Hepa ology. 2022;76:1617–1633.
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wileyonlinelib a y.com/jou nal/hep
ORIGINAL ARTICLE
Cholangioca cinoma p og ession depends on he up ake
and me aboliza ion o ex acellula lipids
Mikel Ruiz de Gauna1 | F ancesca Biancaniello2,3 | F ancisco González- Rome o1 |
Ped o M. Rod igues2,4,5 | Ainhoa Lapi z2 | Bea iz Gómez- San os1 |
Paula Olaizola2 | Sabina Di Ma eo2,3 | Igo Au ekoe xea1,6 | Ibone Labiano2 |
Ane Nie a- Zuluaga1 | Asie Beni o- Vicen e7,8 | Ma ía J. Pe ugo ia2,4 |
Maide Apodaka- Bigu i1 | Nuno A. Pai a2 | Diego Sáenz de U u i1 |
Xabie Buqué1 | Igo z Delgado1 | Césa Ma ín7,8 | Mikel Azka go a9 |
Felix Elo za4,9 | Diego F. Cal isi10 | Jespe B. Ande sen11 |
Domenico Al a o3 | Vincenzo Ca dinale12 | Luis Bujanda2,4 |
Jesús M. Banales2,4,5,13 | Pa icia Aspichue a1,4,6
1Facul y o Medicine and Nu sing, Depa men o Physiology, Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain
2Depa men o Li e and Gas oin es inal Diseases, Biodonos ia Heal h 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
3Depa men o T ansla ional and P ecision Medicine, “Sapienza” Uni e si y o Rome, Rome, I aly
4Na ional Ins i u e o he S udy o Li e and Gas oin es inal Diseases (CIBERehd, Ca los III Heal h Ins i u e), Mad id, Spain
5IKERBASQUE, Basque Founda ion o Science, Bilbao, Spain
6Bioc uces Bizkaia Heal h Resea ch Ins i u e, C uces Uni e si y Hospi al, Ba akaldo, Spain
7Depa men o Molecula Biophysics, Bio isika Ins i u e (Uni e si y o Basque Coun y and Consejo Supe io de In es igaciones Cien í icas (UPV/EHU, CSIC),
Leioa, Spain
8Depa men o Biochemis y and Molecula Biology, Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain
9P o eomics Pla o m, CIC bioGUNE, BRTA (Basque Resea ch and Technology Alliance), P o eoRed- ISCIII, CIBERehd, Bizkaia Science and Technology
Pa k, De io, Spain
10Ins i u e o Pa hology, Uni e si y o Regensbu g, Regensbu g, Ge many
11Bio ech Resea ch & Inno a ion Cen e (BRIC), Depa men o Heal h and Medical Sciences, Uni e si y o Copenhagen, Copenhagen, Denma k
12Depa men o Medico- Su gical Sciences and Bio echnology, "Sapienza” Uni e si y o Rome, Rome, I aly
13Depa men o Biochemis y and Gene ics, School o Sciences, Uni e si y o Na a a, Pamplona, Spain
Recei ed: 3 May 2021
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Re ised: 17 Decembe 2021
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Accep ed: 17 Decembe 2021
DOI: 10.1002/hep.32344
Mikel Ruiz de Gauna and F ancesca Biancaniello sha e i s au ho ship.
Jesús M. Banales and Pa icia Aspichue a sha e senio au ho ship.
Abb e ia ions: ACADM, acyl- CoA dehyd ogenase medium chain; CCA, cholangioca cinoma; CE, choles e yl es e ; CFSE, ca boxy luo escein succinimidyl
es e ; CL, choles e ol; eCCA, ex ahepa ic cholangioca cinoma; FA, a y acid; FABP, a y acid– binding p o ein; FAO, a y acid oxida ion; FASN, a y acid
syn hase; iCCA, in ahepa ic cholangioca cinoma; LPL, lipop o ein lipase; NHC, no mal human cholangiocy e; Nicd1, NOTCH1 in acellula cy oplasmic
domain; PC, phospha idylcholine; PCNA, p oli e a ing cell nuclea an igen; PG, p os aglandin; TG, iglyce ide.
Co espondence
Jesús M. Banales, Depa men o
Li e and Gas oin es inal Diseases,
Biodonos ia Heal h Resea ch Ins i u e
– Donos ia Uni e si y Hospi al, Paseo
del D . Begi is ain s/n, E- 20014, San
Sebas ian, Spain.
Email: jesus.banales@biodonos ia.o g
Abs ac
Backg ound and Aims: Cholangioca cinoma (CCA) includes a he e ogene-
ous g oup o bilia y cance s wi h a dismal p ognosis. We in es iga ed i lipid
me abolism is dis up ed in CCA and i s ole in umo p oli e a ion.
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial License, which pe mi s use, dis ibu ion and ep oduc ion
in any medium, p o ided he o iginal wo k is p ope ly ci ed and is no used o comme cial pu poses.
© 2022 The Au ho s. Hepa ology published by Wiley Pe iodicals LLC on behal o Ame ican Associa ion o he S udy o Li e Diseases
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CATABOLISM OF EXTRACELLULAR LIPIDS PROMOTES CCA PROGRESSION
INTRODUCTION
Cholangioca cinoma (CCA) includes a di e se g oup
o bilia y malignan umo s and ep esen s he second
mos common p ima y li e cance .[1] Acco ding o he
ana omical loca ion, hese cance s a e classi ied as
in ahepa ic (iCCA), pe ihila (pCCA), o dis al (dCCA)
umo s, al hough pCCA and dCCA we e p e iously
conside ed as ex ahepa ic (eCCA).[2] CCA is a a e
cance globally (0.3– 6 cases pe 100,000 people);
howe e , i s incidence and associa ed mo ali y ha e
been signi ican ly ising o e he las ew decades.[1,2]
Cu en ly, he only ea men wi h cu a i e in en is he
su gical esec ion o he umo . Ne e heless, pa ien s
wi h CCA a e usually asymp oma ic un il la e, un esec -
able s ages o he disease. This highligh s he need o
Pa icia Aspichue a, Depa men o
Physiology, Uni e si y o he Basque
Coun y, Ba io Sa iena s/n, E- 48940,
Leioa, Spain.
Email: pa i[email p o ec ed]
Funding in o ma ion
This wo k was suppo ed by “Ayudas
pa a apoya g upos de in es igación
del sis ema Uni e si a io Vasco”
(IT971- 16 o PA), MCIU/AEI/FEDER,
UE (2018- 095134- B- 100 o PA and
by he Uni e si y o Basque Coun y
COLAB20/01 o PA; Spanish Ca los III
Heal h Ins i u e (ISCIII) (FIS PI15/01132,
PI18/01075, PI21/00922, and Miguel
Se e P og am CON14/00129 and
CPII19/00008 o JMB; FIS PI14/00399,
PI17/00022 and PI20/00186 o MJP;
Sa a Bo ell [CD19/00254 o PMR])
co inanced by “Fondo Eu opeo de
Desa ollo Regional” (FEDER); CIBERehd
(ISCIII) o JMB, MJP, PMR, PA and LB);
“Dipu ación Fo al Gipuzkoa” (DFG15/010,
DFG16/004 o JMB and 2020- CIEN-
000067- 01 o PMR), Depa men o Heal h
o he Basque Coun y (2019111024
o MJP, 2017111010 o JMB, and
2020111077 o JMB and PA), “Euskadi
RIS3” (2016222001, 2017222014,
2018222029, 2019222054, 2020333010
o JMB), BIOEF (Basque Founda ion o
Inno a ion and Heal h Resea ch: EiTB
Ma a oia BIO15/CA/016/BD o JMB) and
Depa men o Indus y o he Basque
Coun y (Elka ek: KK- 2020/00008 o
JMB); La Caixa Scien i ic Founda ion
(HR17- 00601 o JMB). “Fundación
Cien í ica de la Asociación Española
Con a el Cánce ” (AECC Scien i ic
Founda ion, o JMB). AMMF- The
Cholangioca cinoma Cha i y (EU/2019/
AMMF /001, o JMB and PMR). MRDG
was unded by “Fundación Cien í ica de la
Asociación Española Con a el Cánce ”
(AECC de Bizkaia), MJP was unded by
he Spanish Minis y o Economy and
Compe i i eness (MINECO: “Ramón y
Cajal” P og am RYC- 2015- 17755), IL, AL
and FG- R by he Basque Go e nmen
(PRE_2016_1_0152, PRE_2018_2_0195
and PRE 2020 2 02500, espec i ely),
AN- Z and BG- S by he UPV/EHU, AB- V
by “P og ama de especialización de
Pe sonal In es igado Doc o ” a he
UPV/EHU (2019- 2020) and MA by he
MCIU/AEI/FEDER. The unding sou ces
we e no in ol ed in he s udy design,
da a collec ion and analysis, decision o
publish, o p epa a ion o he a icle
App oach and Resul s: The in i o and in i o umo igenic capaci y o i e
human CCA cell lines was analyzed. P o eome, lipid con en , and me abolic
luxes we e e alua ed in CCA cells and compa ed wi h no mal human cholan-
giocy es (NHC). The Ak 1/NOTCH1 in acellula cy oplasmic domain (Nicd1)-
d i en CCA mouse model was also e alua ed. The p o eome o CCA cells
was en iched in pa hways in ol ed in lipid and lipop o ein me abolism. The
EGI1 CCA cell line p esen ed he highes umo igenic capaci y. Me abolic
s udies in high (EGI1) e sus low (HUCCT1) p oli e a i e CCA cells in i o
showed ha bo h EGI1 and HUCCT1 inco po a ed mo e a y acids (FA) han
NHC, leading o inc eased iglyce ide s o age, also obse ed in Ak 1/Nicd1-
d i en CCA mouse model. The highly p oli e a i e EGI1 CCA cells showed
g ea e up ake o e y- low- densi y and HDLs han NHC and HUCCT1 CCA
cells and inc eased choles e yl es e con en . The FA oxida ion (FAO) and
ela ed p o eome en ichmen we e speci ically up- egula ed in EGI1, and con-
sequen ly, pha macological blockade o FAO induced mo e p onounced inhi-
bi ion o hei umo igenic capaci y compa ed wi h HUCCT1. The exp ession
o acyl- CoA dehyd ogenase ACADM, he i s enzyme in ol ed in FAO, was
inc eased in human CCA issues and co ela ed wi h he p oli e a ion ma ke
PCNA.
Conclusions: Highly p oli e a i e human CCA cells ely on lipid and lipo-
p o ein up ake o uel FA ca abolism, sugges ing ha inhibi ion o FAO and/
o lipid up ake could ep esen a he apeu ic s a egy o his CCA subclass.
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HEPATOLOGY
de e mine isk ac o s, implemen sc eening policies,
and in es iga e accu a e diagnos ic me hods and he a-
peu ic s a egies o his cance . Howe e , he high he -
e ogenei y o CCAs ex emely comp omises he inding
o common and e ec i e ea men s o all pa ien s bu
opens a doo o p ecision- a ge ed he apies.[2] CCA
subclasses di e no only on hei ana omical loca ion
bu also on hei his ological ea u es,[3,4] isk ac o s,[5]
pu a i e cell o o igin,[6] and mu a ional landscape.[7– 9]
In his sense, wo di e en molecula subclasses (i.e.,
p oli e a i e o in lamma o y) ha e been p oposed o
iCCA based on gene exp ession p o iling.[7] A compa-
able genomic and molecula p o iling o eCCA was
also pe o med, p oposing ou biological subclasses
o eCCA (i.e., p oli e a i e, mesenchymal, me abolic,
o immune).[8]
Me abolic ep og amming is a hallma k o cance ,
and a ge ing me abolism has been p oposed o can-
ce he apy.[10] Cance cells need a g ea amoun o
ene gy and bioma e ials o he abno mal cell g ow h
and di ision ha cha ac e izes he disease. The e o e,
hey adap hei mechanisms o nu ien up ake, en-
e gy p oduc ion, and biosyn hesis o complex mole-
cules o sui hese demands.[11] Gene ic, epigene ic,
and molecula al e a ions in umo cells modula e hei
me abolic s a e. Mo eo e , di e ences in he mic o-
en i onmen and he supply o nu ien s and oxygen
also con ibu e o he me abolic he e ogenei y o he
umo .[11] The cance - speci ic me abolic adap abili y
also con ibu es o he sp ead and su i al o ea -
men s.[11] Thus, he e is a need o iden i y speci ic
me abolic ewi ing o each cance ype. Rega ding
lipid me abolism, cance cells equi e highe amoun
o lipids mainly o he gene a ion o new memb anes
and o ene gy p oduc ion. Consequen ly, mos cance
ypes a e cha ac e ized by inc eased de no o lipogen-
esis h ough o e exp ession o lipogenic enzymes,
such as a y acid syn hase (FASN) o ace yl- CoA
ca boxylase (ACC). Howe e , hey can also p oli e a e
elying on ex acellula lipid sou ces.[11]
Li le is known abou he ewi ing o lipid me ab-
olism in CCA.[12,13] Howe e , p e ious da a showed
a ema kable down egula ion o FASN and ACC le -
els in human iCCA umo s compa ed wi h su ound-
ing non umo ous issue. Fasn exp ession was ound
also diminished in di e en mouse CCA models.[14– 16]
Consis en ly, Fasn silencing did no ab oga e CCA
de elopmen in he Ak 1/NOTCH1 in acellula cy-
oplasmic domain (Nicd1) and Ak 1/N as models.[16]
The e o e, al hough s ill unclea , hese da a sugges
ha he up ake o exogenous lipids, a he han he
de no o biosyn hesis, should p e e en ially play a p e-
dominan ole in CCA p og ession.
The main aim o he p esen s udy was o gain
u he insigh s in o he iden i ica ion o he sou ce,
me abolic a e, and ole o lipids in he p oli e a ion o
CCA cells.
MATERIALS AND METHODS
Animal models
To p omo e he gene a ion o iCCA in mice, 8- week old
male wild- ype mice (mixed backg ound C57BL/6J and
129/S ) we e subjec ed o hyd odynamic ail ein injec-
ion o plasmids, as desc ibed p e iously and de ailed
in Suppo ing Ma e ials and Me hods.[14,15]
Fo he xenog a animal models, CCA cells we e
subcu aneously injec ed in lanks o immunode icien
CD1 nude mice as desc ibed in Suppo ing Ma e ials
and Me hods. E omoxi (30 mg/kg, MedChemExp ess)
was adminis e ed e e y 2 days o 32 days as desc ibed
in Suppo ing Ma e ials and Me hods.
The E hics Commi ee o he Uni e si y o he
Basque Coun y (UPV/EHU) (CEEA M20/2019/60) o
he Biodonos ia Heal h Resea ch Ins i u e (CEEA21- 10/
OH- 21- 027) app o ed all he p ocedu es.
Human coho s
Gene exp ession mic oa ay da a om he Copenhagen
coho including 104 CCA su gical specimens,[17] and
he Jusakul coho including 118 CCA su gical speci-
mens[18] we e used o gene exp ession analysis and
co ela ion. Besides, 23 pai s o ma ched umo s oma
and epi helium we e ob ained.[17] 182 issue samples
om human iCCAs collec ed a he Medical Uni e si y
o Regensbu g (Regensbu g, Ge many) we e used o
he immunohis ochemis y s udies. Ins i u ional Re iew
Boa d app o al was p o ided by he local E hical
Commi ee o he Medical Uni e si y o Regensbu g
(app o al # 17- 1015- 101) in compliance wi h he Helsinki
Decla a ion. In o med consen was ob ained om all
indi iduals. No dono o gans we e ob ained om ex-
ecu ed p isone s o o he ins i u ionalized pe sons.
Cells
Human CCA cell lines (EGI1, TFK1, WITT, HUCCT1
and TKKK) we e used in he expe imen s, as well as
p ima y cul u es o no mal human cholangiocy es
(NHC; i.e., NHC2, NHC3, NHC- SS, and NHC324).
NHCs we e isola ed om no mal li e issue specimens
as p e iously desc ibed.[19– 21] Addi ional in o ma ion
has been added in Suppo ing Ma e ials and Me hods.
Me abolic luxes
Olea e and palmi a e up ake
The analysis o olea e and palmi a e up ake was pe -
o med in i o, in he EGI1 and HUCCT1 CCA cell lines,
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and in he no mal NHC3 cholangiocy es as con ol,
as desc ibed p e iously[22] and de ailed in Suppo ing
Ma e ials and Me hods.
Lipop o ein isola ion, labeling, and up ake
Lipop o ein up ake was de e mined in NHC3, EGI1, and
HUCCT1 cells by luo escen labeling and low cy ome-
y, as desc ibed in Suppo ing Ma e ials and Me hods.
[3H]- olea e inco po a ion in o lipids
The analysis was pe o med ex i o, in esh mice li e
pieces, and in i o, in NHC3, EGI1, and HUCCT1 cells
as p e iously desc ibed.[23] Lipids om li e o cell sam-
ples we e ex ac ed and sepa a ed. Addi ional in o ma-
ion can be ound in Suppo ing Ma e ials and Me hods.
[14C]- palmi a e oxida ion
The analysis o FA oxida ion (FAO) a e was pe o med
ex i o, in esh mice li e pieces, and in i o, in NHC3,
EGI1, and HUCCT1 cells, as desc ibed p e iously.[24,25]
Addi ional in o ma ion can be ound in Suppo ing
Ma e ials and Me hods.
[3H]- olea e blood clea ance and li e up ake
The analysis was pe o med in i o in he Ak 1/Nicd1- d i en
CCA mouse model using un ea ed animals as con ol.
Animals we e injec ed wi h 1 μCi [3H]- olea e. Blood samples
we e collec ed and li e up ake o [3H]- olea e was measu ed
as de ailed in Suppo ing Ma e ials and Me hods.
Lipid quan i ica ion
Lipids om li e samples and om NHC3, EGI1,
and HUCCT1 cells we e quan i ied as desc ibed in
Suppo ing Ma e ials and Me hods.
S a is ical analysis
Da a a e ep esen ed as mean ± SEM. No mal dis ibu-
ion assessmen s we e ca ied ou wi h he Shapi o-
Wilk es . Di e ences be ween g oups we e analyzed
wi h a wo- ailed S uden ’s es , a Tukey’s mul iple com-
pa ison es , Wilcoxon es , o wi h a wo- way ANOVA
es . Associa ion be ween wo a iables was assessed
by Pea son co ela ion coe icien o by Spea man es .
Signi icance was de ined as p < 0.05. Resul s we e s a-
is ically analyzed using G aphPad P ism e sion 8.01
so wa e (San Diego, CA, USA) and SPSS 22 so wa e
(IBM, Ehningen, Ge many).
RESULTS
CCA cells a e en iched in p o eins
in ol ed in lipid and lipop o ein
me abolism
To assess he ele ance o lipid me abolism in CCA, he
p o eomic p o iles o 5 di e en human CCA cell lines
(HUCCT1, TKKK, EGI1, TFK1, and WITT) and 4 p ima y
cul u es o NHCs (NHC2, NHC3, NHC- SS, NHC324)[19– 21]
we e de e mined. The analysis o he p o eome clea ly di -
e en ia ed CCA and NHC cells (Figu e 1A,B and Figu e
S1A) and indica ed ha 2066 p o eins we e up- egula ed
and 1148 down- egula ed in CCA compa ed wi h NHC.
En ichmen analysis showed ha he mos ep esen ed bi-
ological p ocesses ela ed o hose dys egula ed p o eins
in CCA e sus NHC cell cul u es we e “ egula ion o nucleic
acid me abolism,” “me abolism,” and “ene gy pa hways.”
Among he 415 p o eins inside he “me abolism” biological
p ocess, he mos ep esen ed speci ic biological pa hway
was “me abolism o lipids and lipop o eins” (Figu e 1C).
Valida ion o p o eins in ol ed in di e en lipid me -
abolic pa hways by immunoblo ing showed ha le els
o he acyl- CoA syn he ase long chain amily membe
5 (ACSL5), which pa icipa es in he ac i a ion o a y
acids (FAs) o acyl- CoA (Figu e S2A,E), and le els o he
FA- binding p o ein 5 (FABP5), ele an o he malignan
p og ession o CCA[26] and in ol ed in FA up ake, in a-
cellula anspo , and in acellula me abolism (Figu e
S2B,E), we e inc eased in CCA cells when compa ed
wi h NHC (Figu e S2A,B). Rega ding lipop o ein lipase
(LPL) and CD36, he le els o bo h p o eins dec eased
in CCA cell lines compa ed wi h mos NHC, excep in
he EGI1 CCA cell line (Figu e S2C– E).
CCA cells display di e en
p oli e a i e and mig a ion capaci ies
Gi en he di e en o igin and mu a ional p o iles o he
human CCA cell lines (HUCCT1, TKKK, EGI1, TFK1, and
FIGURE 1 The p o ile o p o eins in ol ed in lipid me abolism is dys egula ed in CCA. (A) Volcano plo showing di e en ially exp essed
p o eins be ween CCA and NHCs. (B) Hea map showing di e en ially exp essed p o eins be ween CCA and NHCs. (C) En ichmen analysis
o biological p ocesses was pe o med wi h di e en ially exp essed p o eins be ween NHC p ima y cul u es and CCA cell lines (up) and wi h
me abolism- ela ed, di e en ially exp essed p o eins be ween NHC and CCA cells. Only pa hways wi h p < 0.05 a e shown (hype geome ic
es ). The numbe o p o eins ca ego ized in each p ocess/pa hway is displayed nex o he name.
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CATABOLISM OF EXTRACELLULAR LIPIDS PROMOTES CCA PROGRESSION
WITT),[27] we unc ionally cha ac e ized hem by meas-
u ing he p oli e a ion and mig a ion a es in baseline
condi ions. EGI1 cells displayed he highes g ow h a e
in a subcu aneous xenog a mouse model (Figu e 2A),
leading o inc eased umo size (Figu e 2A,B) and umo
weigh (Figu e 2B). A low cy ome y- based p oli e a ion
assay (ca boxy luo escein succinimidyl es e [CFSE])
also pinpoin ed EGI1 as he mos p oli e a i e CCA cell
line in i o (Figu e 3A). We addi ionally pe o med a
wound healing assay o compa e he mig a ion p ope -
ies o he cells. Among he CCA cell lines, EGI1 cells
displayed he highes mig a ion capaci y (Figu e 3B),
whe eas no signi ican di e ences we e ound among
he o he cell lines (Figu e 3B).
The up ake o ex acellula ee FAs, and
e y low- and high- densi y lipop o eins,
is inc eased mos ly in highly p oli e a i e
CCA cells
The esul s men ioned abo e showed ha EGI1 is he
mos p oli e a i e cell line, wi h he highes mig a ion
and umo igenic capaci y, whe eas he HUCCT1 cell
line is among he less p oli e a i e ones. Thus, aking
in o accoun he e idence sugges ing a ole o ex a-
cellula lipids on CCA p og ession,[16] he up ake o
exogenous ee FAs and lipop o eins was analyzed in
hese CCA cell lines and compa ed wi h NHC (i.e.,
NHC3).
FIGURE 2 In i o umo igenic capaci y o human CCA cell lines. (A) Tumo olume was measu ed 7, 14, 21, 28, 36, and 42 days a e
subcu aneous injec ion o EGI1, WITT, TFK1, HUCCT1, o TKKK CCA cells in he xenog a mouse model. Rep esen a i e images o umo s
in mice a e shown below (n = 5- 7). (B) Tumo weigh was measu ed 42 days a e injec ion o EGI1, WITT, TFK1, HUCCT1, o TKKK cells in
he subcu aneous xenog a mouse model. Rep esen a i e images o esec ed umo s (le ) and quan i ica ion ( igh ) a e shown. Values a e
means ± SEM. S a is ical analysis was de e mined by Tukey’s mul iple compa ison es and by wo- way ANOVA es . Signi ican di e ences
a e deno ed as **p < 0.01, ***p < 0.001
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HEPATOLOGY
No ably, he up ake o ex acellula FAs (oleic acid and
palmi ic acid) was inc eased in bo h EGI1 and HUCCT1
cell lines compa ed wi h NHC3 cells (Figu e 4A).
Di e ences we e also obse ed be ween umo cell
lines, as EGI1 cells p esen ed highe up ake compa ed
wi h HUCCT1 cells (Figu e 4A). To s udy lipop o ein up-
ake, VLDLs, LDLs, and HDLs om se um samples we e
isola ed, pu i ied, and luo escen ly- labeled. The esul s
showed ha VLDL and HDL up ake was ma kedly in-
c eased in EGI1 compa ed wi h HUCCT1 o NHC3 cells,
whe eas LDL up ake was dec eased signi ican ly only in
HUCCT1 cells compa ed wi h bo h EGI1 and NHC3 cells
(Figu e 4B). As choles e yl es e (CE) is a majo com-
ponen o HDLs, we in es iga ed whe he he inc eased
up ake o lipop o eins esul ed in di e ences in he CE
concen a ion in he highly p oli e a i e and mig a i e
EGI1 CCA cells. As expec ed, he CE con en was highe
in EGI1 cells han NHC3 o HUCCT1 cells (Figu e 4C),
closely esembling he obse ed changes in HDL up-
ake. Ne e heless, he inc eased CE le els in EGI1 cells
we e no linked o changes in ee choles e ol (CL) le -
els (Figu e S3A). The same p o ile was obse ed in he
CCA lesions om Ak 1/Nicd1 mice in which li e CE
con en was ound ele a ed (Figu e S3B), whe eas CL
emained unal e ed (Figu e S3B). Gi en ha mos ci -
cula ing lipop o eins in mice a e HDL[28] and ha li e is
a main o gan in ol ed in HDL up ake,[29] o al se um CL
and se um HDL- CL le els we e quan i ied. Acco dingly,
lowe se um le els o o al CL and o HDL- CL we e ob-
se ed in Ak 1/Nicd1 mice compa ed wi h con ol mice
(Figu e S3C), sugges ing an inc eased li e up ake o
se um HDLs in he cance model. The endogenous CE
syn hesis om exogenous oleic acid (olea e) in CCA cell
lines (Figu e 4D) and in li e umo s om Ak 1/Nicd1
mice (Figu e S3D) emained unchanged compa ed wi h
co esponding no mal con ols. The de no o syn hesis o
CL and CE also emained unal e ed (Figu e S3D), sug-
ges ing all oge he ha he inc eased HDL up ake is a
sou ce o CEs in speci ic CCA cell subclasses.
Rewi ing o glyce olipid me abolism in
CCA cells
In hepa ocy es, an inc eased up ake o lipids usually
leads o inc eased s o age o iglyce ides (TGs). Gi en
ha he up ake o FAs and he TG- ich lipop o ein VLDL
was inc eased, mainly in he EGI1 cells, he glyce olipid
con en and he in acellula me abolic luxes ha egu-
la e he es e i ica ion o olea e in o complex lipids, as
an indica o o glyce olipid syn hesis, we e analyzed.
The esul s showed ha he TG con en was highe in
bo h EGI1 and HUCCT1 CCA cells han in no mal chol-
angiocy es (NHC3) (Figu e 5A). Consis en wi h he
inc eased FA up ake in EGI1 compa ed wi h HUCCT1
(Figu e 4A), he luxes ha egula e he syn hesis o TGs
we e also highe in he EGI1 CCA cell line (Figu e 5A).
We[30] and o he s[31] ha e p e iously demons a ed ha
phospha idylcholine (PC) migh be a sou ce o TGs;
hus, he PC con en was also measu ed. The esul s
showed ha PC le els we e dec eased in bo h CCA
cell lines (Figu e 5B), whe eas PC syn hesis was un-
changed (Figu e 5B).
The analysis o li e TG and PC con en and he me -
abolic luxes ha egula e hei syn hesis in he Ak 1/
Nicd1 CCA mouse model (Figu e 6A,B) showed he e
was a as e clea ance o ci cula ing FAs in mice wi h
iCCA lesions, compa ed wi h con ol mice (Figu e 6C).
In conco dance, FA up ake by he li e was inc eased
in he Ak 1/Nicd1 mice compa ed wi h heal hy mice
(Figu e 6D). Acco ding o he esul s ob ained in he
cell lines (Figu e 5A), TG le els and TG syn hesis we e
also inc eased in he mouse CCA lesions when com-
pa ed wi h he con ol mice (Figu e 6E). Me abolomic
analysis e ealed an e iden inc ease in a high numbe
o TG species (Figu e 6E). This was linked again wi h
dec eased PC con en (Figu e 6F). Fu he mo e, in his
in i o iCCA model, li e PC species we e dec eased,
whe eas li e PC syn hesis was ound inc eased com-
pa ed wi h hei no mal con ols (Figu e 6D), which
sugges s he ac i a ion o mechanisms in ol ed in PC
ca abolism, such as phospholipases o SM syn hases,
ha migh con ibu e o he inc eased FA pool,[30,31] and/
o he gene a ion o subs a es o p os aglandin (PG)
syn hesis, al oge he p omo ing p oli e a ion. PC is e-
qui ed o he syn hesis o sphingomyelin (SM) h ough
he ac ion o he SM syn hase (SMS) using ce amide
as a subs a e. SM, a he same ime, can be hyd o-
lyzed by sphingomyelinases (SMases), yielding a ce-
amide and a phospho ylcholine (Figu e S4A). Le els
o ce amides we e signi ican ly dec eased in bo h CCA
cell lines compa ed wi h NHC3, wi h mo e p ominen
al e a ions in HUCCT1 cells (Figu e S4B). On he con-
a y, SM le els we e inc eased only in HUCCT1 com-
pa ed wi h bo h NHC3 and EGI1 (Figu e S4C). These
changes in he lipidome we e in conco dance wi h he
ma ked dec ease in he acid SMase ac i i y ( he mos
abundan SMase in cholangiocy es[32] in bo h EGI1 and
HUCCT1, and he inc ease in he SMS ac i i y only in
he HUCCT1 cell line (Figu e S4D). The esul s he e
sugges ha his emodeling in SM me abolism migh
no be he majo me abolic pa hway esponsible o
he obse ed dec eased PC con en in bo h EGI1 and
HUCCT1 CCA cell lines (Figu e 5B), poin ing ou he
po en ial in ol emen o o he phospholipases.[30,31]
FAO p omo es he hype p oli e a ion o
speci ic CCA cell subclasses
So a , he da a showed ha he inc eased up ake o
ex acellula ee FAs, VLDLs and HDLs esul s in he
accumula ion o neu al lipids (i.e., TGs and CEs), likely
leading o an in acellula pool o FAs ha can be used
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CATABOLISM OF EXTRACELLULAR LIPIDS PROMOTES CCA PROGRESSION
as an ene gy sou ce h ough FAO. These esul s a e
also suppo ed by ou da a (Figu e S5A,B) and p e i-
ous epo s[12– 16] ha showed ha de no o lipogenesis
is no ac i a ed in CCA, hus con i ming a mo e p omi-
nen ole o FA up ake in p omo ing he accumula ion
o FAs.
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HEPATOLOGY
To asce ain whe he he ca abolism o FAs was al-
e ed in he human CCA cell lines, comple e oxida ion
o [14C]- palmi a e in o CO2 was measu ed. We ound
ha he FAO a e was ma kedly inc eased in he EGI1
CCA cells as compa ed wi h he es o he CCA cell
lines o o he NHC (Figu e 7A). Cu iously, FAO a e
dec eased in HUCCT1 cells as compa ed wi h NHC3
cells sugges ing o he sou ces, di e en om lipids,
as majo ene gy subs a es (Figu e 7A). Thus, bo h
glucose and glu amine up ake and comple e oxida-
ion in o CO2 we e measu ed. The esul s showed ha
only HUCCT1 p esen ed a signi ican up egula ion o
glucose up ake compa ed wi h NHC3 (Figu e S6A).
Howe e , comple e glucose oxida ion in o CO2 did no
change in he umo cell lines (Figu e S6B). Rega ding
glu amine, he esul s showed ha i s up ake emained
unal e ed in he CCA cell lines (Figu e S6C), whe eas
he comple e oxida ion in o CO2 was up- egula ed in
HUCCT1 compa ed wi h NHC3 and EGI1, poin ing glu-
amine as a ele an sou ce o uel he ica boxylic acid
(TCA) cycle and ene gy p oduc ion in HUCCT1 CCA
cells.
Conside ing he di e ences in FAO a es be ween
EGI1 and HUCCT1 cells come along wi h di e en ial
p oli e a ion a es (Figu e 3A), we decided o in es iga e
he con ibu ion o FAO o he p oli e a ion capaci y o
EGI1 and HUCCT1 CCA cell lines. As p edic ed, e o-
moxi , a ecognized inhibi o o FAO,[33– 35] e ec i ely
blocked FAO in bo h EGI1 and HUCCT1 cells, eaching
simila oxida ion alues (Figu e S7A). No ewo hy, FAO
blockage wi h e omoxi induced a mo e p onounced in-
hibi ion o p oli e a ion in he highly p oli e a i e CCA
cell line EGI1 han in he less p oli e a i e HUCCT1
(Figu e 7B), sugges ing ha EGI1 elies mo e on FAO
o p oli e a e and g ow. Simila ly, in a subcu aneous
xenog a mouse model, e omoxi adminis a ion de-
c eased umo g ow h in bo h EGI1 and HUCCT1 CCA
cell lines (Figu e 7C), mo e ema kably in he mos p o-
li e a i e one (EGI1).
CCA cells wi h a s em cell- like pheno ype a e mo e
elian on oxida i e phospho yla ion.[36] Acco dingly,
ou esul s showed ha he mRNA le els o he s em-
ness ma ke s EPCAM, ITGA6, CD133 and CD44 we e
ma kedly inc eased in he mos p oli e a i e, highly
lipid- dependen EGI1 cells compa ed wi h bo h NHC3
and HUCCT1 (Figu e S7B). Thus, hese esul s sug-
ges ha inc eased lipid up ake uels he inc eased
mi ochond ial oxida ion obse ed in he CCA s em- like
cells. Lipophagy o au ophagy o cellula lipid d ople s
can also ha e a ole in cance me abolic ep og am-
ming.[37] Gi en he inc eased FAO in he mos p oli -
e a i e CCA cell line, p o ein le els o he au ophagy
ma ke s ATG5 and ATG7, as well as LC3B lipida ion,
we e mainly inc eased in he mos p oli e a i e EGI1
CCA cell line compa ed wi h bo h HUCCT1 and NHC3
(Figu e S7C), which sugges s inc eased au ophagy
luxes in CCA cells, bu pa icula ly in he EGI1 CCA
cell line.
Nex , we compa ed he p o eomic p o ile o bo h
CCA cell lines (EGI1 and HUCCT1) and 1409 p o-
eins we e di e en ially exp essed. Among hem, 1026
we e up- egula ed in EGI1 compa ed wi h HUCCT1
and 383 in HUCCT1 compa ed wi h EGI1 (Figu e S8).
En ichmen analysis on biological pa hways was pe -
o med wi h he 211 di e en ially exp essed p o eins
ca ego ized in o he “me abolism” biological p ocess.
F om hose, 145 showed highe exp ession in EGI1
han in HUCCT1, and 66 in HUCCT1 han in EGI1. The
en ichmen analysis conside ing only pa hways wi h a
p alue smalle han 0.0001 indica ed ha di e en ially
exp essed p o eins we e highly en iched in p ocesses
such as “me abolism o lipids and lipop o eins,” “mi o-
chond ial FA β- oxida ion,” “ he ci ic acid (TCA) cycle,”
and “ espi a o y elec on anspo , ATP syn hesis and
hea p oduc ion,” which usually up- egula e when FAO
a e inc eases (Figu e 7D). Mos o he p o eins inside
hese pa hways showed highe exp ession in EGI1 han
in HUCCT1 (Figu e 7D).
As men ioned, EGI1 cells a e mo e dependen on
FAO o p oli e a ion han HUCCT1, po en ially ex-
plaining hei highe p oli e a i e capaci y. Among he
p o eins in ol ed in FAO showcasing inc eased le -
els in EGI1 when compa ed wi h HUCCT1, acyl- CoA
dehyd ogenase medium chain (ACADM), he i s en-
zyme in ol ed in FAO s ood ou . Valida ion by immu-
noblo ing con i med a highe exp ession o ACADM
in CCA cell lines compa ed wi h NHC, pa icula ly in
EGI1 (Figu e 7E). Acco dingly, he immunos aining
analysis o ACADM in 182 iCCA samples (Figu e 8A)
showed ha he non umo li e issue p esen ed a
s ong cy oplasmic ACADM s aining in hepa ocy es,
whe eas bilia y epi helial cells exhibi ed ain o absen
immuno eac i i y o ACADM (Figu e 8A, uppe pan-
els). On he con a y, 168 ou o 182 (92.31%) iCCA
samples showed obus immunos aining o ACADM
in umo (T) a eas (Figu e 8A, lowe panels). In addi-
ion, ACADM exp ession was up- egula ed in he umo
epi helium compa ed wi h ma ched umo s oma
FIGURE 3 In i o p oli e a i e and mig a ion capaci y o human CCA cell lines. (A) In i o p oli e a ion a es o NHC3, EGI1, WITT,
TFK1, HUCCT1, and TKKK CCA cell lines we e de e mined a 48 h by low cy ome y measu ing loss o luo escence o CFSE- labeled cells.
Rep esen a i e his og ams (ligh - colo ed his og ams, a 24 h; da k- colo ed his og ams, a 72 h) (le ) and quan i ica ion ( igh ) a e shown.
Resul s a e exp essed as ela i e o p oli e a ion a 24 h o each cell line. (B) In i o mig a ion a es o NHC3, EGI1, WITT, TFK1, HUCCT1,
and TKKK CCA cell lines we e measu ed a 12 h by a wound healing assay. Resul s a e exp essed as ela i e o ime 0. Rep esen a i e
images o wound healing a eas (up) and quan i ica ion (down) a e shown. Values a e means ± SEM. S a is ical analysis was de e mined by
Tukey’s mul iple compa ison es . Signi ican di e ences a e deno ed as ***p < 0.001
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CATABOLISM OF EXTRACELLULAR LIPIDS PROMOTES CCA PROGRESSION
acco ding o hei mu a ional and gene ic exp ession
p o ile.(7,8). A “p oli e a ion class,” p esen in bo h iCCA
and eCCA, has been de ined. Ou esul s indica e ha
highly p oli e a i e CCA cells a e cha ac e ized by
highe lipid consump ion, and ea men s ha a ec he
en y o consump ion o lipids could ha e he apeu ic
alue o CCA umo s wi h hese cha ac e is ics.
In summa y, highly p oli e a i e CCA cells a e s ongly
lipid- dependen , as shown by hei up- egula ed lipid
and lipop o ein up ake and ca abolism o p oli e a ion
(Figu e 8E). This obse a ion unde sco es he po en ial
ele ance o a ge ing lipid me abolism o he ea -
men o speci ic sub ypes o CCA.
ACKNOWLEDGMENTS
We hank Jose An onio Lopez om he Depa men
o Physiology, Facul y o Medicine and Nu sing UPV/
EHU, o his help. This a icle is based on wo k om he
COST Ac ion CA18122 Eu opean Cholangioca cinoma
Ne wo k suppo ed by COST (Eu opean Coope a ion
in Science and Technology) www.cos .eu.
CONFLICT OF INTEREST
Au ho s disclose no con lic s ela ed o his s udy.
AUTHOR CONTRIBUTIONS
Mikel Ruiz de Gauna, F ancesca Biancaniello, Jesús M.
Banales, and Pa icia Aspichue a designed he s udy.
Mikel Ruiz de Gauna, F ancesca Biancaniello, F ancisco
González- Rome o, Ped o M. Rod igues, Ainhoa Lapi z,
Sabina Di Ma eo, Igo Au ekoe xea, Ibone Labiano, Ane
Nie a- Zuluaga, Asie Beni o- Vicen e, Ma ía J. Pe ugo ia,
Maide Apodaka- Bigu i, Diego Sáenz de U u i, Bea iz
Gómez- San os, Xabie Buqué, Igo z Delgado, Mikel
Azka go a, Felix Elo za, Jespe B. Ande sen, Luis
Bujanda, Jesús M. Banales, and Pa icia Aspichue a pe -
o med expe imen s and in es iga ions. Césa Ma ín,
Diego F. Cal isi, Jespe B. Ande sen, Domenico Al a o,
Vincenzo Ca dinale, Luis Bujanda, Jesús M. Banales,
and Pa icia Aspichue a designed expe imen al p o ocols.
Mikel Ruiz de Gauna, Jesús M. Banales, and Pa icia
Aspichue a w o e he pape , and all au ho s con ibu ed
o edi ing.
ORCID
Jespe B. Ande sen h ps://o cid.o g/0000-0003-1760-5244
Pa icia Aspichue a h ps://o cid.o g/0000-0002-3553-1755
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SUPPORTING INFORMATION
Addi ional suppo ing in o ma ion may be ound in he
online e sion o he a icle a he publishe ’s websi e.
How o ci e his a icle: Ruiz de Gauna M,
Biancaniello F, González- Rome o F, Rod igues
PM, Lapi z A, Gómez- San os B, e al.
Cholangioca cinoma p og ession depends on he
up ake and me aboliza ion o ex acellula lipids.
Hepa ology. 2022;76:1617– 1633. h ps://doi.
o g/10.1002/hep.32344
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