pharmaceutics
Article
Coincubation as miR-Loading Strategy to Improve the
Anti-Tumor Effect of Stem Cell-Derived EVs
Alessia Brossa 1,2 , Marta Tapparo 2,3, Valentina Fonsato 2,4, Elli Papadimitriou 1,2, Michela Delena 2,
Giovanni Camussi 3 and Benedetta Bussolati 1,2,*
          
       
Citation: Brossa, A.; Tapparo, M.;
Fonsato, V.; Papadimitriou, E.;
Delena, M.; Camussi, G.; Bussolati, B.
Coincubation as miR-Loading
Strategy to Improve the Anti-Tumor
Effect of Stem Cell-Derived EVs.
Pharmaceutics 2021, 13, 76.
https://doi.org/10.3390/
pharmaceutics13010076
Received: 9 December 2020
Accepted: 4 January 2021
Published: 8 January 2021
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1 Department of Molecular Biotechnology and Health Sciences, University of Torino, 10126 Torino, Italy;
alessia.brossa@unito.it (A.B.); elli.papadimitriou@unito.it (E.P.)
2 Molecular Biotechnology Center, University of Torino, 10126 Torino, Italy; marta.tapparo@unito.it (M.T.);
valentina.fonsato@2i3t.it (V.F.); michela.delena@gmail.com (M.D.)
3 Department of Medical Science, University of Torino, 10126 Torino, Italy; giovanni.camussi@unito.it
4 Society for Business Incubator and Tech Transfer, University of Torino, 10126 Torino, Italy
Correspondence: benedetta.bussolati@unito.it; Tel.: +39-011-670-6453
Abstract: Extracellular vesicles are considered a novel therapeutic tool, due to their ability to transfer
their cargoes to target cells. Different strategies to directly load extracellular vesicles with RNA
species have been proposed. Electroporation has been used for the loading of non-active vesicles;
however, the engineering of vesicles already carrying a therapeutically active cargo is still under
investigation. Here, we set up a coincubation method to increase the anti-tumor effect of extracellular
vesicles isolated from human liver stem cells (HLSC-EVs). Using the coincubation protocol, vesicles
were loaded with the anti-tumor miRNA-145, and their effect was evaluated on renal cancer stem cell
invasion. Loaded HLSC-EVs maintained their integrity and miR transfer ability. Loaded miR-145, but
not miR-145 alone, was protected by RNAse digestion, possibly due to its binding to RNA-binding
proteins on HLSC-EV surface, such as Annexin A2. Moreover, miR-145 coincubated HLSC-EVs
were more effective in inhibiting the invasive properties of cancer stem cells, in comparison to naïve
vesicles. The protocol reported here exploits a well described property of extracellular vesicles to
bind nucleic acids on their surface and protect them from degradation, in order to obtain an effective
miRNA loading, thus increasing the activity of therapeutically active naïve extracellular vesicles.
Keywords: extracellular vesicle engineering; microRNA; loading; anti-tumor; cancer stem cells;
exosomes; coincubation
Introduction
Extracellular vesicles (EVs) are nanosized vesicles actively released by many, if not all,
cells and identified in biological fluids [1]. EVs display the ability to deliver active cargo,
including RNA species, to target cells, thus reprogramming their gene expression profile [1].
Therefore, the interest for the exploitation of EVs as therapeutic tool is rapidly increasing [2].
Indeed, EVs appear a highly efficient delivery system, as compared to naked molecules,
as they protect the cargo from degradation by RNases and proteases [3,4]. Moreover, in
comparison to synthetic liposomes, EVs might display an increased efficacy since, being a
natural cell derived product, they show reduced clearance by the macrophagic system, low
immunogenicity and ability to deliver nucleic-acid-based therapeutics across biological
barriers [5,6].
A great deal of effort has been, therefore, dedicated to the pharmacological exploitation
of EVs as carriers of specific cargo of interest [7]. In particular, EV engineering with RNA
species has been proposed for different therapeutic applications, ranging from anti-tumor
strategies to vaccination [4,8,9]. Different technical approaches for EV loading include the
option to modify the originating cell, typically by transfection, or to directly load isolated
EVs using electroporation [4]. The approach of direct EV loading might be of particular
Pharmaceutics 2021, 13, 76. https://doi.org/10.3390/pharmaceutics13010076 https://www.mdpi.com/journal/pharmaceutics
Pharmaceutics 2021, 13, 76 2 of 14
interest in those contexts where the delivery of the identified cargo represents the main
mechanism of action of the therapeutic preparation. For instance, we recently set up a
methodology for direct electroporation of EVs isolated from plasma for the loading of
antitumor microRNAs without perturbation of their integrity and targeting properties [10].
In this context, the use of plasma-derived EVs from an autologous source might appear of
therapeutic relevance.
A different situation can be envisaged when EVs carrying per se a therapeutically
active cargo are loaded with a desired molecule to increase their effect. We recently showed
that EVs from human liver stem cells (HLSC) induced a potent anti-tumor effect in vitro
and in vivo [11–14]. Specifically, HLSC-EVs inhibited the invasion of renal cancer stem cells
(rCSCs) [14], and induced their apoptosis [12]. Preliminary experiments attempting to load
HLSC-EVs with an additional miR cargo through electroporation not only failed to increase
their therapeutic effect, but rather reduced it, suggesting the loss of their endogenous
activity.
The aim of the present paper was, therefore, to identify a strategy to potentiate the
endogenous anti-tumor activity of stem-cell derived EVs avoiding the loss of their endogenous
effect. For this purpose, exploiting the EV ability to bind, protect and transport active
RNA species on their surface, we set up a coincubation protocol able to load microRNAs
on HLSC-EVs and to increase their anti-tumor effects.
Materials and Methods
2.1. Renal Cancer Stem Cells Isolation and Culture
Renal cancer stem cells (rCSCs) were isolated and characterized as previously described
[12,14,15]. Cells were obtained from specimens of renal cell carcinomas from
patients undergoing nephrectomy, according to the Ethics Committee of the S. Giovanni
Battista Hospital of Torino, Italy (168/2014, 16 August 2014). CD105-positive rCSCs were
isolated by magnetic cell sorting from the total tumor cell population, using the magneticactivated
cell sorting (MACS) system (Miltenyi Biotec, Auburn, CA, USA). Single CD105
positive cells were seeded in 96-well plates in presence of the expansion medium, consisting
of DMEM LG (Invitrogen, Carlsbad, CA, USA), supplemented with 2 nM L-glutamine
(Lonza, Basel, Switzerland), insulin-transferrin-selenium, 10-9 M dexamethasone, 100 U
penicillin, 1000 U streptomycin, 10 ng/mL epidermal growth factor (EGF) (all from Sigma-
Aldrich, St. Louis, MO, USA) and 5% fetal calf serum (FCS) (Euroclone, Pero MI, Italy).
A CD105 positive clonal rCSC line was selected and used for all the experiments, as
previously described [12,14]. Mycoplasma absence was routinely tested using RT-PCR.
2.2. Human Liver Stem Cells Isolation and Culture
Human Liver Stem Cells (HLSCs) were generated by Anemocyte International (Gerenzano,
Italy) from a liver donor, according to the standard criteria of Centro Nazionale
Trapianti, as previously described [14,16]. Isolated HLSCs were cultured in the presence
of minimal essential medium ( -MEM; Lonza, Basel, Switzerland) supplemented with
10% FCS (Euroclone), 10 ng/mL human recombinant EGF (Miltenyi, Bergisch Gladbach,
Germany), 10 ng/mL human recombinant basic fibroblast growth factor (Miltenyi, Bergisch
Gladbach, Germany), 2 nM L-glutamine (Lonza, Basel, Switzerland) and 100 U/mL
penicillin/streptomycin (Sigma, St. Louis, MO, USA) and maintained in a humidified 5%
CO2 incubator at 37  C. After 2 weeks, HLSC colonies were expanded and characterized as
previously described [16]. Mycoplasma absence was routinely tested using RT-PCR.
2.3. HLSC-EVs Isolation and Characterization
For EV isolation, sub-confluent HLSCs were cultured overnight in serum-free  -
MEM (Lonza, Basel, Switzerland); the supernatant was then recovered and centrifuged
for 20 min at 3000 g before being filtered (0.22  m filters, Merck-Millipore, Burlington,
MA, USA) in order to remove cell debris and apoptotic bodies. Supernatants were then
ultracentrifuged (Beckman Coulter Optima L-90 K, Fullerton, CA, USA) at 100,000 g for 2 h
Pharmaceutics 2021, 13, 76 3 of 14
at 4  C. HLSC-EVs were resuspended in RPMI supplemented with 1% dimethyl sulfoxide
(DMSO, Sigma-Aldrich, St. Louis, MO, USA) and stored at 􀀀80  C for later use. Nanosight
LS300 system (Malvern Panalytical, Malvern, UK) was used to evaluate EVs concentration
and size distribution. Briefly, EV preparations were diluted (1:200) in sterile saline solution
and analyzed by the Nanoparticle Analysis System using the NTA 1.4 Analytical Software,
as previously described [12,14].
2.4. Electroporation Protocol
HLSC-EVs were electroporated using Invitrogen Neon Kit (Invitrogen, Carlsbad,
CA, USA), as previously described [10]. Briefly, 6   1010 EVs were electroporated with a
voltage of 750 V using a pulse width of 20 ms, for 10 pulses, according to the manufacturer’s
protocol. EVs were then was incubated for 30 min at 37  C, washed by ultracentrifugation at
100,000 g for 2 h at 4  C, resuspended in RPMI (Lonza, Basel, Switzerland) and immediately
used for selected experiments. For each experiment, 6   1010 HLSC-EVs were subjected to
37  C incubation and ultracentrifugation without electroporation to be used as control.
2.5. Coincubation Protocol
EVs (1010) were incubated for 1 h at 37  C with 100 picomoles of the indicated miRNA,
in a final volume of 200  L RPMI (Lonza, Basel, Switzerland). When indicated, coincubation
was followed by RNAse-A (LifeTechnologies, Carlsbad, CA, USA) treatment (0.1 ng/ L) for
3 h at 37  C to digest free miRNA. RNAse digestion was stopped by incubation with 4U of
RNAse inhibitor (Invitrogen, Carlsbad, CA, USA) for 1 h at 37  C. When indicated, RNAse
treatment was followed with trypsin digestion (5 ng/mL, 1 h at 37  C) (Sigma-Aldrich,
St. Louis, MO, USA). Samples were then subjected to centrifugation (4000 RPM, 5 min at
4  C) using 50 kDa filters (Merck-Millipore, Burlington, MA, USA), in order to remove
unbound and undigested miRNAs, and immediately used for indicated experiments.
2.6. Cytofluorimetric EV Analysis
HLSC-EVs were bound to surfactant-free white aldehyde/sulfate latex beads 4%
w/v, 4  m diameter (Molecular Probes, Thermo Fisher, Waltham, MA, USA) for the
cytofluorimetric analysis using Guava instrument (Merck-Millipore, Burlington, MA, USA).
Thirty  g of EVs were incubated with 5  L of beads for 30 min at room temperature and
subsequently for 30 min at +4  C. Adsorbed EVs were then incubated with FITC and
PE labeled antibodies against CD63, CD44, integrin alpha 4 and CD29 (all from Beckton
Dickinson, Franklin Lakes, NJ, USA) with a final dilution of 1:50, for 15 min at +4  C. During
the cytofluorimetric acquisition, the gating strategy was set on the physical parameters
dot plot. Controls corresponded to EVs adsorbed on beads and marked with FITC-, PE- or
APC-conjugated mouse IgG1 Isotypes (all from Miltenyi, Bergisch Gladbach, Germany).
2.7. Apoptosis
Cytofluorimetric evaluation of apoptotic cells was performed using the Muse™ Annexin
V & Dead Cell Kit (Merck-Millipore, Burlington, MA, USA), according to the manufacturer’s
instructions. Briefly, 10   103 cells were incubated with 50   103 EVs/target
cell for 48 h. Cells were then detached and resuspended in Muse™ Annexin V & Dead
Cell Kit (Luminex, Austin, TX, USA), and the percentage of apoptotic cells (Annexin V+)
was detected.
2.8. Invasion
Invasion assay was performed using 24-well cell culture inserts (Beckton Dickinson,
Franklin Lakes, NJ, USA) with a porous membrane (8.0  m pore size) precoated with
100  g growth factor-reduced Matrigel (Beckton Dickinson, Franklin Lakes, NJ, USA) per
well, as previously described [14]. Briefly, 50   103 rCSCs were detached using a nonenzymatic
solution (Sigma-Aldrich, St. Louis, MO, USA), and plated in the presence of
50   103 EVs/target cell in the upper side of the pre-coated transwell in DMEM (Euroclone).
Pharmaceutics 2021, 13, 76 4 of 14
As an attractive stimulus, complete culture medium was added in the well. Every condition
was performed in triplicate. After 48 h, cells that moved from the upper side of the transwell
to the lower one were fixed in MetOH and stained with crystal violet (Sigma-Aldrich,
St. Louis, MO, USA). The total area of invaded Matrigel (original magnification: 100 ) was
evaluated by ImageJ on at least five pictures per transwell.
2.9. Super-Resolution Microscopy
Super-resolution analyses were performed using a Nanoimager S Mark II microscope
from ONI (Oxford Nanoimaging, Oxford, UK) equipped with 405 nm/150 mW,
473 nm/1 W, 560 nm/1 W, 640 nm/1 W lasers and dual emission channels split at 640 nm.
For the preparation of the sample, 10  L of Poly-L-Lysine (Sigma-Aldrich, St. Louis, MO,
USA) was placed on coverslips, in culture wells (Grace Bio-Labs, Sigma-Aldrich, St. Louis,
MO, USA), and left at 37  C in a humidifying chamber for two hours. After removal of
the excess, HLSC-EVs previously coincubated with a Scrambled-FITC RNA sequence and
digested with RNase, were left to attach overnight at +4  C on the coverslips. The next
day, non-attached EVs were removed and 10  L of blocking buffer (PBS-5% Bovine Serum
Albumin) was added into the wells for 30 min. Then, 2.5  g of purified mouse anti-CD29
antibody (Beckton Dickinson, Franklin Lakes, NJ, USA) were conjugated with Alexa Fluor
647 dye, using the Apex Antibody Labeling Kit (Invitrogen, Carlsbad, CA, USA) according
to the manufacturer’s protocol. Anti-CD29 Alexa Fluor 647 antibody was added to the
blocking buffer containing wells at a final dilution 1:10. The antibody was left for overnight
incubation at +4  C. The samples were washed twice with PBS and 10  L ONI BCubed
Imaging Buffer was added for the EV imaging. Two-channel dSTORM data were acquired
sequentially at 30 Hz (Hertz) in total reflection fluorescence (TIRF) mode. Single molecule
data was filtered using NimOS (Version 1.7.1.10213, ONI, Oxford, UK) based on the point
spread function shape, photon count and localization precision to minimize background
noise and remove low-precision localizations.
2.10. EV Incorporation in Target Cells
To evaluate the internalization of coincubated EVs in rCSCs by fluorescent microscopy,
HLSC-EVs were labeled with 1  M Dil dye (ThermoFisher, Waltham, MA, USA) as described
previously [12]. Briefly, HLSC-EVs were resuspended in PBS supplemented with
1  M Dil dye and ultracentrifuged at 100,000 g for 1 h at 4  C. EVs were then washed
with PBS by ultracentrifugation (100,000 g for 1 h at 4  C). The EV pellet was resuspended
in RPMI and processed for coincubation. Coincubated EVs were immediately
used to treat previously plated rCSCs (50   103 EVs/target cell) for 1 h, cells were then
fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and processed for
confocal microscopy.
2.11. miRNA Isolation and Real Time PCR
Total RNA was isolated from different rCSCs or EVs preparations using MirVana kit
(Ambion, ThermoFisher,Waltham, MA, USA), according to the manufacturer’s protocol,
and quantified spectrophotometrically (Nanodrop ND-1000, ThermoFisher, Waltham, MA,
USA). First-strand cDNA was produced from 200 ng of total RNA using the miScript
Reverse Transcription Kit (Qiagen, Hilden, Germany). Real-time PCR experiments were
performed in 20  L reaction mixture containing 5 ng of cDNA template, the sequencespecific
oligonucleotide primers (purchased from MWG-Biotech, Nantes, BRU, Luxembourg)
and the miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany). RNU48 was
used to normalize miRNA inputs.
2.12. Protein Extraction and Western Blot
Different preparations of HLSC-EVs were lysed in RIPA buffer supplemented with
protease and phosphatase inhibitor cocktail and PMSF (Sigma-Aldrich, St. Louis, MO,
USA) immediately after ultracentrifuge. Aliquots of EV lysates containing 30  g proteins,
Pharmaceutics 2021, 13, 76 5 of 14
as determined by Bradford quantification (Biorad, Hercules, CA, USA), were run on 4–20%
SDS-PAGE under reducing conditions and blotted onto PVDF membrane filters using
the iBLOT system (LifeTechnologies, Carlsbad, CA, USA). Membranes were blocked in
Tris-buffered saline-Tween (TBS-T; 25 mM Tris, pH 8.0, 150 mM NaCl, and 0.05% Tween-20)
containing 5% (w/v) non-fat dried milk for 1 h. After blocking, membranes were incubated
overnight with anti-ANAXA2 antibody (LS-C150122, LSBio, Seattle, WA, USA). Blots were
then incubated with Goat anti-Rabbit IgG HRP conjugated (Thermo Scientific,Waltham,
MA, USA) for 1 h at room temperature. Membranes were then probed with ClarityTM
Western ECL substrate (Bio-rad, Hercules, CA, USA), and bands were detected by the
Chemidoc system (Bio-rad, Hercules, CA, USA).
2.13. Statistical Analysis
Statistical analysis was carried out on Graph Pad Prism version 5.04 (GraphPad
Software, Inc, San Diego, CA, USA) by using the Student t-test or ANOVA with Dunnet’s
multi-comparison tests, where appropriate. A p value < 0.05 was considered significant.
Results
3.1. Comparison between Electroporation and Coincubation to Increase the EV Antitumor Effect
We previously demonstrated the anti-tumor effect of HLSC-EVs on renal cancer stem
cells (rCSCs) [12–14]. With the aim of potentiating this effect, we decided to enrich EVs
with anti-tumor miRNAs, known to display a strong anti-invasive and pro-apoptotic effect
in rCSCs [14]. For this aim, we performed parallel experiments in which we compared EV
miRNA loading using electroporation, previously set in our laboratory [10], or coincubation,
already reported to be effective [7], on induction of rCSCs apoptosis.
We first assessed the maintenance of functional properties in EVs after coincubation
with a scrambled RNA sequence in comparison with electroporation, in the absence of
miRNA loading, by testing their biological activity on rCSCs apoptosis induction [12]
(Supplementary Figure S1A). Electroporated EVs showed reduced pro-apoptotic activity,
when compared to naïve HLSC-EVs, as already reported [12]. In addition, electroporated
EVs were unable to induce the endogenous expression of miR-200a and miR-200b, known
to be responsible for the anti-tumor effect of naive EVs [14], suggesting loss of active cargo
in electroporated EVs (Supplementary Figure S1B). At variance, EVs coincubated with a
scrambled sequence maintained their pro-apoptotic effect (Supplementary Figure S1A),
together with the induction of the expression of anti-tumor miRNAs (Supplementary
Figure S1B).
Therefore, we decided to set a coincubation protocol for the direct loading of the
anti-tumor miRNA miR-145, which is present at a low level in naïve HLSC-EVs [14]. In
particular, we focused on assessing whether miR-145 loading could potentiate the effect
of HLSC-EVs on the reduction of the high invasive property of rCSCs [14]. HLSC-EVs
incubated with different doses of a scrambled sequence (EV + SCR 100/30/10 picomol,
corresponding to 6000/2000/600 miR-molecules/EV) maintained the anti-invasive effect
on rCSCs (Figure 1A,B), at levels comparable to naïve EVs. Subsequently, generation of miR-
145 loaded EVs by coincubation with miR-145 potentiated the anti-tumor effect of naive EVs
in terms of invasion (Figure 1A,B). In particular, different doses of miR-145 tested (EV-145
100/30/10 pmol/1010 EVs, corresponding to 6000/2000/600 miR-molecules/EV, red bars),
increased the anti-invasive effect of naïve EVs, with the higher dose (100 picomol/1010 EVs)
being themost efficient. This dose was selected for the following experiments. MiR-145 alone,
used as control of unbound miRNA, at the highest dose (100 picomol/1010 EVs, corresponding
to 6000miR-145molecules/EV), also exerted an anti-invasive effect (Figure 1A,B), since miRNA
molecules could aggregate during EV engineering, as previously shown [17].
Pharmaceutics 2021, 13, 76 6 of 14
higher dose (100 picomol/1010 EVs) being the most efficient. This dose was selected for the
following experiments. MiR-145 alone, used as control of unbound miRNA, at the highest
dose (100 picomol/1010 EVs, corresponding to 6000 miR-145 molecules/EV), also exerted
an anti-invasive effect (Figure 1A,B), since miRNA molecules could aggregate
during EV engineering, as previously shown [17].
Figure 1. Effect of miR145 coincubated HLSC-EVs on rCSC invasion. (A,B) Quantification (A) and representative micrographs
(original magnification: 100×) (B) of rCSCs invasion after treatment for 48 h with HLSC-EVs (EV) loaded with
different doses (100/30/10 pmol/1010 EVs, corresponding to 6000/2000/600 molecules/EV) of a scrambled sequence
(EV-SCR) or with miR-145 (EV-145), or miR-145 alone (145); all at a dose of 10, 30 or 100 pmol/1010 EVs. Data are represented
as mean ± SD of the percentage of invaded area of three experiments. * = p < 0.05 and ** = p < 0.001 vs. CTL; $$ = p <
0.001 vs. 145(100). (C,D) Invasion assay quantification (D) and representative micrographs (original magnification: 100×)
(E) of rCSCs treated for 48 h with miR-145 untreated (miR-145) or digested with 0.1, 1 or 5 ng/μL of RNase-A (miR-145 +
R0.1, 1 or 5, respectively). Data are represented as mean ± SD of the percentage of the invaded area of the three experiments.
** = p < 0.001 vs. CTL.
Figure 1. Effect of miR145 coincubated HLSC-EVs on rCSC invasion. (A,B) Quantification (A) and representative micrographs
(original magnification: 100 ) (B) of rCSCs invasion after treatment for 48 h with HLSC-EVs (EV) loaded with
different doses (100/30/10 pmol/1010 EVs, corresponding to 6000/2000/600 molecules/EV) of a scrambled sequence
(EV-SCR) or with miR-145 (EV-145), or miR-145 alone (145); all at a dose of 10, 30 or 100 pmol/1010 EVs. Data are represented
as mean   SD of the percentage of invaded area of three experiments. * = p < 0.05 and ** = p < 0.001 vs. CTL; $$ = p < 0.001
vs. 145(100). (C,D) Invasion assay quantification (D) and representative micrographs (original magnification: 100 ) (E) of
rCSCs treated for 48 h with miR-145 untreated (miR-145) or digested with 0.1, 1 or 5 ng/ L of RNase-A (miR-145 + R0.1,
1 or 5, respectively). Data are represented as mean   SD of the percentage of the invaded area of the three experiments.
** = p < 0.001 vs. CTL.
3.2. RNAse Treatment of Coincubated EVs
In order to obtain miR-145 coincubated EVs in the absence of unbound miR-145, we
took advantage of RNAse treatment. Indeed, incubation of miR-145 alone with 0.1, 1 or
5  g/mL RNAse abolished the observed anti-invasive effect, indicating its susceptibility
to RNAse treatment at all doses (Figure 1C,D). Therefore, we chose the lowest RNAse
concentration (0.1  g/mL) to digest the unbound fraction of miR-loaded EVs.
We first assessed EV integrity after RNAse treatment by NTA (Figure 2A) and super
resolution microscopy (Figure 2B). As shown in Figure 2A, the size distribution of coincubated
EVs treated with RNAse did not vary with respect to naive EVs. In addition, super
resolution microscopy images of HLSC-EVs, coincubated with a FITC-scrambled sequence,
confirmed the effective RNA loading and maintenance after RNAse treatment (Figure 2B).
Moreover, EV uptake by rCSCs was not affected (Figure 2C). In order to assess whether EV
coincubation and/or RNAse treatment could affect EV protein content, we performed flow
Pharmaceutics 2021, 13, 76 7 of 14
cytometry analysis on HLSC-EVs, evaluating the percentage of EVs positive for different
EV markers (CD44, CD29, integrin alpha4 (A4), CD81, CD63), known to be present on
HLSC-EVs. As shown in Figure 2D, we did not detect any change in marker expression,
suggesting that EV integrity was maintained.
3.3. Anti-Tumor Effect and miR145 Transfer of RNase Treated Coincubated EVs
We applied the above-described protocol of coincubation and digestion of unbound
miRNA with RNAse to enrich HLSC-EVs with the anti-tumor miR-145. In order to assess
whether engineered HLSC-EVs could transfer miR-145 to target cells, we treated rCSCs
with miR-145-loaded EVs (50   103/cell) and we analyzed miR-145 levels in rCSCs after 24
and 48 h of EV treatment. As shown in Figure 2E, naïve HLSC-EVs (EV) did not induce in
rCSCs any detectable change of miR-145 expression with respect to untreated cells (CTL),
while we observed a 100-fold increase in miR-145 levels when rCSCs were incubated with
HLSC-EVs loaded with miR-145 (EV-miR145). RNAse treatment did not interfere with
miR-145 transfer, since the same increase was observed when coincubated EVs were treated
with RNAse (EV-miR145 + RNAse). As suggested by the functional experiments (Figure 1A),
miR-145 levels in target cells were also increased by miR-145 alone, but this effect was
abolished by RNAse treatment, further confirming RNAse digestion of unbound miRNA
(Figure 2E). In order to evaluate if the observed increase of miR-145 in target cells was due
to a miR-145 transfer and not to its induction, we analyzed the levels of pre-miR-145 in
rCSCs treated with HLSC-EVs coincubated with miR-145 (EV-miR145) or with free miR-145.
As shown in Figure 2F, no detectable change in pre-miR145 was observed, confirming
miR-145 transfer in rCSCs by HLSC-EVs coincubated with miR-145. At variance, the
transfer of free unbound miR-145 levels was reduced after RNAse treatment (miR-145 +
RNAse), indicating the degradation of free miR-145 by RNAse (Figure 2E).
3.4. Protection of Surface Loaded miRNAs by RNA Binding Proteins
To understand the mechanism of RNAse protection of miR-145 on HLSC-EV surface,
we hypothesized the presence of surface RNA-binding proteins that could act to protect
bound miRNAs. As shown in Figure 2G, HLSC-EVs expressed Annexin A2 (ANXA2),
known to play an active role in miRNA-loading in EVs [18], and to be present on the EV
surface [19].
To further confirm the possible involvement of surface RNA-binding proteins in
miRNA loading and protection, we treated EVs with trypsin. As shown in Figure 3A,
trypsin treatment did not interfere with EV anti-invasive activity. Therefore, we evaluated
the miR-145 levels in HLSC-EVs (EV) and EV coincubated with miR-145 (EV-miR145),
which were treated for 3 h with RNAse (EV-145 + RNAse) and for an additional 1 h
with trypsin (EV-145 + RNase + TR). As shown in Figure 3B, the enrichment of miR-145
within EVs was high after coincubation and resistant to RNAse treatment but it was
significantly decreased after trypsin treatment, further suggesting that miR-145 is bound to
RNA-binding proteins present on the EV surface.
Pharmaceutics 2021, 13, 76 8 of 14
In addition, we tested the effect of EVs coincubated with miR-145 on the invasion
ability of rCSCs (Figure 3C,D). The effect of naïve EV was increased when EVs were coincubated
with miR-145 as expected (EV-miR145). EV treatment with RNAse did not
reduce the effect of miR-145 coincubated EVs (EV-miR145 + RNAse), while it reduced
that of free miR-145 (miR-145 + RNAse). Trypsin treatment of coincubated EVs
(EV-miR145 + TR) reverted the effect of EV-miR145, confirming at a functional level the
role of membrane RNA-binding proteins on EV loading with anti-tumor miRNAs by
coincubation.
Figure 2. Integrity of miR145 coincubated HLSC-EVs after RNase treatment. (A) NanoSight distribution graph showing
the quantity and size of HLSC-EVs untreated (Pre-RNase) or digested with 0.1 ng/μL RNase-A (RNase 0.1 ng/μL). (B)
Super resolution microscopy micrographs showing the effective loading of a scrambled FITC sequence (green).
HLSC-EVs are labeled with anti-CD29 Ab (red). Scale bar: 100 nm. (C) Representative micrographs of incorporation of
Figure 2. Integrity of miR145 coincubated HLSC-EVs after RNase treatment. (A) NanoSight distribution graph showing the
quantity and size of HLSC-EVs untreated (Pre-RNase) or digested with 0.1 ng/ L RNase-A (RNase 0.1 ng/ L). (B) Super
resolution microscopy micrographs showing the effective loading of a scrambled FITC sequence (green). HLSC-EVs are
labeled with anti-CD29 Ab (red). Scale bar: 100 nm. (C) Representative micrographs of incorporation of DIL-labeled
HLSC-EVs (EV) and DIL-labeled HLSC-EVs treated with 0.1 ng/ L RNase-A (EV-RNase) in rCSCs after 1 h of incubation
detected by confocal microscopy (original magnification 400 ). (D) Immunophenotypic characterization of HLSC-EVs
(EV) loaded with a scrambled sequence (EV-SCR) and treated with 0.1 ng/ L RNase-A (EV-SCR + RNase), expressing the
markers of cells of origin (CD44, CD29 and integrin alpha4 (A4)), together with the exosomal markers CD63 and CD81.
Results are mean   SD of the percentage of positive events of four different independent EV preparations. (E) Real Time
analysis showing miR-145 levels in rCSCs treated for 24 h or 48 h with naïve HLSC-EVs (EV), HLSC-EVs coincubated
with miR-145 untreated (EV-miR145) or digested with 0.1 ng/ L RNase-A (EV-miR145 + RNAse), or with free miR-145
untreated (miR145) or digested with 0.1 ng/ L RNase-A (miR145 + RNAse). Data are represented as mean   SD of four
independent experiments of the Relative Quantification (RQ) normalized to untreated cells (CTL) and to RNU6B. One-way
ANOVA was performed: ** = p < 0.001 vs. CTL. (F) Real Time analysis showing pre-miR-145 levels in rCSCs treated for 24 h
with naïve HLSC-EVs (EV), HLSC-EVs coincubated with miR-145 untreated (EV-145) or digested with 0.1 ng/ L RNase-A
(EV-145 + RNAse), or with free miR-145 untreated (miR145) or digested with 0.1 ng/ L RNase-A (miR145 + RNAse).
Data are represented as mean   SD of three independent experiments of the Relative Quantification (RQ) normalized to
untreated cells (CTL) and to RNU6B. (G) Western blot analysis of three different HLSC-EVs preparations (pool1, pool2,
pool3) showing the presence of Annexin A2 (ANAXA2).
Pharmaceutics 2021, 13, 76 9 of 14
to RNU6B. One-way ANOVA was performed: ** = p < 0.001 vs. CTL. (F) Real Time analysis showing pre-miR-145 levels in
rCSCs treated for 24 h with naïve HLSC-EVs (EV), HLSC-EVs coincubated with miR-145 untreated (EV-145) or digested
with 0.1 ng/μL RNase-A (EV-145 + RNAse), or with free miR-145 untreated (miR145) or digested with 0.1 ng/μL RNase-A
(miR145 + RNAse). Data are represented as mean ± SD of three independent experiments of the Relative Quantification
(RQ) normalized to untreated cells (CTL) and to RNU6B. (G) Western blot analysis of three different HLSC-EVs preparations
(pool1, pool2, pool3) showing the presence of Annexin A2 (ANAXA2).
Figure 3. Anti-tumor effect of miR145 coincubated HLSC-EVs after RNase but not trypsin treatment. (A) Invasion assay
quantification of rCSCs treated for 48 h with naïve HLSC-EVs (EV), or with HLSC-EVs coincubated with a scrambled
sequence (EV-SCR), EV-SCR digested with RNase-A (EV-SCR + RNase) and EV-SCR treated with RNase. Data are represented
as mean of the percentage of invaded area of one experiment performed in triplicate. (B) Real Time analysis
showing miR-145 levels in naïve HLSC-EVs (EV), HLSC-EVs coincubated with miR-145 untreated (EV-145) or digested
with 0.1 ng/μL RNase-A (EV-145 + RNAse), or digested with RNase and treated with trypsin (EV-145 + RNase + TR). Data
are represented as mean ± SD of three independent experiments of the Relative Quantification (RQ) normalized to naïve
EVs (EV) and to RNU6B. * = p < 0.05 vs. EV and $ = p < 0.05 vs. EV-145. (C,D) Invasion assay quantification (D) and rep-
Figure 3. Anti-tumor effect of miR145 coincubated HLSC-EVs after RNase but not trypsin treatment. (A) Invasion assay
quantification of rCSCs treated for 48 h with naïve HLSC-EVs (EV), or with HLSC-EVs coincubated with a scrambled
sequence (EV-SCR), EV-SCR digested with RNase-A (EV-SCR + RNase) and EV-SCR treated with RNase. Data are
represented as mean of the percentage of invaded area of one experiment performed in triplicate. (B) Real Time analysis
showing miR-145 levels in naïve HLSC-EVs (EV), HLSC-EVs coincubated with miR-145 untreated (EV-145) or digested
with 0.1 ng/ L RNase-A (EV-145 + RNAse), or digested with RNase and treated with trypsin (EV-145 + RNase + TR). Data
are represented as mean   SD of three independent experiments of the Relative Quantification (RQ) normalized to naïve
EVs (EV) and to RNU6B. * = p < 0.05 vs. EV and $ = p < 0.05 vs. EV-145. (C,D) Invasion assay quantification (D) and
representative micrographs (E) of rCSCs treated for 48 h with naïve HLSC-EVs (EV), or with HLSC-EVs coincubated with a
scrambled sequence or with miR-145 and digested with RNase-A (EV-SCR + RNase and EV-miR145 + RNase, respectively),
or with EV-SCR + RNase and EV-miR145 + RNase treated with trypsin (EV-SCR + RNase + TR and EV-miR145 + RNase
TR, respectively). Free miR-145, treated with RNase (miR145 + RNase) or with RNase and trypsin (miR145 + RNase +
TR), was used as control. Data are represented as mean of the percentage of invaded area of two experiments performed in
triplicate. An ANOVA analysis was performed: * = p < 0.05 and ** = p < 0.001 vs. untreated rCSCs (CTL).
In addition, we tested the effect of EVs coincubated with miR-145 on the invasion
ability of rCSCs (Figure 3C,D). The effect of naïve EV was increased when EVs were
coincubated with miR-145 as expected (EV-miR145). EV treatment with RNAse did not
reduce the effect of miR-145 coincubated EVs (EV-miR145 + RNAse), while it reduced that
of free miR-145 (miR-145 + RNAse). Trypsin treatment of coincubated EVs (EV-miR145 +
TR) reverted the effect of EV-miR145, confirming at a functional level the role of membrane
RNA-binding proteins on EV loading with anti-tumor miRNAs by coincubation.
Pharmaceutics 2021, 13, 76 10 of 14
miRNAs
we tested the in vitro effect of HLSC-EVs coincubated with all the anti-tumor
miRNAs previously identified as mediators of the anti-tumor effect of HLSC-EVs [14] (miR-
145, miR-200b, miR-200c, 223 and miR-429). Coincubated EVs were subsequently
treated with RNAse in order to exclude any affect due to unbound miRNA. As shown in
Figure 4, we observed an additive anti-invasive effect, with respect to naïve EVs, when EVs
were coincubated with miR-145 or miR-429.
resentative micrographs (E) of rCSCs treated for 48 h with naïve HLSC-EVs (EV), or with HLSC-EVs coincubated with a
scrambled sequence or with miR-145 and digested with RNase-A (EV-SCR + RNase and EV-miR145 + RNase, respectively),
or with EV-SCR + RNase and EV-miR145 + RNase treated with trypsin (EV-SCR + RNase + TR and EV-miR145 +
RNase + TR, respectively). Free miR-145, treated with RNase (miR145 + RNase) or with RNase and trypsin (miR145 +
RNase + TR), was used as control. Data are represented as mean of the percentage of invaded area of two experiments
performed in triplicate. An ANOVA analysis was performed: * = p < 0.05 and ** = p < 0.001 vs. untreated rCSCs (CTL).
3.5. Coincubation Protocol Using Antitumor miRNAs
Finally, we tested the in vitro effect of HLSC-EVs coincubated with all the anti-
tumor miRNAs previously identified as mediators of the anti-tumor effect of
HLSC-EVs [14] (miR-145, miR-200b, miR-200c, miR-223 and miR-429). Coincubated EVs
were subsequently treated with RNAse in order to exclude any affect due to unbound
miRNA. As shown in Figure 4, we observed an additive anti-invasive effect, with respect
to naïve EVs, when EVs were coincubated with miR-145 or miR-429.
Figure 4. Anti-invasive effects of coincubation invasion assay quantification (A) and representative
micrographs ((B), original magnification: 100×) of rCSCs treated for 48 h with naïve HLSC-EVs
(EV), or with HLSC-EVs coincubated with a scrambled sequence or with anti-tumor miRNAs
(miR-145, miR200b, miR200c, miR223 and miR429) and digested with RNase-A (EV-miR + RNase).
Figure 4. Anti-invasive effects of coincubation invasion assay quantification (A) and representative
micrographs ((B), original magnification: 100 ) of rCSCs treated for 48 h with naïve HLSC-EVs (EV),
or with HLSC-EVs coincubated with a scrambled sequence or with anti-tumor miRNAs (miR-145,
miR200b, miR200c, miR223 and miR429) and digested with RNase-A (EV-miR + RNase). miRNAs
alone digested with RNase were used as controls (miR + RNase). Data are represented as mean of the
percentage of invaded area of at least two experiments performed in triplicate. An ANOVA analysis
was performed: * = p < 0.05 vs. EV; $$ = p < 0.001 vs. untreated rCSCs (CTL).
Pharmaceutics 2021, 13, 76 11 of 14
Discussion
In the present study, we successfully set up a coincubation protocol able to load
microRNAs on HLSC-EVs surface. Engineered co-incubated HLSC-EVs efficiently delivered
microRNAs, which were indeed protected by RNase, promoting microRNA-specific
functions while maintaining the desired effect of naïve EVs on rCSC. The ability of EVs
to bind and transport active RNA and DNA species on their surface is a well-known phenomenon
[1,4]. In particular, EVs circulating in serum and present in other biological fluids
contain within their corona surface-bound nucleic acids that are considered contaminants.
In some cases, additional treatment may be required to remove them from the outside
surface of EVs using RNase or DNase [20]. However, membrane-bound RNA species are
likely to be protected from RNase degradation, considering the high levels of RNase in
biological media such as blood plasma. Indeed, recent reports suggest an active effect of
EV surface-associated DNA in horizontal gene transfer of EVs released by mesenchymal
stem cells [21,22].
In the present study, we reasoned to exploit the EV ability of binding, protecting and
delivering nucleic acids to set up a method for EV engineering. The in vitro assessment of
rCSCs invasion was chosen as a readout to compare the effect described for naïve HLSCEVs
[14] with that of engineered HLSC-EVs, in virtue of the simplicity and effectiveness
of this test. Considering preliminary experiments showing the loss of biological effect
of naïve HLSC-EVs using electroporation, we decided to set up a different protocol able
to maintain EV integrity. Indeed, it is conceivable that the electroporation process itself
might generate loss of EV membrane integrity with exit of active components, such as
RNA species, simultaneously to the entrance of the desired miRNAs. Moreover, several
publications have described difficulty in the application of the engineering approach with
electroporation because of a high degree of variability, though an effective silencing of the
target gene was obtained [23].
To increase the therapeutic effect of HLSC-EVs, we here chose miR-145. In fact, miR-
145 is an antitumor miRNA, known to be downregulated in renal cancer [24]. We have
recently shown that transfection of rCSCs with miR-145 results in apoptosis induction and
tumor cell invasion reduction [14]. Moreover, the transfer of anti-tumor miRNAs to rCSCs,
mediated by HLSC-EVs, was able to induce in cancer cells the expression of miR-200 family
members, involved in the inhibition of the metastatic process, both in vitro and in vivo [14].
Therefore, the loading of another anti-tumor microRNA, such as miR-145, in HLSC-EVs,
resulting in the increase of miR-145 levels in HLSC-EVs, could potentiate the observed
anti-tumor effect on rCSCs.
Our protocol successfully preserved the functionality of the HLSC-EVs on the reduction
of CSC invasion and increase of apoptosis, and was able to potentiate those effects
by loading the anti-tumor miR-145. This was not specific for miR-145, as results were
confirmed with miR-429. As expected, miR-145 alone, which was effective per se, was
inactive after RNase treatment, at variance with coincubated EVs. This result excludes
the possible additive effect of contaminating free miR-145, isolated as aggregates [17], on
the effect of coincubated EVs on rCSCs. In addition, miR-145 showed a stable RNAse
insensitive binding to the EV surface, as shown by super resolution microscopy and by the
maintenance of miR transfer to target cells and of functional effects after RNAse treatment.
The observed protection of surface-bound microRNA was likely due to the presence of
RNA-binding proteins able to protect bound miRNAs. In particular, we identified the
expression on the HLSC-EV surface of ANXA2, already described to play an active role in
miRNA-loading in EVs [18]. This RNA binding protein was previously reported on human
pancreatic cancer EV surface [19]. The involvement of surface RNA-binding proteins in
miRNA loading and protection was confirmed by the loss of their activity after trypsin
treatment.
At present, preclinical studies on oligonucleotide administration for cancer treatments
mainly utilize EVs as a delivery system, obtaining them from therapeutically irrelevant
engineered cells [25]. For instance, breast tumor cells were engineered with tumor supPharmaceutics
2021, 13, 76 12 of 14
pressor miR-134 and let-7 and the deriving EVs were shown to display potent anti-tumor
effects [26,27]. Similarly, 293 T cells were successfully transfected with siRNAs against
c-Met, or with the anti-miR214 oligonucleotide to obtain anti-tumor EVs [28,29]. The advantages
of this method are the low cost and the highly efficient EV loading. Furthermore, EVs
have been directly electroporated with desired miRNAs. For instance, HepG2 cell-derived
EVs were electroporated with miR-26 [30] and plasma-derived EVs with miR-31 and miR-
451a [10] to gain anti-tumor activity. Similarly, fibroblast-derived EVs were engineered
with siRNA or shRNA, specific to the oncogenic KrasG12D, gaining strong anti-tumor
effect in models of pancreatic cancer [31]. Indeed, electroporated MSC-EVs with KrasG12D
siRNA are currently being tested in a clinical trial (NCT03608631) on metastatic pancreatic
ductal adenocarcinoma patients harboring a KrasG12D mutation. However, in all studies,
the effect of naïve EVs was not required. This also applies in studies using electroporated
MSC-EVs, in which the effect of naïve MSC-EVs was negligible [32,33]. Considering the
multitude of active cargoes within EVs, it is likely that both cell transfection and direct
electroporation may alter their functionality.
The protocol we propose here has the advantage of maintaining the endogenous
property of EVs while adding the desired effect of the oligonucleotide therapeutic. This
could be of application not only in cancer therapy, but also in regenerative medicine,
considering the potent healing effect of EVs from MSCs and other stem cell types. In
previous experiments, we failed to increase the repairing effect of MSC-EVs using miRNA
loading obtained by MSC transfection, being the advantage only related to a lowering of
the effective dose [34]. At the same time, it is at present unknown whether surface miRNA
binding might be stable during the EV circulation in vivo. Another possible disadvantage
of our protocol is the lower amount of miRNA linked to the EV surface with respect to that
loaded by electroporation [10]. However, it has been recently shown that coincubation of
ineffective serum EVs with miRNA was sufficient to promote angiogenesis in vitro and
in vivo, suggesting that this method of EV engineering could be applied for autologous
therapy (Tapparo M. et al., manuscript under revision).
In conclusion, here, we report a protocol of miRNA loading to engineer EVs with
therapeutically active miRNAs without perturbing their cargo and innate characteristics.
This protocol could be of interest for direct engineering of stem cell-EVs and exploits the
presence of RNA binding proteins on EV surface. Further studies will be required to assess
the miRNA stability and delivery by miRNA co-incubated EVs in in vivo settings.
Supplementary Materials: The following are available online at https://www.mdpi.com/1999-4
923/13/1/76/s1, Figure S1: Comparison of electroporation and coincubation EV protocol on rCSC
apoptosis and miR transfer. A: Percentage of apoptotic rCSCs treated with naïve HLSC-EVs (EV),
or with HLSC-EVs either electroporated (EV-elettr) or coincubated (EV-coinc) with a scrambled
sequence. Results are mean   SD of three independent experiments. A-Nova was performed:
= p < 0.05 vs. untreated rCSCs (CTL). B: Real time analysis of miR-200a and miR-200b levels in
rCSCs treated for 24 h with naïve HLSC-EVs (EV), or with HLSC-EVs either electroporated (EV-elettr),
or coincubated (EV-coinc) with a scrambled sequence. Data are expressed as Relative Quantification
(RQ) normalized to untreated cells (CTL) and to RNU6B.
Author Contributions: Conceptualization, A.B., G.C. and B.B.; experimental procedures A.B., M.T.,
V.F., E.P. and M.D.; data analysis A.B.; writing—original draft preparation, A.B. and B.B.; writing—
review and editing, A.B., G.C. and B.B. All authors have read and agreed to the published version of
the manuscript.
Funding: This study was supported by the Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.),
project IG2015 16973 and by grant no. 071215 from Unicyte to G.C. and B.B.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data is available in the manuscript or the supplementary materials.
Acknowledgments: The authors thank Unicyte AG for providing the HLSCs.
Pharmaceutics 2021, 13, 76 13 of 14
Conflicts of Interest: The authors declare no conflict of interest.
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