Sulconazole inhibi s PD-1
exp ession in immune cells and
cance cells malignan
pheno ype h ough NF-kB and
calcium ac i i y ep ession
Simon Pe no
1,2
‡
, Me cedes Tome
´
1
†‡
, Isabel Galeano-O e o
1
,
Se ge E a d
1,2
, Ike Badiola
3
, F ede ic Delom
1,2
,
Delphine Fessa
1,2
, Ta ik Smani
4
, Ge aldine Sieg ied
1,2
*,
B uno O. Villou eix
5
and Abdel-Majid Kha ib
1,2
*
1
Rep og aming umo ac i i Y and associaTed Mic oen i onmEn (Ry me), Bo deaux Ins i u e o
Oncology (BRIC)-UMR1312 Inse m, Uni e si e
´o Bo deaux, Pessac, F ance,
2
Ins i u Be gonie
´,
Bo deaux, F ance,
3
Depa men o Cell Biology and His ology, Facul y o Medicine and Nu sing,
Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain,
4
G oup o Ca dio ascula
Pa hophysiology, Ins i u e o Biomedicine o Se ille, Uni e si y Hospi al o Vi gen del Rocı
´o/
Uni e si y o Se ille/CSIC, Se ille, Spain,
5
In eg a i e Compu a ional Pha macology and Da a
Mining, INSERM UMR 1141, Rob-e -Deb e
´Hospi al, Pa is, F ance
The o e exp ession o he immunoinhibi o y ecep o p og ammed dea h-1
(PD1) on T-cells is in ol ed in immune e asion in cance . The use o an i-PD-
1/PDL-1 s a egy has deeply changed he he apies o cance s and pa ien
su i al. Howe e , hei e ficacy di e ges g ea ly along wi h umo ype and
pa ien popula ions. The eby, no el ea men s a e needed o in e e e wi h
he an i- umo al immune esponses and p opose an adjunc he apy. In he
cu en s udy, we ound ha he an i ungal d ug Sulconazole (SCZ) inhibi s
PD-1 exp ession on ac i a ed PBMCs and T cells a he RNA and p o ein
le els. SCZ ep essed NF-kB and calcium signaling, bo h, in ol ed in he
induc ion o PD-1. Fu he analysis e ealed cance cells ea men wi h SCZ
inhibi ed hei p oli e a ion, and mig a ion and abili y o media e umo
g ow h in zeb afish emb yos. SCZ ound also o inhibi calcium
mobiliza ion in cance cells. These esul s sugges he SCZ he apeu ic
po en ial used alone o as adjunc s a egy o p e en T-cell exhaus ion
and p omo es cance cell malignan pheno ype ep ession in o de o
imp o e umo e adica ion.
KEYWORDS
PD-1, Ju ka T cells, PBMCs, NF-kB, calcium, cance , zeb afish
F on ie s in Immunology on ie sin.o g01
OPEN ACCESS
EDITED BY
Te esi a Padilla-Bena ides,
Wesleyan Uni e si y, Uni ed S a es
REVIEWED BY
Qibin Liao,
Shenzhen Thi d People’s Hospi al, China
Dalia Hayda ,
Child en’s Na ional Hospi al, Uni ed S a es
*CORRESPONDENCE
Abdel-Majid Kha ib
[email p o ec ed]
Ge aldine Sieg ied
[email p o ec ed]
†
PRESENT ADDRESS
Me cedes Tome
´,
G oup o Me abolism and Cell Signaling,
Andalusian Molecula Biology and
Regene a i e Medicine Cen e-CABIMER-
CSIC, Se ille, Spain
‡
These au ho s sha e fi s au ho ship
RECEIVED 16 Augus 2023
ACCEPTED 30 No embe 2023
PUBLISHED 05 Janua y 2024
CITATION
Pe no S, Tome
´M, Galeano-O e o I, E a d S,
Badiola I, Delom F, Fessa D, Smani T,
Sieg ied G, Villou eix BO and Kha ib A-M
(2024) Sulconazole inhibi s PD-1 exp ession
in immune cells and cance cells malignan
pheno ype h ough NF-kB and calcium
ac i i y ep ession.
F on . Immunol. 14:1278630.
doi: 10.3389/ immu.2023.1278630
COPYRIGHT
©2024Pe no ,Tome
´,Galeano-O e o,E a d,
Badiola, Delom, Fessa , Smani, Sieg ied,
Villou eix and Kha ib. This is an open-access
a icle dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion License (CC BY).
The use, dis ibu ion o ep oduc ion in o he
o ums is pe mi ed, p o ided he o iginal
au ho (s) and he copy igh owne (s) a e
c edi ed and ha he o iginal publica ion in
his jou nal is ci ed, in acco dance wi h
accep ed academic p ac ice. No use,
dis ibu ion o ep oduc ion is pe mi ed
which does no comply wi h hese e ms.
TYPE O iginal Resea ch
PUBLISHED 05 Janua y 2024
DOI 10.3389/ immu.2023.1278630
1 In oduc ion
The inhibi o y ecep o p og ammed cell dea h p o ein-1 (PD-
1, PDCD1) is a membe o he immunoglobulin (lg) supe amily
(1). The exp ession o his ype I ansmemb ane p o ein is induced
in a ious immune cells including T cells, B cells, mac ophages, and
se e al dend i ic cell subse s (2–4). Two main ligands o PD-1,
namely p og ammed cell dea h ligand 1 and 2 (PD-L1/PD-L2) (5)
a e exp essed in a ious umo cell ypes and immune cells (5,6). By
binding o PD-1, PD-L1 inhibi s T cell ac i a ion, leading o
immune escape. The eby, in e e ing wi h PD-1 and PD-L1
in e ac ion o signaling pa hways was p oposed as a he apeu ic
app oach o p e en cance cells om a oiding he an i- umo al
immune esponse. This by eac i a ing he T-cell-media ed umo
cell cy o oxici y and elimina ion (1–5). Indeed, p e ious s udies
indica ed ha he inhibi ion o PD-1 p omo es an e ec i e immune
esponse agains cance cells (1–5) and a ge ing PD-L1 o PD-1
using monoclonal an ibody blocking PD-1 o PD-L1 has been
associa ed wi h significan clinical esponse in a wide ange o
malignancies (7,8). Howe e , al hough blockade o PD-1/PD-L1
wi h hese monoclonal an ibodies shows he apeu ic e ec o
cance pa ien s, hei use displayed some es ic ions. These
include he educed esponse equency and se e al ad e se
e ec s obse ed in some cance pa ien s (7,8). The e o e, o
o e come hese di ficul ies he de elopmen o o he e ficien
s a egies is now necessa y.
An i ungal imidazole de i a i es a e commonly used o he
ea men o opical and sys emic in ec ions including candidal
in ec ions and mycoses (9). Imidazole de i a i es, including
ke oconazole (KET), miconazole (MIC), ioconazole (TIO),
clo imazole (CLO), and sulconazole (SCZ), we e ini ially
iden ified as ligands o he heme i on a om o cy och ome P450
(CYP) (10–12). Fu he mo e, he e ec o hese d ugs ha e been
s udied in he con ex o human pa hologies ea men including
cance (13). O hese, SCZ is an an i ungal agen wi h a b oad
spec um o ac i i y and is p oposed o he ea men o skin
in ec ions such as de ma ophy e in ec ions (14–16). Compa ed o
se e al imidazoles, SCZ shows enhanced an i ungal ac i i y (14–16)
and was epo ed o inhibi he malignan pheno ype o b eas
cance cells (13). In his s udy we demons a e he abili y o SCZ o
inhibi he exp ession o PD-1 on ac i a ed T cells and PBMCs
h ough NF-kB ac i i y and calcium mobiliza ion ep ession.
Sulconazole was also able o inhibi colon cance and b eas
cance cells as well as melanoma p oli e a ion, and mig a ion,
sugges ing he dual he apeu ic e ec o his d ug.
2 Ma e ials and me hods
2.1 PBMCs, T cells and cance cells cul u e
and PBMCs ac i a ion
Human pe iphe al blood mononuclea cells (hPBMCs) we e
ob ained ollowing w i en in o med consen app o ed by Be gonie
Ins i u e (Bo deaux, F ance). PBMCs we e isola ed om heal hy
dono s by densi y g adien cen i uga ion wi h Pancoll
(PANBio ech; human, densi y 1,077g/ml) and we e cul u ed in
RPMI 1640 (PAN-Bio ech) supplemen ed wi h 10% FBS (Gibco),
2mM L-glu amine (Gibco) and penicillin/s ep omycin solu ion
(Dominique Du sche ). Human T cells we e pu ified om blood
using he MACSxp ess®Whole Blood Pan T Cell Isola ion Ki
(Mil enyi Bio ec). The mu ine colon cance cells CT-26, he human
colon cance cells HT29, melanoma cells M10 and Ju ka T and
J.RT3-T3.5 (JRT3) cell lines we e cul u ed in RPMI 1640 comple e
media. The b eas cance cells MDA-MB 231 we e cul u ed in
DMEM comple e media. All he cells we e g own a 37°C in a 95%
ai , 5% CO2 humidified incuba o . Ac i a ion o Ju ka T cells,
pu ified T cells and PBMCs was induced by pho bol my is a e
ace a e (PMA,100ng/ml) and Ionomycin (Io,1ug/ml), as p e iously
desc ibed (17).
2.2 Real- ime qPCR
To al RNA was isola ed using he Nucleospin RNA ki
(Mache ey-Nagel) p io e e se ansc ip ion in a eac ion mix u e
con aining 50mM T is-HCl (pH 8.3), 30mMKCl, 8mM MgCl2, 1mM
dNTPs, and 0.2U Supe sc ip e e se ansc ip ase (In i ogen), as
p e iously desc ibed (17,18) in a Ve i iThe mal Cycle (Applied
Biosys em). Quan i a i e eal- ime PCR was pe o med using specific
TaqMan p ime s and Mas e Mix (Eu ogen ec), in a S epOne Plus
Real-Time PCR sys em ollowing manu ac u e ’s ins uc ions
(Applied Biosys em). GAPDH was used o no maliza ion. The
PDCD1 p ime s used a e F 5’-CTACAACTGGGCTGGCGG-3’
and R 5’-TGTGTTGGAGAAGCTGCAGG-3, espec i ely. The
p ime s o CTLA4 we e de i ed om BioRad Unique Assay
(ID qHsaCED0003794).
2.3 Ca
2+
mobiliza ion measu emen
quenching assay
Indica ed cells we e ea ed o 48 h wi h 10 µM o Sulconazole
o ehicle (DMSO) in hei cul u e media, and hen Ca
2+
influx was
e alua ed using 2 µM Fu a-2 AM (The moFishe Scien ific, US) and
mic ofluo ome y sys em, o adhe en cells, o CLARIOs a ®Plus
mic opla e eade (BMG Lab ech; Ge many), o T cells. The
mic ofluo ome y sys em includes in e ed mic oscope Leica
(We zla , Ge many) wi h a 20×/0.75 NA objec i e, a
monoch oma o (Polych ome V, Till Pho onics, Munich,
Ge many), a CCD came a and HP so wa e (Hamama su
Pho onics, Japan). In bo h cases, he changes o in acellula Ca
2+
concen a ion ([Ca
2+
]
i
) we e shown as he a io o Fu a-2 AM
fluo escence a e exci a ion a 340 and 380 nm ( a io = F
340
/F
380
).
Expe imen s we e done ollowing he pe iod sequence: 4 min in (1)
ee Ca
2+
solu ion (140 mM NaCl, 2.7 mM KCl, 4 mM MgCl
2
, 0.5
mM EGTA, 10 mM HEPES, pH = 7.4) wi h 2 µM o hapsiga gin, in
o de o s imula e S o e Ope a ed Ca
2+
En y (SOCE), and 6 min in
(2) 2-2.5 mM Ca
2+
solu ion (2-2.5 mM CaCl
2
; 140 NaCl, 2.7 KCl, 1
MgCl
2
, 0.5 EGTA, 10 HEPES, pH = 7.4). Then, Ca
2+
influx (D a io)
Pe no e al. 10.3389/ immu.2023.1278630
F on ie s in Immunology on ie sin.o g02
was compu ed as he di e ence be ween he peak a io a e 2 min o
ex acellula Ca
2+
e-addi ion and i s le el jus be o e.
2.4 JRT3 unc ional assay
This unc ional assay, is based on Ju ka T-cell line s ably
exp essing he human LES -gd TCR (JRT3-LES) incuba ed wi h
he colon cance cell line HT29 o e exp essing he endo helial
p o ein C ecep o (HT29-EPCR), as p e iously desc ibed (17).
The ac i a ion o JRT3-LES cells was e alua ed by he exp ession
o CD69.
2.5 P oli e a ion assay
The p oli e a ion o indica ed cells was pe o med using he
IncuCy e li e-cell mic oscopy incuba o (Essen Bioscience). Cells
(2 × 10
5
) we e ea ed wi h indica ed concen a ions o SCZ in he
p esence o 3% and 10% se um and placed in he IncuCy e
incuba o , and phase-con as images we e aken a egula
in e als o e 96 hou s. Resul s we e calcula ed by he IncuCy e
so wa e and p esen ed as confluence ela i e o ime 0. Images we e
aken wi h a ×4 objec i e. Fou images we e aken om each well,
and each condi ion had mo e han 6 wells.
2.6 Wound healing assay
Cells we e seeded in 96-well pla es and allowed o g ow un il
hey eached 90% confluence (2 × 10
6
cells pe well o each cell line).
The cell wound was pe o med using Incucy e®Wound Make 96-
Tool, and cells we e ea ed wi h a ious concen a ion o SCZ
du ing a ious ime pe iods. Pla es we e placed in he IncuCy e
incuba o which ook 4 phase-con as images and calcula ed cell-
ee a ea o each well a egula in e als o e 48 hou s. Resul s we e
shown as ela i e wound densi y.
2.7 Immunocy ochemis y
De ec ion o PD-1 in Ju ka T cells we e moni o ed using an
an i-PD-1 an ibody a 1:100 in TBS wi h 5% BSA, as p e iously
desc ibed (17). Con ocal immunofluo escence images we e aken
using he in e ed mic oscope Nikon C2si Eclipse Ti-S wi h NIS-
Elemen sAR so wa e (NikonIns umen s Eu ope B.V.).
2.8 Flow cy ome y analysis
Cells we e s ained wi h fluo opho e-conjuga ed an ibodies: PE-
an i-PD-1 mAb (MIH4, #560908 eBiosciences) and Flowcy ome y
da a we e acqui ed wi h BD Accu i C6 (BD Biosciences). Flow
cy ome y analyses we e pe o med using BD Accu i C6 so wa e
and FlowJo 9.3.2 (T eeS a ), as p e iously desc ibed (17).
2.9 Wes e n blo ing
Cells we e lysed in PBS con aining 2% NP-40 and lysa es we e
subjec ed o SDS-PAGE (BioRad Minip o ein) and p o eins we e
blo ed on o poly inylidene difluo ide memb ane (PVDF,
Ame sham Pha macia Bio ech). The p ima y an ibodies an i-NF-
kB p65 (#sc-8008, dilu ion 1/500) om San a C uz Bio ech,
phospho-NF-kBp65 (#3033, dilu ion 1/500) om Cell signaling,
TRPC1: abbi an i-TRPC1 (1:500, T8276; Sigma-Ald ich, Uni ed
S a es), STIM1: abbi an i-STIM1 (1:500, 4916S; Cell Signaling,
Uni ed S a es) and O ai1: abbi an i-O ai1(1:250, O8264; Sigma-
Ald ich, Uni ed S a es) we e e ealed by HPR-conjuga ed
seconda y an ibodies (Ame sham Pha macia Bio ech) and
enhanced chemiluminescence (Pie ce ECL Plus, The mo
Scien ific) acco ding o he manu ac u e ’s ins uc ions. Images
we e acqui ed wi h a Genegnome sys em and GeneSysso wa e
(Syngene) (17–19).
2.10 Tumo igenici y assay
All expe imen s pe o med in his s udy we e app o ed by he
uni e si y o Bo deaux Animal E hics. Adul AB zeb afish s ain
(ZIRC, USA) we e main ained ollowing he F ench Di ec i e unde
pe mission numbe A33-063-935. All he p ocedu es we e
conduc ed in compliance wi h he Eu opean Communi ies
Council Di ec i e (2010/63/EU). Following he p oduc ion o
emb yos by adul zeb afish, emb yos we e allowed o g ow in E3
medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM
MgSO4) a 28°C, as desc ibed p e iously (20,21). A e
decho iona ion, 2-day pos e iliza ion (dp ) zeb afish emb yos
we e anaes he ized wi h 0.003% icaine (Sigma, USA) and placed
in 3% me hylcellulose on a dish coa ed wi h 1% aga ose. MDA-
MDB-231 cells we e de ached using Ve saine solu ion and 2.5·10
6
cells we e esuspended in 50 ml o PBS con aining 1% phenol ed.
Immedia ely, cell suspension was loaded in o Fem o ip II capila
needles (Eppendo , Ge many) and injec ions we e pe o med
using a pump (Fem oje 4i; Eppendo , Ge many) and a
mic omanipula o (Phymep). A ound 500 cells pe emb yos we e
inse ed abo e he duc o Cu ie in pe i i elline space o he
emb yo, as p e iously desc ibed (22). A e checking he
implan a ion wi h mammalian cells, zeb afish emb yos we e
main ained in 24-well pla es a 36.3°C. Then, SCZ (1 µM) o
ehicle (DMSO) we added o o each well. Tumo imaging was
done a e 48-h pos injec ion (hpi) using Nikon EclipseTS100
mic oscope. The umo size was e alua ed using he a ea o he
de eloped umo s.
2.11 S a is ical analysis
Unless o he wise indica ed, da a a e p esen ed as mean ± SEM.
A 2- ailed es was used o analyze he da a in G aphPad P ism
(G aphPad So wa e). The s a is ical significance le el is illus a ed
wi h P (p- alues). S a is ical P was se han 0.05.
Pe no e al. 10.3389/ immu.2023.1278630
F on ie s in Immunology on ie sin.o g03
3 Resul s
3.1 Sulconazole inhibi s PD-1 exp ession in
T cells and PBMCs
A po en T cell esponse equi es PKC signaling (23). To
induce PBMCs, human pu ified T cells ac i a ion we fi s used
combina ion o PMA and Io (PMA/Io) ha mimics T-cell
ecep o (TCR) ac i a ion. Indeed, PMA binds o and ac i a es
PKC whe eas ionomycin (Io) is a calcium ionopho e ha
enhances memb ane pe meabili y o calcium. Incuba ion o
indica ed cells wi h PMA/Io o 24h induced PD-1 mRNA
exp ession in all indica ed cells (Figu es 1A,B). In he p esence
o SCZ (5mM), PD-1 mRNA exp ession was conside ably
dec eased a e 24h o ea men , as assessed by eal ime-PCR.
Simila ly, using he Ju ka T cells, hei ea men wi h SCZ, also
a ec PD-1 exp ession (Figu e 1C). These obse a ions indica e
ha PD-1 exp ession on T cells and PBMCs can be ep essed.
To e alua e he exp ession o PD-1 in PBMCs and T cells a
he p o ein le el, we fi s ea ed he PMA/Io ac i a ed-Ju ka T
BC
DE
FG
HIJ
A
FIGURE 1
Inhibi ion o PDCD1 exp ession in PBMCs (A), pu ified T cells (B) and Ju ka T cells (C) by Sulconazole (SCZ). PD-1 mRNA le el upon PMA/Io
s imula ion in he absence and p esence o SCZ (5mM) a 24h, assessed by eal ime-PCR. Da a a e ep esen ed as old change o PMA/Io-s imula ed
cells ha was assigned 100% as mean ± SEM om h ee independen expe imen s. *, P< 0.05. (D, E) Ju ka T cells we e ea ed wi h indica ed SCZ
concen a ions p io hei incuba ion wi h PMA/Io. PD-1 exp ession was analyzed by Flow cy ome y. (F, G) Rep esen a i e con ocal mic oscopy
images o PD-1 immunos aining (g een) o PMA/Io-ac i a ed Ju ka T cells in he absence and p esence o SCZ. (H, I) PD-1 exp ession in PBMCs (H)
and pu ified T (I) cells ea ed wi h SCZ p io hei incuba ion wi h PMA/Io, as assesses by Flow cy ome y. (J) CTLA-4 mRNA le el upon PMA/Io
s imula ion in he absence and p esence o SCZ (5mM) a 24h, assessed by eal ime-PCR. Scale ba , 10 mm. Da a a e ep esen ed as mean ± SEM
om h ee independen expe imen s. MFI, mean fluo escence in ensi y (a bi a y uni ). * P < 0.05.
Pe no e al. 10.3389/ immu.2023.1278630
F on ie s in Immunology on ie sin.o g04
cells wi h indica ed SCZ concen a ions. Flow cy ome y analysis
e ealed ha PD-1 exp ession is also induced a he p o ein le el
in he p esence o PMA/Io ha was ep essed by sulconazole in a
dose-dependen manne (Figu es 1D,E). PD-1 exp ession was
educedbyup o60%wi h5mMo SCZ.Theuseo
immunofluo escence s aining unde hese condi ions confi med
ha PD-1 exp ession was g ea ly dec eased in he p esence o
sulconazole (Figu es 1F,G). The use o PMA/Io- ea ed-PBMCs
and pu ified T cells also e ealed hei educed PD-1 exp ession in
he p esence o SCZ (Figu es 1H,I). These obse a ions indica e
ha SCZ can impede PD-1 exp ession a he RNA and p o ein
le els in PBMCs and T cells. To e alua e whe he SCZ is also able
o a ec he exp ession o o he immune checkpoin inhibi o s
in ol ed in T cell exhaus ion, such as CTLA-4. As illus a ed in
Figu e 1J, ea men o ac i a ed PBMCs wi h SCZ (5mM),
significan ly a ec ed CTLA-4 exp ession.
3.2 Rep ession o NF-kB exp ession and
calcium mobiliza ion by sulconazole
NF-kB ac i a ion and calcium mobiliza ion has p e iously been
epo ed o be in ol ed in PD-1 exp ession (17,24). The eby, we nex
in es iga ed he e ec o SCZ on hese wo PD-1 signaling pa hways.
As illus a ed in Figu es 2A,Bincuba ion o Ju ka T cells a he
indica ed ime poin s wi h PMA/Io induced NF-kB phospho yla ion.
Maximal s imula ion was obse ed a e 10-20 min and was
down egula ed a e 40 min o cells s imula ion. The p esence o
SCZ significan ly educed NF-kB ac i a ion. The inhibi o y e ec o
SCZ was significan a e 20 min and maximal a e 40 min o cells
incuba ion. These esul s show ha SCZ ep esses he ac i a ion o
NF-kB equi ed o PD-1 exp ession. Simila ly, s o e-ope a ed Ca
2+
en y (SOCE) is a mechanism o Ca
2+
influx ac oss he plasma
memb ane ac i a ed in esponse o deple ion o in acellula Ca
2+
B
CD
A
FIGURE 2
Inhibi ion o NF-kB phospho yla ion and calcium mobilisa ion by Sulconazole (SCZ) in Ju ka T cells. (A) Ju ka T cells we e ea ed wi h SCZ p io
hei incuba ion wi h PMA/Io and he phospho yla ion o NF-kB was analyzed by wes e n blo ing a he indica ed ime poin s. (B) Resul s o NF-kB
phospho yla ion quan ifica ion a e shown in he ba g aph and calcula ed, as he a io o p-NF-kB/To al NF-kB and a e ep esen a i e o h ee
independen expe imen s. (C) Rep esen a i e eco dings o hapsiga gin-induced changes in he in acellula calcium concen a ion exp essed as
fluo escence a io (F340/F380). Ju ka cells we e incuba ed o 4 min in a ee Ca
2+
solu ion in he p esence o 2mM o hapsiga gin and Ca
2+
(2.0 mM) was e-added. (D) Ba g aph shows he pe cen age o del a a io inc ease a e and be o e adding Ca
2+
in cells ea ed wi h o wi hou SCZ
(10mM). Da a a e ep esen ed as mean ± SEM om h ee independen expe imen s. * P < 0.05.
Pe no e al. 10.3389/ immu.2023.1278630
F on ie s in Immunology on ie sin.o g05
s o es, mos ly in he ER. To e alua e he e ec o SCZ on Ca
2+
mobiliza ion, Ju ka T cells we e incuba ed wi h sulconazole (10 mM),
and Ca
2+
le els we e measu ed. Cells we e loaded wi h u a-2, and
hen s imula ed wi h 2 mM hapsiga gin o deple e he ER and ac i a e
SOCE. As illus a ed in Figu es 2C,D, SCZ inhibi ed Ca
2+
en y in o
cells. These obse a ions, oge he wi h he finding ha SCZ was able
o egula e NF-kB ac i a ion, indica e SCZ abili y in he ep ession o
di e en pa hways in ol ed in he PD-1 exp ession on T cells.
3.3 Func ional and su i al o Ju ka T cells
in he p esence o sulconazole
To e alua e he e ec SCZ on Ju ka T cells unc ion, we nex
analyzed he exp ession o CD69, a T-cell ac i a ion ma ke , by flow
cy ome y o analyze an igen-TCR binding and he e o e,
downs eam T-cell ac i a ion. The eby we used he TCR-deficien
Ju ka T cells (JRT3), exp essing a specificTCR(LES) ha
ecognizes he EPCR p o ein ha we e cocul u ed wi h EPCR-
exp essing HT29 cance cells. As illus a ed in Figu es 3A,B he
exp ession o CD69 was up egula ed when JRT3-LES cells we e
cocul u ed wi h EPCR-exp essing HT29 cells. Simila ly, Following
JRT3-LES ea men wi h SCZ (5mM) o 24 hou s in he p esence
o EPCR-exp essing HT29 cance cells also s ongly up egula ed
CD69 exp ession (Figu es 3A,B). We nex e alua ed he e ec o
SCZ on cell su i al. The eby, T cells we e incuba ed wi h SCZ in
he absence and p esence o se um and flow-cy ome ic analysis o
cell dea h was pe o med using annexin V and 7AAD as ma ke s.
Flow cy ome ic analysis e ealed ha Ju ka T cells incuba ion wi h
up o 5 mM SCZ had no e ec on cell su i al. In he absence o
B
C
D
A
FIGURE 3
T cell ac i i y and su i al a e no a ec ed by sulconazole. (A, B) Flow cy ome y analysis o TCR-ac i a ion ma ke CD69 in JRT3 cells upon binding o HT29
cance cells in he p esence and absence o SCZ (C, D) Fluo escence-ac i a ed cell so e sca e plo s o Ju ka T cells incuba ed o 24h wi hou o wi h
se um (5%) a indica ed SCZ concen a ions. A e incuba ion, cells we e double s ained wi h annexin V and 7AAD. The use o fluo escence-ac i a ed cell
so e de ec ed iable (nega i e o bo h dyes; lowe le ), ea ly apop o ic (Annexin+/7AAD−,lowe igh ),nec o iccells(Annexin−/7AAD+, uppe le ) and la e
apop o ic (Annexin+/7AAD+, uppe igh ) cells. Da a ep esen ed as mean ± SEM om h ee independen expe imen s. *, P<0.05.NS,no significan .
Pe no e al. 10.3389/ immu.2023.1278630
F on ie s in Immunology on ie sin.o g06
se um, only he concen a ion o 10mM induced cell dea h
(Figu es 3C,D).
3.4 Sulconazole inhibi s calcium
mobiliza ion in cance cells
Calcium is conside ed as a egula o o he malignan
pheno ype o cance cells, he e o e, we u he in es iga ed he
e ec s o SCZ on Ca
2+
influx. Fi s , he b eas cance cells MDA-
MB-231 we e p e ea ed wi h SCZ and hen calcium signals we e
de ec ed. As shown in Figu es 4A,B,Ca
2+
influx induced by
hapsiga gin was blocked by SCZ (10 mM). These esul s implied
ha SCZ blocked calcium mobiliza ion, a signal pa hway equi ed
o a ious p ocesses equi ed o he acquisi ion o he malignan
pheno ype o cance cells, including cell p oli e a ion, and
mig a ion. Fu he analysis e ealed ha he e ec o SCZ on
calcium mobiliza ion is no media ed by changes in he le els o
he calcium channels STIM1, ORAI1 and TRPC1, as assessed by
wes e n blo ing analysis (Supplemen a y Figu e 1).
3.5 Sulconazole inhibi s cance cell
p oli e a ion and mig a ion
To de e mine he e ec o SCZ on he malignan pheno ype, he
p oli e a ion o se e al cance cells, including he melanoma M10,
he b eas MD-MB231 and he colon CT-26 cance cell lines we e
ea ed wi h indica ed concen a ions o SCZ in he p esence o low
(3%) and high (10%) concen a ion o se um and hei p oli e a ion
was measu ed using he IncuCy e Li e Cell Analysis Sys em
(Figu es 5A–I). As illus a ed, ea men o hese cells wi h SCZ
significan ly dec eased hei confluence a e. The e ec o SCZ was
mo e e ficien in he p esence o low concen a ion o se um. We
nex e alua ed he e ec o SCZ on he mig a ion o hese cance
cells in a wound-healing assay. As illus a ed in Figu es 6A–D,
ea men wi h SCZ o indica ed ime pe iods, educed cance cell
mo ili y in a concen a ion and ime-dependen manne .
3.6 Sulconazole inhibi s umo g ow h
in zeb afish
To di ec ly assess he e ec o SCZ on umo g ow h, we used
zeb afish emb yos injec ed wi h MDA-MB-231 cance cells, as in
i o model. Indeed, he adap i e immune sys em o zeb afish
ma u es a 28 dp (25–27). P e iously, he de elopmen o T cells
in zeb afish was epo ed o occu pos 5 days pos - e iliza ion
(dp ). This empo al sequence co esponds o he infil a ion o he
hymus by lymphoid p ogeni o cells a ound c wi h subsequen
eg ess om he hymus occu ing be ween 6 dp and 7 dp ,
acili a ing en y in o he pe iphe al ci cula ion (28). The eby,
mul iple cance models ha e been gene a ed in zeb afish and
p o en simila o hei human coun e pa s molecula ly and
pa hologically (22,29,30). To assess he e ec o SCZ on umo
g ow h we used 2 dp zeb afish emb yos o injec 500 cells/fish (25-
30 fish) and allowed o g ow o 48h in he absence and p esence o
SCZ (1mM). As shown in Figu es 7A,B, he p esence o SCZ in he
emb yo’s media educed up o ~ 4- old ime MDA-MB-231 cance
cells abili y o induce umo g ow h in zeb afish emb yos,
confi ming he an i umo igenic e ec o SCZ in i o.
4 Discussion
The in e ac ion be ween PD-1 on T cells and i s ligand PDL-1
exp essed on umo cells di ec ly a ec s he cy o oxic unc ion o T
cells equi ed o cance cells e adica ion (1,6). Howe e , he umo
B
A
FIGURE 4
Inhibi ion o calcium mobilisa ion by Sulconazole in cance cells.
(A) Rep esen a i e eco dings o hapsiga gin-induced changes in
he in acellula calcium concen a ion exp essed as fluo escence
a io (F340/F380). MDA-MB-231 cells we e incuba ed o 4 min in a
ee Ca
2+
solu ion in he p esence o 2mM o hapsiga gin and Ca
2+
(2.5 mM) was e-added. (B) Ba g aph shows he pe cen age o del a
a io inc ease a e and be o e adding Ca
2+
in cells ea ed wi h o
wi hou SCZ (10mM). Da a a e ep esen ed as mean ± SEM om ou
independen expe imen s.
Pe no e al. 10.3389/ immu.2023.1278630
F on ie s in Immunology on ie sin.o g07
mic oen i onmen (TME) including he componen s o he
ex acellula ma ix o a ious TME cells a ec he exp ession o
PD-1 on T cells leading o immune e asion. The monoclonal
an ibodies used o a ge PD-1 and PD-L1 we e ound o be
po en immune checkpoin inhibi o s and we e used o a ious
cance s ea men such as melanoma, lung cance and gas ic
cance . Indeed, since 2014, FDA has app o ed a ious an i-PD-1
and an i-PD-L1 monoclonal an ibody d ugs. These d ugs ha e
made se ious imp o emen in he clinical ea men o a ious
umo s and p olonged he su i al o cance pa ien s. Fo se e al
cance pa ien s, hese d ugs media ed comple e emission.
Howe e , o he pa ien s wi h solid umo s, including colo ec al
cance s [excep he mic osa elli e-ins able (MSI) subse ] a e
e ac o y o hese ea men s (6,8).
The ailu e o espond o an i-PD-1 d ug is mainly due o he
p esence o i e e sibly exhaus ed T cells. In addi ion, a ious
ad e se esponses a e media ed by hese inhibi o s ha a e
mainly immune- ela ed ad e se eac ions ound o a ec se e al
issues and o gans o he ea ed pa ien s (31,32). O he s udies also
showed ha i is di ficul o an ibody d ugs o success ully infil a e
umo issues and each all a eas o he umo mic oen i onmen in
o de o be accumula ed a an adequa e and e ficien concen a ion
(17). O he s udies e ealed ha he e ficacy o hese d ugs can be
educed wi h ime and was linked o hei immunogenici y ha
induces he p oduc ion o an i-an ibodies du ing he ea men
(32). The e o e, i is necessa y o de elop no el inhibi o s such as
small molecules wi h confi med e ficacy and sa e y o a ge he PD-
1/PD-L1 in e ac ion. Indeed, compa ed o monoclonal an ibodies
ha a e di ficul o p oduce, small molecule p esen a ious
ad an ages o make hem p omising o clinical ea men s (33).
Small molecule inhibi o s a e mo e app op ia e o o al
managemen , and can be used o a oid immune- ela ed ad e se
e ec s due o he acili y o hei changeable hal -li e. In his s udy,
we demons a e ha SCZ is able o ep esses he exp ession o PD-1
BC
DE F
GH
A
I
FIGURE 5
Inhibi ion o cance cells p oli e a ion by Sulconazole. colon cance cells (CT-26) (A–C), Melanoma cells M10 (D–F) and b eas cance cells MDA-
MB-231 (G–I) and we e pla ed a low confluence o ime-lapse phase-con as ideomic oscopy (IncuCy e mic oscope) in he absence and
p esence o di e en concen a ions o SCZ and se um, and cell p oli e a ion was moni o ed by au oma ed confluence analysis a se in e als a e
pla ing. Da a a e ep esen ed as mean ± SEM om h ee independen expe imen s (n= 6 wells pe g oup). * P < 0.05.
Pe no e al. 10.3389/ immu.2023.1278630
F on ie s in Immunology on ie sin.o g08
on T cells and PBMCs and educes cance p oli e a ion, mig a ion
and umo g ow h in i o using zeb afish emb yos model.
P e iously, NF-kB and calcium signaling pa hways we e
epo ed o be equi ed o PD-1 exp ession on T cells (17). We
ound ha SCZ inhibi ed hese pa hways and he eby linking PD-
1 ep ession by SCZ in T cells o NF-kB ac i i y and calcium
mobiliza ion inhibi ion. Fu he analysis e ealed ha SCZ is also
able o ep ess CTLA-4 exp ession in PBMCs. Ou esul s
iden ified SCZ, as small no oxic molecule medicine ha may
cons i u e a s a ing poin o he iden ifica ion o new class o
immune checkpoin inhibi o egula o s ha will ha e he chance
o be used a he clinical se ing since hei none oxici y was
p e iously es ablished. Indeed, cu en p eclinical s udies e ealed
ha small molecule compounds ha e a be e capabili y han
an ibodies o ep ess umo g ow h p og ession (25), sugges ing
hei use o o e come he exis ing p oblems o an ibody d ugs and
allow hem o eplace monoclonal an ibodies o se e as
complemen a y he apies.
BC
D
A
FIGURE 6
Inhibi ion o cance cells mig a ion by Sulconazole. (A–D) cance cell mig a ion was analyzed by sc a ch wound assay. M10 (A, B) CT-26) (C), and
MDA-MB-231 (D) cells we e ea ed wi h a ious SCZ concen a ions and we e subjec ed o sc a ch wounds and imaged using IncuCy e mic oscope
du ing 48h (n= 6 wells pe g oup, 3 independen expe imen s). (B–D) Quan ifica ion o wound closu e du ing indica ed ime pe iods. Da a a e
ep esen ed as mean ± SEM.
Pe no e al. 10.3389/ immu.2023.1278630
F on ie s in Immunology on ie sin.o g09
