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Journal of Controlled Release 266 (2017) 100–108

Contents lists available at ScienceDirect

Journal of Controlled Release


journal homepage: www.elsevier.com/locate/jconrel

Cellular uptake of extracellular vesicles is mediated by clathrin-independent T


endocytosis and macropinocytosis
Helena Costa Verdera1, Jerney J. Gitz-Francois1, Raymond M. Schiffelers, Pieter Vader⁎
Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht 3584 CX, The Netherlands

A R T I C L E I N F O A B S T R A C T

Keywords: Recent evidence has established that extracellular vesicles (EVs), including exosomes and microvesicles, form an
Extracellular vesicles endogenous transport system through which biomolecules, including proteins and RNA, are exchanged between
Exosomes cells. This endows EVs with immense potential for drug delivery and regenerative medicine applications.
Uptake Understanding the biology underlying EV-based intercellular transfer of cargo is of great importance for the
Endocytosis
development of EV-based therapeutics. Here, we sought to characterize the cellular mechanisms involved in EV
Spheroids
Macropinocytosis
uptake.
Internalization of fluorescently-labeled EVs was evaluated in HeLa cells, in 2D (monolayer) cell culture as
well as 3D spheroids. Uptake was assessed using flow cytometry and confocal microscopy, using chemical as well
as RNA interference-based inhibition of key proteins involved in individual endocytic pathways. Experiments
with chemical inhibitors revealed that EV uptake depends on cholesterol and tyrosine kinase activity, which are
implicated in clathrin-independent endocytosis, and on Na+/H+ exchange and phosphoinositide 3-kinase ac-
tivity, which are important for macropinocytosis. Furthermore, EV internalization was inhibited by siRNA-
mediated knockdown of caveolin-1, flotillin-1, RhoA, Rac1 and PAK1, but not clathrin heavy chain. Together,
these results suggest that EVs enter cells predominantly via clathrin-independent endocytosis and macro-
pinocytosis. Identification of EV components that promote their uptake via pathways that lead to functional
cargo transfer might allow development of more efficient therapeutics through EV-inspired engineering.

1. Introduction processes [4]. For this reason, the potential usage of EVs for therapeutic
applications, including cell-free approaches for tissue engineering and
Extracellular vesicles (EVs) are cell-derived membranous nano- drug delivery, is increasingly being explored [5–7]. Very encouraging
particles that enclose and transport complex molecules, including pro- proof-of-concept studies suggest that EVs may bear intrinsic properties
teins, nucleic acids, lipids and sugars, derived from the parent cell [1]. for anti-tumor immunotherapy [8], for treatment of immunological
EVs have been found to function as natural carrier systems that effi- disorders (e.g. graft-versus-host disease [9]) or for protecting or re-
ciently deliver their molecular cargo to recipient cells. EVs are released generating tissue after e.g. kidney injury [10] or myocardial infarction
by organisms ranging from prokaryotic cells to higher eukaryotes, and [11]. Furthermore, EVs may be modified for targeted drug delivery,
in mammals these vesicles have been reported to be released from al- which has already been successfully demonstrated in small animal
most all cell types. EVs are generally classified into three subtypes, studies for therapeutic delivery of RNA [12], protein [13], and small
apoptotic bodies, microvesicles and exosomes, according to their in- molecules [14].
tracellular origin [2]. However, no uniform EV nomenclature exists as As EVs seem naturally capable of crossing biological barriers leading
of yet, due to overlap in vesicle sizes and an absence of subtype-specific to functional delivery of their cargo, it may be hypothesized that they
markers [3]. Therefore, vesicle subtypes are collectively referred to as utilize native mechanisms for internalization and intracellular traf-
EVs. ficking [15]. Endocytosis has been reported to be the major route
EVs have recently been demonstrated to act as a cell-to-cell com- through which EVs are engulfed by cells [16–19]. However, detailed
munication system involved in the natural transfer of macromolecules mechanisms remain unknown. For successful utilization of EVs as
between producer and recipient cells. As such, EVs can affect recipient therapeutic carrier systems, understanding the cellular entry routes that
cell behavior, and modify physiological as well as pathological these endogenous carriers follow to deliver their cargo into recipient


Corresponding author at: Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
E-mail address: pvader@umcutrecht.nl (P. Vader).
1
Authors contributed equally

http://dx.doi.org/10.1016/j.jconrel.2017.09.019
Received 24 July 2017; Received in revised form 12 September 2017; Accepted 13 September 2017
Available online 14 September 2017
0168-3659/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
H. Costa Verdera et al. Journal of Controlled Release 266 (2017) 100–108

cells is of critical importance. anti-CDC42 (1:6000, EPR15620, Abcam), rabbit anti-flotillin-1 (1:6000,
There are numerous mechanisms through which cells may inter- EPR6041, Millipore), rabbit anti-ARF6 (1:1000, EPR8357, Abcam),
nalize endocytic cargo. Generally, endocytosis is divided into two main mouse anti-RhoA (1:1000, 1B12, Abnova), mouse anti-Rac1 (1:1000,
subgroups: phagocytosis and pinocytosis. Phagocytosis is a type of en- 23A8, Millipore), rabbit anti-PAK1 (1:1000, polyclonal, Abcam) or
docytosis that involves internalization of relatively large (> 1 μm) rabbit anti-ANKFY1 (1:1000, polyclonal, Abgent) antibodies in 50% v/v
particles and is typically restricted to specialized professional phago- Odyssey Blocking Buffer in TBS with 0.1% Tween20 (TBS-T). Secondary
cytes. Pinocytosis, in contrast, is exhibited by all cells, and is commonly antibodies included Alexa Fluor 680 goat anti-mouse, Alexa Fluor 680
classified into clathrin-dependent endocytosis (CDE), clathrin-in- goat anti-rabbit (Thermo Fisher Scientific) and IRDye 800 donkey anti-
dependent endocytosis (CIE) and macropinocytosis (MP). CIE can be mouse (LI-COR Biosciences) and were applied at a 1:10000 dilution.
further categorized into caveolae-, Arf-6, flotillin-1-, CDC42- and RhoA- Protein bands were visualized on an Odyssey Infrared Imager (LI-COR
dependent endocytosis [20,21]. Biosciences) at 700 and 800 nm.
The ability of nanoparticles to deliver their cargo and elicit a bio-
logical response is strongly determined by the mechanism through 2.4. Nanoparticle tracking analysis
which they are taken up [22–24]. Here, we set out to identify the en-
docytic routes involved in internalization of EVs. EV size distribution was determined with a Nanosight NS500 na-
noparticle analyser (Malvern Instruments) equipped with a 405 nm
2. Materials and methods laser. A camera level of 15–16 and automatic functions for all post-
acquisition settings except for the detection threshold were used. This
2.1. Cell culture was fixed at 6. Using the script control function, three 30 s videos for
each sample were recorded. Analysis was performed with NTA 3.1
A431 (human epidermoid carcinoma) cells (ATCC) and HeLa software.
(human cervical carcinoma) cells (ATCC) were cultured in Dulbecco's
Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine 2.5. Transmission electron microscopy
serum (FBS) and 100 U/mL penicillin and 100 U/mL streptomycin.
Cells were cultivated at 37 °C and 5% CO2 under humidified conditions. EVs were adsorbed to carbon-coated formvar grids for 15 min at RT.
Cells were routinely checked for Mycoplasma contamination. Unbound EVs were removed by washing with PBS. Grids were fixated in
2% paraformaldehyde, 0.2% glutaraldehyde in PBS for 30 min at RT,
2.2. EV isolation counterstained with uranyl-oxalate and embedded in a mixture of 1.8%
methyl cellulose and 0.4% uranyl acetate at 4 °C. Imaging was done on
A431 cells were seeded in T175 flasks and cultured for 24 h, after a Jeol 1010 microscope (Jeol).
which medium was replaced by serum-free Opti-MEM (Thermo Fisher
Scientific) supplemented with 100 U/mL penicillin, 100 U/mL strepto- 2.6. EV labelling and purification
mycin. After 48 h, EVs were isolated using a recently described size-
exclusion chromatography method [25]. Conditioned medium (CM) EVs were labeled with 5 × 10−6 M PKH67 (Sigma-Aldrich) ac-
was centrifuged for 10 min at 2000 ×g at 4 °C. Subsequently, CM was cording to the manufacturer's instructions. For removal of unin-
vacuum filtered using 0.45 μm bottle top filters (Thermo Fisher Scien- corporated label, XK-16/20 column (GE Healthcare) was packed with
tific) and concentrated to ≤ 5 mL using 100 kD MWCO Amicon Ultra- sepharose CL-4B (Sigma-Aldrich) according to the manufacturer's in-
15 Centrifugal Filter Units (Millipore) at 4 °C. Concentrated CM was structions. Column was connected to a refrigerated ÄKTA Start chro-
loaded onto a HiPrep 16/60 Sephacryl S-400 HR gel filtration column matography system, equilibrated with PBS, and EV/dye mixtures were
(GE Healthcare Life Sciences), equilibrated with phosphate-buffered injected. Pooled EV fractions were concentrated using 100 kD MWCO
saline (PBS) and connected to an ÄKTA Start chromatography system Amicon Ultra-15 Centrifugal Filter Units at 4 °C.
(GE Healthcare), both maintained at 4 °C. EVs were separated from
non-vesicular material using PBS as eluent. EV-containing fractions (as 2.7. Drug treatments
determined by UV absorbance at 280 nm) were pooled, filtered through
a 0.45 μm filter and concentrated using 100 kD MWCO Amicon Ultra- Cells were preincubated with inhibitors before EV addition. For
15 Centrifugal Filter Units at 4 °C. heparin (Sigma-Aldrich), cytochalasin D (Sigma-Aldrich), chlorproma-
zine (Sigma-Aldrich), genistein (Sigma-Aldrich), EIPA (Santa Cruz
2.3. Western blot analysis Biotechnology) and wortmannin (Sigma-Aldrich), cells were pretreated
for 30 min. For simvastatin (Sigma-Aldrich), cells were pretreated for
Cells or EVs were lysed in RIPA buffer (Alfa Aesar) supplemented 20 h. Inhibitors were present throughout experiments.
with Protease Inhibitor Cocktail (Sigma-Aldrich). Lysates were cen-
trifuged at 12000 ×g for 15 min at 4 °C to remove insoluble material, 2.8. Transfections
after which protein concentrations were determined using a MicroBCA
Protein Assay using bovine serum albumin (BSA) as a standard ac- HeLa cells were seeded at a density of 50000 cells/well in 6-well
cording to the manufacturer's instructions. Lysates were mixed with plates. After 24 h, cells were transfected with siRNA (sequences are
sample buffer containing dithiothreitol (or without for CD63 analysis), listed in Table 1) using Lipofectamine RNAiMax (Life Technologies)
heated to 95 °C for 10 min and subjected to electrophoresis on 4–12% according to the manufacturer's instructions. Transfection was repeated
Bis-Tris polyacrylamide gels (Thermo Scientific). Proteins were blotted after an additional 24 h. At 6 h after the second transfection, medium
on Immobilon-FL polyvinylidene difluoride membranes (Millipore), was replaced and cells were incubated for 24 h and reseeded for EV
after which membranes were blocked with 50% v/v Odyssey Blocking internalization assays, or for 48 h for Western blot analysis.
Buffer (LI-COR Biosciences) in Tris-buffered saline (TBS). Subsequently,
membranes were probed with mouse anti-ALIX (1:1000, clone 3A9, 2.9. EV internalization assays
Abcam), mouse anti-CD63 (1:600, clone MEM-259, Abcam), mouse
anti-actin (1:1000, clone JLA20, Millipore), rabbit anti-calnexin For flow cytometric analysis, HeLa cells were seeded at a density of
(1:1000, clone N3C2, Genetex), rabbit anti-CLTC (1:1000, polyclonal, 15000 cells/well in 24-well plates. After 24 h, cells were pretreated
Abcam), rabbit anti-CAV1 (1:6000, clone EPR15554, Abcam), rabbit with inhibitors as described above and then incubated with PKH67-

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Table 1
siRNA sequences.

CAV1 hsc.rnai.n001172895.12.1 Sense 5′-CCUUCACUGUGACGAAAUACUGGtt-3′


Antisense 5′-AACCAGUAUUUCGUCACAGUGAAGGUG-3′
CDC42 hsc.rnai.n044472.12.2 Sense 5′-CCACAAACAGAUGUAUUUCUAGUct-3′
Antisense 5′-AGACUAGAAAUACAUCUGUUUGUGGAU-3′
RhoA hsc.rnai.n001664.12.1 Sense 5′-CCCAGAUACCGAUGUUAUACUGAtg-3′
Antisense 5′-CAUCAGUAUAACAUCGGUAUCUGGGUA-3′
ARF6 hsc.rnai.n001663.12.2 Sense 5′-CCUCUAACUACAAAUCUUAAUGAgc-3′
Antisense 5′-GCUCAUUAAGAUUUGUAGUUAGAGGUU-3′
Flot1 hsc.rnai.n005803.12.8 Sense 5′-GGUGAAUCACAAGCCUUUGAGAAca-3′
Antisense 5′-UGUUCUCAAAGGCUUGUGAUUCACCUG-3′
CLTC hsc.rnai.n004859.12.7 Sense 5′-GCCUUUACAAGGAUGCAAUGCAGta-3′
Antisense 5′-UACUGCAUUGCAUCCUUGUAAAGGCUG-3′
PAK1 hsc.rnai.n002576.12.1 Sense 5′-GCGAUCCUAAGAAGAAAUAUACAcg-3′
Antisense 5′-CGUGUAUAUUUCUUCUUAGGAUCGCCC-3′
Rac1 hsc.rnai.n018890.12.1 Sense 5′-GGAACUAAACUUGAUCUUAGGGAtg-3′
Antisense 5′-CAUCCCUAAGAUCAAGUUUAGUUCCCA-3′
ANKFY1 hsc.rnai.n016376.12.1 Sense 5′-ACAGCGAUCUGAAGAUAAAGGUUgg-3′
Antisense 5′-CCAACCUUUAUCUUCAGAUCGCUGUAC-3′

siRNAs had a single 2-base 3′-overhang on the antisense strand and a blunt end modified with DNA bases (shown in lower case).

labeled EVs (from ~5 mL CM) for 4 h in the presence of inhibitors. 2.11. Statistical analysis
Subsequently, cells were washed with PBS, washed with acid wash
buffer (0.5 M NaCl, 0.2 M acetic acid) to remove membrane-bound EVs Comparisons between two groups were performed using in-
and once more with PBS. Cells were trypsinized, fixated in 0.2% par- dependent two sample t-tests. Multiple-group testing was performed
aformaldehyde in PBS and subjected to flow cytometric analysis on a using one-way ANOVA with Dunnett's post-hoc tests.
FACSCanto (BD Biosciences).
For confocal microscopy analysis, HeLa cells were seeded at a 3. Results
density of 4000 cells/well in 16-well chamber slides (Lab-Tek). After
24 h, cells were pretreated with inhibitors as described above and then EVs were isolated from conditioned medium from A431 cells using a
incubated with PKH67-labeled EVs (from ~20 mL CM) for 4 h in the recently described procedure based on ultrafiltration followed by size-
presence of inhibitors. Subsequently, cells were washed with PBS, wa- exclusion chromatography [25]. EVs were collected in serum-free
shed with acid wash buffer (0.5 M NaCl, 0.2 M acetic acid) to remove medium, thus avoiding potential contamination with FBS-derived EVs.
membrane-bound EVs and once more with PBS. Cells were fixated with Western Blot characterization revealed that EV marker proteins Alix
4% paraformaldehyde in PBS at room temperature for 20 min. After and CD63 were strongly enriched in EVs compared to cell lysate, while
fixation, slides were washed with PBS and mounted using Fluorsave the opposite was observed for calnexin, an integral protein of the en-
(Calbiochem). Confocal fluorescence imaging was done on a LSM700 doplasmic reticulum (Fig. 1A). Actin was found at similar levels in EVs
laser scanning confocal microscope (Zeiss). Images were processed and cell lysate. NTA showed that the majority of EVs were in a size
using LSM Image Browser. Contrast and brightness parameter adjust- range of 60–250 nm, peaking at 100 nm (Fig. 1B). Transmission elec-
ments were applied across whole images and equally across comparison tron microscopy analysis indicated that EVs were spherical, membrane-
groups when necessary. bound particles with most vesicles having a diameter around 100 nm
(Fig. 1C). To study internalization of EVs by HeLa cells, purified EVs
were fluorescently labeled with the membrane dye PKH67. Shortly after
2.10. Spheroid formation and EV internalization assays incubation with EVs, a punctuated pattern of fluorescence could be
observed intracellularly, with increasing signal over time. Uptake was
Spheroids were grown in a 96 well Insphero Gravity TRAP™ ULA inhibited by an excess of unlabeled EVs, as shown by flow cytometry
plate (Perkin Elmer). Wells were prewet with 10 μL of media. HeLa cells (Fig. 1D) and confocal microscopy analysis (Fig. 1E). This indicates that
were seeded at a density of 1000 cells/well, after which plates were EV uptake is mediated by specific processes and also excludes a sig-
centrifuged at 250 × g for 2 min and allowed to incubate for 48 h prior nificant contribution of transfer of free PKH dye to the observed
to treatment. Spheroids were pretreated with inhibitors as described fluorescent signal. EV internalization was completely inhibited at 4 °C
above and then incubated with PKH67-labeled EVs (from ~ 20 mL CM) (Fig. 1F,G), which indicates that it is mediated by active, energy-de-
for 4 h in the presence of inhibitors. Subsequently, cells were washed pendent endocytic processes, as opposed to passive membrane fusion.
with PBS, washed with acid wash buffer (0.5 M NaCl, 0.2 M acetic acid) Furthermore, EV internalization was found to be time-dependent
to remove membrane-bound EVs and twice more with PBS. For flow (Fig. 1H), and significantly inhibited in the presence of heparin
cytometric analysis, cells were trypsinized, fixed in 0.2% paraf- (Fig. 1I), a soluble analogue of heparan sulfate proteoglycans, as de-
ormaldehyde in PBS and subjected to flow cytometric analysis on a scribed previously [26,27]. Cytochalasin D, an inhibitor of actin poly-
FACSCanto (BD Biosciences). For confocal microscopy analysis, spher- merization, significantly inhibited EV uptake, suggesting that endocytic
oids were fixated with 4% paraformaldehyde in PBS at room tem- mechanisms requiring cytoskeletal remodeling are involved.
perature for 20 min. After fixation, spheroids were washed with PBS To unravel the contribution of specific endocytic processes to EV
and transferred to μView clear-bottom 96 well plates (Greiner Bio-One). internalization, recipient cells were pretreated with pharmacological
Confocal fluorescence imaging was done on a Yokogawa Cell Voyager inhibitors known to interfere with different pathways. Chlorpromazine,
7000 (Yokogawa). Images were processed using Fiji. Contrast and which blocks CDE by interfering with the association between clathrin
brightness parameter adjustments were applied across whole images and the plasma membrane [28], did not inhibit, but slightly enhanced,
and equally across comparison groups when necessary. EV uptake, as shown by flow cytometry (Fig. 2A) and confocal micro-
scopy (Fig. 2B). This may be the result of a change in the activity of
other endocytic mechanisms as compensation for alterations in CDE, a

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Fig. 1. EVs are internalized by HeLa cells. (A) Western Blot analysis of Alix, CD63, Actin and Calnexin in A431 cell lysate (CL) and EVs. (B) Size distribution profile of A431 EVs as
determined by NTA. (C) Electron microscopy analysis of A431 EVs. Scale bar represents 100 nm. (D,E) EV uptake by HeLa cells in the absence or presence of a five-fold excess of unlabeled
EVs uptake as quantified by flow cytometry (D) or visualized by confocal microscopy (E). Scale bars represent 10 μm. (F,G) EV uptake by HeLa cells at 37 °C or 4 °C as quantified by flow
cytometry (F) or visualized by confocal microscopy (G). (H) Time dependent EV uptake by HeLa cells. (I) EV uptake by HeLa cells in the absence or presence of 10 μg/mL heparin. (J) EV
uptake by HeLa cells in the presence of increasing concentrations of cytochalasin D. Values represent mean + S.E.M. *p < 0.05.

phenomenon known as cross-regulation [20]. In contrast, chlorproma- inhibitor wortmannin (Fig. 2H). PI(3)K participates in the signaling
zine dose-dependently inhibited uptake of transferrin, an established cascade that stimulates membrane ruffling and closes the macropino-
CDE marker (Fig. S1), showing that chlorpromazine has the expected somes during MP [32]. Together, these findings suggest the importance
activity in these cells. Together, these results suggest that CDE does not of clathrin-independent endocytosis and macropinocytosis for EV up-
significantly contribute to EV internalization. Genistein is a tyrosine take.
kinase inhibitor that causes local disruption of the actin network and Interestingly, none of the inhibitors of either CIE or MP resulted in
can inhibit the recruitment of dynamin to plasma membranes, both of complete inhibition of EV uptake, indicating a contribution from both
which are important in CIE [29]. EV uptake was dose-dependently in- pathways. To evaluate whether inhibition of both pathways had an
hibited by genistein, suggesting a role for CIE in their internalization additive effect, cells were pretreated simultaneously with both EIPA
(Fig. 2C,D). As CIE includes several pathways which share a de- and genistein. While each of the individual inhibitors reduced EV up-
pendency on cholesterol, we next pretreated cells with simvastatin, an take by ~50%, the combination treatment almost completely blocked
inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, an en- internalization (Fig. 3A), suggesting that CIE and MP are processes that
zyme involved in the synthesis of cholesterol [30]. Simvastatin treat- independently contribute. As constitutive MP occurs at very low levels
ment dose-dependently inhibited EV uptake (Fig. 2E), indicating that in epithelial cells, we next evaluated whether EVs themselves could
the presence of cholesterol is indispensable for EV uptake, again sug- induce MP. After incubation with EVs, uptake of the fluid phase marker
gesting the importance of CIE as uptake route. For the study of MP, cells dextran (70 kDa) increased substantially (Fig. 3B), indicating that EVs
were pretreated with 5-(N-ethyl-N-isopropyl)amirolide (EIPA), an in- are capable of stimulating macropinocytosis, and thereby their own
hibitor of Na+/H+ exchange, known to inhibit MP by lowering sub- internalization.
membranous pH, which affects Rac1 activation and actin assembly Due to their reported low specificity in some cell types, chemical
[31]. EIPA dose-dependently restricted EV internalization (Fig. 2F,G). inhibitors alone may not be sufficient to fully elucidate the mechanisms
Similar results were obtained for the phosphoinositide 3-kinase (PI(3)K) that contribute to uptake of EVs. For example, genistein may also

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Fig. 2. EVs are internalized through tyrosine kinase-, cholesterol-, Na+/H+ exchangers-, and PI3K-dependent pathways. EV uptake by HeLa cells in the presence of increasing con-
centrations of (A,B) chlorpromazine, (C,D) genistein, (E) simvastatin, (F,G) EIPA or (H) wortmannin, as quantified by flow cytometry (A,C,E,F,H) or visualized by confocal microscopy
(B,D,G). Scale bars represent 10 μm. Values represent mean + S.E.M. *p < 0.05.

inhibit uptake via CDE by hindering recruitment of F-actin to clathrin- internalization through RNAi-mediated knockdown of specific proteins.
coated pits, while wortmannin may display pleiotropic effects on en- These proteins included clathrin heavy chain (CLTC), a major subunit of
docytosis [33]. Furthermore, cholesterol is not only important for CIE, clathrin, caveolin-1 (CAV-1), CDC42, Flotillin-1 (Flot1), ARF6, and
but also for MP [34]. Therefore, we next sought to confirm our ob- RhoA, key endocytic proteins regulating different subclasses of CIE, and
servations and evaluate the key contributors involved in EV Rac1, PAK1 and ANKFY1, proteins previously shown to regulate MP

Fig. 3. Clathrin-independent endocytosis and macro-


pinocytosis independently contribute to EV internalization.
(A) EV uptake by HeLa cells in the presence of EIPA
(100 μM), genistein (200 μM) or the combination, as
quantified by flow cytometry. (B) Dextran uptake by HeLa
cells in the presence of increasing concentrations of EVs, as
quantified by flow cytometry. Values represent mean
+ S.E.M. *p < 0.05.

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Fig. 4. Knockdown of CAV1, RhoA, Flotillin, Rac1 or PAK1 inhibits EV internalization. (A) Western Blot analysis of HeLa cells transfected with siRNA against key endocytic regulators
(clathrin heavy chain (CLTC), caveolin-1 (CAV1), CDC42, RhoA, ARF6, Flotillin, Rac1, PAK1 and ANKFY1) or negative control siRNA (NS). (B,C) EV uptake by HeLa cells pretreated with
indicated siRNAs, as quantified by flow cytometry (B) or visualized by confocal microscopy (C). Scale bars represent 10 μm. Values represent mean + S.E.M. *p < 0.05.

[21]. After two rounds of transfection, significant downregulation of 4. Discussion


target proteins was observed (Fig. 4A). Depletion of CLTC had no effect
on EV internalization, confirming that uptake of EVs is independent of EVs are gaining increasing interest from both academia and industry
CDE. EV internalization was significantly inhibited after knockdown of as endogenous nanoparticles with properties that makes them highly
caveolin-1, flotillin-1, and RhoA, but not ARF6 and CDC42, suggesting a suitable for therapeutic applications such as regenerative medicine and
contribution of the caveolae-, flotillin-1-, and RhoA-dependent sub- drug delivery [6,36]. Naturally equipped to deliver their biological
classes of CIE (Fig. 4B). Lastly, we observed a significant inhibition of cargo, EVs are capable of modulating or reprogramming recipient cells.
EV uptake upon downregulation of MP regulators Rac1 and PAK1. The However, whether these phenotypic responses are elicited may cru-
effects of knockdown of CLTC, Flotillin and PAK1 as markers of CDE, cially depend on the mechanism of EV uptake, similar to what has been
CIE and MP, respectively, on EV internalization were confirmed by described for viruses [37] and synthetic nanoparticles [22,38].
confocal microscopy analysis (Fig. 4C). In this study, using both pharmacological and RNAi-mediated in-
Compared with 2D monolayers, culturing cells in 3D spheroids may hibition of endocytic pathways, we show that EV internalization occurs
be more representative of the in vivo environment, and has been shown via independent contributions of CIE and MP. Following the assessment
to affect cell metabolism, interaction of cells with each other and the of EV internalization in 2D monolayer conditions, similar evaluations
extracellular matrix, as well as cell polarity and gene profiles (including were performed in 3D spheroid culture. 3D culture more closely mimics
receptor expression) [35]. Therefore, we next evaluated endocytic in vivo cellular organization (e.g. cellular polarization, interaction with
mechanisms involved in EV uptake into 3D spheroids. In contrast to the extracellular matrix), potentially including essential features required
2D monolayer, in which all cells had taken up EVs within a 4 h time for correct EV internalization and processing [39]. Similar inhibitory
frame, uptake into spheroids was mainly restricted to the outer few cell effects of genistein and EIPA, but not chlorpromazine, were observed on
layers. Similar to results obtained using monolayer cultures, genistein 3D spheroids versus 2D cell culture, suggesting that our results may, to
and EIPA, but not chlorpromazine, substantially inhibited EV uptake the best of our knowledge, also be representative for the in vivo si-
(Fig. 5A). EIPA also displayed a slight effect on spheroid compactness tuation.
(brightfield pictures). Flow cytometric analysis revealed that both the To study the involvement of CIE in EV uptake, we employed the
fluorescent signal per cell (Fig. 5B) as well as the percentage of EV- small molecule inhibitors genistein and simvastatin. For both inhibitors,
positive cells (Fig. 5C) were significantly reduced after treatment with a dose-dependent effect on EV uptake was observed. Although often
genistein or EIPA. Taken together, our results show that EVs are in- used for membrane cholesterol depletion, recent work indicates that
ternalized through CIE and MP in 2D monolayers as well as 3D spher- statins may also have other effects on cells, including an effect on Rab
oids. prenylation and glycosphingolipid biosynthesis [40]. Classically, me-
thyl-β-cyclodextrin and filipin, inhibitors known to affect structure and
function of cholesterol-rich membrane domains, are being used to study

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Fig. 5. Inhibition of clathrin-independent endocytosis or macropinocytosis attenuates EV internalization in spheroids. EV uptake by HeLa spheroids in the presence of chlorpromazine
(10 μM), genistein (200 μM) or EIPA (100 μM) as visualized by confocal microscopy (A) or quantified by flow cytometry (B,C). Pictures represent planes at the indicated depths within the
spheroids. Scale bars represents 100 μm. Values represent mean + S.E.M. *p < 0.05.

CIE [33]. However, these drugs may also interact with cholesterol in EV themselves can also stimulate MP, thereby potentiating their own up-
membranes, thereby affecting EV membrane integrity, and thereby in- take. The mechanisms underlying this observation are unclear, but may
directly influencing EV uptake [41]. Therefore, we also used RNAi to involve EV cargo-mediated activation of signaling pathways (e.g.
inhibit expression of proteins with known involvements in specific phosphatidylinositol-3-kinase (PI3K) [46], ras [47], and src [48]) that
subclasses of CIE, and discovered significant contributions of CAV-1, trigger MP induction.
flotillin-1 and RhoA to EV uptake. Caveolins and flotillins constitute a Our data that indicate that EVs are taken up via CIE and MP, but not
group of proteins that are enriched in specific microdomains within the CDE, contrast with some results obtained by others. For example, CDE
plasma membrane, known as lipid rafts. These microdomains contain was shown to be involved in uptake of prostate cancer cell-derived EVs
high concentrations of cholesterol and glycosphingolipids and are by mesenchymal stromal cells [49]. In this study, results from CAV1
known sites for endocytosis. Thus, lipid rafts may be involved in EV knockdown experiments also ruled out an involvement of caveolae-
uptake, as also previously suggested by others [16,42]. Our observation mediated endocytosis in the uptake process. Conversely, our results
that EV internalization was significantly inhibited even after partial coincide with others who revealed a lack of involvement of CDE, but
knockdown of RhoA (Fig.4A) suggests that RhoA-dependent en- support a role for CIE in EV uptake [16,50]. Nevertheless, there are still
docytosis also plays a strong role. However, caution should be taken, as discrepancies in some of the observations, as suppression of CAV1 ex-
possible contributions of RhoA to other internalization mechanisms pression increased EV uptake by embryonic fibroblast cells [16], an
have not been completely ruled out, and interfering with RhoA sig- opposite effect to that observed here (Fig. 4B). Comparison and inter-
naling is likely to affect many other cellular processes [20]. pretation of these different reports is however complicated by the fact
Our experiments using chemical inhibitors EIPA and wortmannin that, at least in some cell types, alterations in CAV1 expression may also
suggest an important contribution for MP in EV internalization. To affect fluid phase as well as CDC42-dependent uptake [51,52]. Simi-
confirm these observations, we silenced Rac1, PAK1 and ANKFY1, larly, while several reports suggested that cells internalize EVs via MP
proteins previously implicated to play important roles in MP. While [18,49,53], others did not find a role for MP in EV uptake [54,55].
Rac1 and PAK1 inhibition attenuated EV internalization, we found no However, these results were based on experiments using small molecule
significant effect after ANKFY1 knockdown. The reason for the apparent inhibitors only. We provide further evidence supporting the involve-
lack of importance for ANKFY1 in this process is unknown. Although ment of MP through RNAi-mediated depletion of proteins that are es-
PAK1 serves as target for both small GTP binding proteins Rac1 and sential for this process. Collectively, these findings demonstrate that EV
CDC42, our data seem to suggest that CDC42 is not involved in EV uptake may rely on a variety of endocytic mechanisms, depending on
uptake. This may be explained by the fact that Rac1 is thought to be the EV donor cell type and recipient cell type.
required for growth factor-induced MP, whereas CDC42 has a role in The observation that multiple, independently contributing path-
constitutive MP, which occurs only at very low levels in HeLa cells ways are involved in EV internalization presents the intriguing possi-
[43,44]. Concordantly, it has previously been shown that induction of bility that distinct EV subpopulations are differentially processed by
MP (through stimulation of growth factor receptors or Ras transfor- cells. While it is generally accepted that cells release at least three types
mation) enhances EV uptake [45]. Here, we demonstrate that EVs of EVs, i.e. apoptotic bodies, microvesicles and exosomes, increasing

106
H. Costa Verdera et al. Journal of Controlled Release 266 (2017) 100–108

evidence suggests that even within these groups, distinct subpopula- microscopy facilities.
tions can be discriminated [56,57]. These subpopulations express un-
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