted using three different techniques; shotgun analysis, SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) and Western blotting. Shotgun analysis was performed to identify common exosomal markers (Table 1). Detected proteins were divided into four categories; tetraspanins, heat shock proteins, enzymes and others. Several common exosomal markers such as CD63, CD81, HSP90 and TSG101 (Tumor susceptibility gene) were shared by all collected exosomes (EVs). However, several other proteins such as CD9, lactate dehydrogenase A (LDHA), flotillin-1, annexin-A2, EGF, lysosomal membrane glycoprotein A (LAMP-2), niemann-pick disease type C1 (NPC1) and clathrin light chain were not detected in exo-S1. To further examine the differential expression of exosomal marker proteins, SDS-PAGE and Western blotting were carried out. SDS-PAGE demonstrated that all exosomes (EVs) samples shared most of main protein bands. But some proteins were missing in exo-S1 samples, especially in the tetraspanin region at 25 kDa (Supplementary Fig. 1). Exosomal proteins that might contribute to exosome (EV) uptake (CD9, annexin-A2, flotillin-1 and EGF) were then screened for by Western blotting analysis. As shown in Fig. 2, these proteins were detected at 25, 35 and 47 kDa corresponding to CD9, annexin-A2 and flotillin-1, respectively, in exo-N and exo-S2 (but not exo-S1) samples. In addition, in exo-N and exo-S2 samples, EGF showed bands at both 51 and 6 kDa, likely cleaved forms of pro-EGF (122.88 kDa). Interestingly, in exo-S1 samples, these proteins were absent or present at very low levels. It is worth noting that TSG101 (band at 47 kDa) was expressed equally in all exosomes (EVs) we tested confirming that equal amounts of exosomes (EVs) were loaded for electrophoretic separation. These results indicate that liposome stimulation caused changes in protein expression in derived exosomes (EVs).

Table 1

Identification of exosomal markers via shotgun analysis.

	Protein name	Protein hits	exo-N	exo-S1	exo-S2
			404	258	552
		Accession number	MW (KDa)	pI	Score (peptide matches)
					exo-N	exo-S1	exo-S2
Tetraspanins	CD9	gi|388912	25.241	6.88	34	0	79
	CD63	gi|976238	25.479	6.69	42	35	99
	CD81 (Tapa-1 protein)	gi|8574076	25.797	5.54	421	394	453
	CD82	gi|148695678	22.373	7.98	114	68	93
	CD151 (SFA-1)	gi|2447007	28.257	7.44	89	62	73
Heat shock proteins	HSPA8 (HSP70.1)	gi|118490060	70.088	5.53	186	107	173
	HSP90 (Endoplasmin)	gi|119362	92.418	4.74	117	117	115
Enzymes	Lactate dehydrogenase A (LDHA)	gi|126048	36.475	7.74	79	0	26
	GAPDH	gi|120702	35.787	8.44	292	226	644
	Alpha enolase 1 (Eno-1)	gi|12832241	47.111	6.37	259	155	454
	Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein (YWHAE or Phopholipase A2)	gi|1304166	27.754	4.73	109	133	138
	L-lactate dehydrogenase B chain isoform Ldhbx	gi|938085832	37.325	5.85	119	92	82
	L-lactate dehydrogenase B chain isoform 2	gi|718551069	27.989		119	85	73
	Fructose-bisphosphate aldolase B	gi|15723268	39.548	8.52	48	79	52
	Phosphoglycerate kinase (PGK1)	gi|202423	44.508	7.53	108	75	345
	Chain A, Modified Glutathione S-Transferase (Pi) Complexed With S (P- Nitrobenzyl)glutathione	gi|4557944	23.471	7.85	107	164	147
Others	Flotillin-1	gi|2149604	47.484	6.71	50	0	86
	Annexin-A2	gi|113951	38.652	7.55	33	0	42
	Epidermal growth factor (EGF)	gi|49523319	122.888	6.79	39	0	24
	lysosomal membrane glycoprotein A (LAMP-2)	gi|293693	45.618	7.05	42	0	67
	NPC1	gi|2251242	142.795	5.52	42	0	64
	Clathrin (light chain)	gi|34785471	23.618	4.43	116	0	116
	TSG101	gi|3184260	44.096	6.28	153	121	184
	Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1)	gi|13278546	50.082	9.1	187	85	255
	Programmed Cell Death 6 Interacting Protein (PDCD6IP)	gi|30048422	96.251	6.15	631	219	537
	Albumin (ALB)	gi|3647327	68.648	5.75	550	504	878
	Gamma actin	gi|6425087	43.572		264	162	451
	Cofilin-1 (CFL1)	gi|116849	18.548	8.22	175	48	142
	Ferritin heavy chain	gi|309233	21.086	5.62	53	41	50
	Alpha-4 integrin	gi|1173604	115.013	6.29	294	381	538
	Clathrin (heavy chain)	gi|51491845	191.435	5.48	327	96	1768
	Lactadherin (MFGE8)	gi|113865979	51.208	6.11	426	80	398

Figure 2

Identification of exosomal marker proteins by Western blotting. Exosomal proteins in each sample (exo-N, exo-S1 and exo-S2) were electrophoretically separated and then blotted in presence of different Abs, including anti-CD9, anti-flotillin-1, anti-annexin-A2, anti-EGF and anti-TSG101. TSG101 was used as a reference (housekeeping) protein. The figure shows the cropped blots of different exosomal marker proteins separately and the full-length blots are presented in Supplementary Fig. 2.

Contribution of exosomal surface proteins to exosome (EV) uptake

The expression of EGF and flotillin-1 was higher in exo-N than exo-S2 samples, while annexin-A2 was higher in exo-S2. In addition, samples from both exosome (EV) types expressed CD9 to a similar degree. To get more insight into the role of certain proteins in uptake of exosomes (EVs) by cancer cells, samples of exosomes (EVs) displaying high protein expression (exo-N and exo-S2, Fig. 2) were selected for cellular uptake inhibition experiments. Briefly, labeled exosomes (EVs) were incubated with different antibodies (Abs) that would inhibit the interaction between surface proteins on exosomes (EVs) and their receptors on recipient cells, and then their uptake by donor B16BL6 (murine melanoma) and allogeneic C26 (Colon 26, murine colorectal carcinoma) cells was evaluated using flow cytometry and confocal laser scanning microscopy. In the exo-N sample (normal exosomes, EVs), anti-CD9 Ab inhibited the uptake of exosomes (EVs) by B16BL6 cells by 24.1% (Fig. 3A), while anti-flotillin-1 Ab inhibited the uptake of exosomes (EVs) by C26 cells by 26.7% (Fig. 3B). These observations were confirmed using confocal laser scanning microscopy, where anti-CD9 Ab and anti-flotillin-1 Abs decreased the accumulation of exo-N (green) into B16BL6 and C26, respectively (Supplementary Fig. 3A). In the exo-S2 samples, anti-CD9, anti-flotillin-1 and anti-EGF Abs inhibited the uptake of exosomes (EVs) by B16BL6 cells by 41.6%, 56.8% and 66.4%, respectively (Fig. 3C). Similarly, confocal microscopy showed the decrease in the internalization of exo-S2 (green) into B16BL6 (Supplementary Fig. 3B). Interestingly, anti-flotillin-1 and anti-EGF Abs substantially restricted the uptake of exo-S2 by C26 by 61.8% and 50.1%, respectively, while anti-CD9 Ab did not inhibit the uptake by C26 (Fig. 3D). Confocal microscopy confirmed that anti-flotillin-1 and anti-EGF Abs inhibited the uptake of exo-S2 by C26 (Supplementary Fig. 3B). These results suggest that different surface markers may be involved in the uptake of exosomes (EVs) by different cells.

Figure 3

Role of certain marker proteins in the uptake of exo-N and exo-S2 by donor cells B16BL6 and other allogeneic cells C26. Labeled exo-N (A and B) or exo-S2 (C and D) were incubated with different Abs in a ratio 1:1 for 2 h at 4 °C and then added to different cancer cell lines, namely B16BL6 (A and C) and C26 (B and D). After 4 h incubation, cancer cells were harvested for analysis by flow cytometry. All data represent the mean ± SD of triplicates. An unpaired t test was applied for each value relative to untreated cancer cells, asterisks indicate different levels of significant difference; *p < 0.05 and **p < 0.01.

Exosome (EV) uptake mechanism

There have been a number of investigations into the mechanisms behind exosome (EV) uptake, in efforts to improve exosome-based (EV-based) drug delivery systems[14]. Since exo-N and exo-S2 showed differences in protein expression (Fig. 2) and cellular uptake (Fig. 3 and Supplementary Fig. 3), the mechanism mediating their uptake was further investigated.

Uptake of exo-N by B16BL6 cells or C26 cells were strongly inhibited at 4 °C, compared to normal conditions (37 °C) (Fig. 4A,B). Similarly, uptake of exo-S2 by B16BL6 cells or C26 cells were strongly inhibited at 4 °C, compared to normal conditions (37 °C) (Fig. 4A,B). These results suggest that the uptake of exo-N and exo-S2 by either B16BL6 cells or C26 cells was mediated by energy-dependent processes. The uptake mechanism was further studied using several chemical inhibitors. The uptake of exo-N by both B16BL6 cells and C26 cells was inhibited by cytochalasin D, chlorpromazine (CPZ) and amiloride in a concentration-dependent manner (Fig. 5A,B and Supplementary Fig. 4A,B). The uptake of exo-N by both cell lines was also inhibited in the presence of filipin and sucrose (Fig. 5A,B and Supplementary Fig. 4A,B). These results indicate that exo-N were taken up by either B16BL6 cells or C26 cells via phagocytosis, clathrin-mediated endocytosis, lipid raft-mediated endocytosis and/or macropinocytosis. In the uptake of exo-S2 by either B16BL6 cells or C26 cells, similar tendencies on inhibitory effect of various inhibitors were observed (Fig. 5C,D and Supplementary Fig. 4C,D). The uptake by B16BL6 or C26 cells was inhibited by cytochalasin D, CPZ, amiloride, filipin and sucrose (Fig. 5C,D and Supplementary Fig. 4C,D). These results indicate that the exo-S2 was also taken up by either B16BL6 cells or C26 cells via the same mechanisms as observed with exo-N. It is noteworthy that the impact of inhibitors on the uptake of both exo-N and exo-S2 was entirely stronger to B16BL6 cells than to C26 cells (Fig. 5A–D). This may suggest that B16BL6 cells highly interact with these exosomes (EVs) somehow via exosome (EV) specific surface protein and thus aggressively internalize them in vitro.

Figure 4

Effect of temperature on exosome (EV) uptake by donor cells (B16BL6) and allogeneic cells (C26). Labeled samples of exo-N and exo-S2 were incubated with different cancer cell lines at 4 °C or 37 °C. After 2 h incubation, cancer cells were harvested for analysis by flow cytometry (A) or imaged by laser scanning confocal microscope after staining the DNA core with Hoechst 33342 (B). All data represent the mean ± SD (A) and one set (B) of triplicates. An unpaired t test was applied for each value relative to untreated cancer cells (***p < 0.001). Exosomes (EVs) were labeled with PHK67 (green) and the DNA core was stained with Hoechst 33342 (blue). Scale bar indicates 20 µm.

Figure 5

Uptake mechanisms for the internalization of exo-N and exo-S2 by donor cells B16BL6 and other allogeneic cells C26. B16BL6 (A and C) and C26 (B and D) cancer cell lines were incubated in the presence of different uptake inhibitors for 30 min and then labeled exo-N (A and B) or exo-S2 (C and D) were added. After 4 h incubation, cancer cells were harvested for analysis by flow cytometry. All data represent the mean ± SD of triplicates. An unpaired t test was applied for each value relative to untreated (NT) cancer cell, asterisks indicate different levels of significant difference; *p < 0.05, **p < 0.01 and ***p < 0.001. NT, untreated cancer cells.

Discussion

In the current study, we found that exosomes (EVs) incubated with different liposome preparations expressed different types of proteins (Table 1). Among the three types of exosomes (EVs) we tested, exo-S1 showed the lowest level of protein expression and lacked several exosome (EV) marker proteins (Fig. 2). CD9, annexin-A2, flotillin-1 and EGF are known to contribute to exosome (EV) uptake by target cells via various mechanisms[14-23]. The lack of relevant proteins in the exo-S1 might be related to the low levels of exosome (EV) uptake by the donor cancer cell line (Fig. 1). Exosome (EV) preparations containing heterogeneous collections of exosomes (EVs) each with different protein expression may contribute to the differences in apparent uptake mechanisms observed in this study (Fig. 5) and various other studies[14,20,24]. Our current study has shown that liposome incubation with donor cells, depending on liposome composition, can lead to substantial changes in the protein expression in the exosomes (EVs), although the mechanism for this is unknown. Our finding suggests a reliable method to control the characteristics of derived exosomes (EVs) by changing physicochemical property of liposomes used for incubation with the donor cells, allowing the fine-tuning of induced exosomes (EVs).

The current study indicated that, not only a single exosomal marker protein, but several proteins are involved in the interaction of exosomes (EVs) with cancer cells (Fig. 3 and Supplementary Fig. 3) and the subsequent cellular uptake of exosomes (EVs) (Figs 4, 5 and Supplementary Fig. 4). As we showed in this study, the involvement of CD9, flotillins and EGF in the adhesion and targeting of exosomes (EVs) to the recipient cells has been highlighted in a number of previous reports[7,15,25,26]. Morelli and colleagues illustrated that exosomes (EVs) taken up by bone marrow dendritic cells is mediated by CD9[15]. Flotillin-1, lipid raft marker of exosomes (EVs), is involved in the clathrin-independent endocytosis pathway[25,26]. Furthermore, EGF-positive exosomes (EVs) are efficiently internalized by breast cancer cells in an EGFR-dependent manner[7]. Although the contribution of other proteins, not investigated in this study, to the exosome (EV) interaction with recipient cells should not be excluded, the exosomal interaction seems to follow a general pattern already reported.

Of note, in the interaction of exo-S2 samples with the donor cell line (B16BL6) or an unrelated cell line (C26), different marker proteins contributed to the interaction of the exosomes (EVs) with the recipient cell lines, i.e., CD-9, flotillin-1 and EGF for B16BL6 cells, and flotillin-1 and EGF for C26 cells (Fig. 3C,D and Supplementary Fig. 3B). This finding indicates that different cells may recognize exosomes (EVs) via different exosome (EV) surface markers. Hence, the specificity of exosomes (EVs) for target cells may be determined not only by characteristics such as the expression pattern of surface marker proteins on the induced exosomes (EVs) but also by the expression pattern of the membrane receptors on the surface of the recipient cells. It has been reported that the exosomes (EVs) that are released by cancer cells[27], can promote tumor development and are involved in mediating intercellular communication within the tumor microenvironment[27,28]. Despite the expected predominance of cancer-derived exosomes (EVs) in tumors, several studies have indicated poor in vivo tumor targetability of tumor-derived exosomes (EVs)[6,7,29]. Differential protein expression, as well as rapid clearance, may account for poor targetability of exosomes (EVs) in vivo[6,7,29].

Confocal laser scanning microscopy allowed us to visualize the internalization of exosome (EV) samples into B16BL6 or C26 cells in the absence of inhibitory Abs (Supplementary Fig. 3A,B, Untreated). The result (Fig. 4) indicates that the internalization is an energy-dependent process, consistent with other studies[15,18,30]. Several endocytosis inhibitors significantly reduced, but didn’t completely inhibit, the uptake of exosomes (EVs) in this study (Fig. 5). Taken together, these results suggest that the endocytosis of exosomes (EVs) occurs through more than one mechanism, again consistent with previous studies[18,19,30]. The heterogeneity of exosome (EV) samples may be one reason for the differential uptake (internalization), in addition to the lack of single clear uptake mechanism. It is possible that a population of exosomes (EVs) can be taken up into cells via a number of different entry pathways, with the initial entry steps depending on the cell type and exosome (EV) composition.

Endocytosis inhibitors inhibited the uptake of exo-N and exo-S2 to a greater degree in donor B16BL6 cells than in C26 cells (Fig. 5). This may suggest that donor cells interact strongly with the exosome (EV) samples via their surface marker proteins, which in turn trigger rapid internalization. It has been reported that the uptake of exosomes (EVs) in vitro occurs as early as 15 min after addition[19], depending on cell type. Exosomes (EVs) may bind to “autocrine” receptors on donor cells that trigger rapid internalization, although further studies would be required to show this.

Nowadays, there is interest in applications of exosomes (EVs) as vehicles for the delivery of therapeutics to diseased cells[4-9]. However, their use is presently restricted by low exosome (EV) yields and exosome (EV) heterogeneity, leading to low targetability. In a previous study, we showed how the release of exosomes (EVs) from donor cancer cells is increased when they are incubated with liposome preparations of varying compositions[13]. In the current study, we report that incubating the donor cancer cells with liposome preparations changes the protein content in the induced exosomes (EVs), which raises the possibility of fine tuning exosome (EV) properties and making them more useful in drug delivery applications. Accordingly, our strategy, to employ and select liposome preparations as stimulators for the production of exosomes (EVs) expressing different surface protein markers, may be useful for engineering exosomes (EVs) for selective targeting to different diseases. Future studies will address these possibilities.

In conclusion, donor cells, when are exposed to liposomes of different physiochemical properties, secrete exosomes (EVs) with varying levels and types of protein expression, leading to their cellular uptake via several uptake pathways, depending on the cell type. Liposome exposure is a promising tool to fine-tune the production of exosomes (EVs) as drug carriers for targeted delivery of therapeutics in vitro and in vivo.

Materials and Methods

Materials

HSPC, DOPE and 1,2-dioleoyl-3-trimethylammonium-propane, chloride salt (DOTAP) were generously donated by NOF (Tokyo, Japan). Cholesterol (CHOL) and sucrose were purchased from Wako Pure Chemical (Osaka, Japan). O,O′-ditetradecanoyl-N-(alpha-trimethyl ammonio acetyl) diethanolamine chloride (DC-6–14) was purchased from Sogo Pharmaceutical (Tokyo, Japan). Cytochalasin D,CPZ, amiloride hydrochloride hydrate and filipin complex were purchased from Sigma Aldrich (MO, US). All Abs were purchased from Abcam (Cambridge, UK), including anti-CD9 (RabMab, ab92726), anti-annexin-A2 (ab41803), anti-flotillin-1 (ab41927), anti-EGF (ab9695), anti-TSG101 (ab30871) and HRP (horseradish peroxidase) conjugated goat anti-rabbit IgG (immunoglobulin G) H&L (ab6721). Exosome-depleted (EV-depleted) fetal bovine serum (FBS) was purchased from System Biosciences (CA, US). All other reagents were of analytical grade.

Cell line and cell culture

Cancer cell lines, C26 and B16BL6, were purchased from the Cell Resource Center for Biomedical Research (RIKEN RBC CELL BANK, Saitama, Japan). Culture medium, consisting of RPMI1640 (Wako Pure Chemical, Osaka, Japan) supplemented with 10% exosome-depleted (EV-depleted) FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin (MP Biomedicals, CA, US) was used to maintain these cells until 80–90% confluency. All incubation processes were carried out using 5% CO2 at 37 °C.

Preparation of liposomes

Two types of cationic liposomes, solid (HSPC-based liposomes) and fluid (DOPE-based liposomes), were prepared by the thin-film hydration method, as previously described[13,31]. The molar ratio of lipid composition was 2/1/0.2, HSPC/CHOL/DC-6–14 and 2/1, DOPE/DOTAP, respectively. The resultant large multilamellar vesicles were extruded through polycarbonate membranes with pore sizes of 400, 200 or 100 nm using an extrusion device (TRANSFERRA Nanosciences, Inc., Burnaby, Canada). The diameters and zeta-potentials of prepared liposomes were determined in phosphate buffered saline (PBS) at 25 °C using a Zetasizer Nano ZS (Malvern Instruments Ltd., WR, U.K.). The average particle size and zeta-potential of the HSPC-based liposomes were 122.26 ± 2.81 nm and +10.21 ± 0.54 mV, respectively. While those of DOPE-based liposomes were 145.89 ± 7.59 nm and +22.18 ± 1.27 mV, respectively. The phospholipid concentration of the resultant liposomes was measured by a colorimetric assay[32].

Exosome (EV) isolation

Exosomes (EVs) were collected from B16BL6 under normal and liposome incubation conditions as previously described[13]. Briefly, cells were maintained in exosome-depleted (EV-depleted) conditioned medium until 80–90% confluence. As a negative control, un-conditioned medium supplement with exosome-depleted (EV-depleted) FBS was tested for contaminating exosomes (EVs), mainly FBS-derived bovine exosomes (EVs), and proved low in contamination of exosomes (EVs) and other particles as probed by Nanoparticle Tracking Analysis (data not shown). For exo-N, the culture medium was replaced with fresh exosome-depleted (EV-depleted) conditioned medium. To obtain exo-S1 or exo-S2, the culture medium was replaced with fresh medium containing 1 mM HSPC-based liposomes or 0.05 mM DOPE-based liposomes, respectively, and further incubated. These liposome concentrations were selected to represent the exosome-inducing (EV-inducing) action of solid and fluid exosomes (EVs) under conditions that did not affect donor cell viability[13]. After a 48 h incubation, the culture medium was collected and centrifuged at 4 °C (200 × g for 10 min, 2,000 × g for 20 min and 12,500 × g for 30 min) to remove cell debris, the apoptotic bodies and microvesicles. Then, exosomes (EVs) were collected from the final supernatant by ultracentrifugation at 100,000 × g for 70 min at 4 °C. The exosome (EV) pellet was washed twice with PBS, and then dissolved in 500 μl of PBS. Gradient ultracentrifugation (0.3–2.5 M sucrose) was conducted to alleviate the possibility of contamination by liposomes. Protein concentrations of the collected exosomes (EVs) was measured by Bio-Rad DC® protein assay (Bio-Rad Laboratories Inc., CA, US) according to the manufacturer’s recommended protocol using a linear standard curve of bovine serum albumin (BSA) to calculate the protein concentration. The concentration and size distribution of the isolated exo-N, exo-S1 and exo-S2 were represented in Supplementary Fig. 5.

Shotgun analysis for exosome (EV) proteins

Shotgun analysis was conducted as described previously[33]. Briefly, six μg protein from exosome (EV) samples was treated with buffer A (8 M urea, 2 mM EDTA, 250 mM Tris-HCl, pH 8.5) containing 10 mM 1,4-dithiothreitol (DTT) for 2 h at 37 °C, followed by incubation with buffer A containing 25 mM iodoacetamide for 1 h at room temperature to conduct carbamidmethylation of the thiol group. The reaction product was mixed with 1/20 amount of trypsin solution (w/w) and incubated overnight at 37 °C. The resultant peptide mixture was passed through a ZipTip μ-C18 (Millipore) for desalting, and 0.4 μg of that was subjected to nanoLC-MS/MS analysis.

For nanoLC-MS/MS analysis, the digested peptides were separated on an Acclaim PepMap RSLC Nano Column (75 μm × 150 mm, 2 μm, C18, Thermo Fisher Scientific Inc., MA, US) operated at a flow rate of 300 nL/min using UltiMate 3000 RSLCnano System (Thermo Fisher Scientific Inc., MA, US). Phase A was 0.1% formic acid, and phase B was 80% acetonitrile containing 0.08% formic acid. After an isocratic step at 4% B for 10 min, B was linearly increased to 55% within 204 min followed by increase to 90% within 10 min. After 4 min washing, B was decreased back to 4% within 1 min. Mass spectrometry (MS) was performed using Orbitrap Elite (Thermo Fisher Scientific Inc., MA, US) operated in positive ion mode (Nanoflow-LC ESI). Capillary voltage was set at 1.7 kV. The mass data was analyzed using Mascot (Matrix Science Inc., MA, US) using the following search parameters; type of search (MS/MS Ion Search), enzyme (Trypsin), variable modifications (Carbamidomethyl (C),Oxidation (M)),mass values (Monoisotopic), protein Mass (Unrestricted), peptide mass tolerance (±10 ppm), fragment mass tolerance (±0.6 Da), max missed cleavages (2) and instrument type (ESI-TRAP). The false discovery rate (FDR) is less than 1%.

SDS PAGE electrophoresis and Western blotting analysis

Exosome (EV) samples were separated on 5–20% gradient gels (epagele-PAGEL; ATTO, Tokyo, Japan) as previously described[33]. In brief, exosome (EV) samples were mixed with 2x sample buffer (0.1 M Tris, 4% SDS, 12% 2-mercaptoethanol, 20% glycerol, slight amount of bromophenol blue) in ratio 1:1 (v/v) and then heated at 95 °C for 5 min. Each lane was loaded with 20 µl of sample containing 15 µg and 60 µg for SDS PAGE and Western blot, respectively. Electrophoresis was run at 25 mA per gel for 70 min. For simple SDS PAGE visualization, Precision Plus Protein All Blue Standard (10–250 kDa, Bio-Rad Laboratories Inc., CA, US) was used as a standard, and the gel was stained with Coomassie brilliant blue dye (0.05%). For Western blotting, the MagicMark™ XP Western Protein Standard (20–220 kDa, Thermo Fisher Scientific Inc., MA, US) was used as a standard, and the protein bands were transferred to nitrocellulose membranes by electrophoresis at 12 V for 30 min. The membrane was then blocked by incubating with 5% BSA in Tris-buffered saline with 0.05% Tween 20 (TBST 0.05%) for 1 h at 37 °C and then incubated with a 1:1,000 dilution of different primary Abs; anti-CD9, anti-annexin-A2, anti-flotillin-1 and anti-EGF in TBST 0.05% overnight at 4 °C. The membrane was then washed three times with TBST 0.05% and incubated with a 1:20,000 dilution of HRP conjugated goat anti-rabbit IgG H&L secondary antibody in TBST 0.05% for 1 h at 37 °C. The membrane was visualized by incubation with Amersham™ ECL™ Prime Western Blotting Detection Reagent (Sigma Aldrich, MO, US) for 5 min at room temperature then imaged using image quant LAS 4000 (GE Healthcare Bio-Sciences, MA, US). Exosome (EV) marker, TSG101, was used as control (housekeeping) antigens.

Evaluation of exosome (EV) uptake

The cellular uptake of different exosomes (EVs) derived from the donor B16BL6 melanoma cell line was evaluated in B16BL6 cells and in the allogeneic colon cancer cell line C26. For exosome (EV) trafficking, the exosomes (EVs) were labeled with PKH67 dye (Sigma Aldrich, MO, US) according to the manufacturer’s protocol with minor modifications[15,34,35]. Under our experimental conditions (sucrose gradient), excess unincorporated dye has been removed efficiently from these preparations as indicated by the absence of any detectable fluorescence signal after incubating the dye-control with cells and analyzed their cellular binding in the same manner as exosome (EV) uptake. To evaluate exosome (EV) uptake by cancer cells, target (recipient) B16BL6 or C26 cancer cells were cultured at 1.5 × 105 cells per well using 6 well plates. After 24 h incubation, labeled exosomes (EVs) were added to the cultured cells to a final concentration of 2 µg exosome (EV) protein/mL. After a further 2–4 h incubation, cells were harvested and exosome (EV) uptake was evaluated using a Gallios flow cytometer (Beckman Coulter, CA, US). The data were analyzed using Kaluza 1.2 software (Beckman Coulter, CA, US). To further confirm exosome (EV) uptake by cancer cells, confocal microscopy was also employed. Briefly, target cancer cells were cultured at 3 × 104 cells in 200 µl of culture medium using Lab-Tek II chamber slides (Thermo Fisher Scientific Inc., MA, US) and incubated overnight. Then, labeled exosomes (EVs) were added and incubated with the cultured cells. After 2–4 h incubation, the culture medium was discarded, adhered cells were washed and then incubated with Hoechst 33342 DNA dye (1.78 µM) (Ana Spec Inc., CA, US) for 5 min. Adhered cells were washed twice and then left to dry for 30 min. Finally, dried cells were fixed with Fluoromount/Plus (Diagnostic Biosystems, CA, US) and examined via confocal laser scanning microscopy at 63x magnification using LSM 700 (ZEISS, Oberkochen, Germany) and LSM-ZEN2012 software (ZEISS, Oberkochen, Germany)[15,34-36].

To study the contribution of surface proteins to exosome (EV) uptake, the inhibitory effect of different Abs against major exosome (EV) surface proteins (anti-CD9, anti-annexin-A2, anti-flotillin-1 and anti-EGF Abs) on exosome (EV) uptake was evaluated. Prior to exosome (EV) incubation with the cultured cells, labeled exosomes (EVs) were mixed with the selected Abs in a ratio 1:1 µg exosome (EV) protein:µg Ab and then incubated at 4 °C for 2 h. To study the exosome (EV) uptake mechanism, the inhibitory effect of different uptake inhibitors on exosome (EV) uptake was evaluated, including cytochalasin D (0.5–10 µM), CPZ (5–50 µM), amiloride hydrochloride (100–1000 µM), filipin complex (1–10 µg/mL) and sucrose (0.45 M). Each uptake inhibitor blocks one or more uptake pathway[14,37]; cytochalasin D blocks phagocytosis, macropinocytosis and other endocytic pathways via disrupting actin polymerization[38-40], CPZ inhibits clathrin-dependent endocytosis by interfering with the assembly of clathrin in plasma membrane[41], sucrose non-specifically blocks clathrin-dependent endocytosis[42], amiloride inhibits macropinocytosis by hindering Na+/H+ exchange[43] and filipin inhibits lipid raft-mediated endocytosis via cholesterol sequestering action[44]. First, the Countess II automated cell counter (Thermo Fisher Scientific Inc., MA, US) was adopted to evaluate cell viability after adding these inhibitors by staining cells with trypan blue dye. The uptake experiment was continued with inhibitor concentrations showing cell viability of not less than 80% (Supplementary Fig. 6). Prior to the incubation of labelled exosomes (EVs) with the cultured cells for 4 h, cells were pre-incubated for 30 min at 37 °C with different concentrations of inhibitors. To evaluate whether exosome (EV) internalization is energy-dependent, the incubation was also done at 4 °C for 2 h.

Statistical analysis

All values were expressed as mean ± S.D. Statistical analysis was performed via unpaired t test or one way ANOVA test (Tukey’s test) using Graphpad Prism 6.01 software (GraphPad Software Inc., CA, US). The level of significance was set at *p < 0.05, **p < 0.01 and ***p < 0.001.

Supplementary Figures