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An i umo e ec o oncoly ic i us and pacli axel encapsula ed in ex acellula esicles
o lung cance ea men
© 2018 The Au ho s. Published by Else ie B.V.
Published e sion
Ga o alo, M.; Saa i, H.; Some salo, P.; C escen i, D.; Ku yk, L.; Aksela, L.; Capasso,
C.; Made oja, M.; Koskinen, Ka a iina; Oksanen, T.; Mäki ie, A.; Jalas uo i, Ma i;
Ce ullo, V.; Ciana, P.; Ylipe ula, M.
Ga o alo, M., Saa i, H., Some salo, P., C escen i, D., Ku yk, L., Aksela, L., Capasso, C., Made oja,
M., Koskinen, K., Oksanen, T., Mäki ie, A., Jalas uo i, M., Ce ullo, V., Ciana, P., & Ylipe ula, M.
(2018). An i umo e ec o oncoly ic i us and pacli axel encapsula ed in ex acellula esicles
o lung cance ea men . Jou nal o Con olled Release, 283, 223-234.
h ps://doi.o g/10.1016/j.jcon el.2018.05.015
2018
Con en s lis s a ailable a ScienceDi ec
Jou nal o Con olled Release
jou nal homepage: www.else ie .com/loca e/jcon el
An i umo effec o oncoly ic i us and pacli axel encapsula ed in
ex acellula esicles o lung cance ea men
M. Ga o alo
a,b,⁎
, H. Saa i
a,1
, P. Some salo
a,b,1
, D. C escen i
b
, L. Ku yk
a,c,d
, L. Aksela
a
, C. Capasso
e
,
M. Made oja
, K. Koskinen
g
, T. Oksanen
a
, A. Mäki ie
h
, M. Jalas uo i
a,g
, V. Ce ullo
e
, P. Ciana
b
,
M. Ylipe ula
a,⁎
a
Di ision o Pha maceu ical Biosciences and Cen e o D ug Resea ch, Uni e si y o Helsinki, Viikinkaa i 5, Helsinki 00790, Finland
b
Depa men o Oncology and Hema o-Oncology, Cen e o Excellence on Neu odegene a i e Diseases, Uni e si y o Milan, Via Balza e i 9, Milan 20133, I aly
c
Na ional Ins i u e o Public Heal h –Na ional Ins i u e o Hygiene, Depa men o Vi ology, 24 Chocimska s , 00-791 Wa saw, Poland
d
Ta go ax Oy, R&D, Clinical Science, R&D, Saukonpaaden an a 2, 00180 Helsinki, Finland
e
Labo a o y o ImmunoVi oThe apy, D ug Resea ch P og am, Facul y o Pha macy, Uni e si y o Helsinki, Viikinkaa i 5, Helsinki 00790, Finland
Made Consul ing, Tykis öka u 4 B, FI-20520 Tu ku, Finland
g
Biological and En i onmen al Science, Nanoscience Cen e , Uni e si y o Jy äskylä, Su on ie 9C, 40500, Finland
h
Depa men o O o hinola yngology –Head and Neck Su ge y, Helsinki Uni e si y Hospi al and Uni e si y o Helsinki, P.O.Box 263, FI_00029 HUS, Helsinki, Finland
ARTICLE INFO
Keywo ds:
Ex acellula esicles
Oncoly ic i uses
Cance he apy
D ug deli e y
Pacli axel
Xenog a animal model
Lung cance
ABSTRACT
S anda d o ca e o cance is commonly a combina ion o su ge y wi h adio he apy o chemo adio he apy.
Howe e , in some ad anced cance pa ien s his app oach migh s ill emaininefficien and may cause many side
effec s, including se e e complica ions and e en dea h. Oncoly ic i uses exhibi diffe en an i-cance me-
chanisms compa ed wi h con en ional he apies, allowing he possibili y o imp o ed effec in cance he apy.
Chemo he apeu ics combined wi h oncoly ic i uses exhibi s onge cy o oxic esponses and oncolysis. He e, we
ha e in es iga ed he sys emic deli e y o he oncoly ic adeno i us and pacli axel encapsula ed in ex acellula
esicles (EV) o mula ion ha , in i o, significan ly inc eased he ansduc ion a io and he in ec ious i e
when compa ed wi h he i us and pacli axel alone. We demons a ed ha he ob ained EV o mula ion educed
he in i o umo g ow h in animal xenog a model o human lung cance . Indeed, we ound ha combined
ea men o oncoly ic adeno i us and pacli axel encapsula ed in EV has enhanced an icance effec s bo h in i o
and in i o in lung cance models. T ansc ip omic compa ison ca ied ou on he explan ed xenog a s om he
diffe en ea men g oups e ealed ha only 5.3% o he diffe en ially exp essed genes we e o e lapping in-
dica ing ha a de no o gene ic p og am is igge ed by he p esence o he encapsula ed pacli axel: his no el
gene ic p og am migh be esponsible o he obse ed enhanced an i umo effec . Ou wo k p o ides a p omising
app oach combining an icance d ugs and i al he apies by in a enous EV deli e y as a s a egy o he lung
cance ea men .
1. In oduc ion
Despi e majo ad ances in con en ional cance ea men s wi h
su ge y, adio he apy, chemo he apy,and hei combina ion, he ou -
come is s ill pa ially ineffec i e agains nume ous cance ypes, like
lung cance [1]. Lung cance is highly in asi e and apidly me as a-
sizing, o en diagnosed a an ad anced s age wi h poo p ognosis and
wi hou efficien ea men op ions [2]. Gi en he poo su i al a e o
pa ien s, new he apeu ic s a egies wi h sys emic d ug deli e y a e
wa an ed. Oncoly ic i o he apy is eme ging as a p omising and
po en ial app oach o ea cance , and he app o al o he fi s onco-
ly ic i us, Imlygic (T-Vec, alimogene lahe pa ep ec), in he Wes e n
wo ld by US Food and D ug Adminis a ion (FDA) and Eu opean
Medicines Agency (EMA) p o ides new pe spec i es o imp o ed
ea men o cance [3,4]. Indeed, i s applica ion can be pa icula ly
ele an o umo s wi hou cu a i e op ions, including me as a ic lung
cance s [1]. In oncoly ic i us he apy, i uses a e specifically en-
ginee ed o p e e en ially in ec , eplica e in and kill cance cells in-
s ead o no mal cells whe e hei no mal unc ions a e es ic ed [5–9].
Vi us eplica ion in umo cells e en ually leads o cell lysis, allowing
h ps://doi.o g/10.1016/j.jcon el.2018.05.015
Recei ed 21 Decembe 2017; Recei ed in e ised o m 10 May 2018; Accep ed 14 May 2018
⁎
Co esponding au ho s a : Di ision o Pha maceu ical Biosciences and Cen e o D ug Resea ch, Uni e si y o Helsinki, Viikinkaa i 5, Helsinki 00790, Finland.
1
Sha ed co-au ho ship.
E-mail add esses: ma iangela.ga o alo@helsinki.fi(M. Ga o alo), ma jo.ylipe ula@helsinki.fi(M. Ylipe ula).
Jou nal o Con olled Release 283 (2018) 223–234
A ailable online 01 June 2018
0168-3659/ © 2018 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/BY/4.0/).
T
he new i us p ogeny o sp ead o su ounding cells and e en o dis an
me as ases h ough ci cula ion [10]. Howe e , as a single he apeu ic
agen oncoly ic adeno i uses ha e no been obse ed o efficien ly
des oy la ge umo mass in pa ien s [11,12]. Thus he e is a need o
enhance hei an i umo efficacy by combining i al he apy wi h o he
an icance agen s [13–15]. Cispla in has imp o ed oncolysis o He pes
simplex i us ype 1 (HSV-1) in non small cell lung cance (NSCLC)
[16]. Addi ionally, combina ion o cispla in wi h adeno i us acili a ed
he eplica ion o he i us and significan ly educed he umo p o-
g ession [17]. Enhanced effec s ha e been also epo ed in a malignan
pleu al meso helioma (MPM) wi h NV1066 (HSV-1 based i us) [18].
Howe e , he use o oncoly ic i uses as a po en ial app oach o ea
cance has also disad an ages [19]; he immune esponse will p e-
sumably limi ongoing i al eplica ion and sp ead in cance cells.
Adminis e ed i uses will be de ec ed by immune sys em and in-
ac i a ed by neu alizing an ibodies, dec easing i s eplica ion and e -
ficacy. Addi ionally, gi en he in a umo al adminis a ion o oncoly ic
i uses [20–22], hey a e eligible only in injec able lesions and hus
limi ing he app oach o ea many solid umo s. Sys emic deli e y o
i us oge he wi h an icance d ugs would ci cum en some o hese
limi a ions. Ex acellula esicles (EVs) a e na u ally occu ing ca go
deli e y agen s wi h he po en ial o be used as d ug deli e y ehicles
[23,24] since hey can ans e biological molecules e en o e long
dis ances wi hin he body [25]. The lipid memb ane o EVs can p o ec
he ca go om deg ada ion by body fluids and u he imp o ed up ake
by he a ge cells [26,27]. In ecen s udies, i has been shown o ha
oncoly ic i uses can be deli e ed in o he nucleus o umo igenic cells
by umo mic opa icles while simul aneously a oiding he p oduc ion
o neu alizing an ibodies and media ing he i us en y in o cance
cells independen ly om he i us-specific ecep o [28].
In his s udy, we se o in es iga e whe he i is possible o en-
capsula e he oncoly ic adeno i us and chemo he apeu ic agen in o
EVs in an a emp o u ilize hem as ca ie s o a ge ed d ug deli e y.
The ob ained o mula ions we e es ed in i o in lung cance cell line
and subsequen ly in i o in lung cance xenog a animal model using
bo h in a umo (i ) and in a enous (i ) injec ions. Ab axane (pa-
cli axel, albumin-bound nanopa icle o mula ion) and EVs wi hou
encapsula ed i us and/o d ug we e used as con ol samples. We ound
ha he sys emic deli e y o bo h oncoly ic i us and pacli axel en-
capsula ed in EVs esul ed in imp o ed d ug efficacy and educed off-
a ge oxici y.
2. Ma e ials and me hods
2.1. Cell cul u e
A549 human lung cance cell line was pu chased om he Ame ican
Type Cul u e Collec ion (ATCC, USA). The cells we e cul u ed a 37 °C
and 5% CO
2
in Dulbecco's modified eagle medium (DMEM, Lonza,
Swi ze land) supplemen ed wi h 10% e al bo ine se um (FBS, Gibco
Labo a o ies, USA), 1% o 100 u/mL penicillin/s ep omycin (Gibco
Labo a o ies) and 1% L-glu amine (Gibco Labo a o ies).PNT2
(Eu opean Collec ion o Au hen ica ed Cell Cul u es, ECACC, UK)
human p os a e cell line was pu chased om Sigma-Ald ich. The cells
we e cul u ed a 37 °C and 5% CO
2
in RPMI 1640 (Gibco Labo a o ies)
supplemen ed wi h 10% FBS (Gibco Labo a o ies), 1% o 100 u/mL
penicillin/s ep omycin (Gibco Labo a o ies) and 2% L-glu amine
(Gibco Labo a o ies). The p os a e cance cell line PC-3 (ATCC), was
cul u ed a 37 °C and 5% CO
2
in Ham's F-12 K (Kaighn's) basal medium
(Gibco Labo a o ies) supplemen ed wi h 10% FBS (Gibco Labo a o ies)
and 1% o 100 u/mL penicillin/s ep omycin (Gibco Labo a o ies).
2.2. Oncoly ic i us
Ad5D24-CpG, was gene a ed acco ding o s anda d p o ocols [29]
by ecombining a CpG- ich shu le plasmid (pTHSN-CpG1) wi h a
plasmid con aining he 24 adeno i us backbone. Vi al s ocks we e ex-
panded in human lung cance cell line A549 and pu ified on cesium
chlo ide g adien s. The i al pa icle concen a ion was de e mined by
OD
260
- eading and s anda d TCID
50
( issue cul u e in ec ious dose 50)
assay was pe o med o de e mine in ec ious pa icle i e . Vi us was
cha ac e ized by PCR and es ic ion enzyme analysis Ad5D24-RFP,
exp essing a ed fluo escen p o ein (RFP) was kindly p o ided by D .
Masa aka Suzuki om Baylo College o Medicine (Hous on, TX [30];).
2.3. Pacli axel (PTX) solu ions
A 50 mM s ock solu ion o Pacli axel (PTX; Selleck Chemicals) was
p epa ed by dissol ing PTX in o di-me hyl sul oxide (DMSO) (Sigma-
Ald ich). This was used as he s ock solu ion in A549 cell expe imen s
as well as in EV-encapsula ion o PTX.Ab axane was p o ided o us by
he Hospi al Pha macy o he Helsinki Uni e si y Hospi al based on he
p esc ip ion o MD A. Mäki ie, School o Medicine, Uni e si y o
Helsinki, Finland. A s ock suspension o Ab axane (Albumin-PTX con-
juga e, Celgene, USA) was p epa ed by suspending he powde , co e-
sponding o 100 mg o PTX, in phospha e buffe saline (PBS, Lonza) o a
final olume o 8.5 mL, esul ing in 11.76 mg/mL o PTX. This s ock
was hen u he dilu ed in PBS o p oduce he solu ions used in he in
i o animal expe imen s exclusi ely.
2.4. P oduc ion o ex acellula esicles (EV) and PTX loaded EVs
o mula ions
In o de o p oduce EVs 2.6 × 10
6
A549 cells we e pla ed in o T-175
flask in medium supplemen ed wi h 5% FBS. The FBS g ow h media
was ul a-cen i uged o e nigh (110,000 ×ga 4 °C o 18 h, Op ima
LE-80 K ul acen i uge, o o ype 50.2, Beckman Coul e ) o emo e
EVs p esen in se um. Cells we e cul u ed a 37 °C and 5% CO
2
un il
cy opa hic effec was seen, whe e upon he media was collec ed.
EVs we e isola ed om he condi ioned medium using diffe en ial
cen i uga ion. Fi s he condi ioned medium was cen i uged a
500 ×gin 4 °C o 10 min o pelle cells (Alleg a X-15R Cen i uge,
Beckman Coul e ). Then, he supe na an was collec ed and ul a-cen-
i uged o 2 h a 100000 ×gin 4 °C, using Op ima L-80 XP ul a-
cen i uge (Beckman Coul e ) wi h o o SW32Ti (Beckman Coul e ).
The supe na an was aspi a ed and EV- con aining pelle s con aining e-
suspended in PBS (Lonza) 100 μL and s o ed a −80 °C.
PTX-loaded EVs we e p epa ed as p e iously desc ibed by us [23]
by incuba ing 1 × 10
8
–5×10
9
EVs in 1 mL o 5 μM PTX-DPBS solu ion
o in i o samples and 10 μM PTX-DPBS solu ion o in i o samples,
o 1 h a 22 °C. Nex , he samples we e cen i uged a 170000 ×g o
2 h o pelle he EVs. The supe na an con aining unbound PTX was
emo ed, and he EV-pelle was washed by suspending i in DPBS and
pelle ing i again a 170000 ×g.
2.5. P oduc ion o EV-Vi us and EV-Vi us-PTX o mula ions
In o de o p oduce EV-encapsula ed i us (EV-Vi us), 2.6 × 10
6
o
A549 cells we e in ec ed wi h wi h 10 i al pa icles/cell o
Ad5D24CpG and we e cul u ed a 37 °C and 5% CO
2
48 h la e when
mos o he cells we e de ached om he cul u e flask, he cul u e
media we e collec ed o EV-Vi us isola ion using diffe en ial cen-
i uga ion. Fi s he condi ioned medium was cen i uged a 500 ×g
and 4 °C o 10 min, o sepa a e he cells (Alleg a X-15R Cen i uge,
Beckman Coul e ). Then, he supe na an con aining EV-Vi us was
collec ed and ul a-cen i uged o 2 h a 100000 ×gand 4 °C, using
Op ima L-80 XP ul a-cen i uge (Beckman Coul e ) wi h o o SW32Ti
(Beckman Coul e ). The supe na an was aspi a ed and pelle s con-
aining EV-Vi us e-suspended in PBS 100 μL and s o ed a −80 °C.EV-
Vi us samples we e incuba ed in 100 mM NaOH a oom empe a u e
o 20 min in o de o inac i a e any ee no EV encapsula ed i us
p esen . F ee i us used as con ols was always inac i a ed o each
M. Ga o alo e al. Jou nal o Con olled Release 283 (2018) 223–234
224
expe imen pe o med as p e iously epo ed [31]. Samples we e sub-
sequen ly neu alized by he addi ion o HCl 0.1 M.
To gene a e EV-Vi us-PTX, he EV-Vi us o mula ion was incuba ed
in a 10 μM PTX solu ion, p epa ed by dilu ing 10 mM PTX in DMSO
wi h PBS wi h he a io o 1:1000. Incuba ion was ca ied ou a RT
wi h mixing o 1 h. Samples we e hen cen i uged a 150000 ×g o
2 h a RT, in o de o pelle EV-Vi us-PTX. The washing p ocedu e was
epea ed using PBS as diluen . The final EV-Vi us-PTX pelle was e-
suspended in 100 μL o PBS and s o ed a −80 °C.
2.6. Quan ifica ion o PTX p esen in EV-Vi us-PTX
50 μL o 1.5 × 10
10
EV-Vi us-PTX/mL as well as supe na an om
he second washing s ep associa ed wi h emo al o ee PTX, o enable
alida ion o he washing p ocedu e, was p ocessed o ul a pe o -
mance liquid ch oma og aphy (UPLC, Acqui y UPLC Sys em) using a
Co ecs UPLC C18+ column, 2.1 × 50 mm, pa icle size 2.7 μm
(Wa e s, USA). Addi ionally, a 10 μM PTX solu ion was p epa ed, and
used as a con ol o sample p ocessing. Sodium dodecyl sul a e (SDS)
was added o samples o a final concen a ion o 5% (w/ ) in o de o
lyse EV-Vi us PTX. Vo exing was ollowed by incuba ion a RT o 1 h.
Ace oni ile was hen added o a final concen a ion o 75% ( / ) and
samples o exed. P ecipi a ed p o eins we e pelle ed by cen i uga ion
a 10000 ×g o 5 min a RT. The supe na an was analyzed by UPLC
using g adien flow o ace oni ile om 30% o 80% in phospha e
buffe , pH = 2, a 30 °C wi hin 3 min. De ec ion and quan ifica ion o
PTX, wi h a e en ion ime o 1.7 min, was pe o med spec o-
pho ome ically a he wa eleng h o 229 nm by using a e e ence
s anda d cu e.
2.7. Size dis ibu ion analysis by nanopa icle acking analysis (NTA)
Size dis ibu ion and concen a ion o EV, EV-Vi us and EV-Vi us-
PTX o mula ions we e analyzed by NTA using Nanosigh model LM14
(Nanosigh ) equipped wi h blue (404 nm, 70 mV) lase and sCMOS
came a. The samples con aining i us we e incuba ed a +95 °C o
10 min in o de o inac i a e he i uses. NTA was pe o med o each
sample by eco ding h ee 90 s ideos, subsequen ly analyzed using
NTA so wa e 3.0 (Nanosigh ). The de ec ion h eshold was se o le el
5 and came a le el o 15.
2.8. Ze a po en ial analysis by elec opho e ic ligh sca e ing
The ze a po en ial was measu ed using Ze aSize Nano (Mal e n,
UK). All he samples we e dilu ed in a olume o 800 L o MilliQ H
2
O
and injec ed wi h a 1 mL sy inge in he capilla y flow (DTS1070 olded
capilla y cell) o he measu emen . An equilib a ion ime o 120 s was
se on he so wa e o allow he samples o s abilize a 25 °C inside he
measu emen chambe . Th ee pa allel measu emen s we e pe o med
on each sample.
2.9. HIM mic oscopy
Fo Helium Ion Mic oscopy, A549 cells we e cul u ed o 70% con-
fluence in DMEM (Gibco Labo a o ies, USA) supplemen ed wi h hea
inac i a ed 10% FBS (Gibco Labo a o ies) and 100 u/mL penicillin-
s ep omycin (Gibco Labo a o ies) on poly-L-lysin (Sigma Ald ich,
Ge many) glass co e slips. Cells in cul u e we e in ec ed wi h EV-Vi us
(5 × 10
9
/mL on 75 cm
2
80% confluen cell cul u e) by eplacing he
cell cul u e medium wi h a solu ion con aining EV-Vi us in DMEM o
2% FBS and 100 u/mL penicillin-s ep omycin. A e 2 h incuba ion,
EV-Vi us solu ion was again eplaced wi h DMEM o 10% FBS and
100 μ/mL penicillin-s ep omycin. Cells we e fixed a diffe en ime
poin s by eplacing he EV-Vi us solu ion wi h 2% glu a aldehyde (GA,
Me ck, USA) in 0.1 M Sodium Cacodyla e (NaCac) buffe (pH 7.4). A e
GA fixa ion, he cells we e washed wice wi h 0.1 M NaCac and u he
fixed wi h 1% OsO
4
in 0.1 M NaCac buffe (pH 7.4). A e 1 h in OsO
4
,
cells we e washed wice wi h 0.1 M NaCac buffe and chemically d ied
in an inc easing E OH concen a ion se ies o 50%, 70%, 96%, and
wice wi h 100%. A e 100% E OH, he cells we e subme ged in 98%
hexame hyldisiloxane (Sigma Ald ich) and le o d y o o e nigh .
A e fixa ion and chemical d ying, co e slips we e moun ed on s ands
and imaged wi h O ion NanoFab Helium Ion Mic oscope (Zeiss,
Ge many) using 30 kV accele a ion ol age wi h beam cu en 0.2–1
pA.
2.10. T ansduc ion assay
Cells we e seeded a a densi y o 1 × 10
4
cells/well in 96-well pla es
and main ained unde app op ia e condi ion. On he ollowing day cells
we e ea ed in iplica es wi h an oncoly ic adeno i us Ad5D24R p
encoding o he ed fluo escen p o ein (10 p/cell) and con ol EVs (10
pa icles/cell), EV-Vi us (10 pa icles/cell), EV-PTX (10 pa icles/cell,
5μM o PTX), EV-Vi us-PTX (10 pa icles/cell, 5 μM o PTX) o Vi us
and PTX sepa a ely (Vi us+PTX) (10 p/cell, 5 μM o PTX). EVs, EV-
Vi us and EV-Vi us-PTX had he same doses o pa icles as i us alone
(10 p we e calcula ed as 10 EV-pa icles pe cell in he well). The cells
we e hen imaged wi h EVOS FL fluo escence mic oscope a 8, 24 and
48 h a e he ea men in o de o coun he po ion o cells exp essing
RFP.
2.11. Immunocy ochemis y s aining (ICC)
The de e mina ion o he in ec i i y was based on he isual quan-
ifica ion o in ec ed cells as p e iously desc ibed [32,33]. Cells we e
seeded a a densi y o 2 × 10
5
cells/well in 24 well pla es, and main-
ained unde app op ia e condi ion in DMEM, comple ed wi h 5% FBS,
1% L-glu ammine and 1% o penicillin/s ep omycin. All he chemicals
we e pu chased om Gibco Labo a o ies. On he ollowing day cells
we e ea ed wi h Vi us (10 p/cell), Vi us + PTX (10 p/cell and 5 μM
PTX solu ion), EV-Vi us o mula ions (10 pa icles/cell) and EV-Vi us-
PTX o mula ions (10 pa icles/cell, 5 μM o PTX).
Pla es we e cen i uged o 90 min wi h 1000 ×gin 37°C and in-
cuba ed o 48 h be o e s aining a 37 °C and 5% CO
2
.48 h ollowing he
incuba ion cells we e fixed by adding 250 μL o ice-cold me hanol pe
well and incuba ed 15 min. Then cells we e washed h ee imes wi h
PBS 1%-BSA (Bo ine se um albumin, 9048-46-8 Sigma-Ald ich) solu-
ion and incuba ed in he da k o one hou wi h 1s an ibody, mouse
monoclonal an i-hexon 1:2000 (No us Biological, NB600-413). A e
he incuba ion ime, cells we e washed h ee imes wi h PBS 1%-BSA
and incuba ed in he da k o o he 1 h wi h 2nd an ibody: Bio in-SP-
conjuga ed goa an i-mouse 1:500 (Jackson Immuno Resea ch, 115-
065-062). A e he incuba ion ime, cells we e washed h ee imes wi h
PBS1%-BSA and incuba ed in he da k o 30 min wi h ex a idin-pe -
oxidase (Sigma Ald ich, E2886). Finally cells we e washed h ee imes
as indica ed ea lie and ea ed wi h Dab pe oxidase subs a e solu ion
(Sigma Ald ich, A7284-50ML). To quench he eac ion, cells we e
ea ed once wi h PBS. The de ec ion o he in ec ious i e was pe -
o med using mic oscope EVOS, and each well was pho og aphed wi h
5 pcs a 5 non-o e lapping si es. The ollowing o mula was used o
de e mine he in ec ious i e :
L
mL
In ec ious i e :x A(well)
A( ield)
11
Whe e x = numbe o in ec ed (s ained cells).
A(24 well) = 190 mm
2
.
A(field) = su ace a ea o he field.
L = dilu ion.
= olume o i us dilu ion applied pe well.
M. Ga o alo e al. Jou nal o Con olled Release 283 (2018) 223–234
225
2.12. MTS cell iabili y assay
A549, PNT2 and PC-3 cells we e seeded a a densi y o 1 × 10
4
cells/
well in 96-well pla es and main ained unde app op ia e condi ion. On
he ollowing day cells we e ea ed in iplica es wi h Vi us (10 p/
cell), Vi us + PTX (10 p/cell + 5 μM PTX solu ion), con ol EVs (10
pa icles/cell), EV-PTX (10 pa icles/cell, 5 μM o PTX), EV-Vi us o -
mula ions (10 pa icles/cell), EV-Vi us-PTX o mula ions (10 pa icles/
cell, 5 μM o PTX). Cell iabili y was de e mined by MTS assay ac-
co ding o he manu ac u e 's p o ocol (Cell Ti e 96 AQueous One
Solu ion Cell P oli e a ion Assay; P omega, Nacka, Sweden). The ab-
so bance was measu ed wi h a 96-wells pla e spec opho ome e
Va ioskan Flash Mul imode Reade (The mo Scien ific) a 490 nm. The
expe imen s we e independen ly pe o med h ee imes wi h iplica es
o each condi ion in each expe imen .
2.13. Analysis o apop o ic and nec o ic cells
A549 cells we e pla ed in o 6 well pla es, 2 × 10
5
cells/well. Cells
we e ea ed wi h an oncoly ic adeno i us Ad5D24CpG 10 p/cell,
Vi us + PTX (10 p/cell + 5 μM PTX solu ion), con ol EVs (10 pa i-
cles/cell), EV-PTX (10 pa icles/cell, 5 μM o PTX), EV-Vi us o mula-
ions (10 pa icles/cell), EV-Vi us-PTX o mula ions (10 pa icles/cell,
5μM o PTX). The amoun o apop o ic and nec o ic cells was measu ed
a e 24 h pos - ea men wi h a TACS Annexin V-FITC ki (T e igen
Inc., Gai he bu g, MD, US) and BD LSRII flow cy ome e acco ding o
he manu ac u e 's ins uc ion.
2.14. In i o xenog a animal expe imen s
FIRST DAY OF
TREATMENT
Day 0
SECOND DAY OF
TREATMENT
Day 2
THIRD DAY OF
TREATMENT
Day 4
FOURTH DAY OF
TREATMENT
Day 15
EVs
(1x109pa icles/ umo )
XXX
Vi us
(1x108 p/ umo )
XXPBS
Ab axane
(10 mg o PTX/kg)
PBS PBS X
Vi us+Ab axane
(1x108 p/ umo +10
mg o PTX/kg)
XXX
EV-Vi us
(1x108pa icles/ umo
+ 1x108 p/ umo )
XXPBS
EV-Vi us-PTX
(1x108pa icles/ umo
including 1x108
p/ umo , and 10 mg
o PTX/kg)
XXX
All he animal expe imen s pe o med unde he e hical pe mission
(ESAVI/10482/04.10.07/2015) o he Na ional Labo a o y Animal
Boa d o Finland (Ca e and Use Commi ee) by Made Consul ing L d. Oy
(Tu ku, Finland) in GLP le el animal acili y.
Mice we e ob ained om Jan ie Labs (Ba ie 4E-1, F ance) a
4 weeks o age. The acclima iza ion pe iod was 13 days p io o A549
cance cell injec ions, he cell line was he same used o he in i o cell
expe imen s. The A549 cell line pu i y was es ed usingby IDEXX
BioResea ch - IMPACT III panelbe o e inocula ion and he cells iabili y
o 99% we e de ec ed 50 min p io o fi s inocula ion and 90% 20 min
a e he las inocula ion using NucleoCoun e NC-200. Heal h s a us o
he mice was moni o ed daily and as soon as signs o pain o dis ess
we e e iden hey we e eu hanized. Fo he efficacy expe imen , human
xenog a s we e es ablished by injec ing 1.5 × 10
6
A549 cells s.c. in o
he flanks o 6-week old emale BALB/c nude mice. The ea men
g oups we e as ollows: Vi us (n = 6); Vi us+Ab axane (n = 6);
Ab axane (n = 6); EV (n = 6) and EV-Vi us (n = 9); EV-Vi us-PTX
(n = 9). T ea men g oups we e adminis e ed i. (100 μL) and i.
(50 μL) o mice wi h umo s (one umo pe mouse abou 5 mm in
diame e ). The dosing days we e 0, 2, and 4 o Vi us (acco ding o
p e ious p o ocols [29]) and EV-Vi us; 0 and 15 o Ab axane as p e-
iously epo ed [34]; 0, 2, 4 and 15 o EV and EV-Vi us-PTX ( able
abo e and he Supplemen a y Table 1). The equa ion:
0.52 × leng h × (wid h)
2
, was used o calcula e he umo olumes o
s udy he efficacy o he used EV- o mula ions. Howe e , he a e age
diame e o 15 mm was used as a limi o eu hanize mice.Tumo s, li e s
and spleens om each mouse we e collec ed o his opa hological ex-
amina ions.
2.15. Quan i a i e PCR
qPCR o adeno i us E4 copy numbe was ca ied ou acco ding o
he p o ocol p e iously desc ibed [35] (p ime FW:50-GGA GTG CGC
CGA GAC AAC-30, p ime RV: 50-ACT ACG TCC GGC GTT CCA T-30,
p obe E4: 50-(6FAM)-TGG CAT GAC ACT ACG ACC AAC ACG ATC T-
(TAMRA)230). To al DNA was ex ac ed om BALB/c nude mu ine
samples ( umo s, li e s, blood) using he QIAamp DNA Blood Mini Ki
(Qiagen, Hilden, Ge many) acco ding o manu ac u e 's p o ocol. Sub-
sequen ly isola ed DNA was analyzed o adeno i al E4 copy numbe
no malized o mu ine be a-ac in (li e , blood) and human be a-ac in
( umo ), espec i ely ((p ime FW: 50-CGA GCG GTT CCG ATG C-30,
p ime RV: 50-TGG ATG CCA CAG GAT TCC AT-30, p obe mu ine be a-
ac in: 50-(6FAM)-AGG CTC TTT TCC AGC CTT CCT TCT TGG-(TAMRA)
230; (p ime FW: 50-CAG CAG ATG TGG ATC AGC AAG-30, p ime RV:
50- CTA GAA GCA TTT GCG GTG GAC-30, p obe human be a-ac in: 50-
(6FAM)- AGG AGT ATG ACG CCG GCC CCT C-(TAMRA)230). Samples
we e analyzed using LighCycle qPCR machine (LighCycle 480, Roche,
Basel, Swi ze land).
2.16. His opa hological s udies
Le la e al lobe o li e , hal o spleen and one lobe o lungs we e
M. Ga o alo e al. Jou nal o Con olled Release 283 (2018) 223–234
226

aken in o 4% buffe ed o malin a nec opsy. Finnish Cen e o
Labo a o y Animal Pa hology, Helsinki, Finland pe o med he his o-
pa hological e alua ions o he lung, li e , spleen and umo in i o
samples o nude (a hymic) mice, implan ed wi h human umo xeno-
g a and ea ed wi h i us alone, con ol EVs and EV-Vi us-PTX by
his o echnology. The samples we e embedded in o pa affin, cu a 4 μm
(spleen 3 μm), and s ained wi h haema oxylin and eosin. The his o-
pa hological e alua ion was pe o med as a blind using 40×magnifi-
ca ion using he mic oscope Zeiss Axio Image .A2, Ca l Zeiss
Mic oscopy GmbH, Jena, Ge many.
2.17. To al RNA-sequencing
RNA om umo issues, om mice ea ed wi h con ol EVs, EV-
Vi us and EV-Vi us-PTX, we e ex ac ed using RNeasy Plus Mic o Ki
(Qiagen) acco ding o he manu ac u e 's ins uc ions. Indexed lib a ies
we e p epa ed om 10 ng/ea. pu ified RNA wi h SMARTe S anded
To al RNA-Seq Ki - Pico Inpu Mammalian (Clon ech Labo a o ies,
Inc.) acco ding o he manu ac u e 's ins uc ions. Lib a ies we e
quan ified using he TapeS a ion 4200 (Agilen Technologies) and
pooled such ha each index- agged sample was p esen in equimola
amoun s, wi h final concen a ion o he pooled samples o 2 nM. The
pooled samples we e subjec o clus e gene a ion and sequencing using
an Illumina Nex Seq 500 Sys em (Illumina) in a 2 × 150 single ead
o ma a a final concen a ion o 1.8 pmol.
2.18. RNA-Seq Analysis
The aw sequence files gene a ed ( as q files) unde wen quali y
con ol analysis using Fas QC (h p://www.bioin o ma ics.bab aham.
ac.uk/p ojec s/ as qc/).To analyze RNA-Seq da a we used he s a egy
called “Di ec mapping”as p e iously desc ibed [36]. Reads we e fi s
mapped on human genome (assembly hg38) using STAR [37]. The
quan ifica ion o ansc ip s exp essed o each eplica e o he se-
quenced samples was pe o med using HTSeq-coun [38]. R was used o
c ea e a ma ix o all ansc ip s exp essed in all samples wi h he
co esponding ead-coun s and he Bioconduc o package limma [39]
was used o no malize he da a and hen o pe o m he diffe en ial
exp ession analysis: an Empi ical Bayes mode a ion - es was pe o -
med.Da a we e also no malized in FPKM (F agmen s Pe Kilobase O
Exon Pe Million F agmen s Mapped) using Cuffno m [40]. Genes up-
egula ed (log2FC ≥1) and down- egula ed (log2FC ≤−1) wi h a P-
alue < 0.05 we e selec ed as diffe en ially exp essed. F om hese lis s
o DEGs, Genesis so wa e [41,42]was used o gene a e hea maps and
o in es iga e he gene on ology (GO) e ms in he wo gene se s o
diffe en ially exp essed genes.
2.19. S a is ical analysis
S a is ical analysis was pe o med by using one-way ANOVA ol-
lowed by Bon e oni pos -hoc es . Su i al cu es and hei s a is ical
analysis we e pe o med using Kaplan–Meie es . The in i o he -
apeu ic syne gy was calcula ed using ac ional umo cell iabili y
(FTV) me hod [43,44]. Adjus ed P- alues in Supplemen a y Table 3
we e calcula ed pe o ming Fishe 's exac es and he co ec ion o
mul iple hypo hesis es ing using he Benjamini-Hochbe g me hod [45].
All s a is ical analysis, calcula ions and es s we e pe o med using
G aphPad P ism 5 (G aphPad So wa e, San Diego, CA).
3. Resul s
3.1. Oncoly ic adeno i uses can be encapsula ed inside EVs wi h PTX
Fo in es iga ing he possibili y o c ea e a new ype o sys emic
d ug deli e y s a egy o lung cance , we encapsula ed chemo he apy
d ug and oncoly ic i us in o EVs. The size dis ibu ions o EV-Vi us and
EV-Vi us-PTX o mula ions we e de e mined by using NTA (Fig 1AB).
Size dis ibu ion o bo h con ol EVs and EV-Vi us o mula ions we e
de ec ed o be in he ange o 50–1000 nm. The size dis ibu ion o EV-
Vi us o e laps wi h he size o he Vi us (93.8 ± 4.3 nm) wi h mos o
he EVs being smalle o he same size as he i us (Fig. 1A). In addi ion
e en hough hea ing was used o inac i a e any ee i uses in samples,
he inac i a ed ee i us pa icles a e s ill p esen and his may affec
he size dis ibu ion o EV-Vi us, b inging i close o he size dis-
ibu ion o ee i us (Fig. 1A). The effec o hea ing on he size dis-
ibu ion o EVs was also assessed wi h A549 con ol EVs: while he
pa icle coun o hea ed EVs was wi hin he s anda d e o o non-
boiled EVs, he size dis ibu ion shi ed o sligh ly smalle (app oxi-
ma ely 20 nm) a e boiling, which can affec he esul o EV-Vi us as
well (unpublished da a no shown). In any case, he size dis ibu ions o
EV-Vi us and con ol EVs we e e y simila , wi h EV-Vi us being
sligh ly mo e o ien ed owa ds i s peak a 75 nm. Wi h EV-Vi us-PTX
and EV-PTX (EVs loaded wi h PTX), no significan size diffe ences we e
seen due o he addi ion o PTX (Fig. 1B). Fu he mo e, con ol EVs and
EV-Vi us bo h had a simila s ongly nega i e ze a-po en ial o ap-
p oxima ely −40 mV, while he ee i us had a ze a-po en ial o
−20 mV (Fig. 1C), sugges ing ha EV-Vi us p epa a ion consis ed
mos ly o EVs, o he wise i ze a-po en ial should ha e shi ed owa ds a
Fig. 1. Oncoly ic adeno i us encapsula ed in o he
Ex acellula esicle allows complex o ma ion. (A-
B) Size dis ibu ion o i us alone, EV- i us, EV-pa-
cli axel, EV-Vi us-pacli axel and Vi us we e de-
e mined by using Nano acking analysis (NTA). (C)
The su ace cha ge o he i us alone, EV- i us, EV-
pacli axel, EV-Vi us-pacli axel and Vi us was mea-
su ed using Ze aSize Nano Mal e n. (D) Helium Ion
Mic oscopy pic u es imaged wi h O ion NanoFab
Helium Ion Mic oscope (Zeiss, Ge many) using 30 kV
accele a ion ol age wi h beam cu en 0.2–1 pA.
Images a e o non-in ec ed (le ) and EV-V in ec ed
( igh ) cells. The de o med pheno ype wi h mul iple
p o usions ex ending om he cell su ace was o en
obse ed in in ec ed cells a leas a e 24 h pos -in-
ec ion.
M. Ga o alo e al. Jou nal o Con olled Release 283 (2018) 223–234
227
less nega i e alue. The ze a-po en ial was also unal e ed in EV-Vi us-
PTX, and EV-PTX o mula ions, as is o be expec ed since PTX is a
cha geless molecule (Fig. 1C).
Helium Ion Mic oscopy (HIM) is an imaging echnique compa able
o Scanning Elec on Mic oscopy, wi h he dis inc ion ha samples do
no equi e conduc i e coa ing. HIM was used o image unin ec ed cells
as well as EV and EV-Vi us in ec ed cells in an a emp o obse e po-
en ial changes due o he EV-Vi us in e ac ions on he cell su ace. EV
exposed cells did no appea o diffe om he un-in ec ed cells ( esul s
no shown). Ye , occasionally he su ace o EV-Vi us in ec ed cells was
co e ed wi h esicle-like p o usions (Fig. 1D). This phenomenon may
de i e om i us-induced changes inside he cell, esul ing in pheno-
ypic al e a ions on he su ace.
The amoun o PTX encapsula ed in o he EVs was de e mined by
UPLC as p e iously desc ibed [23] (Supplemen a y Table 1). The
washing p o ocol used in he p oduc ion o EV-Vi us-PTX o mula ions
we e success ul, since he PTX concen a ion o he second washing s ep
supe na an was below 0.05 μM, and hus insignifican when compa ed
o he PTX concen a ion o EV-Vi us-PTX (4.7 μM). The UPLC assessed
concen a ion o he 10 μM PTX con ol sample, shows a 38% loss o
PTX (Supplemen a y Table 1). Howe e , he concen a ion o he
10 mM PTX DMSO s ock solu ion was confi med by UPLC analysis. PTX
p ecipi a ion is mos likely no he eason o he PTX loss seen in he
con ol sample, since ace oni ile was added o a final concen a ion o
75% o samples p io o UPLC analysis. Howe e he analysis o PTX in
EV-Vi us-PTX was jus mean as a quali a i e p oo ha PTX is indeed
encapsula ed in he esicles.
3.2. In i o and in i o enhanced an i umo effec o i us and PTX in EV-
Vi us and EV-Vi us-PTX o mula ions
The esponsi eness o solid umo s o chemo he apeu ic agen s
depends o g ea ex en o he op imiza ion o he d ug deli e y. As o
such, we he e se o e alua e in a ac o ial expe imen he in i o effi-
cacy o se e al combina ions o EV-Vi us-PTX o mula ions o al e
umo g ow h in a lung xenog a animal model. Ab axane is a clinically
app o ed nano o mula ion [46] chosen acco ding o he clinical se -
ings p e iously desc ibed [34] and i was in oduced, since p e ious
s udy showed ha pa ien s wi h non small cell lung cance may benefi
om he ea men [47,48]. Nude mice bea ing A549 cells o igina ing
umo in he igh flank we e ea ed by in a enous (i ) injec ions on
day 0, 2, 4 and 15 wi h: i) EVs alone (1 × 10
9
pa icles/ umo ), Vi us
alone (1 × 10
8
p/ umo ), Ab axane (10 mg o PTX/kg);
Vi us + Ab axane (1 × 10
8
p/ umo + 10 mg o PTX/kg); EV-Vi us
(1 × 10
8
pa icles/ umo + 1 × 10
8
p/ umo ); EV-Vi us-PTX o mula-
ion (1 × 10
8
pa icles/ umo including 1 × 10
8
p/ umo , and 10 mg o
PTX/kg) (Supplemen a y Table 2). EVs alone we e no able o con ol
he umo g ow h, and we e hus used as nega i e con ol in ou ex-
pe imen s. The in a umo al (i ) ea men did no show significan
diffe ences be ween EV-Vi us, EV-Vi us-PTX and Ab axane ea men s
(Supplemen a y Fig. 1).
In e es ingly, he i injec ion o he EV-Vi us-PTX o mula ion sig-
nifican ly educed (P< 0.001) umo g ow h in compa ison o naked
i us and Vi us + Ab axane (Fig. 2A). The highes su i al a e was
obse ed in EV-Vi us and EV-Vi us-PTX ea men s (90% a 60 days)
(Fig. 2B). The bes su i al a e (Fig. 2B) was obse ed in he combi-
na o y g oup: EV-Vi us-PTX o e o he s udied o mula ions, sugges ing
ha he bes an i- umo efficacy esponse was posi i ely co ela ed
wi h he su i al.
The local eplica ion o he i us was quan ified by he adeno i us
E4 copy numbe in umo , li e and se um by qPCR analysis.
Adeno i al pa icles we e no de ec ed in se um and li e in any o he
es ed g oups (Fig. 2C), sugges ing ha EV-Vi us adminis e ed in-
a eniously in ec s and eplica es only in umo cells.
In o de o e i y he in i o cell dea h by he EV-Vi us and EV-
Vi us-PTX due o he apop o ic e en s, he flow cy ome y
measu emen s we e ca ied ou by measu ing he amoun o Annexin-V
(ea ly apop o ic s age) and p opidium iodide (la e apop o ic s age) o
posi i e cells a 24 h pos ea men . By ha we we e able o confi m
ha EV-Vi us and Vi us+PTX ea men s induced bo h ea ly and la e in
i o apop o ic effec in A549 cells (Fig. 2 DE) (Supplemen a y
Fig. 2).The in i o he apeu ic syne gy be ween EVs and Vi us was
calcula ed using ac ional umo cell iabili y (FTV) me hod and de-
mons a ed syne gis ic an i umo effec in he EV-Vi us-PTX ea men
g oup (Fig. 2 FG).
His opa hological analysis o he li e , spleen and umo samples
om mice demons a ed no subs an ial changes (Fig. 3), wi h he ex-
cep ion o EV-Vi us ea men , which showed mode a e-sized in-
flamma o y ocus (mos ly neu ophils) in o he wise no mal li e lobule
(Fig. 3 A4). Spleen samples displayed gene al his ological pa e n y-
pical o nude mice such as pe ia e iola lympha ic shee a eas (PALS)
and in some cases mild lymphocy e hype plasia (Fig. 3 B1–B4) [49].
Tumo samples exhibi ed ypical ea u es o a lung ca cinoma (malig-
nan epi helial umo ) and hey we e e y uni o m in hei g ow h
pa e n and cellula ea u es (Fig. 3 C4).
3.3. Oncoly ic adeno i uses encapsula ed in he EVs show inc eased
ansduc ion efficacy and enhanced in ec ious i e
The ansduc ion assay was conduc ed by using he ed fluo escen
p o ein [30] exp essing i us Ad5D24RFP encapsula ed in EVs. The
ansduc ion efficacy o he i us Ad5D24RFP alone was compa ed wi h
EV-Vi us and EV-Vi us-PTX.T ansduc ion was assessed a 8, 24 and 48 h
pos -in ec ion. In e es ingly, al eady a 8 h om in ec ion, he amoun
o ed fluo escen cells in EV-Vi us and EV-Vi us-PTX ea ed cul u es
was highe compa ed o cells ea ed wi h Vi us Ad5D24RFP a e 48 h
(Fig. 4AB). The in ec i i y o he diffe en o mula ions (Vi us,
Vi us + PTX, EV-Vi us and EV-Vi us-PTX) we e u he in es iga ed by
Immunocy ochemis y Assay (ICC). The in ec ious i e was ound o be
significan ly highe o EV-Vi us and EV-Vi us-PTX o mula ions when
compa ed o cells ea ed wi h i us Ad5D24RFP alone o wi h
Vi us + PTX (Fig. 4 CD).
3.4. Oncoly ic adeno i uses encapsula ed in he EVs show enhanced
cy o oxici y
To ensu e ha he cance de i ed EVs did no affec heal hy cells a
leas du ing he pe iod o 48–96 h, in which we saw a clea an icance
effec in cance cells, we pe o med he expe imen s also wi h he PNT2
cells, which is a non-cance ous cell line o p os a e epi helium. I was
shown by MTS cell iabili y assay (Fig. 5A).
Then cy o oxici y o he EV- o mula ions was s udied by MTS cell
iabili y assays on he A549 cell line. EV-Vi us and EV-Vi us-PTX o -
mula ions educed cell iabili y significan ly mo e when compa ed o
cells ea ed wi h he i us alone (Fig. 5B) (p< 0,001). Indeed, he cell
killing ac i i y o EV-Vi us and EV-Vi us-PTX o mula ions was in-
es iga ed by he MTS cell iabili y assays on A549 and PC-3 cell lines
ea ed wi h samples ob ained om PC-3 and A549 cells, espec i ely.
The EV- o mula ions ob ained om PC-3 cells (EVs, EV-PTX, EV-Vi us,
EV-Vi us-PTX) ha e been used o ea A549 cells, while EV- o mula-
ions ob ained om A549 cells (EVs, EV-PTX, EV-Vi us, EV-Vi us-PTX)
ha e been used o ea PC-3 cells in o de o ca y ou he c oss-ex-
pe imen s. In bo h expe imen s, i was obse ed ha he cell killing
effec o he EV o mula ions is no cance cell line dependen (Fig 5CD)
(p < 0,001).
3.5. Diffe en molecula mechanisms unde lying he an i-neoplas ic effec s
p oduced by EV-Vi us and EV-Vi us-PTX ea men s
Fo he e alua ion o he molecula mechanisms unde lying he
enhanced an i umo effec obse ed be ween i al and pacli axel
ea men s deli e ed wi h EVs, we ca ied ou an RNA-SEQ
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ansc ip omic analysis on he RNAs ex ac ed om umo xenog a s
g own in EV, EV-Vi us and EV-Vi us-PTX ea ed mice. The analysis was
ca ied ou o 3 samples/g oup wi h he only excep ion o he EV-Vi us
g oup o which only 2 samples we e o sufficien quali y o be analyzed
due o he small a io o human e sus mouse mRNA ex ac ed om he
xenog a . The analysis iden ified 615 and 317 ansc ip s o EV-Vi us-
PTX and EV-Vi us ea men s, espec i ely, as diffe en ially exp essed
in he wo condi ions, when compa ed o he EV con ol ea men .
Mos o he diffe en ially exp essed genes we e up- egula ed in he EV-
Vi us, while he majo i y we e down- egula ed in he EV-Vi us-PTX
g oups (Fig. 6A). Among he diffe en ially exp essed genes only 47
we e diffe en ially modula ed by bo h ea men s (Fig. 6B) as also de-
ailed in he hea -maps (Fig. 6C). The wo dis inc gene ic p og ams
igge ed by EV-Vi us and EV-Vi us-PTX indica ed ha a diffe en ial
cellula esponse was p oduced by he wo ea men s. Indeed, al hough
he ypes o biological p ocesses in ol ed in he esponse appea ed
ema kably simila o bo h ea men s (Supplemen a y Table 3), he
genes dys egula ed we e diffe en . A gene al conside a ion on he ne
effec p oduced by he diffe en ial exp ession in e ms o up- and down-
egula ion o each pa hway is somehow hampe ed by he complexi y o
signals like “me abolic p ocess”o “cellula componen o ganiza ion”.
Howe e , we e he analysis was possible like o example o he mi-
ogenic pa hway, i was clea ha bo h ea men s igge ed an an i-
cance effec while egula ing diffe en se o genes (Supplemen a y
Fig. 3). In he mi ogenic pa hway, o example among he genes
modula ed by EV-Vi us-PTX ea men , USP37, SNX33 and POLE we e
down egula ed, whe eas o e exp ession o hese genes was shown o
induce p oli e a ion by p omo ing G1/S phase ansi ion [50]; S-phase
p og ession and mi osis espec i ely [51]. A ne an i-p oli e a i e effec
was sugges ed also conside ing he genes diffe en ially exp essed by he
EV-Vi us ea men , which in ol es down egula ion o BRSK2 ex-
p ession, known o inc ease he pe cen age o cells in G2/M when
o e exp essed [52], and he up egula ion o E2F4, a ac o nega i ely
influencing he G1 p og ession h ough cell cycle [53]. In conclusion,
Fig. 2. In i o and in i o enhanced an i umo effec s
o oncoly ic i us and pacli axel encapsula ed in
Ex acellula esicles o lung cance ea men .(A)
A549 cell line was implan ed subcu aneously in o
he igh flank o BALB/c nude mice. All ea men s
we e adminis e ed in a enously (i. .). Tumo
g ow h was ollowed o e ime. (B) Kaplan-Meie
es was used o calcula e he su i al p ofile. (C)
Adeno i al copies owa ds E4 gene we e measu ed
by qPCR om eu hanized mice's o gans ( umo , li e
and se um) a he end o he ea men . (D-E) Ea ly
and la e apop o ic o nec o ic cell dea h we e mea-
su ed in A549 cells a e 24 h pos - ea men . The
amoun o ea ly and la e apop o ic o nec o ic cells
we e analyzed by flow cy ome y 24 h a e he
ea men s. FITC-labeled Annexin-V was used o in-
dica e he ea ly apop o ic cell and PI o he nec o ic
o la e apop o ic cells, ***P< 0.001. (F-G) The as-
sessmen o he in i o he apeu ic syne gy was
calcula ed wi h FTV me hod. Obse ed FTV (mean
alue o expe imen al cell iabili y)/(mean alue o
cell iabili y con ol). Expec ed FTV (mean FTV o
Expe imen al condi ion)/(mean FTV o expe imen al
con ol). A a io > 1 indica es a syne gis ieffec ,
and a a io < 1 indica es a less han addi i e effec ).
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229
Fig. 3. His opa hological examina ion on li e ,
spleen and umo . (A-C) Li e samples om mice
ea ed wi h Vi us (A1), EVs (A2), EV-Vi us-PTX (A3)
o EV-Vi us (A4) exhibi ed no significan his o-
pa hological findings.A li e sample om an EV-
Vi us - ea ed mouse (A4) shows a backg ound le-
sion; a mode a e-sized inflamma o y ocus (mos ly
neu ophils; a ow) in o he wise no mal li e lobule.
(B1) Spleen samples om mice ea ed wi h i us
alone exhibi mild lymphocy ehype plasia (lym-
pha ic ollicles wi h lymphoblas -like cells, mi o ic
figs and ingible body mac ophages), while spleen
samples (B2) om mice ea ed wi h ex acellula
esicles alone show no significan findings. Typical
o nude mice, he PALS a ea (ligh ly-s aining zone
in whi e pulpa su ounding blood essels) is spa se
and ma ginal zone inconspicuous. In addi ion, no
seconda y ollicles a e p esen . (B3) A ep esen a i e
spleen sample om a mice ea ed wi h EV-Vi us-
PTX displays mild hype plasia o he ma ginal zone
wi hou lympha ic hype plasia and a sample (B4)
om mouse ea ed wi h EV-Vi us mild hype plasia
o he ma ginal zone wi h mild lymphocy e hype -
plasia. (C1) Tumo samples om mice ea ed wi h
i us alone show nec o ic emnan s o cells in a small
nec o ic a ea and apop o ic cell emnan s in degene a i e a ea. (C2) A la ge ca i y is filled wi h p o einacous fluid and lined by cubic o fla ened cell. No nec osis.
Tumo capsule (C3) A umo sample ea ed wi h EV-Vi us-PTX exhibi s a la ge ca i y wi h in aluminal blood and p o einacous fluid.Tumo issue g ows in densely
packed nes s o packe s, and pe iphe al co ds. No nec osis is p esen . (C4) Tumo sample ea ed wi h EV-Vi us displays la ge numbe o neu ophils (a owheads;
examples) among oamy neoplas ic cells.
Fig. 4. Effec o oncoly ic adeno i us encapsula ed
in o ex acellula esicles on cell ansduc ion and
in ec i i y. (A) The ansduc ion efficacy was e al-
ua ed by in ec ion wi h an oncolyi c adeno i us en-
coding o he ed fluo escen p o ein (RFP) en-
capsula ed in o he ex acellula esicles loaded o
no wi h PTX.RFP was measu ed using Va ioskan
pla e eade a e 8, 24 and 48 h pos in ec ion, ***
P< 0.001. (B) Mos ep esen a i e fluo escen mi-
c oscope pho og aph (400 μm) o he in ec ed cells.
(C) The in ec i i y o i us alone, Vi us + PTX, EV-
Vi us and EV-Vi us-PTX we e assessed by ICC assay.
(D) Mos ep esen a i e mic oscope pho og aphs
(400 μm) o he in ec ed wells a e p esen ed. Figs
ep esen diffe ence in hexon p o ein exp ession
( i us assembling), wi hou dis inguishing in-
ec i i y, eplica ion, o gene exp ession manne , ***
P < 0.001. (Fo in e p e a ion o he e e ences o
colou in his fig legend, he eade is e e ed o he
web e sion o his a icle.)
Fig. 5. Effec o oncoly ic adeno i uses encapsula ed
in o ex acellula esicles on cell iabili y. (A-B) Cell
iabili y was pe o med by MTS assay on PNT2 and
A549 cell lines. The abso bance was measu ed wi h a
96-wells pla e spec opho ome e Va ioskan Flash
Mul imode Reade a 490 nm. (C-D) Cell iabili y
was pe o med by MTS assay on A549 and PC-3,
espec i ely ea ed wi h samples om PC-3 cells
and A549 cell lines. EV- o mula ions om PC-3 cell
line es ed in A549 cell line: con ol EVs, EV-PTX,
EV-Vi us, EV-Vi us-PTX. O he o mula ions es ed
in A549 cell line: Vi us alone, PTX, Vi us + PTX. EV-
o mula ions om A549 cell line es ed in PC-3 cell
line: con ol EVs, EV-PTX, EV-Vi us, EV-Vi us-PTX.
O he o umula ions es ed in PC-3 cell line: Vi us
alone, PTX, Vi us + PTX.
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