Sulfisoxazole inhibits the secretion of small extracellular vesicles by targeting the endothelin receptor A

Article abstract of DOI:10.1038/s41467-019-09387-4

Inhibitors of the secretion of cancer exosomes, which promote cancer progression and metastasis, may not only accelerate exosome biology research but also offer therapeutic benefits for cancer patients. Here we identify sulfisoxazole (SFX) as an inhibitor

Full text of DOI:10.1038/s41467-019-09387-4

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https://doi.org/10.1038/s41467-019-09387-4
OPEN
S u l fis o x a z ol e i n h i b i t s t he s e c r e ti o n o f s m a l l
e x t r a ce l l ul a r v e s i c l es b y t a r g e t i ng t h e e n do th e l in
r e c e pt o r A
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E u n - J u I m , C h a n - H y e o n g L e e , P y o ng - Go n M o o n , G u n a ss e k a r a n G o w r i R a n g a s w a m y , B y u n g h e o n L ee ,
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S e u n g - Yo n g S e o
, Y ou n g- E un C ho , B y o u n g - J o o n S o n g & M o o n - C h a n g B a e k
I n hi b i t o r s o f t h e s e c r e t i o n o f c a n c e r e x o s o m e s , w h i c h p ro m ot e c a n c e r p ro g r e s s i o n a n d
m e t a s t a s i s, m ay n o t o n l y a c ce l e r a t e e x o s o m e b i o l o g y r e s e a r c h b ut a l s o o ff e r t h e r a p eu t i c
b e ne fit s f o r c a n c e r p a t i e n ts . H e r e w e i d e n t i f y s u l fis o x a z ol e ( S F X ) a s a n i n h i bi to r o f s m a l l
e x t r a c e l l ul a r v e si c l e s ( s E V ) s e c r e ti o n f r o m b r e a s t c a n c er c e l l s t h ro u g h i n t er fe re n c e w i t h
e n do t he l in r e c e pt or A ( E T A ) . S F X , a n F D A - a p p r o v e d o ra l a n t i bi ot i c , s h o we d s i g ni fic a n t a n t i -
t u m o r a nd a nt i- me ta st a t i c e f f e c ts i n m o u s e m o d e l s o f b re a s t c a n c e r x en o g ra f t s , t h e r e d u c e d
e x p r e s s io n o f p r ot e in s i n v o l v e d i n b i o g e n e si s a n d s e c r e t i o n o f s E V , a n d t ri g g er e d c o -
l o c a l i z at io n o f m u lt i ve s i cu l a r e n d o s o me s w i th l y so s o m e s f or d e g r a d a t i o n. W e d em on s t r a t e
t h e i m p o r ta n t r o le o f E T A, a s t a r g e t o f S F X , b y g a i n - a n d l o s s - o f- f u nc ti on s t u d i e s o f t h e
E T A p r ot e in , t h r o ug h a d i r e c t b i n d i n g a s s a y , a n d p h a rm a c o l o g i c a l a n d g e ne ti c a p p ro a c h e s .
T h e s e fin d in g s m ay p r o v i d e a f o u n d a t i o n f o r s E V - ta rg e t e d c a n c e r t h er a p i es a n d t h e
m e c h an is t i c s t u d i e s o n s E V b i o l o g y .
1 Department of Molecular Medicine, CMRI, Exosome Convergence Research Center (ECRC), School of Medicine, Kyungpook National University, Daegu
41944, Republic of Korea. 2 Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Daegu 41944, Republic of
Korea. 3 Department of Microbiology, School of Medicine, Kyungpook National University, Daegu 41944, Republic of Korea. 4 Research Institute of
Pharmaceutical Researches, College of Pharmacy, Kyungpook National University, Daegu 41566, Republic of Korea. 5 Department of Immunology and School
of Medicine, Keimyung University, Daegu 42601, Republic of Korea. 6 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National
University, Seoul 08826, Republic of Korea. 7 Chuncheon Center, Korean Basic Science Institute, Gangwon-do 24341, Republic of Korea. 8 Department of
Biochemistry, College of Natural Sciences, Kangwon National University, Gangwon-do 24341, Republic of Korea. 9 Drug & Disease Target Group, Korea Basic
Science Institute, Cheongju 28119, Republic of Korea. 10 College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea. 11 College of Pharmacy
and Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon 21936, Republic of Korea. 12 Section of Molecular Pharmacology and
Toxicology, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism (NIAAA), Bethesda 20892 MD,
USA. Correspondence and requests for materials should be addressed to M.-C.B. (email: mcbaek@knu.ac.kr)
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etastasis is the main cause of mortality in cancer sEV secreted from the cancer cells treated with each drug. During
patients, but clinical options against advanced metas- the initial primary screening, we tested drugs at 30 μM, and the
tasis stage of cancer remain limited owing to high top 26 drugs (that inhibited sEV secretion by up to 30%) were
M
complexity of the biological events of metastasis, leading to selected as potential candidates for the secondary assessment
inefficient drug development and poor treatment outcomes1,2
.
at 50 and 100 μM concentrations (Supplementary Fig. 2a, b).
Exosomes are 50–150 nm small extracellular vesicles (sEV) that However, some drugs were not further studied based on a range
harbor proteins, lipids, RNAs, and DNA, and thereby act as of exclusion criteria, as described (Fig. 1a). Finally, the antibiotic
important mediators of cell–cell communications in various sulfisoxazole (SFX) was selected for further in-depth evaluation
physiological and pathological pathways3. Cancer-cell-derived because it is a cheap, orally administered FDA-approved drug
sEV prepare a favorable microenvironment at future metastatic without cytotoxicity at effective doses (Fig. 1a and Supplementary
sites as well as the primary tumor4–7. Hence, the clearance of Fig. 2b). This drug was originally known to be a competitive
these malicious sEV in circulating system has emerged as a novel inhibitor of the enzyme dihydropteroate synthetase through
and potentially useful therapeutic strategy for anti-metastatic preventing the condensation of pteridine with p-aminobenzoic
drug development8. Many reports have already demonstrated that acid, a substrate of the enzyme in prokaryotic systems15.
the reduction of sEV secretion (or secreted sEV), achieved by
using a chemical inhibitor9,10, genetic engineering11, or anti-
SFX inhibits sEV secretion quantitatively and qualitatively. We
body12, can enhance the efficiency of cancer chemotherapy and
systematically analyzed the inhibitory effects of SFX on the
inhibit cancer metastasis. However, further work is required to
secretion of sEV in three representative human breast cell lines:
determine whether these inhibitors can affect the secretion of
MCF10A (normal), MCF7 (weakly invasive), and MDA-MB231
other EVs or soluble proteins, or the pathophysiological features
(highly invasive). Notably, SFX treatment quantitatively inhibited
of donor cells, as reviewed previously13. Moreover, the underlying
sEV secretion from MCF7 and MDA-MB231 cancer cells, as
mechanisms of the already-identified inhibitors that have been
determined by the ultracentrifugation method (Supplementary
demonstrated to control exosome biogenesis and secretion have
Fig. 3a, b), in a dose-dependent manner without morphological
still not been clearly elucidated while their safety/toxicity profiles
change or cytotoxic effect (Fig. 1b, c and Supplementary
are unknown.
Fig. 3c–e). However, no significant inhibition was observed in
Drug repurposing, the process of finding new indications for
normal MCF10A cells, which rarely secrete sEV, suggesting that
existing drugs, is a faster, cheaper, and safer drug development
SFX shows a strong inhibitory effect on the secretion of sEV from
strategy. In this process, the new indication can be derived from
breast cancer cells (Fig. 1b and Supplementary Fig. 3c). Con-
the same target (on-target) or a newly-recognized target (off-
sistent with this reduction of secreted sEV, the amounts of sEV
target) of the original drug14. A significant advantage of drug
proteins, such as CD9, filotillin-1, Alix, Tsg101, and CD63, were
repurposing is that regulatory agency-approved drugs have
decreased (Supplementary Fig. 3f). Moreover, the number of sEV
already passed toxicity and safety tests in humans. One of major
and amounts of sEV proteins were reduced in SK-MEL-28
concerns for the development of a new drug to inhibit the
human melanoma cancer cells, which secrete a large number of
secretion of sEV is the toxicity, probably caused by any partial or
sEV (Supplementary Fig. 3g).
temporary inhibition of exosome secretion from normal cells
Next, we investigated whether SFX affects other secretion
when a drug candidate inhibits the secretion of sEV from cancer
pathways, such as microvesicles (MVs) and various soluble
cells. We believe that drug repurposing could reduce the risk of
protein secretions. SFX neither significantly affected the secretion
failure by saving valuable time and efforts during the identifica-
of MVs from cells nor altered the activity of acidic sphingomye-
tion and development of a new inhibitor of sEV secretion as a
linase (aSMase), which is involved in the formation of large MV16
novel anti-cancer therapeutic agent.
(Fig. 1d and Supplementary Fig. 3h). In addition, the classical
In this study, by screening the library of FDA-approved drugs,
secretion pathway was not significantly affected by SFX treatment
we identified sulfisoxazole (SFX), an oral antibacterial drug, as a
(Fig. 1e). Furthermore, we performed miRNA microarray
specific inhibitor of the biogenesis and secretion of sEV from
(Supplementary Fig. 4a) and proteomics analyses (Supplementary
breast cancer cells, resulting in the effective suppression of breast
Fig. 4b) of MDA-MB231 sEV, and confirmed that SFX affected
cancer growth and metastasis without significant toxicity. Fur-
the components of sEV cargo, including various miRNAs and
thermore, we found that endothelin receptor A (ETA), a member
proteins (EDIL3, HSP90, and GPC117–19), known to be present in
of GPCR family, is critically associated with sEV biogenesis and
sEV derived from MDA-MB231 cells20 (Fig. 1f, g).
secretion in breast cancer cells, and that ETA is a newly-identified
target (off-target) of SFX, as evidenced by gain- and loss-of-
function studies of the ETA protein through pharmacological and SFX inhibits breast cancer progression. We also examined the
genetic approaches. Our findings may provide a foundation for anti-cancer effect of SFX on breast cancer cells because cancer-
sEV-targeted cancer therapies and the mechanistic studies on sEV cell-derived sEV are known to play important roles in cancer
biology.
progression and metastasis. SFX significantly halted cellular
proliferation, colony formation, and cancer cell invasion/migra-
tion activities (Supplementary Fig. 5a–e), without obvious cyto-
toxicity (Supplementary Fig. 3c), compared with the control cells.
Results
Discovery of a drug for inhibition of EV secretion. To identify Based on these in vitro data, we examined the anti-cancer effect of
drugs that reduce sEV secretion, we developed cell-based high- SFX by using two different mouse cancer xenograft models
throughput assay system with 1163 FDA-approved drugs, (Fig. 2a). In our previous pharmacokinetic study21, SFX showed
according to the flow chart for primary and secondary screenings excellent oral bioavailability and maintained desirable exposure
(Fig. 1a). To accomplish this task, MDA-MB231 triple-negative levels at 200 mg kg−1 day−1 in mice after an oral administration.
human breast cancer cells were engineered to stably secrete sEV Before the validation of chemotherapeutic effect of SFX, we stu-
that contain CD63-GFP (MDA-MB231 CD63-GFP (+)) and died the subacute oral toxicity profile of SFX. Any pathological
grown in 96-well plates (Supplementary Fig. 1a–h). Inhibitory signs, including abnormal behaviors, body weight changes, and
effect on sEV secretion was determined by decreased fluorescence unexpected death, were evaluated after daily oral administrations
from the individual culture supernatant, which should contain of SFX (100, 300, and 900 mg kg−1 day−1 for 28 consecutive days.
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(Screening flow chart)
Screening library : 1163 FDA-approved drugs
a
Selection of
Sulfisoxazole (SFX)
Secondary screening
Primary screening
(two points)
(single point)
Exception criteria
- Known chemo-drug
- Cytotoxicity
- Drug dosage form
& administration route
≥ 30 %
inhibition
of sEV
N
- Drug CONC: 50 & 100 μM
- sEV secretion
(GFP measurement)
- Cell viability (MTT assay)
O
- Drug CONC: 30 μM
- Small EV (sEV) secretion
(GFP measurement)
O
O
CH3
S
N
H
CH3
H2N
b
c
MDA-
MB231
(×103)
15
MDA-MB231
MCF7
MCF10A
MDA-MB231
MCF7
MCF7
***
60
***
***
**
10
5
40
20
0
***
***
***
***
n.s.
0
(μM)
SFX
SFX
SFX
d
e
(×102)
MDA-MB231
n.s.
MCF7
IL8
IL2
IL6
CX3CL1 CXCL13
CCL2
TGFB1
n.s.
50
40
9
6
3
0
n.s.
30
10
n.s.
n.s.
n.s.
n.s.
n.s.
5
0
n.s.
(μM)
SFX
SFX
f
g
SFX
1.5
1.0
0.5
0
+ SFX
EDIL3 HSP90
GPC1
Alix
(μM)
–
70
***
*
EDIL3
HSP90
GPC1
Alix
**
**
**
*
1
0
*
105
*
*
**
**
**
**
55
100
MDA-MB231
SFX
SFX
SFX
SFX
Fig. 1 S FX i n h i b i t s t h e s e cr e t i o n o f s E V f ro m b r e a s t c a n c e r c e l l s q u a n t i t a t i v e l y a n d q u a l i t a t i v el y . a S c r e en i n g flo w c h a r t o f p r i m a r y a n d s e c o n d a ry s c r e e ni ng s
w i t h s o m e e xc l u s i o n c r i t e r ia t o i d e n t i fy a n i n h i b i t o r o f s E V s e c r e ti o n , s u l fis o x a z o l e . b T h e n u m b e r o f s e c r e t e d s E V w i t h t h e i n d i c a t e d c o n c e n t ra t i on s o f S F X .
n = 3 . c L e f t, e l e c tr o n m i c r o s c o p y i m a g e . S c a le b a r , 1 00 n m . R i g h t , Q u a n t ific a t i o n o f t h e n u m b e r o f s e c r e t e d s E V . R a n d o m i ze d fie l d s w e r e c a p t ur e d a nd
c o un t e d . n = 3 0 . d M e a su r e me n t o f t h e m i c r o v es i c le c o n c e n t r a t io n b y n a n o p a r ti c l e t r a ck i n g a n a l y s i s ( N T A ) . n = 3 . e M e a s u r e m en t o f s o l u b l e c y t o k i ne s
s e c re t ed f r o m M D A - M B 23 1 c e l l s . n = 3 . f q R T- P C R a n a l y s is o f t h e i n d i c a t e d m i R N A s i n M D A - M B2 3 1 c e l l s a f t e r t r e a tm e n t o f 1 0 0 μM S F X . n = 3 . T he G E O
a c c e ss i o n n u m b e r o f m i R NA m i c r o a r r a y s e t i s G S E 12 4 3 20 . g I m m u n o b lo t o f v a r i o u s p r o t e i n s i n M D A - M B2 3 1 c e l l - d e r i v e d s E V . E q u a l a m o u n t s o f s E V
p ro t e i n ( 1 0 μg ) w e re l o a d e d p e r l a n e . n = 3 . E xp er i m e nt s w er e p e r f o r m e d w i t h 9 5 % c o nflu e n t c e l l s . S i g n ific a n c e w a s d e t e r m i n ed u s i n g a n u n p ai r e d t w o -
t a il e d S t u d e n t’s t- t es t . * * * p < 0 . 00 1 , * *p < 0 . 00 5 , a n d * p < 0 . 05 . E r r o r b a r , S D . S o u r c e d a t a (b–g) a r e p r o v i d e d a s a S o u r c e D a t a fil e
Our subacute toxicity study revealed that mice appeared healthy
Therefore, we evaluated the effect of SFX on the growth rates of
and statistically significant differences in the body weights and MDA-MB231 cells orthotopically implanted into female nude
various parameters of serum chemistry were not observed mice. Docetaxel (DOC), currently used intravenously as an anti-
between SFX-treated mice and controls, suggesting that SFX up to cancer agent, was used for control and combination therapy
900 mg kg−1 daily administrations did not cause any obvious experiments. The growth of MDA-MB231 cells was significantly
clinical indications in both males and females (Supplementary suppressed by oral administrations of SFX, compared to the
Fig. 6a, b).
vehicle-control group (Fig. 2b). SFX also effectively delayed the
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3

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a
b
Anti-proliferation study
Anti-proliferation study
Anti-metastasis study
Vehicle
SFX
DOC
Combi
Luminescence
6
MDA-MB231-luci (+)
Human breast cancer
4T1-luci (+)
Murine breast cancer
4
2
Balb/c nude
Balb/c WT
Counts
(×102)
10
8
Vehicle
SFX
DOC
1. Vehicle
Combi (SFX+DOC)
2. SFX
Groups
3. Docetaxel
4. SFX + Docetaxel
(Combination)
6
Start
treatment
4
2
0
5
10
15
Days after treatment
c
d
Anti-metastasis study
1.0
0.8
0.6
0.4
0.2
0
Luminescence
1.5
Vehicle
SFX
DOC
Combi
0.5
Vehicle
SFX
DOC
Combi (SFX+DOC)
Radiance
(×103)
6
4
2
0
Vehicle
SFX
DOC
10
20
30
40
50
Days after treatment
Combi (SFX+DOC)
f
55
40
Circulating sEV
***
*
1
0
Start
treatment
35
55
40
35
10
20
30
Days after treatment
e
Lung
***
Liver
**
8
6
4
2
0
30
20
10
0
*
metastasis of mouse 4T1 breast cancer xenografts on day 29 determined the amounts of circulating human cancer cells-
relative to the vehicle group (Fig. 2c), thus survival rates were derived sEV in the sera of host mice by using the specific antibody
significantly increased (Fig. 2d). In addition, SFX treatment recognizing the human CD63 but not the mouse CD63 (ref. 12).
markedly reduced the countable colonies of metastatic foci of 4T1 Densitometric analysis revealed that the expression of human
cancer cells in the lung and liver (Fig. 2e) with massive growth of CD63 exosomal protein was significantly decreased in the SFX-
tumor nodules of the experimental mice, compared with those treated animals and DOC-combination group compared to
of control animals. To further study whether SFX can inhibit vehicle-exposed controls (Fig. 2f). To further validate whether
the secretion of sEV from transplanted cancer cells in vivo, we the anti-cancer effect of SFX is mediated through the inhibition of
4
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N A T UR E C O M M UN I CA T I O NS | h t t p s : //d o i . o r g / 10 . 1 0 38 / s 4 1 4 6 7 - 01 9 - 0 9 3 8 7 - 4
A R T I C L E
Fig. 2 S FX s u p p r e sse s b r e a s t c a n c e r c e l l p r o l i f e r a ti o n a n d m e t a s t a s i s i n t w o c a n c e r x e n o gr a f t m o d e l s . a S c h e m a t i c i l l u s t r a t i o n o f t h e i n v i v o e x pe r i m e n t al
d e s ig n s w i th d i f f e re n t t r ea t m e n t s. b T o p , R e p r e s e n t a t iv e i m a g e o f c a n c e r c e l l s t r a ck e d w i t h t h e I V I S i m a gi n g s y s t em f o l l o w i n g t h e i n j e ct i o n o f m i c e w i t h
l u c if e r a s e- ex p r e ss i n g M D A -M B 2 3 1 c e l l s . B o t t o m , T u m o r m a s s v o l u m e o f i n B A LB / c nude f e m a l e m i c e i n o cu l a t e d w i t h M D A - M B23 1 - l u c i ( +) c e l ls a nd t h e n
t re a t e d w i th a n i n d i ca te d d r u g o r v e h i c l e . n = 7 p e r g ro u p . c T o p , i n v i v o i m a ge s o f 4 T1 c a n c e r c e l l - b e a r i n g m i c e u s i n g a n I V I S i m a gi n g s y s t e m . B ot t o m ,
t um o r m a s s v o l u m e o f 4 T 1 -l u c i (+) c e l l s i n o c u l a t ed B A L B / c nude f e m a l e m i c e t r e a te d w i t h a n i n d i c a t e d d r u g o r v e h i c l e n = 9 p e r g r o u p . d M e a s u r e m e nt o f
t he s u r vi v a l r a t es i n t u m o r- b e a r i n g m i c e a f t e r d i f f e r en t t r e a t m e n ts . T h e s u r v i v a l c u r v e s w e r e g e n e r a t e d a c c o r di n g t o t h e K a p l a n–M e i e r m e t h o d n = 5 p e r
g r ou p . e Q u a n t i t a t i ve r e p r e s e nt a t i o n o f l u n g a n d l iv e r n o d u l es f ro m m i c e b e a r i n g 4 T1 - l u ci ( +) c e l l s . n = 5 p e r g r o u p . f L e ft t o p , i m m u n o b l o t o f h u m an C D6 3
i n c i rc u l a ti n g c a n c e r- c e l l - d e ri v e d s E V f r o m M DA - M B 23 1 -l u c i (+) - b e a r i n g m i c e . n = 3 p e r g r o u p w h e r e s i m i l a r a m o u n t s o f p r o t e i n / l a n e w e r e v e r i fie d b y
P on c e a u S s t a i n i n g ( Bo tt o m ) . R i gh t , A n a l y s i s o f t h e r e la t i ve p r o t e i n i n t e n s i t y o f C D 6 3 m e a s u r ed u s i n g a d e n s i t o m e t er s y s t em . S i g n i fic a n c e w a s d e t e r m in e d
u si n g a n u n p a i r ed t w o - t a i l e d S t u d e n t’s t- te s t . * * *p < 0 . 0 01 , * * p < 0 .0 0 5 , a n d *p < 0 .0 5 . E r r o r b a r , S D . S o u r c e d a t a ( b–f) a r e p r o v i d ed a s a S o u r c e D at a fil e
sEV secretion, we performed rescue experiments using two regulated by a calcium-dependent mechanism28 (Supplementary
animal models. The anti-proliferation and anti-metastasis effects Fig. 9d). These results suggest that SFX would influence both sEV
of SFX and DOC combination groups were significantly reduced biogenesis and secretion through the ESCRT-dependent
by sEV treatment, suggesting that the anti-cancer effects of SFX mechanism.
are mostly mediated through the inhibition of sEV secretion
(Supplementary Fig. 7a–e).
SFX inhibits sEV secretion via ETA. Our data described above
strongly indicate that a newly-recognized off-target of SFX exists
SFX influences ESCRT-dependent multivesicular endosome in cancer cells to exhibit its inhibitory effect on the secretion of
biogenesis and secretion. To investigate whether SFX inhibits sEV. First, to determine whether the active site (the NH2-group
sEV secretion through altered expression of the genes involved in on the N4 position) of SFX as an antibiotic entity is essential for
sEV biogenesis, we performed a microarray analysis of mRNAs the inhibition of sEV secretion, three SFX derivatives consisting
obtained from MDA-MB231 and SK-MEL-28 cells after SFX of two mono-acetylated (N1-acetylated SFX, N1AS and N4-
treatment. Strikingly, most genes down-regulated by SFX treat- acetylated SFX, N4AS) and one diacetylated SFX (DAS) by
ment in both cell lines encode the regulatory proteins associated modifying the two nitrogen atoms on SFX were synthesized
with transport and small GTPase-mediated signal transduction (Fig. 4a). As expected, an antibiotic effect completely disappeared
(Fig. 3a, b and Supplementary Fig. 8a–c). This result strongly in two derivatives (N4AS and DAS) in which the active site (N4)
suggests that an upstream factor (a potentially newly-recognized was blocked by acetylation, as indicated by the minimum inhi-
off-target of SFX), which regulates the transcriptions of various bitory concentration (MIC) against two microorganisms (MIC
genes associated with sEV biogenesis and secretion, is likely >512 in both Staphylococcus aureus and Escherichia coli)
present in these cancer cells and that this upstream factor could (Fig. 4b). Surprisingly, however, all three derivatives still reduced
be regulated by SFX, even though this drug was originally the sEV secretion (Fig. 4c) and the expression of Rab27a in
developed as an antibiotic to kill bacteria.
MDA-MB231 (Fig. 4d), similar to the changes induced by SFX.
Several RABs (RAB5, RAB7, and RAB27A), VPS4B, and MITF These results strongly suggest that the NH2 group at the N4 site is
genes were down-regulated by SFX treatment, as confirmed by not critical for inhibition of sEV secretion although this group is
both quantitative reverse transcription polymerase chain reaction essential for antibiotic function.
(qRT-PCR) (Fig. 3c) and western blotting (Fig. 3d). Greater
To identify a direct protein target of SFX in cancer cells, we
amounts of RAB5 and RAB27A proteins were expressed in cancer applied an in silico approach based on a Similarity Ensemble
cells, compared to normal cells (Supplementary Fig. 9a). More- Approach (SEA), a method developed by our group29. Using data
over, the expressed amounts of RAB5 and RAB27A in breast from the BindingDB, SEA identified four proteins (Fig. 4e) as
cancer cells (Fig. 3d) and SK-MEL-28 melanoma cells (Supple- target candidates, which are: endothelin receptor type A (ETA),
mentary Fig. 9b) were significantly reduced after treatment with kynurenine 3-monooxygenase (KMO), carbonic anhydrase 13
SFX in a dose-dependent manner. The ESCRT machinery is (CA13), and angiotensin II type 1a receptor (AGTR1). To
important in the multivesicular endosomes (MVE) maturation22. determine whether each candidate protein can bind directly to
VPS4B23, an important ESCRT-related component to regulate SFX, a pull-down assay was conducted using biotinylated SFX
intraluminal vesicles formation, and Alix24, an ESCRTIII-binding with the lysate or membrane fraction of MDA-MB231 cells. Our
partner, were decreased by SFX (Fig. 3d). Moreover, cellular analysis revealed that only the antibody recognizing ETA showed
CD63, ESCRT-dependent or -independent sEV biogenesis a signal in elution parts from the membrane fraction but not the
regulator25, was also down-regulated (Fig. 3d). However, neutral lysate (Fig. 4f). Consistent with these results, the expressions of
sphingomyelinase (nSmase) enzyme activity, which is related to RAB27A, RAB5, and RAB7 proteins were significantly decreased
ceramide-regulated events and a target of GW4869 (ref. 26), was by siRNA-mediated knockdown of the ETA gene (Fig. 4g), but
not affected by SFX (Fig. 3d and Supplementary Fig. 9c). In not by suppression of the other three genes, in MDA-MB231 cells
addition, SFX significantly suppressed the levels of a transcription (Fig. 4h). To demonstrate direct binding of SFX with ETA,
factor MITF (Fig. 3d), which can increase the expression of late radioactive binding assay was performed and an IC50 value of
endosomal proteins, such as RAB7 and CD63, and a main sEV 11.0 μM SFX was subsequently calculated (Fig. 4i). This binding
secretion regulator, RAB27a, in melanoma cells27. Hence, the assay result was consistent with an earlier result reported over two
decreased levels of RAB7, CD63, and RAB27a might be due to the decades ago, where SFX could be an ETA selective endothelin
down-regulation of MITF by SFX. RAB-interacting lysosomal antagonist30.
protein (RILP), which is the RAB7 effector required for transport
It has been reported that the ETA activation by its agonists
to lysosomes, was markedly upregulated by SFX in a dose- (endothelin-1, ET1, and endothelin-2, ET2) promotes cancer
dependent manner (Fig. 3c, d). SFX did not alter the intracellular progression through a network of cellular pathways and
calcium concentration when compared to dimethyl amiloride interactions with the tumor microenvironment31. Consistently,
(DMA), an inhibitor of the secretion of exosome via reducing we observed that ETA and its agonists were strongly expressed in
intracellular calcium levels, although sEV secretion can be highly invasive MDA-MB231, compared to MCF7 and MCF10A
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5

A
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-
0
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3
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-
4
a
b
MDA-MB231
(SFX vs Vehicle)
MDA-MB231
(SFX vs Vehicle)
GO biological process
70
50
30
10
0
SPACA1
RILP
CHRNB1
KCNH8
MYH14
GRIN3B
SAR1B
DNAJC6
GJA1
1. Transport
1
CLCA4
TP73
2. Small GTPase mediated signal transduction
ATP2A3
2
3
4
SLC25A17
AP4M1
BTC
5
6
7
8
9
10
3. Regulation of transport
FGD4
MCM3AP
CCHCR1
ADCY6
SLC10A2
APOBR
PNLIP
SLC38A8
IFT74
c
MDA-MB231
PPBP
RAB5A
RAB7 RAB27A
VPS4B
MITF
RILP
GNA13
IPO8
3.0
2.0
1.0
0
1.5
ROCK1
CASQ2
CEL
*
1.0
0.5
0
ZPBP2
SCG5
**
**
CELSR1
RSAD2
ARHGAP3
STAB2
CIB1
TSNAX
RAB28
SOX11
BUB1B
HAMP
**
**
**
***
***
MLC1
RAB27A
SDAD1
SLC16A1
SRGN
SFX
SFX
SFX
SFX
SFX
SFX
(μM)
ADCY7
SRSF12
MASP1
RHEBL1
TACR2
ORAI3
SK-MEL-28
VPS26A
TCAF1
VPS4B
SLC30A1
PDE4B
IL18
RAB5A
RAB7 RAB27A
VPS4B
MITF
RILP
1.5
1.0
0.5
0
6.0
4.0
**
KCNK6
LCN12
STX10
*
*
**
1.0
0.5
0
*
**
NOX5
SCN7A
SETE2
STOM
***
***
***
BANK1
SLC6A20
SLC22A18
SCNA
***
BTN3A2
LYPLA1
RAB5A
MITF
SFX
SFX
SFX
SFX
SFX
SFX
(μM)
SLC2A9
RASL11B
CYGB
d
SFX
SCN2A
RPL4
(μM)
2
1
0
SNX22
VANGL2
NETO2
ASIC2
26
RAB27A
RAB5
26
26
70
*
** ***
*
RAB7
nSMase2
Alix
***
TMEM38A
RASD1
MFI2
***
*
**
*
100
ARHGAP2
USP20
GAS1
70
50
35
50
35
CD63
Beta-actin
FBLN5
LYST
SLC6A15
SLC29A4
ARHGAP2
RLBP1
FGFR3
SLC9A9
PSD4
SFX
***
***
2
1
0
(μM)
RILP
50
50
VPS4B
Beta-actin
MITF
AR
***
**
50
35
Vehicle
GW4869
50 μM
–1
0
1
50
70
Fold change
(LOG2)
Lamin B
100 μM
cells (Fig. 4j). Small-molecule antagonists of ETA have been (Knockdown, K/D) or an ETA ORF (over-expression, OE).
developed as anti-cancer drugs32; however, to our knowledge, Similar to SFX treatment, ETA K/D significantly decreased the
there has been literally no report on the relationship between secretion of sEV (Fig. 5a), with a reduction of RAB27A, RAB5,
ETA and sEV secretion.
and RAB7 proteins (Fig. 5b). Conversely, ETA-OE significantly
We therefore studied the underlying mechanisms by which increased the secretion of sEV (Fig. 5a), with elevated levels of
ETA interferes with sEV secretion. First, to achieve this, MDA- RAB5 and RAB7 proteins (Fig. 5b). To further investigate the
MB231 cells were stably transfected with an ETA shRNA relationship between ETA and sEV secretion, we studied the
6
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N A T UR E C O M M UN I CA T I O NS | h t t p s : //d o i . o r g / 10 . 1 0 38 / s 4 1 4 6 7 - 01 9 - 0 9 3 8 7 - 4
A R T I C L E
Fig. 3 S FX i m p a i r s s E V s e c r et i o n t h r o u g h i n t e r fe r in g w it h t h e E SC R T - d ep en d e n t M V E b i o ge n e s i s. a H e a t m a p o f t h e s e l e c t e d t r a n s c r i pt o m e o f S F X -t r e a t e d
M D A - M B 23 1 c e l l s c o m p a r e d t o u n t r e a te d c o n t r o l c e l l s . H ea t m a p r e p r e s e n t s p r o b e s e t s f o r t r a n s c r i p ts e x p r e s s e d a t s i gn i fic a n t l y h i g h e r o r l o w e r l e v e ls a ft e r
e x p o s u r e t o 1 0 0 μM S FX , r e s p e c t iv e l y ( F C = 1 .3 p v a l u e = 0 . 0 5 ) . n = 3 . b A d v a n c e d b u b b l e c h a r t s h o w s e n r i c h m e n t o f d i f f e r e n ti a l l y e x p r e s s e d g e n e s i n
t he i n d i c at e d s i g n a l i n g p a t h w a y s . T h e x- a x i s l a b e l i n d i c a t es g en e o n t o l o gy ( G O) b i o l o g i c a l p r o ce s s e s , a n d t h e y- a x i s l a b e l r e p r e s e n t s p v a l u e . c q R T -P C R
a n al ys i s o f v a ri o u s g e n e s i n 2 4 h S F X - tr e at e d M DA - MB 2 3 1 ( u p pe r ) a n d 6 h S F X - t r e a t e d S K- M E L - 2 8 h u m a n m e l a n o m a c e l l s ( l o w e r ) . n = 3 . d I m m un ob l ot
f o r t h e i n d i ca te d p r o te i n s i n M DA - MB 2 3 1 c e l l s . B e t a - a c t i n a n d L a m i n B w e r e u s e d a s l o a d i n g c o n t r o l s f o r c e l l l y s a t e s a n d n u c l e a r f r a c t i o n , r e s pe c t i v e l y.
R i g h t , A n a l y s i s o f t h e r el a t i v e p r o t e i n i n t e n s i t y o f p r o t e i n s m e a s u r e d u s i n g a d e n s i t o m e te r s y s t em . n = 3 . E x p e r i me n t s w e r e p e r f o r m e d w i t h 9 5 % c o n flu e n t
c e l l s. S i g n ific a n c e w a s d e t e r m in e d u s in g a n u n p a i r e d t wo -t a il e d S t u d e n t ’s t- t e s t . * * * p < 0 .0 0 1 , * * p < 0 .0 0 5 a n d *p < 0 .0 5 . E r r o r b a r , S D . S o u r c e d at a ( c, d)
a r e p ro vi d e d a s a S o u rc e D a t a fil e
effects of several ETA-specific agonists ET1 and ET2 or by SFX treatment relative to the control (Fig. 6e). Importantly,
antagonists (zibotentan, BQ123, and PD159701) on sEV secre- these observations have led to the hypothesis that SFX initially
tion. ET2 significantly increased sEV secretion, which was slightly influences ESCRT-dependent MVE biogenesis, followed by the
elevated by ET1, compared to control (Fig. 5c and Supplementary imperfect MVEs moving to the lysosomes for degradation.
Fig. 10a). In contrast, the three antagonists tested in this study
Next, we investigated whether the ETA signaling pathway is
potently inhibited sEV secretion with respect to the number and related to the fusion of lysosomes and MVEs. Similar to the
the protein amounts of secreted sEV (Fig. 5c and Supplementary effects in SFX-treated cells, autolysosomes or AVs were detected
Fig. 10a). Moreover, the number and the protein amounts of the in antagonist-treated MDA-MB231 cells (Fig. 7a). Moreover,
secreted sEV were still decreased by the combined treatment of confocal microscopy verified co-localization of MVEs with
SFX and ET1 or ET2 agonist (Fig. 5d and Supplementary lysosomes, the increase in lysosomal activity, and the up-
Fig. 10b), indicating a potency of SFX in decreasing the sEV regulation of LAMP-1 in antagonist-treated or ETA-siRNA-
secretion despite the presence of ETA agonists. Similar to the transfected MDA-MB231 cells (Fig. 7b–e). These results strongly
SFX-treated cells (Fig. 1e), ETA antagonists did not significantly suggest that one of the major targets of SFX in MDA-MB231 cells
alter the classical cytokine secretion pathway (Fig. 5e). Consis- is ETA, which could be a GPCR protein newly-recognized for the
tently, the mRNA expression levels of RAB27a, RAB5, RAB7, regulation of sEV biogenesis and secretion.
RILP, MITF, and VPS4B, as shown in SFX-treated cells (Fig. 3c),
were down-regulated by ETA antagonists (Supplementary
Fig. 10c). Additionally, we confirmed the relationship between
ETA and cancer progression in a breast cancer model. ETA
antagonists, zibotentan, BQ123, and PD156707, significantly
repressed the invasion and migration activities of cancer cells,
MDA-MB231 and 4T1 in vitro (Supplementary Fig. 11a–e).
Furthermore, the potent anti-proliferation effects of ETA
antagonists through the inhibition of sEV secretion were observed
in mice inoculated with MDA-MB231-ETA K/D cells or
pretreated with ETA antagonists (Fig. 5f, g and Supplementary
Fig. 11a). Taken together, these results suggest that ETA is one of
critical elements of the complex machinery for sEV secretion and
constitutes an upstream protein for regulating sEV in MDA-
MB231 cells.
Discussion
Breast cancer is the most prevalent cancer in women and the
leading cause of death worldwide, up to half-a-million deaths
annually, despite surgical treatments combined with advanced
radiotherapy and chemotherapy. Metastasis is a major cause for
deaths of many patients with various types of cancer, including
breast cancer. Metastatic dissemination of breast cancer can occur
in a late stage of cancer progression35 as well as in preinvasive
stages of tumor progression, by various systemic factors secreted
from the tumors36. The exosomes and/or sEV, emerging players
of systemic factors, play an important role in establishing pre-
metastatic niche in future metastatic organs and furthermore in
dictating organotrophic metastasis by integrin repertoire of sEV
in breast cancer cells37. Therefore, early detection as well as
prevention of metastasis by conventional treatments and/or sEV
ETA antagonists increase fusion of MVE with lysosomes. To secretion could become very important in reducing the cancer-
further investigate morphological changes inside cancer cells after related pathologies and deaths.
SFX treatment, we performed transmission electron microscopy
Despite significant advancement in exosome/sEV research in
(TEM) analysis of MDA-MB231 cells at 6, 12, and 24 h post- recent decades, no drugs targeting to inhibition of sEV secretion
exposure. Interestingly, the structure of degraded autophagic have been approved for human usage. In this study, we identified
vacuoles (AVs) following lysosomal fusion could be observed 12 h SFX as an inhibitor of the secretion of sEV by high-throughput
after SFX treatment (Fig. 6a). The most abundant type of vesicles screening of a library of FDA-approved drugs. SFX affects the
occupying the cytosol of MDA-MB231 cells appeared to be expression of many genes associated with the pathways of sEV
“empty”. In addition, much larger structures occupied extensive biogenesis and secretion in cancer cells (Fig. 3). Our results
areas of the cytosol of MDA-MB231 and MCF7 cells at 24 h after suggest that an upstream target, as a newly-recognized off-target,
SFX treatment, but the control groups looked normal (Fig. 6a). of this old drug might regulate the sEV-associated pathways in
More importantly, lysosomes or autolysosomes densely filled with breast cancer cells. From our computational approach, ETA was
multi-lamellar structures were also observed, while the number of selected as the prime target of SFX with the highest priority and
these sub-organelle structures and the expression of lysosome- was confirmed by a direct binding study, genetic and pharma-
associated membrane proteins 1 (LAMP-1), which is critical in cology approaches. Therefore, we showed that SFX exhibits a
lysosome biogenesis and autophagy33, were increased in SFX- potential anti-cancer effect that is mediated through ETA
treated cells (Fig. 6b).
It has been reported that the balance between autophagy and
dependent-inhibition of sEV biogenesis/secretion.
For physiologically relevance, we simply compared the con-
sEV biogenesis is important for the maintenance of cellular centration of SFX as an inhibitor of sEV secretion with that of
homeostasis34. Hence, we investigated whether the relationship SFX as an antibiotic in vitro and in vivo. As shown in Supple-
between MVE and lysosomes could be affected by SFX. We mentary Fig. 11, each MIC of SFX was 16 and 32 μg ml−1 in E.
observed that lysosome activity was strongly increased by SFX coli and S. aureus in this study, converted to 60 and 120 μM,
(Fig. 6c, d) and the fusion of MVE with lysosomes was accelerated respectively, and thus the concentration (up to 100 μM in this
N A T U R E C OM M U N I C A T I ON S |
(2 0 1 9 ) 1 0 : 1 3 8 7 | h t t p s :/ / d o i . o r g / 1 0 . 1 0 3 8 / s 4 1 4 6 7 - 0 1 9 - 0 9 3 8 7 - 4 | w ww .n a t u r e . c o m / n a t u re c o m m u n ic a t io n s
7

A R T I C L E
N
A
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3
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4
1
4
6
7
-
0
1
9
-
0
9
3
8
7
-
4
(×103)
15
a
b
c
8
6
4
2
0
WT-SFX
MIC (μg/ml)
N
O
O
O
10
5
CH3
S
WT
32
N1AS N4AS DAS
*
N
**
**
**
H
CH3
S. aureus
E. coli
512
128
>512
>512
>512
>512
*
***
***
***
H2N
***
16
0
N1AS
N4AS
DAS
CH3
CH3
CH3
d 26
50
N
O
N
SFX derivatives
SFX derivatives
N
RAB27A
CH3
CH3
CH3
O
O
Beta-actin
1.0
e
35
N
NH
O
N
N1
O
O
O
N1
Similarity search
S
S
S
O
O
Target candidates
Value (Tcs)
O
O
**
ETA
KMO
113.5
10.7
14.2
73.1
0.5
0
**
***
**
NH
NH
NH2
N4
N4
CA13
AGTR1
O
O
Active site for anti-bacterial effect
*
SFX derivatives
f
g
h
si-ATGR1
#1 #2
si-CA13
#1 #2
SFX
Biotinylated SFX
ETA siRNA
26
50
26
26
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RAB27A
RAB27A
#1 #2 #3 #4
ETA
26
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AGTR1
CA13
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RAB5
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35
50
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Beta-actin
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(ETA + SFX)
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ETA
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**
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6.80 μM
0.86
n.s.
ET2
IC50
Ki
50
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Beta-actin
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0
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2
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Fig. 4 I d en tific a t i o n o f t h e e n d o t h e li n r e c e p t o r a s t h e n o v e l t a r g e t o f S F X. a S c h e m a t i c i l l u s t r a t i o n o f t h r e e a c e t y l a t e d S F X d e r i v a t i v es . N 1 , N 1 - a ce t yl at i o n s i t e
( b l u e ) . N 4 , N 4 - a c et y l a ti o n s i t e ( g r e en ) . b T h e m i n i m a l i n h i b i t o r y c o n c e n t ra t i o n ( M IC ) o f S F X a n d i t s d e r i v a t i v es a g a i n s t Staphylococcus aureus A T CC 2 9 2 1 3
a n d Escherichia coli A T C C 2 5 9 2 2 . c M ea s u r e m e n t o f t h e n u m b er o f s E V a n d t h e a m o u n ts o f s E V p r o t ei n s . L e ft , t h e n u m b e r o f s E V . R i gh t , t h e a m ou nt s o f
s E V p r o t e i n s. n = 3 . d I m m u n o b l o t ( T o p) a n d q R T - P C R a n a l y s is ( b o t t o m ) o f R A B 2 7 A i n M D A - M B2 3 1 c e l l s t r e a te d w i t h 1 0 0 µM S F X o r i t s s t r uc t ur a l
d e r i v a t i v es . e C a n d i d a t e p r o t e i n s f o r b i n d i n g t o S F X b y s e a r c h i n g b i n d i n g D B. T a n i m a t o c o e f fic i e n t s ( T c s) a s t h e m e a s u r e o f s i m i l a ri t y a r e s u m m ar iz e d .
f D e t e c ti o n o f E T A b y i m m u n o b l o t . E lu t e d p a r ts f ro m t h e m e m b r a n e f r a c t i o n o f M D A - M B23 1 c e l l s w e r e u s e d f o r b i o t i n - b a s e d p u l l - d o wn a s s ay . W T - S F X
T
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w a s u s ed a s a n e g a t i v e c o n t r o l . n = 3 . g I m m u n o b lo t o f v a r i o u s p r o t e i n s i n M D A - M B2 3 1 c e l l s a f t e r t r a n s f e ct i o n o f d i f f e r e n t E T A s i R NA s . A c c u T ar get
s iR N A w a s u s e d f o r c o n t r o l s i R N A . n = 3 . h I m m u n o b l o t o f R a b 2 7 a p r o t ei n a f t e r t r a n s f e ct i o n o f e a c h s i R NA s p e c ific t o A G T R 1 , C A 1 3 , o r K M O i nt o M D A -
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t he i n h i b i t o r y c o n s ta n t, nH H il l c o e ffic i e n t . j I m m u n o b l o t o f E TA a n d i t s l i ga n d , E T 1 a n d E T 2 i n M C F 1 0A , M C F 7 , a n d M D A - M B2 3 1 c e l l s . n = 3 p e r c e l l l in e .
E x p e r i me n t s w e re p e r f o rm ed w it h 9 5 % c o n flu e n t c e l l s . S i gn ific a n c e w a s d e t e r m i n e d u s i n g a n u n p a i r e d t w o - ta i l e d S t u d e n t ’s t- t e s t . * * *p < 0 .0 0 1 , * * p < 0 . 0 0 5
a n d *p < 0 . 0 5 . E r r o r b a r , S D. S o u r c e d a t a ( b–d, i–j) a r e p r o v i d e d a s a S o u r c e D a t a fil e
study) for inhibition of sEV secretion in cancer cells could be in this study, as an inhibitor of sEV secretion in cancer cells and
reasonable. Furthermore, to evaluate whether the dose for mouse as an anti-cancer agent in two different mouse models of breast
study can be physiologically relevant in humans, we used a for- cancer xenografts, could be physiologically relevant.
mula for dose translation from animal to human studies38. The
Gut microbiota is a critical factor for many pathophysiological
dose (200 mg kg−1 day−1) used in this study was about 4.1–8.2 conditions such as immune reaction and inflammatory pro-
times lower than the maximal dosage as an antibiotic in human cesses39. SFX, an antibiotic, may decrease gut microbiome density
usage. Therefore, we believe that the concentrations of SFX used and/or modify its composition. Several bacteria phyla are shown
8
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MDA-MB231
MDA-MB231
WT
ETA O/EETA O/EETA K/D
*
10
5
50
26
ETA
15
10
5
RAB27A
RAB5
26
**
**
26
50
RAB7
*
*
Beta-actin
35
70
0
0
CD63
35
50
35
Beta-actin
(×103)
(×103)
15
d
c
Agonists
Antagonists
15
10
5
**
**
***
*
*
***
10
5
***
*
*
**
**
***
***
0
0
40 10 50
1
10
(nM)
Zibo
BQ
PD
e
f
IL8
IL2
CXCL13
CCL2
CX3CL1
TGFB1
IL6
60
(×102)
50
40
8
6
4
2
0
WT
ETA K/D
n.s.
n.s.
n.s.
n.s.
15
10
5
*
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s
n.s
0
1
5
10
Days after cell inoculation
WT ETA K/D
15
g
Circulating sEV
ETA K/D
WT
(×104)
*
55
40
2
1
0
WT
ETA K/D
Fig. 5 I d e n t i fic a t i o n o f E T A a s t h e n o v e l r e gu l a t o r o f s E V b i o g e n e s i s. a M e a s u r e m en ts o f t h e n u m b e r o f s E V s e c r e t e d ( l ef t ) a n d t h e a m o u n t s o f s EV p r ot e in s
( r i g h t ) f r o m W T , E T A - o ve r e x p r e ss e d ( E T A -O E ) , a n d E TA k n o c k d o w n ( K / D) M D A - M B2 3 1 c e l l s . n = 3 . b I m m u n o b l o t o f v a r i o u s p r o t ei n s i n E T A - O E a nd
E T A - K/ D M D A - M B 2 3 1 c e l l s . n = 3 . c M ea s u r e m en t o f t h e n u m b e r o f s E V s e c r e t e d a n d t h e a m o u n t o f s E V p r o t ei n s f r o m E T A a g o n i s t- o r a n t ag o ni s t-
t re a t e d M D A - M B 23 1 c e l l s . n = 3 . d M ea s u r e m e n t o f t h e n u m b er o f s E V a n d t h e a m o u n ts o f s E V p r o t ei n s f r o m M D A - M B23 1 c e l l s t r e a te d w i t h 1 0 n M E T 1 o r
1 0 n M E T 2 i n t h e p r e s e n c e o f 1 00 μM S F X . n = 3 . e M ea s u r e m en t o f s o l u b l e p r o t e i n s s e c r e t e d f r o m M D A - M B23 1 c e l l s i n t h e p r es e n ce o f P D 1 56 7 0 7 o r
1
1
z i b o te n t an f o r 2 4 h . T h e u n i t o f I L 8 , I L 2, C X C L 1 3 , C C L 2, T G F B 1, a n d I L6 i s p g m l − . T h e u n i t o f C X 3 C L 1 i s n g m l − . n = 3 . f T u m o r m a s s v o l u m e o f i n B A L B / c
nude f e m a l e m i c e i n o c u l a t ed w it h M DA - MB 2 3 1 o r M DA - MB 23 1 E T A K / D. B a r , 1 c m . n = 8 p e r g r o u p . g L e ft t o p , i m m u n o b l o t o f h u m a n C D 6 3 i n c i r c ul at i n g
c a nc e r- c e l l - d e r i v e d s E V f r o m M DA - MB 23 1 o r M DA - M B 23 1 E TA K / D- b e a r i n g m i c e (n = 8 p e r g r o u p) w h e r e s i m i l a r a m o u n t s o f p r o t e i n /l a n e w e r e v e r ifie d
b y P on ce a u S s t a i n i n g ( B o t t o m ) . R i g h t , d e n s it o m etr i c a n a l y s is o f t h e r e l a t i v e p r o t ei n i n t e n s i t y o f C D 6 3 . E x p e r i m e nt s w e r e p e r f o r me d w i t h 9 5 % c o n flu e n t
c e l l s. S i g n ific a n c e w a s d e t e r m in ed u s in g a n u n p a i r e d t wo -t a il e d S t u d en t’s t- t e s t . * * *p < 0 .0 0 1 , * * p < 0 .0 0 5 a n d * p < 0 .0 5 . E r r o r b a r , S D . S o u r c e d a t a ( a, c–g)
a r e p ro vi d e d a s a S o u rc e D a t a fil e
N
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(2 0 1 9 ) 1 0 : 1 3 8 7 | h t t p s :/ / d o i . o r g / 1 0 . 1 0 3 8 / s 4 1 4 6 7 - 0 1 9 - 0 9 3 8 7 - 4 | w ww .n a t u r e . c o m / n a t u re c o m m u n ic a t io n s
9

A R T I C L E
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a
MDA-MB231
MCF7
12 h
24 h
24 h
d
b
c
12 h
24 h
Lysosome
Autolysosome
Lysosome
MVE
Overlap
6
4
2
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Lysotracker
CD63-GFP
***
**
***
10
5
*
140
LAMP1
100
50
Beta-actin
35
0
e
Lysosome
MVE
Overlap
LAMP1
RAB7
300
200
100
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*
***
Fig. 6 S FX i n d u c es f u s i o n o f M VE w it h l y s o s o m e s i n b r e a s t c a n c e r c e l l s . a T E M a n a l y s i s o f S F X - t r e at e d M D A - M B23 1 c e l l s . R e d a r r o w s i n d i c a t e d u nu s ua l
s tr u c tu r es i n S FX - t r ea te d M DA - M B 23 1 a n d M C F7 , a n d w h it e a r r o w s i n d i c a t e d M V E s i n c o n t r o l c e l l s . S c a l e b a r , 5 μm . b T o p , I m a g e s o f l y s os om e a nd
a u t o l y s o so m e s t ru c tu re s o b s e r ve d i n S F X - tr e at e d M DA - MB 2 3 1 c e l l s . B o t t o m l e f t , t h e n u m b e r s o f d i s t r i b u t e d l y s o s o m e a n d a u t o l y s o so m e s t r uc t ur e s w e r e
c o un t e d . n = 2 0 . B o t to m r i gh t , L A MP - 1 p r o t e i n e x p r e s s i o n w a s u p r e gu l a t e d i n S F X - t r e a t e d M D A - M B2 3 1 c e l l s . n = 3 . c C o n f o c a l i m a ge o f C D 6 3 -G F P
(+) - MD A - M B 2 3 1 i n t h e p r e s e n c e o r a b s e n c e o f S F X . R e d , l y s o t r a c ker . G r ee n , C D 6 3 - G F P . S c a l e b a r , 2 μm . d M e a s u r e m en t o f L y s o t r a c ke r ( r e d ) i nt e ns i t y i n
r a pa m y c i n -t r ea t e d ( p o si t i ve c o n t r o l ) o r S F X - tr e a t ed M DA - MB 2 3 1 c e l l s . L y s o t r a ck e r i n t e n s i t y m e a s u r e m e n t > 1 7 0 c e l l s p e r g r o u p . e L e ft , I m a g e o f M D A -
M B 2 3 1 t r ea t e d b y 1 0 0 μM S F X . G r e e n , C e l lL ig h t- L y s o s o me -G FP ( G F P - L A MP 1 ). R e d , C e l l Li g h t- l a t e e n d o s o m e/M V E - R F P ( R F P - R A B 7) . S c a l e b a r , 2 μm .
R i g h t , Q u a n t i t at i v e c o l o c a l i za ti o n r a t e s o f l y s o s o m es a n d M VE i n S F X - t r e a t ed M D A - M B23 1 . C o l o ca l i z a t i o n p u n c t a c o u n t > 6 0 c e l l s p e r g r o u p u s i ng I m ag e J
p ro g r a m . B afil o m y c i n A 1 ( B a f A 1) w a s u s ed a s a n e g a t i ve c o n t r o l . E x p e r i m e n t s w e r e p e r f o r m e d w i t h 9 5 % c o n flu e n t c e l l s . S i g n ific a n c e w a s d e t e r m in ed u s in g
a n u n p a i re d t w o - t a i l e d S t u d e n t’s t- te s t . * * *p < 0 . 00 1 , * *p < 0 . 00 5 a n d *p < 0 .0 5 . E r r o r b a r , S D . S o u r c e d a t a ( b, d, e) a r e p r o v i d e d a s a S o u r c e D a ta fil e
to critically important in cancer development during changes might be partially responsible for the anti-cancer effect of
dysbiosis40,41. Based on the recent reports, gut microbiota is SFX. However, antagonists (not antibiotics) to ETA, a target of
considered as a holistic hub point for cancer development, thus SFX, also showed the anti-proliferation effect through the inhi-
antibiotics-mediated microbiome modulation can be a novel anti- bition of sEV secretion (Supplementary Fig. 5a). Therefore, these
cancer strategy42,43
.
Antibiotics-mediated gut microbiome results suggested that the inhibitory effect of SFX on cancer cell
10
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PD156707
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100
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Overlap
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**
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Fig. 7 A n t a g o n i st s a g a i n s t t h e e n d o t h e l i n r e c e p t o r i n d u c e f u s i o n o f M V E s w i t h l y s o s o m es i n M D A - M B2 3 1 c e l l s . a T E M a n a l y s i s o f E T A - a n t a gon is t- t r e a t e d
M D A - M B 23 1 c e l l s . S c a l e b a r , 5 μm . b M ea s u r e m en t o f l y s o t r a c k e r ( r e d ) i n t e n s i t y i n r a p a m y c i n - t re a t e d o r a n t a go ni s t ( Z i b o t e n ta n a n d P D 1 5 6 7 0 7 )- t re a t e d
M D A - M B 23 1 . L y s o t r a c ke r i n t e n s i t y m e a s u r e me n t > 1 0 0 c e l l s p e r g r o u p . c T o p , I m m u n o b l o t o f L A M P - 1 p r o t ei n i n a n t a go n i s t ( Z i b o , B Q- 1 2 3 , a nd
P D 1 5 67 0 7) - t r ea t e d M D A - M B 2 3 1. B o t t o m , I m m u n o b lo t o f L A MP - 1 p r o t e i n i n E T A - s i R N A - tr a n s f e ct e d M D A - M B23 1 a n d i n E T A - K / D M D A - M B 2 31. n = 3 .
d L e f t , I m a g e o f M D A - M B 2 3 1 t r e a t e d b y S F X . G r e e n , C e l lL ig h t- L y s o s o m e - G FP ( G F P - L A MP 1 ). R e d , C e l l Li g h t- l a t e e n d o s o m e/ M V E- R F P ( R F P - R A B7 ). S c a le
b a r, 2 μm . R i g h t , Q u a n t i t a ti v e c o lo c a li z a ti o n r a t e s o f l y s o s o m es a n d M V E i n M D A - M B2 3 1 t r e a te d w i t h Z i b o t e n t a n , P D 1 5 6 7 0 7 , o r B Q1 2 3 . C o l oc al i z at i on
p u n ct a c o u n t > 65 c e l l s p e r g ro u p . e I m a ge s o f E TA K / D M DA - M B 2 3 1 o r W T M D A - M B23 1 c e l l s . D i f f e r e n t c o l o r s r e p r e s e nt t h e s a m e m a r ke r s a s d e s c r ib e d
i n d. S c a l e b a r , 2 μm . E x p er i m e nt s w er e p e r f o r m e d w it h 9 5 % c o n flu e n t c e l l s . S i g n ific a n c e w a s d e t e r m in e d u s i n g a n u n p a i r e d t w o - ta i l e d S t u d e n t ’s t- t e s t .
* * *p < 0 . 0 0 1 , * *p < 0 . 0 0 5 a n d * p < 0 . 05 . E rr o r b a r , S D. S o u r c e d a t a ( b, d) a r e p r o v i d ed a s a S o u r c e D a t a fil e
proliferation and metastasis mostly depends on the inhibition of mGluRs increase the secretion of exosomes by calcium release
sEV although its effect may come from other properties.
from the endoplasmic reticulum via the secondary messenger,
Recent reports suggested that the stimulation of GPCR by IP345. The activation of histamine H1 receptor in HeLa cells also
its agonists initiates the signaling cascade to regulate exosome increases the secretion of exosomes through the promotion of
formation and secretion. For instance, the activation of GPR143 MVB-PM fusion46. Some GPCRs, such as A2A receptors, can
by its ligand, L-DOPA, halted the secretion of exosomes for be transferred via exosomes from the source to the recipient
intercellular communication in the eye44. Activated group I target cells47.
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A R T I C L E
N A T U R E C O M MU N IC A T I ON S | h t t p s : / / d o i . o r g / 1 0 .1 0 3 8 / s 4 14 6 7 - 0 1 9 -09 3 8 7 -4
SFX
4.Small EVs secretion
ETA
3.Exosome secretion
Early
endosome
-RAB27A
Lysosome
MVEs
2.Fusion of MVB with lysosome
-LAMP1
1.Exosome biogeneis
-RAB proteins
-MITF
-ESCRT-mediated components
Breast cancer cell
Fig. 8 P r o p o s ed m o d e l f o r S F X - m ed i at e d i n h i b i t io n o f s E V s e c r et i o n i n M D A - M B2 3 1 c a n c e r c e l l s . S F X, t h r o u gh E T A b i n d i n g a n d s u b s e q u e n t i n t e r f e r e n c e,
r e d u ce d t h e e xp r es s i o n o f p r o t e i n s i n v o l v ed i n t h e s E V b i o g e n e s i s a n d s e c r e t i o n , i n c r e a s e d t h e f u s i o n o f M V E s w i t h l y s o s o me s , a n d fin a l l y d e c r e as e d t h e
n u m be r o f s e cr e t e d s E V f r o m b r e a s t c a n c er c e l l s
In this study, we first demonstrated that ETA is associated and this finding will accelerate the development of a novel class
with sEV biogenesis and secretion in breast cancer cells. The of drugs through mechanistic studies on the regulation of
endothelin receptors (ETRs), which are Family A (Class 1) sEV biogenesis or secretion (Fig. 8). Furthermore, SFX and
GPCRs, consist of two receptor subtypes, ETA and ETB. In ETA antagonists can interfere with the ETA function, suppress
particular, the activation of the ETA pathway, by ET1 or ET2, the secretion of sEV, and change the components of sEV cargo
induces various effects in cancer cells, such as growth, metastasis, from cancer cells, contributing to anti-cancer effects. Therefore,
and angiogenesis32,48. Thus, ETRs have emerged as key targets these ETA-related SFX results are novel and very important
for cancer therapy, and small-molecule antagonists, including for clinical implications although we only tested the effects of
zibotentan and BQ123, have been tested in humans for cancer SFX and ETA antagonists in breast cancer cells. We expect
drug development49.
The increased
that these compounds can also beneficially affect other types
the of cancer cells possibly through preventing sEV biogenesis
degradation
of
MVEs
via
autophagy–lysosome pathway was demonstrated by ultra- and secretion, although this needs to be confirmed by future
structural analysis when breast cancer cells were treated with SFX experiments.
or specific ETA antagonists (Figs. 6a and 7a). Another report
showed that ETA is related with autophagy regulation in H9C2
myoblasts50. Therefore, we additionally studied whether the
inhibitory effect of SFX and ETA antagonists depend on the
autophagy pathway, even if the elevation of autophagosome
structures was not clearly observed in cancer cells by treatment of
these drugs. Through the expression of LC-3BII protein, one of
the critical markers in macroautophagy pathway, and the addi-
tional LC3B puncta study, SFX and ETA antagonists did not fully
induce the LC3B-dependent autophagy pathway. Furthermore,
Methods
Cells and cell culture. All breast cancer and other cells were obtained from the
American Type Culture Collection (ATCC) and grown at 37 °C under humidified
atmosphere with 5% CO2 and 95% air using the recommended culture medium.
MCF7, MDA-MB231, and HEK293T cells were cultured in Dulbecco’s modified
Eagle’s medium (Hyclone) with 10% fetal bovine serum (FBS) and 1% antibiotics.
MCF10A cells were grown in mammary epithelial cell growth medium (MEGM,
Lonza) with 5% FBS, 52 μg ml−1 bovine pituitary extract, 0.5 μg ml−1 hydro-
cortisone, 10 ng ml−1 EGF, and 5 μg ml−1 insulin. SK-MEL-28 cells were cultured
in Minimum Essential Medium with Earle’s Balanced Salts (MEM/EBSS; Hyclone).
these drugs still inhibited sEV release in LC3B K/D cells. These For drug treatment experiments, cells were washed and incubated with FBS-free
medium after cell cultures reached 90% confluency, except invasion, migration,
colony formation, and proliferation assays.
results suggested that the degradation of MVEs by SFX or ETA
antagonists might not be related to the LC3B-dependent autop-
hagy pathway. However, we could not exclude the possibility that
other non-canonical autophagy pathways might function partially
Chemicals. An FDA-approved drug library was purchased from Selleckchem
in cancer cells following treatment with ETA antagonists.
Therefore, further work is required to identify the detailed
mechanism of the degradation pathway via ETA antagonists for
the reduction of sEV biogenesis and secretion.
(L1300) and used for in vitro screening study. The following chemicals were
purchased from Sigma Aldrich, including Sulfisoxazole (SFX, S6377), 5-(N,N-
dimethyl)amiloride hydrochloride (DMA, A4562), GW4869 (D1692), Rapamycin
(R8781), FTY720 (SML0700), ET1 (E7764), BQ123 (B150), PD156707 (PZ0141),
zibotentan (SML1550), and Bafilomycin (B1793). ET2 (#1164) was obtained from
Tocris Bioscience. Docetaxel (DOC) was a gift from professor Keon-Wook Kang
Here, we found that ETA, selected as a newly-recognized
off-target of SFX, is associated with sEV biogenesis and secretion, (Seoul National University).
12
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N A T UR E C O M M UN I CA T I O NS | h t t p s : //d o i . o r g / 10 . 1 0 38 / s 4 1 4 6 7 - 01 9 - 0 9 3 8 7 - 4
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Fluorescence-based high-throughput screening assay. MDA-MB231 cells
were transfected with pCT-CD63-GFP (pCMV, Exosome/Secretory, CD63 Tetra-
spanin tag) plasmid (System Bioscience) to construct MDA-MB231-CD63-GFP
stable cell line. For high-throughput screening assay, MDA-MB231-CD63-GFP (+)
cells (1 × 104/well) were seeded in 96-well culture plates and grown overnight.
These cells were subsequently incubated with 1163 individual FDA-approved
drugs (Selleckchem) for 24 h under serum-depleted condition. The culture
supernatants were then transferred into 96-well black plates and fluorescence
from each supernatant was measured at 485 nm (excitation) and 538 nm
(emission) using a fluorescence microplate reader (GeminiEM; Molecular Devices,
San Jose, CA).
the ELISA. For the measurement of cytokine, the following ELISA kits from R&D
systems were used: IL8 (D8000C), IL2 (D2050), IL6 (D6050), CX3CL1 (DCX310),
CXCL13 (DCX130), CCL2 (DCP00), and TGFB1 (DB100B).
RNA extraction and qRT-PCR. Total RNA from different cells was extracted
by using TRIzol reagent (#15596026; Invitrogen) according to the manufacturer’s
recommendation. A total of 2 μg RNA was reverse-transcribed using the RT-
premix (K2041; Bioneer). qRT-PCR gene expression analysis was performed in
three biological replicates using gene-specific qRT-PCR oligonucleotides. RT-
PCR reactions were monitored on an ABI stepOne Plus instrument (Applied
Biosystems) using the SYBR premix (#4368577; ThermoFisher). Each sample
was PCR-amplified from the same amount of cDNA template. Following an
initial step in the thermal cycler for 15 min at 95 °C, the PCR amplification was
proceeded for 40 cycles of 15 s at 95 °C and 1 min at 60 °C, and completed by
melting curve analysis to confirm specificity of the PCR products. The baseline
and threshold values were adjusted according to the manufacturer’s instructions.
Primer sequences used for RT-PCR (5′ to 3′) are summarized in Supplementary
Table 1.
FACS. For green fluorescence protein (GFP) detection, cells were re-suspended in
FACS buffer (phosphate-buffered saline (PBS) with 5% fetal calf serum). The GFP
intensities in MDA-MB231 cells were determined by FACSCalibur (BD
Bioscience).
Proliferation and cytotoxicity assay. Concentration- and time-dependent
effects on cell proliferation and cytotoxicity were measured using the MTT
[3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide] assay. In the
cytotoxicity assay, cells were seeded in a 24-well plate at a density of 2 × 104 cells/
well and grown for 24 h. Then, the culture medium was replaced with a fresh
medium containing 2% FBS and different concentrations of SFX for another 24 h.
At the end of the incubation, 500 μg ml−1 MTT reagent was added to each
well and incubated for 3 h at 37 °C. After removal of the MTT containing
supernatant solution, 100 μl dimethyl sulfoxide (DMSO) was added to each
well to dissolve violet formazan crystals and optical density of each well was
assessed with a microplate reader at 550 and 570 nm. The proliferation assay
followed the same procedure, except that the low confluency (starting with 8%)
of cells were treated with SFX every 24 h to measure the change in proliferation
over time.
Microarray analysis. The Affymetrix Whole-Transcript Expression Array
process was executed according to the manufacturer′s recommended protocol
(GeneChip Whole Transcript PLUS reagent kit). cDNA was synthesized using a
GeneChip WT (Whole Transcript) amplification kit according to the manu-
facturer’s instructions. The sense cDNA was fragmented and biotin-labeled with
TdT (terminal deoxynucleotidyl transferase) using a GeneChip WT Terminal
Labeling kit. Approximately 5.5 μg of labeled DNA target was hybridized to the
Affymetrix GeneChip Clariom S Human Array at 45 °C for 16 h. The hybridized
arrays were washed, stained on a GeneChip Fluidics Station 450, and scanned
on a GCS3000 Scanner (Affymetrix). Signal values were computed using
Affymetrix® GeneChip™ Command Console software. The data were summarized
and normalized using the robust multi-average (RMA) method implemented
in Affymetrix® Power Tools (APT). Gene-enrichment and functional annotation
analysis for the significant probe list was performed using Gene Ontology
(http://geneontology.org) and KEGG (http://kegg.jp) technology. For sEV miRNA
microarray, the Affymetrix miRNA Expression Array process was executed
according to the manufacturer’s recommended protocol (miRNA 4.0 chip).
Raw data were extracted automatically in Affymetrix data extraction protocol
using the software provided by Affymetrix GeneChip® Command Console®
Software (AGCC). The CEL files import, miRNA level RMA + DABG-All analysis,
and result export were performed by using Affymetrix® Power Tools (APT)
Software.
Isolation of MVs and sEV. The MVs were isolated by following the method
reported by Rong Xu. et al.51. Briefly, the individual supernatants from MCF7,
MDA-MB231, and SK-MEL-28 cells were serially centrifuged at 300 × g/3 min and
2500 × g/20 min. Then, the supernatants were filtrated using a 0.8 μm syringe filter
and centrifuged at 10,000 × g/40 min again. The MV pellets were re-suspended with
PBS and examined with the nanoparticle tracking analysis (NTA). To isolate sEV,
individual supernatants obtained from the indicated cells were serially centrifuged
at 300 × g/3 min, 2500 × g/20 min, and 10,000 × g/30 min. Then the supernatants
were filtrated using a 0.2 μm syringe filter, and centrifuged at 120,000 × g/90 min.
The sEV pellets were re-suspended with PBS and centrifuged at 120,000 × g/90 min
again. The purified sEV pellets were re-suspended with PBS or 1× RIPA buffer for
further experiments.
For miRNA microarray assay, ExoQuick-TC exosome precipitation solution
(System Bioscience) was used for sEV isolation with the method optimized by our
group52. Briefly, MDA-MB231 cells were treated with vehicle or SFX in FBS-free
media, and incubated for 24 h. Next, conditioned media was sequentially
centrifuged at 300 × g/3 min, 2500 × g/20 min, and 10,000 × g/30 min, and then
conditioned media were filtrated using 0.2 μm filter. These medium were incubated
with ExoQuick-TC solution at 4 °C overnight. After incubation, sEV were isolated
by centrifugation at 1500 × g/30 min and washed with PBS three times. Finally,
sEVs were re-suspended in nuclease-free water (Promega) for total RNA
extraction.
Proteomics. sEV proteins were digested by trypsin52 and the tryptic peptides were
analyzed by nano-ultra-high-performance LC (UPLC) (Waters) and tandem mass
spectrometry using a Q-Tof Premier (Waters)53,54. Digested peptides were injected
into a 2 cm × 180 μm trap column and resolved in a 25 cm × 75 μm nanoACQUITY
C18 column (Waters) using the LC system. All samples were analyzed in triplicate.
For protein identification, MS raw data were converted into peak lists using MASCOT
Distiller version 2.1 (Matrix Science, London, UK) with default settings. All MS/MS
raw data were analyzed using MASCOT version 2.2.1 (Matrix Science)53. Mascot was
used to search the SwissProt database (release 2018_07) with human taxonomy.
Quantification was performed using PEAKS Studio version 10.0 (Bioinformatics
Solution Inc., Waterloo, Canada). For Label-free protein quantification, identified
peptides were filtered based on False Discovery rate <1%. The abundance of each
peptide was determined using ion chromatography extraction and the protein
ratio was calculated using the average abundance among the corresponding
peptides. Protein ratios were considered acceptable when the proteins contained
more than one unique peptide.
Nanoparticle tracking analysis. Cell culture supernatants containing MVs and
sEV were analyzed using a NanoSight LM10 device (Nanosight, Malvern). A
monochromatic laser beam at 405 nm was set to analyze the nanoparticles, and a
video with a 30-s duration was taken at a rate of 30 frames/s and a camera level of 7.
Approximately 30–100 particles were analyzed in each field of view, and then
particle brown-movement was assessed using NTA software (version 2.3, Nano-
sight). NTA post-acquisition settings were optimized and kept constant between
samples, and recorded video was then analyzed to measure particle sizes and
concentrations.
Western blotting. Cellular or sEV proteins were resolved by sodium dodecyl
sulfate polyacrylamide gel electrophoresis, transferred onto nitrocellulose
membranes, probed with the respective primary antibody, and incubated with a
horseradish peroxidase-linked secondary antibody. Images were visualized using
enhanced chemiluminescence (ECL) detection reagents (#34580; Thermo
Scientific) and quantified using ECL hyper-film (AGFA; Morstel).
The following primary antibodies were used: anti-CD63 (ab68418, 1:1000;
Abcam), CD9 (ab2215, 1:1000; Abcam), CD81 (ab109201, 1:1000; Abcam), Alix
(ab56932, 1:1000; Abcam), Tsg101 (ab30871, 1:1000; Abcam), Filotillin-1 (#3253,
1:1000; CST), LAMP-1 (ab25630, 1:1000; Abcam), RILP (ab128616, 1:1000;
Abcam), beta-actin (4670, 1:3000; Cell Signaling Technology (CST)), Rab27a
(ab55667, 1:1000; Abcam), Rab5 (ab18211, 1:1000; Abcam), Rab7 (ab50533,
1:1000; Abcam), EDIL3 (ab88667, 1:1000; Abcam), Glypican-1 (PA5-28055,
1:1000; ThermoFisher), HSP90a (#8165, 1:1000; CST), ASMase (#3687, 1:1000;
CST), nSMase2 (ab68735, 1:1000; Abcam), LC3B (NB600-1384, 1:1000; Novus
Biologicals), Endothelin receptor type A (ab117521, 1:2500; Abcam), Angiotensin
II type I receptor (ab18801, 1:1000; Abcam), Carbonic anhydrases-13 (ab135986,
1:1000; Abcam), Kynurenine 3-monooxygenase (ab130959, 1:1000; Abcam),
Endothelin-1 (ab2786, 1:1000; Abcam), Endothelin-2 (sc293248, 1:1000; SCBT),
Determination of sEV protein concentration. Concentration of sEV proteins
was determined using the microBCA assay (#23235; ThermoFisher). To
measure protein concentrations, sEV were isolated by differential ultracentrifuga-
tion. sEV proteins were extracted using 1× RIPA buffer (#50–188; Merch
Millipore). The microBCA assay was performed according to the manufacturer’s
instructions.
ELISA. The levels of IL8, IL2, IL6, CX3CL1, CXCL13, CCL2, and TGFB1 cytokines/
chemokines were measured by sandwich enzyme-linked immunoassay (ELISA).
Briefly, MDA-MB231 cells (3 × 105/well) were seeded in six-well plates overnight,
and treated with SFX, PD156707, or BQ123 for additional 24 h. Subsequently, the
supernatants were centrifuged at 300 × g/3 min to remove the debris and used for
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13

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VPS4B (ab102687,1:1000; Abcam), MITF (#12590, 1:1000, CST), and Lamin B
(#13435, 1:1000; CST). For the detection of specific human cell-derived sEV, the
following primary antibodies were used: the anti-human CD63 (SHI-EXO-M02,
1:1000; Cosmo Bio Co., Ltd).
using DAPI-containing mounting solution. For labeling of lysosomes and late
endosomes, 1 × 105 cells were transfected with red fluorescence protein (RFP)-
RAB7 vector (CellLight Late Endosomes-GFP, BacMam 2.0, C10588; Molecular
Probes) and GFP-LAMP-1 vector (CellLight-lysosome-GFP, BacMam 2.0, C10596;
Molecular Probes) and grown overnight. Fluorescence images were obtained with a
Ziess LSM5 laser scanning fluorescence confocal microscope. Colocalization puncta
was measured using ImageJ.
miRNA expression analysis. sEV miRNA was extracted according to the man-
ufacturer’s instruction (total exosome RNA isolation kit, #4478545; ThermoFisher).
Briefly, after ultracentrifugation, the sEV pellets were re-suspended with the exo-
some resuspension buffer, and then added 2× denaturating solution and Acid-
Phenol:Chloroform to separate into aqueous and organic phases. The total RNA-
containing aqueous phase was then transferred to filter cartridge for miRNA iso-
lation. Extracted miRNA was washed using the miRNA wash solution and eluted
using the elution buffer. TagMan® MicroRNA system was used for miRNA
expression analysis. First, RT master mix [extracted miRNA, dNTPs (with dTTP),
reverse transcriptase and RNase inhibitor] was loaded into the thermal cycler to
perform reverse transcription using following parameter values: 30 min at 16 °C,
30 min at 42 °C, and 5 min at 4 °C. Then, the fluorescent signals of specific miRNAs
by real-time PCR system were detected and recorded. The reaction mixture
contained: TagMan MicroRNA assay reagent (ThermoFisher), RT product, and
TagMan universal PCR master mix (#4304437; ThermoFisher). U6 snRNA was
used as reference. miRNA sequence used for miRNA expression analysis is sum-
marized in Supplementary Table 2.
Radioligand binding assay. Radioligand binding assay was performed in Eurofins
Panlab Discovery Service Center (Taiwan). To evaluate the binding activity of SFX,
assay was performed under the following conditions. Human recombinant CHO-
K1 cells were used as the source, and ligand concentration was 0.030 nM [125I]
endothelin-1. IC50 values were determined by a non-linear, least-squares regression
analysis using MathIQTM (ID Business Solutions Ltd, UK). The inhibition con-
stants (Ki) were calculated by the equation of Cheng and Prusoff55 using the
observed IC50 of the tested compound, the concentration of radioligand employed
in the assay, and the historical values for the KD of the ligand (obtained experi-
mentally at Eurofins Panlab, Ltd). The Hill coefficient (nH), defining the slope
of the competitive binding curve, was calculated by using MathIQTM.
Synthesis of a sulfisoxazole-based affinity probe. First, we synthesized 4-((4-(N-
(3,4-dimethylisoxazol-5-yl)sulfamoyl)phenyl)amino)-4-oxobutanoic acid (SP-1). To
a solution of SFX (557 mg, 2.08 mmol), Et3N (0.58 mL, 4.16 mmol) and DMAP
(127 mg, 1.04 mmol) in CHCl3 (4 mL) were added dropwise into methyl 4-chloro-
4-oxobutanoate (0.257 mL, 2.08 mmol) at 0 °C. The reaction mixture was then
stirred at room temperature for 2 h and washed with saturated aqueous NaHCO3
(2 × 50 mL). The reaction mixture was extracted three times with ethyl acetate and
the combined organic phase was washed with brine, dried over anhydrous MgSO4,
and concentrated under reduced pressure. The residue was purified by flash col-
umn chromatography on a silica gel (ethyl acetate/n-hexane = 1:1) to yield the
amide (310 mg, 39%). 1H-NMR (600 MHz, CDCl3) δ 9.12 (bs, 1H), 7.67 (d, 2H,
J = 13.2 Hz), 7.53 (d, 2H, J = 12.6 Hz), 3.64 (s, 3H), 2.68 (d, 2H, J = 7.2 Hz), 2.66
(d, 2H, J = 7.2 Hz), 2.01 (s, 3H), 1.65 (s, 3H); HRMS (FAB+): mass calculated for
RNAi analysis. Transient transfection was performed by using Lipofectamine 3000
(L3000015; ThermoFisher) according to the manufacturer’s instructions. Briefly,
cells were seeded in 60 mm dishes at 70–80% confluence, and then were transfected
with 2.5 μg siRNA with Lipofectamin 3000 under serum-reduced condition. After
1 day, culture media were replaced with a serum-containing medium, and then
RNA and proteins were extracted after 48–72 h post-transfection. siRNAs of ETA,
AGTR1, KMO, and CA13 were purchased from Dharmacon; negative control
siRNA was purchased from Bioneer (AccuTargetTM Negative Control siRNA,
SN-1001-CFG). The siRNA sequence used for RNAi interference analysis is
summarized in Supplementary Table 3.
C
16H20N3O6S [M + H]+, 382.0995; found, 382.1072. Additionally, to the solution
of the amide compound (105 mg) in tetrahydrofuran (10 mL), lithium hydroxide
monohydrate (105 mg) in H2O (10 mL) was added at 0 °C. The reaction mixture
was stirred for 48 h and concentrated under reduced pressure. The residue was
purified by flash column chromatography on a silica gel (MeOH/CH2Cl2/AcOH =
1:10:0.1) to yield acid (1) (81 mg, 80%). 1H-NMR (600 MHz, DMSO-d6) δ 12.15
(bs, 1H), 10.90 (bs, 1H), 10.40 (s, 1H), 7.77 (d, 2H, J = 9 Hz), 7.68 (d, 2H, J = 8.4
Hz), 2.68 (t, 2H, J = 6.6 Hz), 2.54 (t, 2H, J = 6.6 Hz), 2.07 (s, 3H), 1.61 (s, 3H); 13C-
NMR (150 MHz, DMSO-d6) δ 174.1, 174.0, 171.4, 161.7, 143.8, 128.3, 119.0, 118.9,
31.6, 29.2, 29.0, 10.8, 6.3; HRMS (FAB): mass calculated for C15H18N3O6S [M + H]
Sphingomyelinase (sMAse) activity measurements. Acidic sphingomyelinase
activity (acidic sMAse, ab190554; Abcam) and sphingomyelinase activity (neutral
sMAse, ab138877; Abcam): acidic and neutral sphingomyelinase activities were
determined by using an assay kit. Briefly, cells were co-cultured with SFX for 24 h
and then lysed with 1× mammalian lysis buffer. These samples were then reacted
with the acidic sMAse assay reagents according to the manufacturer’s recom-
mended protocol. After the incubation, fluorescence from each sample was
obtained by using a microplate reader (GeminiEM; Molecular Devices) at Ex/Em
= 540/590 nm (the cut off was 570 nm). The fluorescence in a blank well was used
as a negative control; GW4869 and FTY720 were used for positive controls of
neutral SMase and acidic SMase, respectively.
+, 368.0838; found, 368.0913. Next, we synthesized N-(17-azido-3,6,9,12,15-pen-
taoxaheptadecyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)
pentanamide (SP-2). To a solution of biotin-ONp (300 mg, 0.82 mmol) in THF
(5 mL), 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-amine (250 μL, 0.82 mmol)
and Et3N (350 μL, 2.46 mmol) were added at room temperature. After stirring for
12 h at room temperature, the solution was concentrated under reduced pressure.
The residue was purified by flash column chromatography on a silica gel (MeOH/
CH2Cl2 = 1:10) to yield the azide (2) (401 mg, 92%). 1H-NMR (600 MHz, CD3OD)
δ 4.40 (m, 1H), 4.22 (m, 1H), 3.58–3.51 (m, 18H), 3.45 (t, 2H, J = 5.4), 3.28–3.25
(m, 4H), 3.12 (m, 1H), 2.84 (dd, 1H, J = 13.2 and 5.4 Hz), 2.62 (d, 1H, J = 13.2 Hz),
2.13 (t, 2H, J = 13.2 Hz), 1.67–1.47 (m, 4H), 1.37–1.31 (m, 2H); 13C-NMR (150
MHz, CD3OD) δ 174.6, 164.6, 70.2, 70.1, 69.8, 69.7, 69.2, 61.9, 60.2, 55.6, 50.4, 39.7,
39.0, 35.3, 28.4, 28.1, 25.5; HRMS (FAB+): mass calculated for C22H41N6O7S [M +
H]+, 533.2679; found, 533.2879. Then, we synthesized N-(17-amino-3,6,9,12,15-
pentaoxaheptadecyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-
4-yl)pentanamide (SP-3). A solution of azide (4) (29 mg, 0.054 mmol) and 10%
Pd/C (6 mg, 0.005 mmol) in MeOH (2 mL) was placed under an atmosphere of
hydrogen. After stirring for 24 h, the reaction mixture was diluted with ethyl
acetate, filtered through a short pad of celite, and concentrated under reduced
pressure. The residue was purified by flash column chromatography on a silica gel
(MeOH/CH2Cl2 = 1:10) to yield amine (3) (24 mg, 88%). 1H-NMR (600 MHz,
CD3OD) δ 4.41 (m, 1H), 4.22 (m, 1H), 3.55–3.52 (m, 16H), 3.45–3.41 (m, 4H),
3.27–3.24 (m, 3H), 3.21 (bs, 2H), 3.12 (m, 1H), 2.84 (dd, 1H, J = 12.6 and 4.8 Hz),
2.62 (d, 1H, J = 12.6 Hz), 2.13 (t, 2H, J = 13.2 Hz), 1.66–1.46 (m, 4H), 1.37–1.33
(m, 2H); 13C-NMR (150 MHz, CD3OD) δ 174.8, 164.6, 72.0, 70.0, 69.9, 69.7, 69.7,
69.4, 61.9, 60.2, 55.6, 40.6, 39.6, 38.8, 35.3, 28.3, 28.1, 25.4; HRMS (FAB+): mass
calculated for C22H43N4O7S [M + H]+, 507.2774; found, 507.2856. Finally, we
synthesized N1-(4-(N-(3,4-dimethylisoxazol-5-yl)sulfamoyl)phenyl)-N4-(19-oxo-
23-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9,12,15-
pentaoxa-18-azatricosyl) succinamide (SP-4): To a dimethylformamide (DMF)
solution (0.5 mL) of carboxylic acid (1) (22 mg, 0.059 mmol), 1-[(1-(cyano-2-
ethoxy-2- oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]
methanaminium hexafluorophosphate (COMU) (26 mg, 0.06 mmol) and diiso-
propylethylamine (DIPEA) (22 μL, 0.129 mmol) were added at 0 °C. After stirring
for 30 min, the DMF solution (0.5 mL) of the amine (3) (30 mg, 0.059 mmol) was
added to the reaction mixture. After stirring for 24 h, the reaction mixture was
Intracellular calcium concentration measurements. [Ca2+]i concentration was
measured using fluo-3/AM (F1242; Invitrogen). For the experiments, MDA-
MB231, MCF7, and SK-MEL-28 cells were loaded with 5 μM fluo-3/AM for 30 min
at 37 °C. Subsequently, the cells were scanned using a fluorimeter (GeminiEM;
Molecular Devices) and the collected data were analyzed according to the manu-
facturer’s instructions.
Transmission electron microscopy (TEM) analysis. For TEM analyses to eval-
uate cell morphologies and sub-organelle structures, drug-treated or control MDA-
MB231 CD63-GFP fusion cells were pelleted by centrifugation, and pelleted cells
were fixed with 2.5% glutaraldehyde in a 0.1 M phosphate buffer. After washing
several times with 0.1 M carcodylate buffer, cells were dehydrated by gradient series
of ethanol (50%, 60%, 70%, 80%, 90% ethanol for 20 min each step, 100% 20 min
twice) followed by propylene oxide for twice. Afterwards, cells were infiltrated with
progressively concentration Eponate 812, and then polymerized in fresh Eponate
812 for 2 days at 60 °C. Samples were sectioned using an Ultra microtome (Ultracut
UCT; Leica) and stained with uranyl acetate and lead citrate. Sections were
examined with energy filtering TEM (LEO-912AB OMEGA; Carl Zeiss) at the
Korean Basic Science Institute Chuncheon Center.
For TEM analyses to detect sEV, MDA-MB231 and MCF7 cells were pelleted by
serial ultracentrifugation, and the pellets were deposited on pure carbon-coated
EM grids. After staining with 1% uranyl acetate, the grids were dried at room
temperature and viewed at ×12,000 magnification using a Biotransmission electron
microscope (HT7700; Hitachi) operated at 120 kV.
Immunofluorescence staining and confocal microscopy. Cells were seeded onto
glass coverslips at 2 × 104 cells/well in a six-well confocal chamber overnight and
were treated with SFX for 24 h. For the detection of lysosomal activity, 500 nM
Lysotracker (L7528; ThermoFisher) was added into the culture medium for 1 h
before fixing with 4% paraformaldehyde in PBS for 30 min at room temperature.
Fixed cells were washed with PBS, and the coverslips were stained and mounted
14
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N A T UR E C O M M UN I CA T I O NS | h t t p s : //d o i . o r g / 10 . 1 0 38 / s 4 1 4 6 7 - 01 9 - 0 9 3 8 7 - 4
A R T I C L E
concentrated under reduced pressure. The residue was purified by flash column
chromatography on a silica gel (MeOH/CH2Cl2 = 1:10) to afford the sulfisoxazole
affinity probe (4) (25 mg, 49%). 1H-NMR (600 MHz, CD3OD) δ 7.67 (d, 2H,
J = 8.4 Hz), 7.63 (d, 2H, J = 8.4 Hz), 4.39–4.37 (m, 1H), 4.20 (m, 1H), 3.53–3.50
(m, 16H), 3.43 (d, 4H, J = 5.4 Hz), 3.27–3.25 (m, 4H), 3.10 (m, 1H), 2.83 (dd, 1H,
J = 12.6 and 4.2 Hz), 2.62–2.59 (m, 3H), 2.49 (t, 2H, J = 6.6 Hz), 2.12 (t, 2H,
J = 7.2 Hz), 2.04 (s, 3H), 1.67 (s, 3H), 1.63–1.47 (m, 4H), 1.35–1.33 (m, 2H); 13C-
NMR (150 MHz, CD3OD) δ 174.7, 173.3, 171.9, 164.6, 161.7, 156.9, 143.2, 134.7,
127.8, 118.9, 107.4, 70.1, 70.0, 70.0, 69.8, 69.2, 61.9, 60.2, 55.6, 39.6, 39.0, 38.9, 35.3,
31.6, 30.1, 28.3, 28.1, 25.4, 9.2, 5.0; HRMS (FAB+): mass calculated for
The cell invasion study was performed using a cell culture insert chamber
(CLS3364; Corning). Chamber was coated with the basement membrane Matrigel
(100 μl of 20% matrigle/filter, #354248; Corning). The cancer cells were seeded at
10,000 cells/well into upper chamber, and invading cells were fixed and stained
with crystal violet. The membranes were mounted on glass slides, and images of the
cells were captured using a microscope (×4 magnification).
2D colony-forming assay. Cells were seeded in a six-well plate (1 × 103/well) and
treated SFX immediately. Then, cells were washed using PBS and treated with the
indicated concentrations of SFX every 24 h. After 6 days, cell colonies were fixed
with glutaraldehyde (6.0% v/v) and stained with crystal violet (0.5% w/v). Stained
colonies were dissolved in 25% methanol, incubated for 10 min, and then measured
at 590 nm with a spectrometer (MultiskanTM GO Microplate spectrometer;
ThermoFisher).
C
37H58N7O12S2 [M + H]+, 856.3507; found, 856.3580. Chemical structure of
sulfisoxazole-based affinity probe is summarized in Supplementary Fig. 13.
Target identification. To predict the functional targets of SFX, an in silico
approach termed Similarity Ensemble Approach (SEA) was used. Of the data
registered in BindingDB (56), only those measured through the protein-based
assays were extracted. Tanimoto coefficient (Tc) calculated based on Morgan
circular fingerprint using the RD Kit (http://www.rdkit.org) was used to quantify
the chemical similarity between a test molecule and SFX. The value of Tc was
between 0 and 1, indicating that the two molecules with the value closer to
1 shared greater chemical similarity. After retrieval of the protein targets that
were small-molecule modulators with the higher Tcs to SFX, SEA was used to
compare the similarities. In SEA, the pairwise Tc values between two molecules are
summed to form ΣTc. The value of the ΣTc in a test molecule and the distribution
of ΣTc in a set of small molecules were drawn with a bar and histogram for
comparison.
Cancer xenograft studies in mice. All animal research was performed in accor-
dance with protocols approved by the Kyungpook National University (KNU)
Institutional Animal Care and UCommittes (IACUCs. Approve number:
2017–0146). In the proliferation study, MDA-MB231-luci (+) cells suspended in
PBS were orthotopically injected into the left fat pad of 5-week-old female BALB/c
nude mice. SFX (200 mg kg−1 day−1) was orally administered for 14 days or
zibotentan (10 mg kg−1 day−1) was intraperitoneally administrated for 14 days.
Docetaxel (8 mg kg−1 week−1) was intravenously administered once a week for
two times. Tumor growth was measured every 2 days by using a caliper. After
14 days, mice were euthanized, and luciferase signals were measured using an
IVIS imaging system. In rescue experiments, MDA-MB231-luci (+)-derived
sEV (10 µg) were intravenously injected once every two days for seven times.
In addition, MDA-MB231-ETA K/D cells were orthotopically injected into the
left fat pad of 5-week-old female BALB/c nude mice to monitor the cancer
progression.
In the metastasis study, 4T1-luci (+) cells suspended in PBS were orthotopically
injected into the left fat pad of 5-week-old female BALB/c wild-type mice; other
procedures were as described for the proliferation study. Tumor attenuation was
monitored every week for 5 weeks using an IVIS imaging system. In each group, five
mice were used for the survival test. In addition, Zibotentan (10 mg kg−1 day−1) or
BQ123 (1 mg kg−1 day−1) were intraperitoneally administrated for 21 days to
validate the anti-cancer effect of ETA antagonists. Final attenuation of tumor
metastasis was monitored using an IVIS imaging system. In rescue experiments,
4T1-luci (+)-derived sEV (5 μg) were intravenously injected once every 2 days
for 11 times.
Biotin-based pull-down assay. The biotin-based pull-down assay was performed
according to the manufacturer’s instructions (#21115; Thermo Scientific). Briefly, a
streptavidin ligand-bound agarose gel was immobilized on the biotin-tagged SFX,
while the unbound SFX was washed away. Next, membrane proteins of MDA-
MB231 cell lysates were incubated with biotin-tagged SFX-bound agarose gel to
isolate the prey proteins that were selectively eluted with a low-pH elution buffer
and used for western blot analyses.
Antimicrobial susceptibility testing. The minimal inhibitory concentrations
(MICs) of SFX and three structural derivatives were determined by microdilution
in Mueller–Hinton agar (#225250; Difco Laboratories) according to the guidelines
of the Clinical and Laboratory Standard Institute (CLSI, 2015). Staphylococcus
aureus (ATCC 29213) and Escherichia coli (ATCC 25922) were used as quality
control strains.
Statistical analysis. Unpaired two-tailed students t-test was used for experiments
comparing two sets of data. The error bars in the graphical data represent means
standard deviation. All in vitro experiments were performed in triplicates unless
otherwise stated. p values less than 0.05 were considered to denote statistically
significant differences. *, **, and *** denote p value of <0.05, 0.005 and 0.001,
respectively. NS denotes not significant. Data were analyzed using PRISM 6 soft-
ware (GraphPad Software, Inc.).
Toxicology study and blood chemistry. Seven-week-old male and female ICR
mice were supplied by Hanabio Corporation (Seoul, Korea). Animals were
bred under SPF conditions and maintained in barrier housing during the experi-
ment. They were maintained in animal care facilities at Chung-Ang University
(22 °C 1 °C, humidity 60% 10%, and a 12 h/12 h light/dark cycle). Nutritionally
complete rodent chow and water were provided ad libitum. All protocols were
approved by the Ethics Committee of Animal Experiments of Chung-Ang Uni-
versity. This present study was performed according to the general principles of
OECD guideline 407 (repeated daily doses for 28-day oral study in rodents) with
some modifications. ICR male and female mice were randomly divided into four
groups (vehicle control; and low, middle, and high SFX dose groups) according to
weight (n = 12/subgroup). SFX was suspended in corn oil, and orally administered
using an oral gavage needle once a day for 28 days. In a single administration,
vehicle control (corn oil), or 100, 300, or 900 mg kg−1 day−1 of SFX was given to
the mice. Mice were weighed every 2 days during the experiment. Individual body
weights were used to calculate the correct SFX doses. After collection of blood from
the orbital socket, serum from each mouse was stored at −20 °C. Later, serum
samples were characterized on a Biochemical Autoanalyzer (Type 7170; Hitachi,
Tokyo, Japan) using the following blood chemical parameters: total protein,
albumin, globulin, alanine aminotransferase, aspartate aminotransferase, glucose,
urea nitrogen, creatinine, low-density lipoprotein, cholesterol, triglyceride, and
total bilirubin.
Reporting Summary. Further information on experimental design is available in
the Nature Research Reporting Summary linked to this article.
Data availability
All microarray data that support the findings of this research have been deposited in the
Gene Expression Omnibus (GEO) and are accessible through the GEO accession number
GSE117991 (mRNA microarray) and GSE124320 (miRNA microarray). The proteomics
data have been deposited to the ProteomeXchange Consortium via the PRIDE with the
dataset identifier PXD012689. The source data underlying Figs. 5 (b–g), 2 (b–f), 3 (c, d),
4b–d, i, j), 5 (a, c–g), 6 (b, d, e), 7 (b, d) as well as those underlying Supplementary figs. 1
(b, d–h), 2 (a, b), 3 (c–e, g, h), 5 (a–c), 6 (a), 7 (c–e), 9 (a–d), 10 (a–c), and 11 (b, c, e) are
provided as a Source Data file. All other relevant data of this study are available from the
corresponding authors upon reasonable request. A reporting summary is available as
a Supplementary Information file.
Cellular assays. For wound healing assay, MDA-MB231 cells were seeded in a 24-
well plate and grown to confluence overnight. The next day, the monolayer was
wounded by repeated scratches with a 200 μl pipette tip, and the media was
changed to remove cell debris. Each wound was imaged at 0 h, and again after 24 h.
The average wound healing was assessed by the average of three measurements of
the wound areas.
Received: 21 August 2018 Accepted: 6 March 2019
A transwell migration assay was performed using the Costar transwell system
(CLS3364; Corning). Briefly, MDA-MB231 cells (2 × 103 cells) were suspended in
200 μl serum-free medium and seeded in the upper insert chamber and 500 μl
medium was added to the lower chamber. At 4 h after the cells were seeded, media
in both the upper insert and lower chambers were removed. Cells that had
migrated into the lower chamber through the 8-μm pore membrane were stained
by crystal violet solution and then the migrated cells were visualized using a
microscope (×4 magnification).
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Acknowledgements
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critical suggestions; we also thank members of this laboratory for helpful discussion and
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Author contributions
E.-J.I. designed, performed, and analyzed most experiments, and wrote the manuscript.
C.-H.L. performed confocal analysis. P.-G.M. conducted ELISA and TEM analysis.
G.R.G. and B.-H.L. conducted mouse work. J.-C.L. conducted MIC analysis. J.G.J.
conducted in silico modeling. M.-S.J., J.-E.L., H.-S.J., H.-J.R., and S.J. conducted TEM
analysis. W.K. conducted mouse toxicology study. S.-Y.S. synthesized chemical deriva-
tives. J.-S.B., J.-M.L., T.-K.K., K.-W.K., Y.-E.C. discussed the hypothesis and contributed
to data interpretation. B.-J.S. contributed to data interpretation and wrote the manu-
script. M.-C.B. designed and analyzed experiments, wrote the manuscript, and conceived
the idea and supervised the study.
16
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Additional information
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