REVIEW
published: 12 Oc obe 2018
doi: 10.3389/ immu.2018.02270
F on ie s in Immunology | www. on ie sin.o g 1Oc obe 2018 | Volume 9 | A icle 2270
Edi ed by:
Ch is oph Kahle ,
Uni e si ä sklinikum Ca l Gus a
Ca us, Ge many
Re iewed by:
Mallika jun Bida ima h,
Co nell Uni e si y, Uni ed S a es
Niels Halama,
Na ionales Cen um ü
Tumo e k ankungen (NCT), Ge many
*Co espondence:
Ma ia J. Pe ugo ia
[email p o ec ed]
Jesus M. Banales
[email p o ec ed]
†These au ho s sha e senio
au ho ship
Special y sec ion:
This a icle was submi ed o
Immunological Tole ance and
Regula ion,
a sec ion o he jou nal
F on ie s in Immunology
Recei ed: 18 Feb ua y 2018
Accep ed: 12 Sep embe 2018
Published: 12 Oc obe 2018
Ci a ion:
Lapi z A, A belaiz A, Olaizola P,
A anbu u A, Bujanda L, Pe ugo ia MJ
and Banales JM (2018) Ex acellula
Vesicles in Hepa obilia y Malignancies.
F on . Immunol. 9:2270.
doi: 10.3389/ immu.2018.02270
Ex acellula Vesicles in
Hepa obilia y Malignancies
Ainhoa Lapi z1, Ande A belaiz1, Paula Olaizola1, Ai zibe A anbu u1, Luis Bujanda1,2,
Ma ia J. Pe ugo ia1,2,3*†and Jesus M. Banales1,2,3*†
1Depa men o Li e and Gas oin es inal Diseases, Biodonos ia Resea ch Ins i u e, Donos ia Uni e si y Hospi al, Uni e si y
o he Basque Coun y (UPV/EHU), San Sebas ian, Spain, 2“Cen o de In es igación Biomédica en Red de En e medades
Hepá icas y Diges i as” (CIBERehd), Ca los III Na ional Ins i u e o Heal h, Mad id, Spain, 3IKERBASQUE, Basque
Founda ion o Science, Bilbao, Spain
P ima y hepa obilia y malignancies include a he e ogeneous g oup o cance s wi h dismal
p ognosis, among which hepa ocellula ca cinoma (HCC), cholangioca cinoma (CCA),
and hepa oblas oma (HB) s and ou . These umo s mainly a ise om he malignan
ans o ma ion o hepa ocy es, cholangiocy es (bile duc epi helial cells) o hepa oblas s
(emb yonic li e p ogeni o cells), espec i ely. Ea ly diagnosis, p ognosis p edic ion and
e ec i e he apies a e s ill a u opia o hese diseases. Ex acellula esicles (EVs) a e
small memb ane-enclosed sphe es sec e ed by cells and p esen in biological luids.
They con ain mul iple ypes o biomolecules, such as p o eins, RNA, DNA, me aboli es
and lipids, which make hem a po en ial sou ce o bioma ke s as well as egula o s o
human pa hobiology. In his e iew, he ole o EVs in he pa hogenesis o hepa obilia y
cance s and hei po en ial use ulness as disease bioma ke s a e highligh ed. Mo eo e ,
he he apeu ic alue o EV egula ion is discussed and u u e di ec ions on basic and
clinical esea ch a e indica ed.
Keywo ds: ex acellula esicles, hepa ocellula ca cinoma, cholangioca cinoma, hepa oblas oma, pa hogenesis,
he apy, diagnosis
INTRODUCTION
Li e cance is a majo heal h p oblem wo ldwide, ep esen ing he second leading cause o all
cance - ela ed dea hs (1). This cance in ol es a he e ogeneous se o hepa obilia y malignancies
including hepa ocellula ca cinoma (HCC), cholangioca cinoma (CCA), and hepa oblas oma
(HB), which mainly a ise om he malignan ans o ma ion o hepa ocy es, cholangiocy es,
and hepa oblas s, espec i ely (1,2). Ea ly non-in asi e diagnosis, p edic ion o p ognosis
and ea men - esponse, as well as e ec i e pe sonalized he apies a e s ill a challenge, highly
comp omising pa ien ou come (3–5).
HCC is he six h mos p e alen malignan umo (10:100,000 incidence) and is s ongly
associa ed (∼90%) wi h he p esence o li e ci hosis (LC) caused by alcohol, i al in ec ions
[hepa i is B (HBV) o C (HCV) i uses], and/o s ea osis, among o he s (5,6). CCA is a a e cance ,
bu i s incidence (∼5/100.000) is inc easing wo ldwide. Al hough he e iology o he majo i y o
CCAs is unknown, se e al isk ac o s may p edispose o i s de elopmen , including he p esence
o p ima y scle osing cholangi is (PSC), li e luke in ec ions (endemic om Eas Asia), ci hosis
and congeni al bilia y diso de s (3). On he o he hand, HB is he mos common pedia ic li e
malignancy, p incipally a ec ing child en be ween 6 mon hs and 3 yea s o age. HB is esponsible
Lapi z e al. EVs in Li e Cance
o up o ∼1% o all pedia ic cance s, wi h an annual incidence
o 0.5–1.5 cases (4,7). Despi e mos HB cases a e spo adic, some
o hem ha e been associa ed wi h he edi a y cance synd omes
including amilial adenoma ous polyposis (FAP) and Beckwi h-
Widemann synd ome (BWS), as well as wi h p ema u i y o low
bi h weigh (4,7). Since hepa obilia y malignances a e usually
diagnosed in la e s ages and a e highly chemo esis an , he
comple e su gical esec ion o he umo s o li e ansplan a ion
cons i u e he only po en ial cu a i e op ions. Howe e , hese
he apeu ic s a egies a e exclusi ely indica ed unde ce ain
s ic and conse a i e clinical c i e ia (3,5,6). The e o e,
he e is an u gen need o de e mine new accu a e non-in asi e
bioma ke s o he ea ly diagnosis o hese diseases, as well
as o moni o and p edic disease p og ession and ea men
esponse. Mo eo e , new e ec i e pe sonalized ea men s a e
desi able in o de o imp o e he ou come and li e quali y o
pa ien s.
Du ing he las decade, ex acellula esicles (EVs) ha e
opened new oppo uni ies o non-in asi e diagnosis and
moni o ing o human diseases. Thei p esence in biological luids
(se um, u ine, bile, sali a, e c.) and hei unique and di e se
biomolecula composi ion (p o eins, RNA, DNA, me aboli es,
and lipids) make EVs excellen candida es as a sou ce o
bioma ke s (8,9). Fu he mo e, since EVs pa icipa e in
in e cellula communica ion in human heal h and disease, hey
ha e been pos ula ed as po en ial ools o a ge s o he apy.
EVs a e small memb ane-encapsula ed sphe es p oduced and
sec e ed by cells h ough complex and p ecise molecula
Abb e ia ions: 4F2hc, 4F2 cell-su ace an igen hea y chain; A1AG1, alpha-
1-acid glycop o ein 1; A2MG, alpha-2-mac oglobulin; ADAM10, disin eg in
and me allop o einase domain-con aining p o ein 10; ADMSCs, adipose issue-
de i ed mesenchymal s em cells; AFP, alpha- e op o ein; AMPN, aminopep idase
N; ASGPR1, asialoglycop o ein ecep o ; APCs, an igen p esen ing cells;
ASPGPR1, asialoglycop o ein ecep o 1; AUC, a ea unde he ecei e ope a ing
cu e; BWS, Beckwi h-Widemann synd ome; CAF, cance associa ed ib oblas ;
CBD, common bile duc ; CCA, cholangioca cinoma; CCL2/MCP-1, chemokine
(C-C mo i ) ligand 2; CCNG1, cyclin G1; CDC, cell di ision p o ein; CDK,
cyclin-dependen kinase; CHB, ch onic hepa i is B; CME, cla h in-media ed
endocy osis; CXCL1, chemokine (C-X-C mo i ) ligand 1; DC, dend i ic cell;
DMA, dime hyl amilo ide; EGFR, epide mal g ow h ac o ecep o ; EIPA, 5-
e hyl-N-isop opyl amilo ide; EPCAM, epi helial cell adhesion molecule; ESCRT,
endosomal so ing complex equi ed o anspo ; EVs, ex acellula esicles;
FAP, amilial adenoma ous polyposis; FCN2, icolin-2; FIBG, ib inogen gamma
chain; HB, hepa oblas oma; HBV, hepa i is B i us; HCV, hepa i is C i us;
HCC, hepa ocellula ca cinoma; HGF, hepa ocy e g ow h ac o ; HSPs, hea shock
p o eins; HSPG, hepa in sul a e p o eoglicans; ICAM-1, in e cellula adhesion
molecule 1; IGF1R, insulin-like g ow h ac o 1 ecep o ; IL, in e leukin; ILVs,
in aluminal esicles; ITGB4, in eg in β4; ITIH4, in e -alpha- ypsin inhibi o
hea y chain H4; LC, li e ci hosis; LG3BP, galec in-3-binding p o ein; linc-
ROR, long in e genic non-p o ein coding RNA egula o o ep og amming;
lncRNA, long non-coding RNA; MβCD, me hyl-b-cyclodex in; MHC, majo
his ocompa ibili y complex; MICB, MHC class I polypep ide- ela ed sequence
B; MMP, ma ix me allop o einase; MSC, mesemchymal s em cell; MVs,
mic o esicles; MVE, mul i esicula endosome; NK cell, na u al kille cell; NKT
cell, na u al kille T cell; nSMase, neu al sphyngomyelinase; PAFs, pa a-
cance ib oblas s; PBX3, p e-B-cell leukemia ansc ip ion ac o 3; PI3K,
phosphoinosi ide-3-kinase; PIGR, polyme ic immunoglobulin ecep o ; PLD2,
phospholipase D2; PS, phospha idylse ine; PSC, p ima y scle osing cholangi is;
SNARE, soluble N-e hylmaleimide-sensi i e ac o a achmen p o ein ecep o ;
TAMPs, umo -associa ed mic opa icles; VEGF, ascula endo helial g ow h
ac o ; VNN1, pan e heinase; VTDB, i amin-D binding p o ein.
mechanisms (10–13). T adi ionally, EVs a e classi ied acco ding
o hei biogenesis in o exosomes, mic o esicles (MVs) o
mic opa icles, and apop o ic bodies (11,12). Exosomes a e
e e ed o hose EVs p oduced inside he mul i esicula
endosomes (MVEs) o he cells. Thei mo phology is sphe ical
and he size anges be ween 40 and 150 nm in diame e
(11,14,15). Cell MVEs a e esicula en i ies gene a ed in
he ma u a ion p ocess o he ea ly endosomes, and whe e
in aluminal esicles (ILVs) a e o med by he in agina ion
o he MVE memb ane. ILVs a e he incipien exosomes ha
a e eleased o he ex acellula media upon he usion o he
MVEs wi h he plasma memb ane o he cell (11). On he
o he hand, MVs o mic opa icles o igina e om he di ec
budding o he cell plasma memb ane. Thei size (40–1000 nm)
and mo phology a e he e ogeneous (15,16). Apop o ic bodies
a e esicles p oduced by cells unde going apop osis. Thus, hei
size (∼40 o 2000–5000 nm) and mo phology a e di e se (15,
16). Al hough his classi ica ion is widely accep ed, o da e,
he e a e no speci ic bioma ke s o di e en ia e exosomes om
o he ypes o nano-sized esicle popula ions, limi ing hei
speci ic isola ion om bio luids. The e o e, he smalles esicles
(nano- esicles) p esen and isola ed om biological luids
comp ise a mix o exosomes and plasma memb ane-de i ed
esicles.
EVs ha e changed he pa adigm o in e cellula
communica ion, which was adi ionally es ic ed o he
au oc ine, pa ac ine and endoc ine in e ac ion h ough soluble
p o eins and lipids, o h ough di ec cell- o-cell con ac
media ed by p o eins, gap junc ions, o unneling nano- ubes in
plu icellula o ganisms (17,18). Acco dingly, EVs con ain an
aqueous lumen and a speci ic subse o memb ane and soluble
p o eins, nucleic acids (DNA and RNA), lipids and me aboli es
ha can be ho izon ally ans e ed o local o dis an cells by
di ec EV-cell memb ane con ac , usion o in e naliza ion (12).
The impo ance o EVs is highligh ed by he ac ha hei
composi ion is speci ic depending on he cell s a us and on he
ecei ed s imuli (19,20), which indica es a ce ain deg ee o
selec i e packaging. EVs con e p o ec ion o he biomolecules
enclosed inside he lipid bilaye , p e en ing hei enzyma ic
deg ada ion (21).
EVs pa icipa e in he egula ion o mul iple cance hallma ks.
They can ansmi oncogenic signals by ans e ing p o- umo
RNAs and p o eins ha egula e di e se key p ocesses in umo
p og ession such as p oli e a ion, su i al, di e en ia ion, and
in asion/mig a ion o cance cells (22–30). Addi ionally, EVs
a e in ol ed in he c oss alk be ween umo cells and s oma,
p omo ing in lamma ion (31), cell ma ix emodeling (32),
neo ascula iza ion o angiogenesis (33,34), chemo esis ance
(35–37), o ma ion o he me as a ic niche (38,39), and
inhibi ion o he an i- umo immune esponse (40–42).
The e o e, EVs ep esen key a ge s o he apy a a ious
le els, including p oduc ion, elease and up ake by a ge cells.
Addi ionally, he blockage o emo al o umo -de i ed EVs by
aphe esis wi h speci ic de ices cons i u es a po en ial he apeu ic
app oach. O no e, EVs a e also excellen candida es o he
deli e y o new an i-cance p o eins, RNAs, me aboli es, d ugs
o cance accines.
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Lapi z e al. EVs in Li e Cance
EV BIOGENESIS AND REGULATION
EV p oduc ion is a highly egula ed and complex cellula p ocess
whe e se e al p o ein ne wo ks and di e se in acellula signals
a e in ol ed (Figu e 1). Among he di e en EV popula ions, he
exosome p oduc ion machine y is he bes s udied. Ne e heless,
bo h exosomes and MVs sha e nume ous mechanisms ha
pa icipa e in hei biogenesis, elease, and up ake (43).
Rega ding he mechanisms in ol ed in he biogenesis o
exosomes, he endosomal so ing complex equi ed o anspo
(ESCRT) machine y has been epo ed o pa icipa e in MVE and
ILV gene a ion (44). Howe e , ESCRT independen mechanisms
ha e also been desc ibed in he o ma ion o exosomes, including
ce amide p oduc ion by neu al ype II sphingomyelinase
(nSMase2) (45,46), lipid a s (47), phospholipase D2 (PLD2)
(48,49), and e aspanin amily o p o eins (e.g., CD9, CD63, and
CD81) (50,51), which o m dynamic memb ane mic odomains
ha p omo e hei budding and assu e exosome o ma ion.
In acellula a icking o MVEs is coo dina ed by he
cy oskele on and mo o p o eins such as dynein (52) and
Rab amily o GTPases (Figu e 1) (53). MVEs use wi h he
plasma memb ane ia SNARE p o eins, inally allowing exosome
sec e ion (53,54). In con as , MVs a ise as a esul o he di ec
budding o he cell plasma memb ane. Thei biogenesis equi es
Ca2+-dependen memb ane phospholipid and cy oskele on
ea angemen s, which enable MV blebbing and elease (55).
Released EVs a e ecognized by he ecipien cells h ough
speci ic in e ac ions be ween hei memb ane componen s.
These include in eg ins, lipids, e aspanins, p o eoglycans,
among o he s (56). Plasma memb ane-bound EVs can be
in e nalized h ough cla h in-media ed endocy osis (CME)
o cla h in-independen p ocesses ha include phagocy osis,
mac opinocy osis and lipid a s.
Se e al expe imen al s a egies ha e a ge ed he
a o emen ioned mechanisms o in e e e wi h EV p oduc ion,
elease and up ake a di e en le els (Figu e 1). Among hese,
in e en ion on ce amide p oduc ion has been he mos widely
used s a egy o dec ease exosome p oduc ion in cance cells
and he eby abolish he mul iple oncogenic e ec s o umo -
de i ed exosomes in se e al cance s. Expe imen al inhibi ion
o nSMase2, esponsible o ce amide p oduc ion, wi h i s
inhibi o GW4869 educes exosome sec e ion (45,57) and
sensi izes cance cells o chemo he apy (58). O no e, he
p esence o GW4869 inhibi s he mig a o y capaci y o CCA
cells (31). Se e al in e cellula signals in ol ed in he egula ion
o he EV p oduc ion a e also unde in es iga ion, including
he educ ion o in acellula Ca2+concen a ion. In ac , he
Na+/Ca+2exchange inhibi o dime hyl amilo ide (DMA) leads
o diminished EV p oduc ion in lymphoma cells, esul ing in
an enhanced an i- umo immune esponse (40). Rega ding
he p o eins ha pa icipa e in he anspo o exosomes,
Rab amily p o eins a e key media o s o MVE ansi o he
plasma memb ane, hei inhibi ion being linked o a dec ease
o exosome elease (59). Acco dingly, he ep ession o Rab27a
diminished g ow h and dissemina ion o cance cells in i o
(39,60).
Di e en complex mechanisms, including p o ein and/o lipid
in e ac ions be ween EV and ecipien cell su ace componen s,
a e equi ed o he EV cell up ake (Figu e 1). These mechanisms
include phagocy osis, mac opinocy osis, cla h in, and ca eolin
dependen endocy ic pa hways, as well as lipid a -media ed
and memb ane usion p ocesses (56). The e o e, aiming o
block umo EV up ake p ocesses, di e en componen s o
hese machine ies ha e been a ge ed. Memb ane p o eins
including e aspanins, in eg ins, lec ins, p o eoglycans, majo
his ocompa ibili y complex (MHC) molecules, glycop o eins and
o he ecep o s a e in ol ed in EV- ecipien cell in e ac ion.
Te aspanins, en iched p o eins p esen in EVs and well-
es ablished ma ke s o hese esicles (61,62), pa icipa e in EV-
cell su ace adhesion media ing hei up ake. Thus, an ibody-
based inhibi ion o he CD81 and CD9 e aspanins as well
as he blockade o αV and β3 in eg ins, hampe s EV up ake
(63,64). Lec ins, such as galec in-5, can also be a ge ed
wi h he glycop o ein asialo e uin o in e e e wi h EV-cell
in e ac ion and he subsequen cellula in e naliza ion (65).
Likewise, a ge ing he lec in ecep o s DC-SIGN o DEC-205
wi h speci ic an ibodies also esul s in a educ ion o EV up ake
(66,67). Hepa in can also block he in e naliza ion o cance
EVs by binding o he cell su ace hepa an sul a e p o eoglycans
(68,69). Fu he mo e, he pi o al in e ac ion be ween EVs and
he plasma memb ane o he cell can be pa ially inhibi ed
by p o einase K ea men , blocking EV ecogni ion and he
subsequen endocy ic p ocess in cance cells (70).
The bes s udied EV in e naliza ion mechanisms a e
ela ed wi h he endocy ic pa hway (63,70,71). Since hese
p ocesses depend on he cy oskele on, he inhibi ion o
ac in polyme iza ion by cy ochalasin D educes EV up ake
by phagocy osis (56,70,71). In addi ion, he up ake o
EVs by mac ophages can be ab oga ed by he inhibi ion
o phosphoinosi ide-3-kinase (PI3K) wi h wo mannin o
LY294002 (72). Mo eo e , inhibi o s o mac opinocy osis
o CME [5-e hyl-N-isop opyl amilo ide (EIPA) and
chlo p omazine, espec i ely] educe umo -de i ed EV
in e naliza ion (70). Dynaso e can also impai CME h ough he
inhibi ion o dynamin 2, needed o cla h in-coa ed endosome
memb ane ission (65,73–76). On he o he hand, ce ain
endocy ic p ocesses a e closely ela ed o lipid a s, and he
in e en ion on hei composi ion impai s he up ake o EVs.
Thus, he use o he glycosphingolipid syn hesis inhibi o s [i.e.,
umosinin B1 and N-bu yldeoxynoji imycin hyd ochlo ide (also
known as CAS72599)] educes EV up ake (77). Choles e ol
educing agen s including me hyl-be a-cyclodex in (MβCD)
(70,78,79), ilipin (71,79) and sim as a in, as well as he
inhibi ion o ERK1/2 signaling by U0126 may also impai he
up ake o EVs (79). Masking phospha idylse ine (PS), p esen
in he memb ane su ace o EVs, by Diannexin and blocking i s
ecep o TIM4 inhibi s epide mal g ow h ac o ecep o (EGFR)
ans e om umo EVs o endo helial cells esul ing in educed
umo g ow h and mic o ascula densi y in i o (80). EVs can
also elease hei con en in o he ecipien cells by he di ec
usion o plasma and EV memb anes. This usion is enhanced in
acidic condi ions, a gene al ea u e o cance cells (81). In his
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Lapi z e al. EVs in Li e Cance
FIGURE 1 | Regula o y mechanisms o EV biogenesis, elease, and up ake. Exosome elease can be inhibi ed by in e e ing hei biogenesis (e.g., ce amide
p oduc ion) o he memb ane usion o he mul i esicula endosome (MVE) wi h he plasma memb ane (e.g., Rab27). Once EVs a e eleased o he ex acellula milieu,
hei up ake can be blocked by in e e ing he EV-plasma memb ane p o ein in e ac ions (e.g., Te aspanins), cla h in- and ca eolin-dependen endocy osos (e.g.,
Dynaso e), phagocy osis (e.g., Wo mannin), and by inhibi ing lipid- a media ed endocy osis (e.g., Filipin). DFMO, di luo ome hylo ni hine; DMA, dime hyl amilo ide;
ESCRT, endosomal so ing complex equi ed o anspo ; EVs, ex acellula esicles; HSPG, hepa an sul a e p o eoglycans; ICAM-1, in e cellula adhesion molecule
1; MβCD, me hyl-β-cyclodex in; nSMase, neu al sphingomyelinase; PS, phospha idylse ine.
sense, p o on pump inhibi ion leads o educed EV up ake by
cance cells (82).
EVs IN HEPATOBILIARY CANCERS
Hepa ocellula Ca cinoma
HCC cell-de i ed EVs pa icipa e in au oc ine and/o
pa ac ine cellula communica ions, egula ing umo g ow h,
chemo esis ance, angiogenesis, and dissemina ion. Se e al lines
o e idence indica e ha HCC cell-de i ed EVs p omo e umo
esis ance agains chemo he apeu ic d ugs such as so a enib,
doxo ubicin o camp o hecin. Fo ins ance, an en ichmen o
long in e genic non-coding RNA egula o o ep og amming
(linc-ROR) in EVs de i ed om so a enib- ea ed HCC
cells p e en s chemo he apy-induced apop osis h ough p53
ep ession and inc eases he exp ession o umo -ini ia ing
li e cance s em cell CD133 ma ke (83). Ano he molecula
mechanism in ol ed in HCC cell-de i ed EV-induced so a enib
esis ance includes he ac i a ion o he hepa ocy e g ow h
ac o (HGF)/c-Me /Ak signaling pa hway in li e cance
cells (84).
EVs de i ed om HCC cells may also egula e angiogenesis
(85). Expe imen al in i o models indica e ha EVs de i ed
om CD90+li e cance cells (i.e., cance s em-like cells p esen
in p ima y umo s and blood o HCC pa ien s, associa ed wi h
me as asis as well as bad p ognosis) a e en iched in long non-
coding RNA (lncRNA) H19, which p omo es he exp ession
o ascula endo helial g ow h ac o (VEGF) and i s ecep o
VEGF-R1 in endo helial cells. Mo eo e , lncRNA H19 s imula es
ube o ma ion as well as cell-adhesion p ope ies in endo helial
cells, inducing he exp ession o in e cellula adhesion molecule
1 (ICAM-1) in his cell- ype.
Conce ning he ole o EVs in me as asis, se e al s udies ha e
epo ed ha EVs sec e ed om HCC cells o om adjacen
cells a e also in ol ed in he p omo ion o umo cell me as asis
(86). T ansc ip omic and p o eomic p o iling e ealed ha EVs
de i ed om me as a ic HCC cells ca y a la ge numbe o p o-
umo igenic RNAs and p o eins, such as MET p o o-oncogene,
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Lapi z e al. EVs in Li e Cance
S100 amily membe s (S100A4, S100A10, and S100A11) and
he ca eolins (CAV1 and CAV2). HCC-de i ed EVs igge he
ac i a ion o PI3K/Ak and MAPK signaling pa hways and he
sec e ion o ac i e MMP2 and MMP9 ma ix me allop o einases
(MMPs) in hepa ocy es, which in u n enhance hei mig a o y
and in asi e abili y. On he o he hand, cance -associa ed
ib oblas (CAF)-de i ed EVs may also con ibu e o HCC cell
p oli e a ion and me as asis (87). Thus, a educ ion in he miR-
320a le el was obse ed in CAF-de i ed EVs compa ed o pa a-
cance ib oblas s (PAFs). This miR-320a di ec ly a ge s p e-B-
cell leukemia ansc ip ion ac o 3 (PBX3), supp essing HCC cell
p oli e a ion, mig a ion, and in asion. The an i- umo e ec s o
miR-320a we e con i med in i o using HCC umo xenog a
models, in which umo g ow h was inhibi ed when HCC cells
we e co-injec ed wi h miR-320a o e -exp essing CAFs in o nude
mice. Besides EVs de i ed om CAFs, inna e immune cell-
de i ed MVs ha e also been epo ed o enhance HCC me as asis
h ough CD11b and CD18, also known as in eg in αMβ2(88).
Cholangioca cinoma
The p esence o EVs in bile and hei ole egula ing
cholangiocy e physiology was i s desc ibed in mu ine models
(89). Howe e , EVs also play a ole in bilia y pa hobiology. In
CCA umo s, se e al epo s ha e emphasized he impo ance
o EVs in he egula ion o he in e play be ween CCA cells
and he cells p esen in he umo s oma. CCA cell-de i ed
EVs a o he ib oblas ic di e en ia ion o bone ma ow-
de i ed mesenchymal s em cells (MSCs) and he sec e ion
o p o-in lamma o y cy okines and chemokines, including
in e leukin (IL)-6, chemokine (C-X-C mo i ) ligand 1 (CLXC1),
and chemokine (C-C mo i ) ligand 2 (CCL2/MCP-1), which
ul ima ely s imula e CCA cell p oli e a ion ia IL6/STAT3
signaling pa hway (31).
CCA-de i ed EVs may con ain oncogenic biomolecules no
only in ol ed in modula ing in lamma o y and p oli e a i e
esponses bu also con olling mig a o y and me as a ic
p ocesses. Two s udies employing compa a i e p o eomic
app oaches ha e explo ed he p o ein con en o CCA-
de i ed and cholangiocy e-de i ed EVs in i o, iden i ying
signi ican di e ences and a pa icula oncogenic p o ein p o ile
ela ed o p oli e a ion and mo ili y in cance cell-de i ed
EVs (90,91). Di e en ially exp essed p o eins in ol ed in
cholangioca cinogenesis included EGFR, Mucin-1, in eg in
β4 (ITGB4), and epi helial cell adhesion molecule (EPCAM)
(90). EGFR pa icipa es in CCA p og ession, a o ing he
dedi e en ia ion and in asi eness o umo cells and ep esen s
a bad p ognos ic ac o (92,93). Simila ly, Mucin-1 and EPCAM,
which a e also up egula ed in CCA, co ela e wi h poo ou come
in pa ien s wi h CCA (94–96). In e es ingly, ITGB4 has ecen ly
been desc ibed as an EV in eg in ha dic a es u u e me as a ic
si es, con ibu ing o p e e en ial o gano opism o umo
cells (38). On he o he hand, EVs sec e ed by li e - luke
associa ed CCA cells induce cholangiocy e p oli e a ion (97)
and in asion (91), e en s ha a e associa ed wi h an en ichmen
o oncop o eins in EVs, including galec in-3 binding p o ein
(LG3BP), p os aglandin F2 ecep o nega i e egula o , 4F2
cell-su ace an igen hea y chain (4F2hc), in eg in-β1 and
EPCAM (91).
NON-INVASIVE BIOMARKERS
The p esence o EVs in biological luids and hei di e se
molecula ca go has ecen ly placed EVs as a new sou ce o
non-in asi e disease bioma ke s. Indeed, po en ial bioma ke
candida es (miRNAs and p o eins) ha e been desc ibed in se um-
and bile-de i ed EVs o he diagnosis and/o he p ognosis
p edic ion o HB, HCC, and CCA (Table 1).
In HB pa ien s, se um EV miR-21 le els we e highe han in
heal hy child en, and nega i ely co ela ed wi h pa ien su i al
(98). On he o he hand, dec eased le els o miR-34a, miR-
34b, and miR-34c we e epo ed in se um EVs om HB in an s
compa ed o heal hy indi iduals. Combina ion o hese miRs
showed highe diagnos ic alue han he gold s anda d alpha
e op o ein (AFP) (99). Fu he mo e, educed le els o he miR-
34 panel in EVs o HB we e associa ed wi h lowe o e all su i al
(99).
In HCC pa ien s, le els o miRs 18a, 221, 222, and 224 in
se um EVs we e ound up egula ed compa ed o pa ien s wi h
ch onic hepa i is B (CHB) o li e ci hosis, pa ien s, whe eas
miR-101 le el was down egula ed (105). Likewise, inc eased
exp ession o miR-21 was iden i ied in se um EVs om pa ien s
wi h HCC compa ed o CHB pa ien s o heal hy indi iduals, and
co ela ed wi h ci hosis and ad anced umo s age (106). MiR-
665 in se um EVs may also be a po en ial p ognos ic bioma ke
o HCC, as high miR-665 le els posi i ely co ela ed wi h la ge
umo size, local in asion and ad anced clinical s ages (s age
III/IV), and nega i ely wi h o e all su i al (107). Mo eo e ,
diminished le els o se e al miRNAs in se um EVs ha e been
sugges ed as p edic o s o HCC ecu ence o o e all su i al
(108,109). MiR exp ession p o iling in se um EVs iden i ied he
umo supp esso miR-718 down egula ed in pa ien s wi h la ge
umo diame e s and ecu ence. Reduced miR-718 exp ession
also co ela ed wi h poo his ological umo cell di e en ia ion
(108). Fu he mo e, low le els o miR-125b in se um EVs
ha e been linked o ad anced TNM s ages and encapsula ion,
sugges ing his miR as a po en ial p ognos ic candida e o
ecu ence and o e all su i al (109). Besides miRNAs, di e en
p o eins p esen in se um EVs such as LG3BP, polyme ic
immunoglobulin ecep o (PIGR) and alpha-2-mac oglobulin
(A2MG) we e ound up egula ed in HCC pa ien s compa ed o
heal hy indi iduals, wi h a be e diagnos ic alue han AFP (90).
Apa om changes in he EV ca go, he EV concen a ion i sel
could also se e as a disease bioma ke . In ac , s age I and II HCC
pa ien s showed highe EV concen a ion in se um compa ed o
pa ien s wi h li e ci hosis (100).
In CCA, a panel o miRs (191, 486-3p, 1274b, 16, 484)
was ound up egula ed in bile EVs o pa ien s wi h CCA
compa ed o a con ol g oup con aining PSC, bilia y obs uc ion
and bile leak synd ome pa ien s (102). The analysis o he
lncRNA p o ile in bile EVs om CCA pa ien s s. pa ien s
wi h bilia y obs uc ion iden i ied he up egula ion o wo
lncRNAs (i.e., ENST00000588480.1 and ENST00000517758) in
F on ie s in Immunology | www. on ie sin.o g 5Oc obe 2018 | Volume 9 | A icle 2270
Lapi z e al. EVs in Li e Cance
TABLE 1 | EVs as non-in asi e bioma ke s o hepa obilia y malignancies.
Disease Name Bioma ke
ype
EV
sou ce
Numbe o pa ien s Exp ession SEN (%) SPE (%) AUC Re e ences
HB miR-21 miRNA Se um HB (n=32) s. Heal hy
indi iduals (n=32)
Up — — 0.861 (98)
miR-34a* Se um HB (n=63) s. Heal hy
indi iduals (n=63)
Down — — 0.963 (99)
miR-34b* — —
miR-34c* — —
HCC LG3BP P o ein Se um HCC (n=29) s. Heal hy
indi iduals (n=32)
Up 96.6 71.8 0.904 (90)
PIGR 82.8 71.8 0.837
A2MG 92.9 56.2 0.796
MV (ug/mL) Mic o esicle
concen a ion
Blood S age I HCC (n=28) s.
Ci hosis (n=40)
Up — — 0.83 (100)
S age II HCC (n=20) s.
Ci hosis (n=40)
Up — — 0.94
AnnexinV+EpCAM+
(mic opa icle/mL)
TAMP
concen a ion
Se um HCC (n=86) s. Heal hy
indi iduals (n=58)
Up — — 0.77 (101)
AnnexinV+EpCAM+
ASGPR1+
(mic opa icle/mL)
Se um HCC (n=86) s. Ci hosis
(n=49)
Up — — 0.73
CCA FIBG P o ein Se um iCCA (n=12) s. HCC (n=29) Up 83.3 89.6 0.894 (90)
A1AG1 83.3 82.1 0.845
VTDB 75 89.2 0.823
AMPN Se um CCA (n=43) s. Heal hy
indi iduals (n=32)
Up 90.7 65.6 0.878
VNN1 72.1 87.5 0.876
PIGR 83.7 71.8 0.844
PIGR Se um CCA I-II (n=13) s. Heal hy
indi iduals (n=22)
Up 75 95.4 0.905
AMPN 91.7 72.7 0.833
FIBG 100 68.1 0.833
FIBG Se um CCA (n=43) s. PSC (n=30) Up 88.4 63.3 0.796
A1AG1 76.7 70 0.794
S10A8 69.8 66.6 0.759
FCN2 Se um CCA I-II (n=13) s. PSC
(n=30)
Up 100 80.9 0.956
ITIH4 91.7 80.9 0.881
FIBG 91.7 80.9 0.881
miR-191* miRNA Bile CCA (n=46) s. Con ol
(n=50; including PSC, bilia y
obs uc ion and bile leak)
Up 67 96 — (102)
miR-486-3p*
miR-1274b*
miR-16*
miR-484*
ENST00000588480.1* lncRNA Bile CCA (n=35) s. Con ol (n=56) Up 82.9 58.9 0.709 (103)
ENST00000517758.1*
Nanopa icles/L EV
concen a ion
Bile Malignan CBD s enoses
(panc ea ic cance ; n=10 and
CCA; n=5) s. nonmalignan
CBD s enoses (ch onic
panc ea i is; n=15)
Up — — 1 (104)
(Con inued)
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Lapi z e al. EVs in Li e Cance
TABLE 1 | Con inued
Disease Name Bioma ke
ype
EV
sou ce
Numbe o pa ien s Exp ession SEN (%) SPE (%) AUC Re e ences
AnnexinV+EpCAM+
ASGPR1+
(mic opa icle/mL)
TAMP
concen a ion
Se um CCA (n=38) s. Ci hosis
(n=49)
Up — — 0.63 (101)
Li e
cance
(HCC/CCA)
AnnexinV+EpCAM+
ASGPR1+
(mic opa icle/mL)
TAMP
concen a ion
Se um Li e umo (HCC; n=86 and
CCA; n=38) s. Ci hosis
(n=49)
Up — — 0.7 (101)
A1AG1, alpha-1-acid glycop o ein 1; A2MG, alpha-2-mac oglobulin; AMPN, aminopep idase N; ASPGPR1, asialoglycop o ein ecep o 1; AUC, a ea unde he ecei e ope a ing cu e;
CBD, common bile duc ; CCA, cholangioca cinoma; EpCAM, epi helial cell adhesion molecule; FCN2, icolin-2; FIBG, ib inogen gamma chain; iCCA, in ahepa ic cholangioca cinoma;
ITIH4, in e -alpha- ypsin inhibi o hea y chain H4; HB, hepa oblas oma; HCC, hepa oca cinoma; LG3BP, galec in-3-binding p o ein; lncRNA, long non-coding RNA; miR, mic oRNA;
MV, mic o esicle; PIGR, polyme ic immunoglobulin ecep o ; PSC, p ima y scle osing cholangi is; SEN, sensi i i y; SPE, speci ici y; TAMP, umo -associa ed mic opa icle; VNN1,
pan e heinase; VTDB, i amin-D binding p o ein. *bioma ke panel.
CCA pa ien s (103). The combined exp ession o bo h lncRNAs
showed ele an diagnos ic and p ognos ic alue, being inc eased
in ad anced TNM s ages (III-IV) and showing wo se o e all
su i al a high lncRNA concen a ions. On he o he hand,
di e en p o eins p esen in se um EVs exhibi ed high diagnos ic
alues when compa ing CCA pa ien s wi h heal hy indi iduals,
such as aminopep idase N (AMPN), pan e heinase (VNN1),
and PIGR (90). Some p o eins p esen in se um EVs, such as
icolin-2 (FCN2), in e -alpha- ypsin inhibi o hea y chain H4
(ITIH4) and ib inogen gamma chain (FIBG), displayed be e
diagnos ic alues han CA19-9 (a non-speci ic umo ma ke o
he diagnosis o CCA) in he di e en ial diagnosis be ween CCA
(s age I-II) and PSC (90). Nowadays, he di e en ial diagnosis
be ween in ahepa ic CCA (iCCA) and HCC by non-in asi e
me hods is no easible and comp omises adequa e ea men .
In his ega d, p o eins p esen in se um EVs—such as FIBG,
alpha-1-acid glycop o ein 1 (A1AG1) and i amin-D binding
p o ein (VTDB)—exhibi ed highe accu acy han CA19-9 and
AFP o he di e en ial diagnosis o iCCA s. HCC (90). As
a o emen ioned, he EV concen a ion analysis could also be
ele an o he diagnosis o malignan bilia y diseases. In
his ega d, bile EV concen a ion was epo ed o accu a ely
disc imina e be ween malignan common bile duc (CBD)
s enosis and nonmalignan CBD s enosis (104). In addi ion,
ele a ed concen a ion o AnnexinV/EpCAM/ASGPR1 posi i e
umo -associa ed mic opa icles (TAMPs) allowed he diagnosis
o pa ien s wi h li e cance (HCC and CCA) compa ed o
ci ho ic pa ien s, while no changes we e de ec ed be ween HCC
and CCA (101). No ably, he le els o hese TAMPs dec eased 7
days a e he su gical esec ion o li e umo s, closely ela ing
his mic opa icle popula ion wi h umo p esence.
THERAPEUTIC IMPLICATIONS
The use o EVs in an i-cance he apy is cu en ly unde
in es iga ion. As EVs ca y di e en ypes o molecules, hey can
be used as ehicles o deli e he apeu ic ca go in o cance cells
(110). Mo eo e , EVs ha e shown he abili y o modula e he
immune sys em, and o s imula e he immune esponse agains
umo cells (111).
Molecule Ca ie s
EVs as he apeu ic deli e y sys ems p o ide bene i s o
he ca ied he apeu ic molecule. Hence, encapsula ion o
he apeu ic compounds (such as chemicals, RNAs, DNAs,
p o eins, o lipids) inc eases hei bioa ailabili y by p ese ing
hei in eg i y and biological ac i i y, as well as p o ec ing
hem om enzyma ic deg ada ion in biological luids (112).
In compa ison o o he he apeu ic ec o s such as syn he ic
nano-pa icles, liposomes o ecombinan i al ec o s, EVs
a e gene ally non-immunogenic in na u e, which enhances
hei esis ance o as clea ance om ci cula ion (112). EVs
also display low oxici y and a e qui e s able in issues and
ci cula ion, ep esen ing adequa e he apeu ic deli e y sys ems
agains cance (113). Fu he mo e, cell ype-speci ic p o eins
wi hin EVs seem o p o ide ce ain cell opism (112).
The s a egy o using EVs as he apeu ic molecule deli e y
ehicles is s a ing in li e cance , mainly ocusing on miRNAs.
S ella e cell-de i ed EVs loaded wi h miR-335-5p, a umo
supp esso miR down egula ed in HCC, inhibi s HCC cell
in asi eness in i o and induces HCC umo sh inkage in
i o h ough he ep ession o p oli e a ion and s imula ion
o apop osis (114). Mo eo e , miR-122 en iched EVs ob ained
om adipose issue-de i ed mesenchymal s em cells (ADMSCs)
inc eases HCC cell sensi i i y o he chemo he apeu ic agen s
so a enib and 5-FU (115). The unde lying mechanism egula ing
chemosensi i i y consis s on he down egula ion o miR-122
a ge genes including cyclin G1 (CCNG1), disin eg in and
me allop o einase domain-con aining p o ein 10 (ADAM10),
and insulin-like g ow h ac o 1 ecep o (IGF1R), which induce
apop osis and cell cycle a es in i o. Fu he mo e, in a-
umo injec ion o miR-122-en iched EVs in a HCC xenog a
mouse model syne gized he inhibi o y e ec o so a enib in i o,
educing umo size (115).
In CCA, s ella e cell-de i ed EVs ca ying miR-195 inhibi ed
CCA g ow h and in asi eness in i o (116). Tail ein injec ion
o miR-195 loaded EVs in o an o ho opic a model o CCA
educed umo size and imp o ed he o e all animal su i al
(116). These an i-neoplasic e ec s a e likely media ed ia
a ge ing VEGF, cell di ision con ol (CDC) p o eins 25 and 42,
as well as cyclin-dependen kinases (CDK) 1, 4, and 6.
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Lapi z e al. EVs in Li e Cance
Immuno he apy
An al e na i e he apeu ic s a egy con empla es he use o
EVs as s imula o s o he immune sys em in o de o elici
a non oxic, sys emic, and long-li ed an i- umo immune
esponse. Di e en s udies ha e desc ibed he po en ial use
o EVs as immunos imula o y en i ies agains HCC (117–
121). Fo ins ance, HCC cells unde s ess condi ions, such as
hea shock o chemo he apeu ic an i-cance d ug ea men ,
inc eased EV sec e ion and su ace exp ession o hea shock
p o eins (HSPs) (117). HSP-bea ing EVs can boos na u al
kille (NK) cell-media ed cy o oxic esponse agains HCC
cells in i o (117). Simila ly, his one deace ylase inhibi o
MS-275 enhanced he p o ein le els o immunos imula o y
molecules [MHC class I polypep ide- ela ed sequence B (MICB)
and HSP70] in EVs de i ed om HCC cells, inc easing
he cy o oxici y o NK cells and an i- umo esponse (118).
The an i-HCC umo immune esponse can also be induced
by ADMSC-de i ed EVs, which p omo e na u al kille T
cell (NKT) an i- umo esponse, he eby acili a ing HCC
supp ession (119).
Al e na i ely, HCC cell-de i ed EVs display HCC an igens
AFP and glypican 3. Cap u e o hese EVs by dend i ic cells
(DCs) igge s a s ong DC-media ed T cell dependen an i-
umo immune esponse bo h in i o and in ec opic and
o ho opic in i o mouse models (120). EVs om an igen
p esen ing cells (APCs) can also induce an i- umo immune
esponses agains HCC. EVs de i ed om AFP-exp essing DCs
a e able o igge po en an igen-speci ic an i- umo immune
esponses and eshape he umo mic oen i onmen om an
immunoinhibi o y o an immunos imula o y se ing in di e se
HCC mice models including ec opic, o ho opic and ca cinogen-
induced HCC (121). Thus, AFP-exp essing DC-de i ed EVs
s imula e an igen-speci ic an i- umo immune esponses in i o,
elici ing supp ession o umo g ow h and p olonging mice
su i al (121).
CONCLUDING REMARKS AND FUTURE
DIRECTIONS
Ea ly diagnosis and ea men o hepa obilia y malignancies
is s ill a om being manageable. The de elopmen o non-
in asi e diagnos ic and disease moni o ing ools ep esen s
a majo challenge. The p esence o EVs in biological luids,
as well as hei capaci y o ca y umo -associa ed molecules,
make EVs excellen candida es o clinical applica ion. He eo ,
ce ain p og ess is being made in he po en ial use o EVs as
a sou ce o non-in asi e disease bioma ke s. EV concen a ion
as well as hei speci ic ca go can se e as indica o s o
he di e en pa hological s ages o a disease, including he
disc imina ion be ween ea ly and la e phases, and es ima ion o
ecu ence and me as asis isk. Fo ha ma e , he applica ion
o omic echnologies has p o ided some po en ial candida e
bioma ke s. Howe e , in o de o ans e knowledge in o
he clinical p ac ice, se e al limi a ions, and conce ns should
be conside ed: (i) di e en EV isola ion p ocedu es (i.e.,
ul acen i uga ion, size exclusion, immune-a ini y isola ion,
polyme ic p ecipi a ion, and mic o luidics) a e cu en ly used,
p o iding di e se EV popula ions and yield depending on he
na u e o he isola ion p o ocol (ii) a p ope cha ac e iza ion
o he EVs ac ion should be pe o med. The e a e minimal
expe imen al equi emen s de ined by he In e na ional Socie y
o Ex acellula Vesicles (ISEV) (122), which include he
analysis o he EV quan i y [e.g., nanopa icle acking analysis
(NTA), IZON qNano echnique, low cy ome y], size [e.g.,
NTA, IZON qNano echnique, elec on mic oscopy, dynamic
ligh sca e ing (DLS)], and p esence o speci ic su ace ma ke s
(e.g., immunoblo , immune-gold elec on mic oscopy) (122,
123), (iii) speci ic EV ma ke s o dis inguish EV subpopula ions
acco ding o hei o igin (e.g., exosomes, MVs, apop o ic
bodies) a e s ill missing (122), (i ) app op ia e clinically-
ele an con ol g oups wi h biopsy-p o en diagnosis, as well
as a ep esen a i e numbe o samples should be included o
ensu e he accu acy (sensi i i y, speci ici y, AUC, p edic i e
and likelihood a io alues) and signi icance o he esul s
(124), ( ) candida e bioma ke s iden i ied in a disco e y phase
mus be in e na ionally alida ed using easily ans e able
me hodologies in o he clinical se ings (e.g., ELISA, qPCR),
ideally using aw biological luids (i.e., se um, u ine, sali a)
and a oiding he cos ly and ime consuming EV isola ion
echniques.
EVs ep esen a new oppo uni y o cance he apy.
They pa icipa e in he de elopmen and p og ession o
cance , including he o ma ion o a p o- umo igenic
mic oen i onmen , angiogenesis, chemo esis ance, and he
gene a ion o a me as a ic niche, p omo ing umo g ow h,
and agg essi eness. The e o e, in e e ing he EV biogenesis
and/o elease may be a po en ial he apeu ic s a egy. Se e al
inhibi o s a ge ing hese c ucial s eps ha e been de eloped
(Figu e 1), al hough hei sa e y and e icacy should be clinically
e alua ed in he u u e. Ne e heless, addi ional egula o y
mechanisms o EV gene a ion (e.g., loading), a icking and
au oc ine/pa ac ine signal ansduc ion (e.g., ecipien cell
in e naliza ion ou es o speci ic EV subpopula ions) need o be
elucida ed, which could p o ide o he a ge s o he apy (125).
On he o he hand, EVs could be used as d ug deli e y sys ems
and as immunomodula o s p omo ing an i- umo esponse.
Fo d ug deli e y, a majo challenge ep esen s he speci ic cell
a ge ing in i o, as well as he use o immunologically ine and
biocompa ible EVs. In con as , he capaci y o EVs o egula e
he immune sys em opens new oppo uni ies o a ge ing
malignancies and o de eloping an i- umo accines (126).
In conclusion, EVs ep esen an eme ging and s imula ing
ield o esea ch in li e cance wi h mul iple po en ial
applica ions, om bioma ke disco e y o he apy. None heless,
ho ough esea ch is s ill needed o gain knowledge on hei
in insic ole in li e heal h and disease, and o e alua e hei
po en ial clinical applica ion.
AUTHOR CONTRIBUTIONS
All au ho s lis ed ha e made a subs an ial, di ec and in ellec ual
con ibu ion o he wo k, and app o ed i o publica ion.
F on ie s in Immunology | www. on ie sin.o g 8Oc obe 2018 | Volume 9 | A icle 2270
Lapi z e al. EVs in Li e Cance
FUNDING
Spanish Minis ies o Economy and Compe i i eness [JB (FIS
PI12/00380, FIS PI15/01132 and Miguel Se e P og amme
CON14/00129); MP (FIS PI14/00399, FIS PI17/00022) and
Ramon y Cajal P og amme RYC-2015-17755] co inanced
by Fondo Eu opeo de Desa ollo Regional (FEDER); ISCIII
[CIBERehd: JB, LB, and MP], Spain; Dipu ación Fo al
Gipuzkoa (JB: DFG15/010,DFG16/004), BIOEF (Basque
Founda ion o Inno a ion and Heal h Resea ch: EiTB Ma a oia
BIO15/CA/016/BD o JB); Depa men o Heal h o he Basque
Coun y (JB: 2013111173 and 2017111010; MP: 2015111100),
and AECC Scien i ic Founda ion (JB). AL and PO we e unded
by he Basque Go e nmen .
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F on ie s in Immunology | www. on ie sin.o g 9Oc obe 2018 | Volume 9 | A icle 2270
