Uptake profiles of human serum exosomes by murine and human

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Uptake profiles of human serum exosomes by murine and human tumor cells through combined use of colloidal nanoplasmonics and flow cytofluorimetric analysis Sara Busatto, Arianna Giacomini, Costanza Montis, Roberto Ronca, and Paolo BERGESE Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04374 • Publication Date (Web): 05 Jun 2018 Downloaded from http://pubs.acs.org on June 5, 2018

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Analytical Chemistry

Uptake profiles of human serum exosomes by murine and human tumor cells through combined use of colloidal nanoplasmonics and flow cytofluorimetric analysis Sara Busatto,1,3 Arianna Giacomini,1 Costanza Montis,2,4 Roberto Ronca,1 Paolo Bergese1,3,4* 1. Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Brescia, Italy 2. Department of Chemistry “Ugo Schiff” e CSGI, University of Florence, via della Lastruccia 3, 50019, Florence, Italy. 3. INSTM, National Interuniversity Consortium of Materials Science and Technology, Via Giusti, 9 50121 Florence, Italy. 4. CSGI, Research Center for Colloids and Nanoscience, Via della Lastruccia 3, 50019, Sesto Fiorentino, Florence, Italy * Corresponding author: Paolo Bergese, Associate Professor of Chemistry and Nanotechnology. Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123, Brescia, Italy. Tel. +39 030 3717555-7543, e mail [email protected], ORCID Paolo Bergese: 0000-0002-4652-216

KEYWORDS Uptake, exosomes, interspecies, nanoplasmonics, flow cytofluorimetric analysis. ABSTRACT: Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capital importance for both EV basic biology and clinical translation but still presents analytical hurdles, such as undetermined purity grade and/or concentration of the EV samples and lack of standard protocols. We report an accessible, robust and versatile method for resolving dose-dependent uptake profiles of exosomes − the nanosized (30-150 nm) subtypes of EVs of intracellular origin which are more intensively investigated for diagnostic and therapeutic applications − by cultured cells. The method is based on incubating recipient cells with consistently increasing doses of exosomes which are graded for purity and titrated by a COlorimetric NANoplasmonic (CONAN) assay followed by cell flow cytofluorimetric analysis. The proposed method allowed to evaluate and compare the uptake of human serum exosomes by cancer cell lines of murine (TRAMP-C2) and human (LNCaP, DU145, MDA-MB-231, A375) origin, setting a firmer footing for better characterization and understanding of exosome biology in different in vitro and (potentially) in vivo models of cancer growth.

as the next generation tools for precision and nano-medicine

INTRODUCTION

(e.g. as multiplexed biomarkers, therapeutic targets, smart drug Extracellular micro- and nano-vesicles (EVs) are secreted by

nanocarriers) 9-11.

cells to mediate long distance communication between cells and

Realization of EV diagnostic and therapeutic potential requires

organs, supporting both physiological and pathological pro-

robust and reliable procedures which allow to obtain EV formu-

1-3

. They include microvesicles (or ectosomes), budded

lations with narrow physicochemical properties – including

from the plasma membrane with a size ranging from 100 to

high purity separation from biological fluids and tissues and

cesses

4,5

1000 nm, and exosomes, of intracellular origin , with a size

mesoscale characterization – and in turn study of EV biogenesis

6

ranging from 30 to 150 nm . EVs transport and protect molec-

and function. In particular, the search for analytical strategies

ular information such as proteins and regulatory RNAs and, ac-

for accurate quantification of EV-cell docking and uptake is a

cording to a growing number of studies, have innate targeting

field of lively multidisciplinary endeavor 12,13.

capabilities7,8. These features are garnering an increasing inter-

EVs share physicochemical characteristics, including size,

est from academia and biotech companies, which envision EVs

buoyant density and surface charge, with other nanoscale

1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 10

objects present in biological matrixes, including high-density

medium (DMEM) supplemented with 10% fetal bovine serum

lipoproteins (HDLs), low-density lipoproteins, fat globules and

(FBS) (Lonza), 1% penicillin/streptomycin (Lonza), 1% gluta-

14

protein aggregates . This poses severe challenges in EV sepa-

mine (Lonza); DU145 (ATCC HTB-81, prostate cancer derived

ration and quantification, often leading to remarkable constitu-

from brain metastasis) and LNCaPs (ATCC CRL-1740; pros-

tive differences between EV formulations obtained from differ-

tate cancer derived from supraclavicular lymph node metasta-

15

ent biological matrixes or using different isolation methods .

sis) were grown in RPMI 1640 supplemented as DMEM, at

The presence of exogenous contaminants changes the overall

37°C, 5% CO2. Murine cell line TRAMP-C2 (ATCC CRL-

physicochemical properties of the formulation (posing handling

2731, prostate cancer) were grown in supplemented DMEM

and processing issues) and may affect its final biological activ-

added with 1% 1M HEPES solution, 1% 2-mercaptoethanol so-

ity. Contaminants can exert their biological function in concom-

lution 50mM, 0.05% Bovine Insulin and 0.005% of Testos-

itance with EVs, thus emphasizing or hiding EV function and

terone.

causing artifacts and misleading results

12-16

.

Cells were initially maintained at 37°C at 5% CO2 in their re-

This work proposes a robust method to accurately determine

spective proper medium supplemented with 10% FBS (Thermo

dose-dependent uptake profiles of nanosized EVs by cells. The

Fisher Scientific). During conditioned media production and

method is based on the integration of a COlorimetric NANo-

exosome incubation protocol, cells were cultured in media

17

plasmonic (CONAN) assay , which ensures consistent relative

without FBS. Viability of cells was tested by the Trypan blue

dosing of EVs, and flow cytofluorimetric analysis. In particular,

assay (Luna II automated cell counter, Logos).

we explore the uptake of nanosized EVs with a diameter ranging from 30 to 150 nm enriched in intraluminal vesicle proteins

Purity and concentration of the EV formulations (CONAN

(which are traditionally referred as exosomes, see Results and

assay)

Discussion for further nomenclature details) separated from se-

Exosome preparations were checked for purity and titrated by

rum of healthy individuals, by different preclinical in vitro cel-

the COlorimetric NANoplasmonic (CONAN) assay, which ex-

lular models: a murine prostate cancer cell line (TRAMP-C2,

ploits the nanoplasmonic properties of colloidal gold nanopar-

derived from the Transgenic Adenocarcinoma of the Mouse

ticles (AuNPs) and their peculiar interaction with proteins and

Prostate model) and four human tumor cell lines representative

lipid bilayers (see Results and Discussion Section and Ref 17).

of triple negative breast cancer (MDA-MB 231), melanoma (A375) and androgen-dependent (LNCaP) or independent (DU145) prostate cancer. These experimental models have been designed and selected also in view of their inherent biomedical and translational interest. In fact, they allowed to systematically explore for the first time if “wild-type” human serum exosomes are internalized by both mouse and human cells and to draft the specific uptake profiles for each cell line. This is a crucial step to face in vivo targeting/biodistribution approaches based on EVs in the field of cancer therapy and imaging18.

Purity assessment. The CONAN assay used in this work consisted in a 6 nM MilliQ water solution of 14 nm diameter AuNPs. AuNPs were synthesized by the Turkevich’s method19. The experiments were conducted, and data analyzed adapting the protocols described in20,21. All the UV/vis/NIR absorption spectra were collected with an Ensight multimode plate reader (Perkin Elmer), which allowed to collect the spectra on samples of 100 µL final volume. Titration. In the case of pure exosome preparations, the AI of the CONAN assay is proportional to the exosome concentra-

EXPERIMENTAL SECTION

tion, which allows researchers to exploit the assay to determine molarity titration. Full details are given here 17-21. For this work,

Cell lines

we built a calibration line by using PBS solutions of 70 nm hy-

Human cell lines A375 (ATCC CRL-1619, malignant melanoma) and MDA-MB-231 (ATCC HTB-26, triple negative breast cancer) were grown in Dulbecco’s modified eagle’s

drodynamic diameter POPC liposomes at known molar concentrations, ranging from 0.8 nM to 12.5 nM22. POPC liposome synthesis and characterization and typical UV/vis/NIR

ACS Paragon Plus Environment

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absorption spectra of POPC liposome calibration solutions are

scaled with respect to the number of adherent cells to match the

reported as SI. AI data points were calculated from the

EV/cell ratio of the uptake experiment at 2.5·1012 EVs, see sec-

UV/vis/NIR absorption spectra of the CONAN assays mixed

tion “Uptake experiments”). TRAMP-C2 cells were then

with the POPC liposome dispersions. All the exosome formula-

washed twice with PBS buffer and added with fresh complete

tions are diluted in sterile PBS and mixed with a 6 nM AuNP

medium. Finally, cells were examined under a Zeiss Fluores-

water solution with a 1:1 ratio.

cence Axiovert 200M microscope (Carl Zeiss, Milan, Italy, EU).

EV track We have submitted all relevant data of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV180010)23 Incubation with cells and flow cytofluorimetric analysis Bodipy-labeled exosome pellets were dissolved and pooled into 500 μL of sterile PBS buffer. Growing doses of labeled exosomes purified by sucrose gradient fractionation (SGF) from serum of healthy individuals were incubated with the same number (2·106) of seeded cells of human and mouse cell lines, namely: MDA-MB231, A375, DU 145, LNCaP and TRAMPC2. All the uptake experiments were performed with the same

RESULTS AND DISCUSSION Separation and characterization Exosomes were isolated by differential centrifugation (DC) followed by SGF or size exclusion chromatography (SEC) with qEV columns from iZON science (qEV), see SI and ref

24

for

further details. Finally, EV formulations purified by SGF were checked for purity by the CONAN method. The assay consists of an aqueous solution of bare AuNPs at 6 nM concentration. When mixed with pure EV-formulations, the AuNPs cluster on the EV membrane. Whereas, in EV formulations which contain exogenous protein contaminants (EPCs) the AuNPs are prefer-

pool of fluorescently labeled exosomes. Incubation was performed at 37°C with 5.5% of CO2 for 3 hours. Elapsed the incubation time cells were washed twice with PBS and detached with Trypsin. Cells were then pelleted at 1000 rpm for 10 minutes. The pellet was resuspended in 1 ml of PBS added with 0.2% of FBS. Cell samples were counted

entially cloaked by such EPCs (an AuNP-EPC corona forms), which prevents AuNPs from clustering to the EV membrane. When AuNPs cluster (are in tight proximity) their localized surface plasmon resonance (LSPR) red shifts and broadens, resulting in a color change of the AuNP solution from red to blue,

with Luna II automated cell counter (Logos) and a total of 2·105

which can be accurately monitored through UV-vis spectros-

cells in 300 μL of buffer volume were collected and analyzed

copy. The assay red shift is therefore directly related to the pu-

by flow cytofluorimetric analysis. The percentage of fluorescent cells after exosomes uptake and the relative fluorescent intensity were measured using the MACSQuant® Analyzer (Miltenyi Biotec, Bergisch-Gladbach, Germany, EU) and data analyzed using the FlowJo vX.0.7 software (Tree Star, Inc., Ash-

rity grade of the added EV formulation and can be conveniently quantified by describing the AuNP UV/Vis/NIR absorption spectra with the nanoparticle aggregation index (AI), defined as the ratio between the absorbance intensity at the LSPR peak and the intensity at 650 nm

25,26

. For all the analyzed formulations

isolated as described in the SI, the AI values resulted lower than

land, OR, USA).

20% of the reference AI of the initial assay (i.e. the dispersed Fluorescent microscopy

AuNP solution). This proves the EV formulations contained 5

A total amount of 1.5·10 TRAMP-C2 adherent cells were

negligible amounts of EPCs 17-19,21 (EV sample UV/vis/NIR ad-

stained with Cell Tracker Red CMPTX. CMPTX was firstly re-

sorption spectra and related AI are given as SI).

suspended in DMSO (10 mM), then diluted 10 µM in warmed

The EV formulations isolated through SGF (fractions 6 to 9)

SF cell culture medium and incubated for 30 minutes with

were then inspected by AFM 27 (protocol details given as SI). A

TRAMP-C2 cells.

sample topography image sprinkled with EVs with size ranging

After the cytoplasm staining TRAMP-C2 cells were incubated

from tens to few hundreds of nm is shown in Fig. 1a. The black

at 37°C with 5.5% of CO2 for 3 hours with 1.9·1011 Bodipy FL

background indicates the absence of any relevant nanoscale fea-

C5-HPC (Bodipy) labeled EVs (note: the number of EVs was

ture other than the EVs (with reference to the color code bar



placed on the image side), confirming negligible presence of

made of nanosized EVs ranging from 30 to 150 nm which are

EPCs. The analysis of several AFM topography images allowed

enriched in ILV markers. Such EV populations are tradition-

estimating the EV size distribution reported in the bar chart of

ally referred to as exosomes 34, 35,36 and in the following we

Fig. 1b. The size distribution is Poisson-like, indicating the pop-

also will stick to this nomenclature.

Page 4 of 10

ulation is prevalently composed by EVs with size included between 30 and 150 nm, with a mean value of about 60 nm. Remarkably, both size distribution and its mean value match those typical of exosomal populations 2,3,6. Purity of the EV formulation was also corroborated by running the EV preparations versus bovine serum albumin (BSA) on agarose gel electrophoretic assays in native conditions for the separation of mixed colloidal solutions. The related agarose gel is shown in the panel of Fig. 1c, where the EV formulation run versus the BSA one is imaged by direct Coomassie staining. Coomassie is detectable only in the BSA lane whereas in the EV lane is not detectable, confirming in agreement with the CONAN assay and AFM imaging that the EV formulation contains negligible amount of EPCs. Finally, EV presence and purity was biochemically proved by Western Blot analysis (WB) of typical EV proteins of intraluminal vesicle (ILV) origin, Golgi matrix proteins and lipoproteins. In particular, we checked the presence of two tetraspanins CD63 and CD81; of one intracytoplasmatic protein (involved in the regulation of the endosomal trafficking) Alix 28 and of a membrane associated protein Annexin XI (belonging to Annexin protein family, known to play pivotal role into exocytosis and endocytosis processes)29-31. GM130, a cis-Golgi matrix protein known to be absent in any exosome population32, was analyzed to exclude the presence of other intracytoplasmic nanosized components in the isolated formulation30. Finally, APO-A1 antibody was used to prove the absence of HDLs6.

Figure 1. EV characterization. (a) AFM topography image of the

As a WB positive control, we analyzed the pellet (P3) ob-

EV preparation adsorbed onto mica, scale bar 1µm. (b) Size dis-

tained after the DC protocol of a serum sample, containing a

tribution obtained from analysis of AFM images such as in (a). (c)

heterogeneous population of EVs together with EPCs and

Agarose gel electrophoresis of the EV preparation and of control

HDLs.

BSA solution stained with Coomassie. (d) Western Blot analysis

The Western Blot results were acquired as reported in

33

and

of P3 sample and of exosomes isolated by SGF (SGFexo).

are summarized in Fig.1d, which indicates that the EV sample purified by SGF protocol carries EV markers, while does not carry the ones of intracytoplasmic nano-residues and of HDLs. These data were counterchecked and confirmed by replicates separated with SEC (experimental given as SI). Overall, the above results indicate that the final EV formulation is pure and

Titration (dosing) Titration of the total molar concentration by the CONAN assay grounds on the fact that in a pure solution of vesicles, either liposomes or exosomes, the LSPR red shift, viz. the AI, of the AuNPs has a linear dependence on the AuNP/vesicle molar ratio17. The calibration line was built by using POPC liposomes


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of about 70 nm mean diameter (according to DLS data and analysis, see SI), which fairly mimic both the size distribution and lipid composition of the analyte exosomes − in other words, since AuNP aggregation at membranes is driven by non-specific electrostatic forces, the NPs “see” no difference between POPC liposomes and exosomes37 − The UV/vis/NIR spectra of POPC liposome aliquots of increasing molar concentration (from 0.8 nM to 12.5 nM) were collected in triplicate and the related AIs determined (see SI Figure S2). The calibration line reported in Fig. 2 was then built by plotting all the obtained AIs versus the POPC liposome concentrations (filled black circles) and fitted by linear regression (black line). A least-squares minimization procedure based on the Levenberg-Marquardt algorithm was implemented, which resulted in a fitting line of slope equal to (0.61 ± 0.05) nM−1 and

Figure 2. Calibration line. The AIs obtained from each single POPC spectra are plotted versus the POPC liposome concentrations (filled black circles) and fitted by a linear regression line (black

ordinate axis intercept equal to (1.7 ± 0.4) a.u. (fitting salient

line); the empty red squares represent the exosome formulation ex-

parameters: R = 0.893, p < 0.0001).

trapolated data points.

The empty red squares represent the extrapolated concentration of one exosome aliquot purified from 4 ml of healthy individuals pooled serum sample. Interpolating the AI value of unknown sample allowed the determination of the exosome formulation relative concentration to be 4.7 nM and of the related experimental error, evaluated through the error propagation theory, to be ± 0.6 nM (about 10 %). The concentration correspondent to the AI value is referred to the reaction volume tested. The final concentration of the exosome solution, with a volume 150 μL, was 58.8 nM, corresponding to 5.3⋅1012 exosomes in the overall volume (8.9⋅10-12 moles). Serial isolation protocols have been performed until a total exo13

some number of 9⋅10 was reached and then pooled. Careful titration of the exosome content in the formulations represents a fundamental step for the determination of dose-dependent exosome cell uptake. It is worth to note that the determined concentrations rely on the liposome calibration line. Indeed, the use of these much simpler exosome synthetic mimics may hide systematic errors and caution should be used in considering the determined values in absolute terms. Nevertheless, the calibration line straight linearity ensures high relative (internal) accurateness, which turn into reliable and dose-dependent experiments.

Uptake experiments

The interplays between incoming exosomes and recipient cells entails both membrane-to-membrane interactions and whole exosome endocytosis (phagocytosis, pinocytosis, membrane fusion), also determining the incorporation by the recipient cell of any fluorescent molecule that labels the exosomes 38. This can be readily exploited to evaluate the extent of exosome uptake by cells through flow cytofluorimetric analysis

7,39

. Starting

from the exosome stock preparations previously isolated and characterized, four sets of exosome doses with increasing concentration were prepared. Increasing concentrations of exosome sets (from 5⋅1011 to 5⋅1012) were added to 2⋅106 tumor cells (MDA-MB-231, A375, DU145, LNCaP, TRAMP-C2) plated in a cell culture dish. After 3 hours of incubation at 37°C cells were washed with PBS buffer, gently detached, and analyzed by flow cytofluorimetric analysis. The extent of the exosome-cell uptake was evaluated by calculating the percentage of fluorescent cells and their fluorescent signal intensity, assuming that the acquisition and the increase of any fluorescent signal by cells entails the uptake of growing doses of fluorescently labeled exosomes. For each analysis, a suspension of not-fluorescent wild type cells was run in parallel and analyzed as negative control. For each point, two replicates using the same exosome stock solution were performed.



Page 6 of 10

Flow cytofluorimetric analysis shows that each cell line is

exosome uptake between the five cell lines is marked at the

characterized by a specific dose-dependent correlation between

2.5·1012 exosome dose whereas it flattens at the highest dose.

increasing doses of Bodipy-labeled exosomes and the

This suggests that at the highest exosome concentration the

percentage of resulting fluorescent cells (Fig. 3a) as well as their

uptake is mainly determinated by unspecific mechanisms due to

fluorescence intensity (Fig. 3b and Supplementary Fig. S3). The

the saturating conditions of the in vitro exosome-cell system.

possibility of artifacts due to unspecific direct uptake of the dye

For instance specific uptake mechanisms should be studied at

by the cells and dye background was controlled and excluded in

lower, not saturating doses.

dedicated control experiments, which will be discussed later. Overall, the data indicate that serum exosomes from healthy individuals are internalized by both mouse and human cells with specific dose-depended profiles. Differences may have several origins, depending by both cell and exosome phenotypes, that finally determine the uptake dominant mechanisms. Indeed, resolving the uptake profiles is a powerful analytical tool to get a first empirical insight in this complex process and set the optimal conditions for further investigations. The difference in

Figure 3. (a) Dose-dependent uptake profiles of human serum exosomes by tumor derived murine and human cells (% of fluorescent cell represents the % of cells which have taken up a detectable number of labelled exosomes). (b) Dose-dependent fluorescent intensity signal of TRAMP-C2 cells. For each cell line two replicate using the same exosome preparation were performed.


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Further discussion on exosome uptake, dye background and

As shown in Fig. 4, the red cytoplasmic dye (CMTPX) effi-

unspecific signals

ciently stained all cells, whereas a lower number of cells present

Despite the standardization protocol and the accurate titration

the green fluorescent signal upon incubation with green (Bod-

and dosing of exosomes, we can’t exclude passive/unspecific

ipy) labeled exosomes. This result confirms flow cytofluorimet-

diffusion of the fluorescent lipophilic Bodipy dye between ex-

ric data, where about 90% of cells incubated with 2,5·1012 exo-

osomes and cell membranes during exosome-cell contacts that

somes displayed a green fluorescent signal (Figure 3a). Overall,

40

eventually did not evolve into exosome internalization . In or-

these control experiments ensure that background noise due to

der to evaluate the relevance of this phenomenon, the following

unspecific passive probe diffusion is negligible with respect to

experiment was performed. The same amount of Bodipy used

the specific signal due to a selective exosome uptake.

to label the whole exosome stock solution employed in cell uptake experiments was processed following the same protocol but without exosomes. The final pellet was assumed to represent in first excess approximation the dye background signal, that is the dye molecules that could escape from intercalating the EV membrane and coprecipitate with the labeled EVs. The pellet was then resuspended into 500 µl of sterile PBS and divided into four different aliquots, each corresponding to an exosome dose featured in the uptake of previous experiments (3.5 µl, 7 µl, 17.5 µl and 35 µl). After 3 hours of incubation, cells were analyzed by flow cytofluorimetry and for each analysis a suspension of not-fluorescent wild type cells was run in parallel and analyzed as negative control. Two replicates of all the analysis were performed. Results (given as SI, Fig. S8) show overlapping slightly dosedependent Bodipy background for the TRAMP-C2, A 375 and DU145 cell lines, which are significantly lower and unrelated

Figure 4. Fluorescence microscopy imaging of the exosome up-

to the exosomes signals. Instead, the exosome signal is low and

take process performed on adherent TRAMP-C2 cell line.

comparable to the dye background at low doses and at all doses

CMTPX column represents TRAMP-C2 cytoplasmic staining,

for the MDA-MB-231 and LnCap cell lines, respectively. This

Bodipy column represents intracellular Bodipy-labeled exosome

can be due to many different reasons, also unrelated to exosome

signal, Merge column represents a merge of fluorescent red and

uptake, including different affinity of the dye for different cell

green signals. Objective lens: 63x oil; Scale bar: 30 μm.

line membranes.41,42 What matters here is how the control ex-

CONCLUSIONS

periment resulted pivotal to frame and understand the experimental results and their reliability. They suggest that finer direct

The present study shows the possibility to determine dose-de-

and control experiments are needed to analyze the exosome up-

pendent uptake profiles of human serum exosomes by murine

take of MDA-MB-231 and LnCap cell lines.

and human tumor cells through combined use of a titration as-

Exosome cell uptake was finally counterchecked by optical mi-

say based on colloidal nanoplasmonics (CONAN) and flow cy-

croscopy. Fig. 4 shows the fluorescence microscopy images ac-

tofluorimetric analysis.

quired after three-hour incubation performed using green (Bod-

Titration by CONAN allowed to prepare an exosome stock so-

ipy) labeled exosomes and red cytoplasmic labeled TRAMP-C2

lution (volume 500 µl) with a concentration of 300 nM (9·1013

cells.

total exosomes), subsequently aliquoted in increasing doses and administrated to murine (TRAMP-C2) and human (LNCaP, DU145, MDA MB 231, A375) tumor cell lines. Prior to



incubation, exosomes were labeled with Bodipy, a green fluorescent lipophilic probe, and the amount of recipient cells were determined by flow cytofluorimetric analysis. Interestingly, different cell lines displayed different uptake pro-

ORCID identification number (s) for the authors of this article: Sara Busatto https://orcid.org/0000-0002-9128-5579 Arianna Giacomini https://orcid.org/0000-0002-9984-9033 Costanza Montis https://orcid.org/0000-0001-6960-3772 Roberto Ronca https://orcid.org/0000-0001-8979-7068 Paolo Bergese https://orcid.org/0000-0002-4652-2168

files. In particular, at an intermediate not-saturating dose of ex-

Notes

osomes (2.5·1012) TRAMP-C2, A 375, DU145 and MDA-MB-

The authors declare no competing financial interest.

231 showed a specific signature of uptake ratio (expressed as

ACKNOWLEDGMENT

percentage of fluorescent positive cells), whereas at the highest exosome dose, when saturating conditions arise, internalization occurred for a percentage of cells greater than 50%. Experiments resulted not sensitive/significant only for LnCap cells, indicating they need tuning of the protocols, such as exploration of different dose ranges and/or use of other label dyes.

Page 8 of 10

The authors gratefully acknowledge Marco Presta, who substantially contributed to lay down and start this study; Lucia Paolini for help in performing size exclusion chromatography isolation and subsequent WB of exosomes and Stefania Mitola and Debora Berti for fruitful discussions on the manuscript. This work was supported by Fondo ex 60% Un. of Brescia P.B., MFAG 18459 grant from Associazione Italiana per la Ricerca sul Cancro to R.R. and Fondazione Cariplo grant n° 2016-0570 to A.G.

In conclusion, “one dose does not fit all”. Determination of cell line specific dose-dependent uptake profiles (i.e. which is the

REFERENCES

best working exosome/cell ratio) is a diriment starting point to significantly undertake any more sophisticated targeting/delivery experiments. In contrast, using a fixed EV dose for all cell lines 43,44 may entail misleading results. “Exosome dose working range” becomes a requirement for the correct setup and interpretation of any in vitro and in vivo model of translational relevance. Indeed, even though it cannot still be concluded that human serum exosomes display a different tropism for murine or human cancer cells, the method allowed to show that human exosomes can be directly used in inter-species studies, opening the possibility for their direct use in pre-clinical murine models. Overall, the proposed method results accessible, robust and versatile, contributing to implement the EV analytical toolbox necessary for exosomes fundamental and translational studies.

ASSOCIATED CONTENT Supporting Information The Supporting Information reporting detailed materials and methods and additional data is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *Paolo Bergese, [email protected]

Author Contributions All authors contributed to the conception and realization of the experiments and the analysis and discussion of results. SB and PB wrote the first draft of the manuscript. All authors contributed to writing and gave approval to the final manuscript.

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