tumor-stroma mechanics coordinate amino acid availability ...glutamine/glutamate metabolism (n=3)....
TRANSCRIPT
![Page 1: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/1.jpg)
Cell Metabolism, Volume 29
Supplemental Information
Tumor-Stroma Mechanics Coordinate
Amino Acid Availability to Sustain
Tumor Growth and Malignancy
Thomas Bertero, William M. Oldham, Eloise M. Grasset, Isabelle Bourget, EtienneBoulter, Sabrina Pisano, Paul Hofman, Floriant Bellvert, Guerrino Meneguzzi, Dmitry V.Bulavin, Soline Estrach, Chloe C. Feral, Stephen Y. Chan, Alexandre Bozec, and CedricGaggioli
![Page 2: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/2.jpg)
Tumor-stroma mechanics coordinate amino acid availability to sustain tumor
growth and malignancy
Thomas Bertero, William M. Oldham, Eloise M. Grasset, Isabelle Bourget, Etienne Boulter,
Sabrina Pisano, Paul Hofman, Floriant Bellvert, Guerrino Meneguzzi, Dmitry V. Bulavin,
Soline Estrach, Chloe C. Feral, Stephen Y. Chan, Alexandre Bozec and Cedric Gaggioli
![Page 3: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/3.jpg)
Glutamate
Glutamine
Aspart
ate
Gln/glu
Succinate
a-keto
malateCitr
ate
NA
D+/
NA
DH
Glutamate
Glutamine
Aspart
ate
Gln/glu
Succinate
a-keto
malate
Citrate
0Nor
mal
ized
intr
acel
lula
r m
etab
olite
leve
l (A
.U)
GLS1
LDHA
Tubulin
1kPa 8kPa
SCC12 SCC12
**
***
GLS1 LDHA0
2
4
6
8
Den
sito
met
ry
(Gen
e/tu
bulin
)
1kPa8kPa
*
***
** *
Nor
mal
ized
G
LS a
ctiv
ity (A
.U.)
GDH activity
2
4
6
** *
***
*
SCC12
1kPa8kPa
Nor
mal
ized
intr
acel
lula
r m
etab
olite
leve
l (A
.U)
0
1
2
3
**
Soft (1kPa)
Stiff(8kPa)
Tubulin
LDHA
GLS1
CAF
GLS1 LDHA
CAF
01234
Den
sito
met
ry
(Gen
e/tu
bulin
)
**
**
Soft (1kPa)Stiff (8kPa)
nmol
/min
/10
cel
ls6
0
2
4
6
*
**
0
1
2
3
4
5
GDH activity
0
5
10
15
20
Nor
mal
ized
G
LS a
ctiv
ity (A
.U.)
0
1
2
3
4
5nm
ol/m
in/1
0 c
ells
6
NA
D+/
NA
DH
1
0
0
1
1.5
0.5
SCC12
CAFCAF
CAF1kPa8kPa
0.5
1.5
*
% o
f Ki6
7+ c
ells
*
0
1
2
3
GDHGLS1
LDHA
SLC1A5
Fold
cha
nge
**
****
1kPa8kPa
CAF 1kPa b
oiled C
M
CAF 1kPa b
oiled C
M
+Asp
(5mM)
CAF 8kPa b
oiled C
M
SCC12 C
M
CAF free
ze/th
aw C
M
SCC12 boile
d CM
CAF_brea
st CM
CAF_brea
st boile
d CM
CAF_lung C
M
A459 C
M
MDA-MB-46
8 CM
MDA-MB-46
8 boile
d CM
A549 b
oiled
CAF_lung boile
d CM
SCC12 MDA-MB-468 A549
CAF CM
CAF boiled C
M
SCC12 fr
eeze
/thaw
CM
SCC12 1k
Pa boile
d CM
SCC12 8k
Pa boile
d CM
SCC12 1k
Pa boile
d CM
+Glu (5
mM)
** **
0
1
2
3
Fold
cha
nge
0
10
20
30
*** ********
***
0
10
20
30
% o
f Ki6
7+ c
ells *** ***
0
10
20
30
% o
f Ki6
7+ c
ells *** ***
0
0.5
1
1.5
2 CAF (8kPa)***
*
*** ******
***CAF_breast (8kPa)
0
0.5
1
1.5
2
MDA-MB-46
8 CM
MDA-MB-46
8 boile
d CM
CAF_brea
st boile
d CM
CAF_brea
st CMFo
rce
mag
nitu
de/a
rea
(kPa
)
Forc
e m
agni
tude
/are
a (k
Pa)
******
*
CAF_lung C
M
CAF_lung boile
d CM
A459 C
M
A549 b
oiled
*** ***
*
0
0.5
1
1.5
2CAF_lung (8kPa)
GDHGLS1
LDHA
SLC1A5
Forc
e m
agni
tude
/are
a (k
Pa)
GOT1
GOT1
1kPa8kPa
CAF
SCC12
A D
E F G H I
L
N OP Q R
S T U
V W X
Bertero et al., Supplemental figure 1
SCC 1kPa
00.2
5101520 SCC 8kPa
0.40.60.8
1
Fold
cha
nge
10 20 30 40 5000
0.2
5101520
0.40.60.8
1
Fold
cha
nge
10 20 30 40 500
Gln
Glu
Asp
TryIso
TyrPhe
MetThr
GlyVal
Leu
SerHisAsn
TryIsoPheTyrProGluGlnCysArg
Asp
SerHisAsn
CAF 1kPa CAF 8kPa
00.2
5101520
0.40.60.8
1
00.2
5101520
0.40.60.8
1Fold
cha
nge
Fold
cha
nge
10 20 30 40 500 10 20 30 40 500
Asp
Gln
Glu
TryIso
PheTyr
ProVal
Asn
Met
CysHis
TryIsoPheTyr
ProAsn
AspLysGlnGluHisSer
Ser
Glu
cose
rate
(fm
ol/h
/cel
l)
Lact
ate
rate
(fm
ol/h
/cel
l)
0100200300400
***0
-50
-100
-150***
SCC12
1kPa8kPa
1kPa8kPaSCC12
B C
0
-30
-60-90
-120Glu
cose
rate
(fm
ol/h
/cel
l)
Lact
ate
rate
(fm
ol/h
/cel
l)
0
100
200
300
400
**
***
CAF CAF
1kPa8kPa
1kPa8kPa
J K M
-200
500
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Figure S1 related to figure 1: Tumor niche mechanics differentially reprogram tumor
niche cells metabolism to sustain their pro-tumoral behaviors. A-G) SCC12 cells were
plated on soft (1kPa) or stiff (8kPa) hydrogels. A-B) Glucose and lactate fluxes were
determined. Mean of 9 wells from 3 independent experiments. C) Intracellular levels of the
indicated metabolites were analyzed by LC-MS (n=5). Data normalized to 1kPa. D)
Extracellular amino acid flux analyses of SCC12 plated on 1kPa or 8kPa hydrogel. Mean
expression (n=3) at t0 was assigned a fold change of 1, to which relevant samples were
compared E) RT-qPCR expression levels analysis of the indicated genes related to the
glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1
and LDHA protein levels. Representative images of 3 independent experiments were shown
G-I) GLS and GDH activity as well as the NAD+/NADH ratio were measured (n=3). J-R) CAF
were plated on soft (1kPa) or stiff (8kPa) hydrogels. J-K) Glucose, and lactate fluxes were
calculated. Mean of 9 wells from 3 independent experiments. L) Intracellular levels of the
indicated metabolites were analysed by LC-MS (n=5). Data normalized to 1kPa. M)
Extracellular amino acid flux analyses of CAF plated on 1kPa or 8kPa hydrogel. Mean
expression (n=3) at t0 was assigned a fold change of 1, to which relevant samples were
compared N) RT-qPCR expression levels analysis of the indicated genes related to the
glutamine/glutamate metabolism (n=3). O) Westernblot analysis and quantification of GLS1
and LDHA protein levels. Representative images of 3 independent experiments were shown
P-R) GLS and GDH activity as well as the NAD+/NADH ratio were measured (n=3). S-U)
Quantification showing change in per cent of proliferative (Ki67+ cells) SCC12 (S), MDA-
MB468 (T) and A549 (U) plated on stiff (8kPa) substrate 24h after treatment with the
indicated conditioned media (CM; n=3). V-X) Quantification of contractile forces generated by
CAF from HNSCC (V), breast (W) or lung (X) tumors plated on 8kPa hydrogel following
treatment with indicated CM. Mean of n=6 wells from 3 independent experiments. In all
panels, mean expression in control groups (Soft) was assigned a fold change of 1, to which
relevant samples were compared. Data are expressed as the mean ± SD (*P < 0.05, **P <
0.01, ***P < 0.001) of at least 3 independent experiments performed in triplicate. Paired
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samples were compared by 2-tailed Student’s t test, while 1-way ANOVA and post-hoc
Tukey’s tests were used for group comparisons.
![Page 6: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/6.jpg)
Ala
nine
(P
eak
area
)
Stiff (8kPa)
01E+82E+83E+84E+8
Soft (1kPa)
01E+82E+83E+84E+8
CAF-CMSCC-CM
Double CM(SCC-CM on CAF)
01E+82E+83E+84E+8
01E+82E+83E+84E+8
Arg
inin
e (P
eak
area
)
0
1E+9
2E+9
1E+7
2E+7
3E+7
0
Asp
arag
ine
(Pea
k ar
ea)
1E+7
2E+7
3E+7
0
0
1E+9
2E+9
Asp
arta
te(P
eak
area
)
1E+7
2E+7
0
1E+7
2E+7
0
Cys
tine
(Pea
k ar
ea)
0
1E+8
2E+8
3E+8
Glu
tam
ate
(Pea
k ar
ea)
0
1E+8
2E+8
3E+8
0
1E+8
2E+8
3E+8
0
1E+8
2E+8
3E+8
0
2E+9
4E+9
6E+9
0
2E+9
4E+9
6E+9
Glu
tam
ine
(Pea
k ar
ea)
Gly
cine
(ion
curr
ent)
01E+72E+73E+74E+7
01E+72E+73E+74E+7
His
tidin
e(io
n cu
rren
t)
02E+84E+86E+88E+8
0
2E+8
4E+8
6E+8
Isol
euci
ne(io
n cu
rren
t)
0
2E+9
4E+9
6E+9
Leuc
ine
(ion
curr
ent)
02E+94E+96E+98E+9
02E+84E+86E+88E+8
10E+8
0
4E+8
8E+8
12E+8
Lysi
ne(io
n cu
rren
t)
1E+9
2E+9
0
Met
hion
ine
(ion
curr
ent)
1E+9
2E+9
0
3E+9
0
5E+8
10E+8
Phen
ylal
anin
e(io
n cu
rren
t)
0
5E+8
10E+8
Prol
ine
(ion
curr
ent)
0
2E+9
4E+9
6E+9
0
2E+9
4E+9
6E+9
02E+94E+96E+9
01E+72E+73E+74E+75E+7
Serin
e(io
n cu
rren
t)
01E+72E+73E+74E+75E+7
01E+82E+83E+84E+8
Thre
onin
e(io
n cu
rren
t)
01E+82E+83E+84E+8
02E+84E+86E+88E+8
10E+8
Tryp
toph
an(io
n cu
rren
t)
0
5E+8
10E+8
15E+8
3E+8
6E+8
9E+8
0
Tyro
sine
(ion
curr
ent)
3E+8
6E+8
9E+8
0
01E+92E+93E+94E+9
Valin
e(io
n cu
rren
t)
01E+92E+93E+94E+9
02E+94E+96E+98E+9
*** ***
***
*****
Ala
nine
(io
n cu
rren
t )
***
****
**
**
*
**
** ***
*
***
** **
*****
*
*
**
**
**
*
*
0
1E+9
2E+9
0
1E+9
2E+9
Arg
inin
e (io
n cu
rren
t)
1E+7
2E+7
3E+7
0Asp
arag
ine
(ion
curr
ent)
1E+7
2E+7
3E+7
0
Asp
arta
te(io
n cu
rren
t)
1E+7
2E+7
0
1E+7
2E+7
0
Cys
tine
(ion
curr
ent)
0
1E+8
2E+8
3E+8
0
1E+8
2E+8
3E+8G
luta
mat
e(io
n cu
rren
t)
01E+82E+8
4E+83E+8
01E+82E+8
4E+83E+8
0
2E+9
4E+9
6E+9
Glu
tam
ine
(ion
curr
ent)
0
2E+9
4E+9
6E+9
Gly
cine
(ion
curr
ent)
01E+72E+73E+74E+7
01E+72E+73E+74E+7
His
tidin
e(io
n cu
rren
t)
0
2E+8
4E+8
6E+8
0
2E+8
4E+8
6E+8
CAF-CMSCC-CM Double CM
(CAF-CM on SCC)
02E+94E+96E+98E+98E+9
Isol
euci
ne(io
n cu
rren
t)
0
2E+9
4E+9
6E+9
Leuc
ine
(ion
curr
ent)
02E+94E+96E+98E+9
02E+94E+96E+98E+9
02E+84E+86E+88E+8
10E+8
Lysi
ne(io
n cu
rren
t)
02E+84E+86E+88E+8
10E+8
1E+9
2E+9
0
Met
hion
ine
(ion
curr
ent)
1E+9
2E+9
0
3E+9
0
5E+8
10E+8
0
5E+8
10E+8
Phen
ylal
anin
e(io
n cu
rren
t)Pr
olin
e(io
n cu
rren
t)
0
2E+9
4E+9
6E+9
0
2E+9
4E+9
6E+9
01E+72E+73E+74E+75E+7
01E+72E+73E+74E+75E+7
Serin
e(io
n cu
rren
t)
01E+82E+83E+84E+8
Thre
onin
e(io
n cu
rren
t)
01E+82E+83E+84E+8
0
5E+8
10E+8
15E+8
02E+84E+86E+88E+8
10E+8
Tryp
toph
an(io
n cu
rren
t)
3E+8
6E+8
9E+8
0
Tyro
sine
(ion
curr
ent)
3E+8
6E+8
9E+8
0
01E+92E+93E+94E+9
01E+92E+93E+94E+9
Valin
e(io
n cu
rren
t)
******
******
******
******
***
*
*
*
*
***
**
***
***
***
**
**
**
**
*
****
*
***** *
A B
Bertero et al., Supplemental figure 2
Stiff (8kPa)Soft (1kPa) Stiff (8kPa)Soft (1kPa) Stiff (8kPa)Soft (1kPa)
C D1kPa
Asp Glu
amin
o ac
id
conc
entr
atio
n (µ
M)
0
100
200
3008kPa
Asp Glu0
200
400
800
600
1000
1200
amin
o ac
id
conc
entr
atio
n (µ
M)
***
*** **
***
***
**
***
*
******t0
CAF-CMSCC-CM
t0CAF-CMSCC-CM
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Figure S2 related to figure 1: Amino acid flux within the tumor niche is reprogrammed
by matrix stiffening. A-B) LC-MS/MS analysis of amino acids in SCC12 conditioned media,
CAF conditioned media, double conditioned media (SCC12-conditioned medium added to
CAF, A; CAF-conditioned media added to SCC12, B). The aspartate and glutamate data
here are also presented in Fig. 1J-K. Error bars represent the s.d. of n = 3 independent
experiments. C-D) Amino acid concentration is displayed in standard DMEM containing 10%
FBS at the starting point (T0) and after conditioning by CAF or SCC12 cells plated on 1kPa
(C) or 8kPa (D). Error bars represent the s.d. of n = 3 independent experiments. 1-way
ANOVA and post-hoc Tukey’s tests were used for group comparisons (*P < 0.05, **P < 0.01,
***P < 0.001).
![Page 8: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/8.jpg)
pGFP
pGLS1_1
pGLS1_2
GLS1
Tubulin
SCC12
0
0.5
1
1.5
* *
Nor
mal
ized
intr
acel
lula
r as
part
ate
leve
l (A
.U)
siNCsiGLS1_1siGLS1_2
DA
PI/P
CN
AD
API
/PC
NA
siNC siGLS1 siGLS1+Asp
Vehicle CB839 CB839+Asp
1
1.5
0.5
0
***N
orm
aliz
ed in
trac
ellu
lar
aspa
rtat
e le
vel (
A.U
)
VehicleCB839
Vehicle CB839 CB839+GluCAF
0
0.5
1
1.5
2
Forc
e m
agni
tude
/are
a (k
Pa)
CAF
***
******
Veh.
CB839
CB839+
Glu
siNC siGLS1_1 siGLS1_1+Glu
CAF
0 1 3kPa
2 0 1 3kPa
2
0
5
10
15
20
0
5
10
20
15
CAF pGFP 1kPa CM
CAF pGLS1_1 1kPa CM
CAF pGLS1_2 1kPa CM
siNCsiGLS1_1siGLS1_2
+Asp
VehicleCB839CB839+Asp
0 1 2 3Days
0 1 2 3Days
CAF pGFP 1kPa C
M
CAF pGLS1_1 1
kPa C
M
CAF pGLS1_2 1
kPa C
M0
10
20
30
% o
f Ki6
7+ c
ells
*** ***
CAF pGFP
Asp
arta
te ra
te
(fmol
/h/c
ell)
1kPa
CAF pGLS1_1CAF pGLS1_2
** **
DA
PI/K
i67
****
* ***
******
**
***
0
1
2
3
4
1kPaSCC12 pGFPSCC12 pGLS1_1SCC12 pGLS1_2
*** **
0
4
8
Glu
tam
ate
rate
(fm
ol/h
/cel
l)
siNC
siGLS1_
1
siGLS1_
1
+Glu
0
0.5
1
1.5
2
Forc
e m
agni
tude
/are
a (k
Pa)
CAF
siGLS1_
2
siGLS1_
2
+Glu
*** ***
** ***
VehicleAspartate (5mM)
01020304050
% o
f PC
NA
+ ce
lls
siNC
siGLS1_
1
siGLS1_
2
*** ***
Vehicle CB8390
1020304050
% o
f PC
NA
+ ce
ll
***
VehicleAspartate (5mM)
*** ***
***
0 1 3kPa
2
SCC12 pGFP 1kPa CMSCC12 pGLS1_1
1kPa CMSCC12 pGLS1_2
1kPa CM
4
0
0.5
1
1.5
2
Forc
e m
agni
tude
/are
a (k
Pa)
*** ***
SCC12 pGFP
1kPa C
M
CAF pGLS1_1
1kPa C
M
CAF pGLS1_2
1kPa C
M
5
pGFP
pGLS1_1
pGLS1_2
pGFP
GLS1
Tubulin
CAF
A B C
D
E
F
G H I
J K
L M N
Bertero et al., Supplemental figure 3
Fold
cha
nge
in c
ell n
umbe
rFo
ld c
hang
e in
cel
l num
ber
12
![Page 9: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/9.jpg)
Figure S3 related to figure 2: Tumor niche metabolic rewiring is dependent on
glutamine metabolism and sustains CAF and SCC pro-tumoral activities. A-F) In
SCC12 cultivated on stiff matrix, siRNA knockdown of GLS1 or pharmacological inhibition of
GLS1 decreased the intracellular level of Aspartate (A-B) and decreased cell proliferation as
measured by cell counting (C-D) and quantification of percentage of PCNA+ cells (E-F).
Upon GLS1 inhibition, aspartate supplementation rescued SCC12 proliferation.
Representative pictures showing change in per cent of proliferative (PCNA+ cells) SCC12
cells. Error bars represent the S.D. of 3 independent experiments. G) Immunoblot analysis
confirmed the overexpression of GLS1 in SCC12. H) Glutamate rate of SCC12 cells
overexpressing GFP (control) or GLS1 and plated on soft matrix. Error bars represent the
S.D. of 3 technical replicates from independently prepared samples from individual wells. I)
Representative heat map and quantification showing contractile forces generate by CAF
plated on 8kPa hydrogel following treatment with indicated SCC12 conditioned medium
(CM). Mean of n=6 wells from 3 independent experiments. J-K) Representative heat map
and quantification showing contractile forces generated by CAF plated on 8kPa hydrogel
following siRNA knockdown (J) or pharmacological inhibition (K) of GLS1. Upon GLS1
inhibition aspartate supplementation rescued SCC12 proliferation. Mean of n=6 wells from 3
independent experiments. L) Immunoblot analysis confirmed the overexpression of GLS1 in
CAF. M) Aspartate rate of CAF cells overexpressing GFP (control) or GLS1 and plated on
soft matrix. Error bars represent the S.D. of 3 technical replicates from independently
prepared samples from individual wells. N) Representative pictures and quantification
showing change in per cent of proliferative (Ki67+ cells) SCC12 plated on stiff (8kPa)
substrate 24h after treatment with the indicated CAF conditioned media (CM; n=3). In all
panels, paired samples were compared by 2-tailed Student’s t test, while 1-way ANOVA and
post-hoc Tukey’s tests were used for group comparisons (*P < 0.05, **P < 0.01, ***P <
0.001)
![Page 10: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/10.jpg)
ASLC1A3
Tubulin
siNC
siSLC1A
3_1
siSLC1A
3_2
siSLC1A
3_1
+siG
LS1_1
siSLC1A
3_2
+siG
LS1_2
SCC12B SCC12
siNC
GLS1
SLC1A3
Tubulin
E
SLC1A3
Tubulin
siNC
siSLC1A
3_1
siSLC1A
3_2
siSLC1A
3_1
+siG
LS1_1
siSLC1A
3_2
+siG
LS1_2
CAF
siNC
GLS1
SLC1A3
Tubulin
FCAF
M
Veh.
CAF
0
20
40
60
80
% o
f con
trac
tion
**
*
******
**
**
**
CB839 TFB-TBOA CB838TFB-TBOA
Veh.Glu (5mM)
Veh.Glu (5mM)* *
* *
****** ******
0
20
40
60
siNC
% o
f con
trac
tion
siSLC1A
3_1
siSLC1A
3_2
siGLS1_
1+
siSLC1A
3_1
siGLS1_
2+
siSLC1A
3_2
CAF
0
-2
-4* *
Asp
arta
te ra
te
(fmol
/h/c
ell)
siNCsiSLC1A3_1siSLC1A3_2
CAF CM on SCC12:
0
-2
-4
-6
-8
siNCsiSLC1A3_1siSLC1A3_2
SCC12 CM on CAF:
Glu
tam
ate
rate
(fm
ol/h
/cel
l)
Vehicle CB839 CB839+TFB-TBOA
Asp
(5m
M)
Glu
(5m
M)
Vehi
cle
TF-TBOA ON
1
1.5
2
2.5
Veh. CB839 TF-TBOA CB839+TFB-TBOA
*
***
***
*** ***
***
Inva
sion
inde
x
- + - + - +- +Asp+Glu:
***
CAF: siNCsiSLC1A3_1siSLC1A3_2
Nor
mal
ized
intr
acel
lula
r g
luta
mat
e le
vel (
A.U
.)
CAF CM SCC12 CM
SCC12: siNCsiSLC1A3_1siSLC1A3_2
SCC12 CM CAF CMNor
mal
ized
intr
acel
lula
r a
spar
tate
leve
l (A
.U.)
0
1
2
3
0
1
2
3***
*
****
****
***** **
pGFP
pGLS1
pGLS1+pSLC1A
3
Tubulin
GLS1
SLC1A3
pSLC1A3
pGFP
0
4
8
12
16SCC12
0 1 2 3Days
SCC12
Fold
cha
nge
in c
ell n
umbe
r siNCsiSLC1A3siSLC1A3+AspsiGLS1
+siSLC1A3siGLS1
+siSLC1A3+Asp
*****
**
***
***
0
4
8
12
16
Fold
cha
nge
in c
ell n
umbe
r
SCC12
0 1 2 3Days
VehicleTFB-TBOATFB-TBOA+AspCB839+TFB-TBOACB839+
TFB-TBOA+Asp**
***
***
C D
G H
I J K
L
Bertero et al., Supplemental figure 4
-6
![Page 11: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/11.jpg)
Figure S4 related to figure 3: Amino-acids crosstalk through SLC1A3 enables CAF and
SCC pro-tumoral activities. A-B) Immunoblot analysis confirmed the knockdown of
SLC1A3 (A) and both SLC1A3 and GLS1 (B) by 2 independent siRNA sequences in SCC12.
C-D) SCC12 cells were transfected with the indicated siRNA and treated with indicated CM.
Aspartate rate (C) and intracellular aspartate level (D) were determined by LC-MS/MS. Error
bars represent the S.D. of 3 independent wells from representative experiments (of 3
experiments). E-F) Immunoblot analysis confirmed the knockdown of SLC1A3 (E) and both
SLC1A3 and GLS1 (F) by 2 independent siRNA sequences in CAF. G-H) CAF were
transfected with the indicated siRNA and treated with indicated CM. Aspartate rate (G) and
intracellular aspartate level (H) were determined by LC-MS/MS. Error bars represent the s.d.
of 3 independent wells from representative experiments (of 3 experiments). I-J) SCC12 cells
were cultivated on stiff matrix and transfected with the indicated siRNA or treated with the
indicated pharmacological inhibitors. Cell proliferation was monitored over a 72h –period.
Upon SLC1A3 inhibition, aspartate supplementation failed to rescue SCC12 proliferation.
Error bars represent the S.D. of 3 independent experiments performed in triplicate. K)
Immunoblot analysis confirmed the overexpresion of a control vector (GFP), or GLS1 or
SLC1A3 and both SLC1A3 and GLS1 in SCC12. L-M) CAF were transfected (L) with the
indicated siRNA or treated with the indicated pharmacological inhibitors (M) and tested for
their ability to remodel/contract the ECM. Upon SLC1A3 inhibition glutamate
supplementation failed to rescue CAF contractility. Error bars represent the S.D. of 3
independent experiments performed in triplicate. N-O) In three-dimensional co-culture assay
and in presence of aspartate and glutamate pharmacological inhibition of both GLS1 and
SLC1A3 was necessary to blunt cell invasion. In panels D,H, I and J mean expression in
control groups (siNC or Vehicle) was assigned a fold change of 1, to which relevant samples
were compared. Paired samples were compared by 2-tailed Student’s t test, while 1-way
ANOVA and post-hoc Tukey’s tests were used for group comparisons (*P < 0.05, **P < 0.01,
***P < 0.001).
![Page 12: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/12.jpg)
FAK
ROCK
YAPYAP
ECM
Integrin
Actin remodelling
Nucleus
Verteporfin
Y27632
PF573228
YAP-dependant genes
YAP-independant genes
GLS1
LDHA
Tubulin
Veh. PF57 Y27 VP
SCC12
SLC1A3
GLS1
LDHA
Tubulin
Veh. PF57 Y27 VP
SLC1A3
GLS1 LDHA SLC1A30
0.5
1
1.5
Den
sito
met
ry
(Gen
e/Tu
bulin
)
VehiclePF573228Y27632Verteporfin
** **** **
** **
**** **
GLS1 LDHA SLC1A3
0
-40
-80
-120
Glu
tam
ine
rate
(fm
ol/h
/cel
l)
VehiclePF573228Y27632Verteporfin
VehiclePF573228Y27632Verteporfin
**** **
* **
**
* * * ** *
0
0.5
1
1.5
Den
sito
met
ry
(Gen
e/Tu
bulin
)
0
5
10
15
Glu
tam
ate
rate
(fm
ol/h
/cel
l)
0
-2
-4
-6
Asp
arta
te ra
te (f
mol
/h/c
ell)
*** *
**** **
Glu
tam
ine
rate
(fm
ol/h
/cel
l)
0
-20
-40
-60
-80
0
5
10
15
Glu
tam
ate
rate
(fm
ol/h
/cel
l)
** ***
** **
0
-2
-4
-6
Asp
arta
te ra
te (f
mol
/h/c
ell)
**
siNCsiYAP/TAZ_1siYAP/TAZ_2
0-10-20-30-40-50
Glu
tam
ine
rate
(fm
ol/h
/cel
l)
**** **
Glu
tam
ate
rate
(fm
ol/h
/cel
l)
* * *
0
-4
-8
-12
Veh.PF57Y27VP
0
2
4
6
Asp
arta
te ra
te (f
mol
/h/c
ell)
* * *
0
-10
-20
-30
-40
Glu
tam
ine
rate
(fm
ol/h
/cel
l)
* *
0
-4
-8-10G
luta
mat
e ra
te (f
mol
/h/c
ell)
** **
0
2
45
Asp
arta
te ra
te (f
mol
/h/c
ell)
siNCsiYAP/TAZ_1siYAP/TAZ_2
* *
SCC12CAF siNC CM CAF siYAP/TAZ_1 CM
+Asp (5mM)
CAF siYAP/TAZ_2 CM CAF siYAP/TAZ_1 CM CAF siYAP/TAZ_2 CM
Ki6
7/F-
actin
/DA
PI
SCC12 siNC CM SCC12 siYAP/TAZ_1 CM
SCC12 siYAP/TAZ_2 CM
SCC12 siYAP/TAZ_2 CM
SCC12 siYAP/TAZ_1 CM
+Glu (5mM)CAF
3
0
1
2
kPa
% o
f Ki6
7+ c
ells
0
10
20
30
*** ***
******
CAF siNC CMCAF siYAP/TAZ_1 CMCAF siYAP/TAZ_2 CMCAF siYAP/TAZ_1 CM+AspCAF siYAP/TAZ_2 CM+Asp
SCC12 siNC CMSCC12 siYAP/TAZ_1 CMSCC12 siYAP/TAZ_2 CMSCC12 siYAP/TAZ_1 CM+GluSCC12 siYAP/TAZ_2 CM+Glu
0
0.5
1
1.5
2
Forc
e m
agni
tude
/are
a (k
Pa)
*** ***
******
Bertero et al., Supplemental figure 5
A
D
B
E
H
G
I
J
K
C
F
VehiclePF573228Y27632Verteporfin
Glu
cose
rate
(fm
ol/h
/cel
l)
0
-50
-100
-150
SCC12
SCC12
SCC12
CAF CAF
**
** *****
*** ***
0100200300
Lact
ate
rate
(fm
ol/h
/cel
l)
VehiclePF573228Y27632Verteporfin
0
-100
-40
Glu
cose
rate
(fm
ol/h
/cel
l)
0
100
200
300
Lact
ate
rate
(fm
ol/h
/cel
l)
1
-200
400500
-100
-20
-60-80
**** ** *** *** ***
-2
-63
1
![Page 13: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/13.jpg)
Figure S5 related to figure 4: YAP/TAZ-dependent mechanotransduction pathway
inhibition in tumor cells blunts metabolic reprogramming and pro-tumoral activities.
A) Schematic dependent of key molecular mediators and/or sensors of mechanotransduction
in cells. B-C) Glucose and lactate rate (B) as well as glutamine, glutamate and aspartate rate
(C) of SCC12 plated on stiff hydrogel and treated with the indicated inhibitors. Error bars
represent S.D. of n = 4 technical replicates from independently prepared samples from
individual wells. D) Immunoblot analysis and quantification of GLS1, LDHA and SLC1A3
protein level in SCC12 cells plated on stiff hydrogel and treated with the indicated inhibitors.
Error bar represent the S.D. of 3 independent experiments. E) Glutamine, glutamate and
aspartate rate of SCC12 plated on stiff hydrogel and transfected with the indicated siRNAs.
Error bars represent S.D. of n = 4 technical replicates from independently prepared samples
from individual wells. F-G) Glucose and lactate rate (F) as well as glutamine, glutamate and
aspartate rate (G) of CAF plated on stiff hydrogel and treated with the indicated inhibitors.
Error bars represent S.D. of n = 4 technical replicates from independently prepared samples
from individual wells. H) Immunoblot analysis and quantification of GLS1, LDHA and SLC1A3
protein level in CAF plated on stiff hydrogel and treated with the indicated inhibitors. Error bar
represent the S.D. of 3 independent experiments. I) Glutamine, glutamate and aspartate rate
of CAF plated on stiff hydrogel transfected with the indicated siRNAs. Error bars represent
S.D. of n = 4 technical replicates from independently prepared samples from individual wells.
J) Representative pictures and quantification showing change in per cent of proliferative
(Ki67+ cells) SCC12 plated on stiff (8kPa) substrate 24h after treatment with the indicated
conditioned media (CM; n=3). K) Representative heat map showing contractile forces
generate by CAF plated on 8kPa hydrogel following treatment with indicated CM. Mean of
n=6 wells from 3 independent experiments. In panels D and H, mean expression in control
groups (Vehicle) was assigned a fold change of 1, to which relevant samples were
compared. Paired samples were compared by 2-tailed Student’s t test, while 1-way ANOVA
and post-hoc Tukey’s tests were used for group comparisons (*P < 0.05, **P < 0.01, ***P <
0.001).
![Page 14: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/14.jpg)
A
Asp
arta
te (A
.U.)
67NR 410.4 4T1
B C
0
2
4
6GlutaminolysisGlycolysis
* **
*
67NR 410.4 4T1
aggressiveness
0
2
4
6
8
10
Gln
/Glu*
**
**
***
*
*La
ctat
e/Py
ruva
te
67NR 410.4 4T10
1
2
3
* *
*
**
*
1kPa8kPa
D
67NR 410.4 4T1
GLS1
0
1
2
3
4
Fodl
cha
nge
** ****
***
**
** LDHA
0
1
2
3
4
Fodl
cha
nge
***
67NR 410.4 4T1
E
** **
***
*
**
** SLC1A3F
HGLox activity (Mammary tumor)
012345
Fold
cha
nge
67NR Veh. BAPN VP4T1
***
***
******
67NR 410.4 4T10
2
4
6
8
Fodl
cha
nge
***
** *
*****
CTGF CYR61 GLS1 SLC1A30
0.5
1
1.5
Fold
cha
nge
VehicleBAPNVerteporfin
Bertero et al., Supplemental figure 6
**
* **
*****
* *
1kPa8kPa
1kPa8kPa
1kPa8kPa
1kPa8kPa 1kPa
8kPa
![Page 15: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/15.jpg)
Figure S6 related to figure 5: Manipulation of mechanotransduction affects metabolic
reprogramming and breast cancer progression in vivo.
A-C) In well-established Balb/c mammary tumor cell lines 67NR (non metastatic), 410.4
(locally invasive) and 4T1 (metastatic) targeted LC-MS experiments revealed that matrix
stiffness increased lactate/pyruvate ratio and aspartate production while
Glutamine/glutamate ratio was decreased. Error bar represent the s.d. of 4 independent
experiments. D-F) In these cells, metabolic reprogramming correlated with increased GLS1
(D), LDHA (E) and SLC1A3 (F) gene expression as assessed by RT-qPCR. Error bars
represent the S.D. of 3 independent experiments. G-H) Following tumor cell implantation,
mice were treated daily with BAPN (n = 14) ; with daily i.p. injections of verteporfin (n = 26) or
vehicle (B; n = 26). RT-qPCR analysis (G) confirmed a decrease of known YAP/TAZ target
genes CTGF and CYR61 as well as GLS1 and SLC1A3 upon BAPN and Verteporfin
treatments. Both BAPN and verteporfin decreased Lox activity (H) in mice mammary tumors.
Data were normalized to the 67NR group; each dot represents a mouse. In all panels, mean
expression in control groups (Soft or Vehicle) was assigned a fold change of 1, to which
relevant samples were compared. In A-F panels, data are expressed as the mean ± SD,
while in G-H panels, data are expressed as the mean ± SEM (*P < 0.05, **P < 0.01, ***P <
0.001). Paired samples were compared by 2-tailed Student’s t test, while 1-way ANOVA and
post-hoc Tukey’s tests were used for group comparisons.
![Page 16: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/16.jpg)
D0
D-2 D2 D18
D18D-2 D2
D0
+ Dox - Dox
+ Dox- Dox
I.V. (4T1 2.10 cells)5
I.V. (4T1 2.10 cells)5
0
Num
ber o
f nod
ules
shNC
shGLS1
shSLC1A
3
shGLS1
+SLC1A
3
0
10
20
30
40
Num
ber o
f nod
ules
10
20
30
shNC
shGLS1
shSLC1A
3
shGLS1
+SLC1A
3
E F G
*** ***
***
*
***
***
***
shNC
shGLS1
shSLC1A
3
shGLS1
+SLC1A
3
Metastatic index
*** *
***
***
Lung
met
asta
sis
area
(%) ***
0
5
10
15
20
25
Gls1
Slc1a3
Tubulin
4T1
CAF
Gls1
Slc1a3
Tubulin
shNC
shGLS1+
shSLC1A3
shNC
shGLS1+
shSLC1A3
-Dox +Dox
shNC
shGLS1+
shSLC1A3
shNC
shGLS1+
shSLC1A3
-Dox +Dox
A
C
Den
sito
met
ry
(Gen
e/tu
bulin
)
shNC
shGLS1+
shSLC1A
3sh
NC
shGLS1+
shSLC1A
3
-Dox +Dox
******
0
0.5
1
1.5
Gls1Slc1a3
Gls1Slc1a3
shNC
shGLS1+
shSLC1A
3sh
NC
shGLS1+
shSLC1A
3
-Dox +Dox
Den
sito
met
ry
(Gen
e/tu
bulin
)
0
0.5
1
1.5
*****
shNC
shGLS1
shSLC1A
3
shGLS1
+SLC1A
3
Lung
met
asta
sis
area
(%)
0
5
10
15
20
25Metastatic index
*** ***
****
0
10
20
30
40
Lung
met
asta
sis
area
(%) Metastatic index
Veh.
CB839
TFB-TBOA
CB839+
TFB-TBOA
*** ***
***
******
67NR+CAF_shNC 67NR+CAF_shGLS1+shSLC1A3α-SMA/GLS1/SLC1A3/DAPI
α-SMA/GLS1/SLC1A3/DAPI
4T1_shNC 4T1_shGLS1+shSLC1A3
B
D
H I J
K
Bertero et al., Supplemental figure 7
α-SMA+ cells
GLS1
SLC1A30
1.5
0.5
1
Nor
mal
ized
inte
nsity
shNCshGLS1+
shSLC1A3
** **
GLS1
SLC1A30
1.5
0.5
1
Nor
mal
ized
inte
nsity
α-SMA- cells
shNCshGLS1+
shSLC1A3
*** **
0.01
Num
ber o
f nod
ules
/Pr
imar
y tu
mor
vol
ume
0.02
0.03
0.04
0
**
**
Veh.
CB839
TFB-TBOA
CB839+
TFB-TBOA
L M
**Met
asta
tic in
dex/
Prim
ary
tum
or v
olum
e
Veh.
CB839
TFB-TBOA
CB839+
TFB-TBOA
0
0.05
0.1
0.15
![Page 17: Tumor-Stroma Mechanics Coordinate Amino Acid Availability ...glutamine/glutamate metabolism (n=3). F) Westernblot analysis and quantification of GLS1 and LDHA protein levels. Representative](https://reader033.vdocuments.us/reader033/viewer/2022050302/5f6b853ac5a2997a3641c9d7/html5/thumbnails/17.jpg)
Figure S7 related to figure 6: Inhibition of metabolic reprogramming by inducible
shRNA in either CAF or cancer cell. A) Following Doxycycline induction (+Dox),
immunoblot analysis and quantification confirmed the knockdown of GLS1 and SLC1A3 by
shRNA in CAF. Error bars represent the S.D. of 3 independent experiments. B)
Representative pictures and quantification showing GLS1 and SLC1A3 staining in α-SMA
positive cells in mice injected with the indicated cells and treated with Doxycycline. Error bars
represent the S.E.M. of 5 mice. C) Following Doxycycline induction (+Dox), immunoblot
analysis and quantification confirmed the knockdown of GLS1 and SLC1A3 by shRNA in 4T1
cells. Error bars represent the S.D. of 3 independent experiments. D) Representative
pictures and quantification showing GLS1 and SLC1A3 staining in α-SMA negative cells in
mice injected with the indicated cells and treated with Doxycycline. Error bars represent the
S.E.M. of 5 mice. E-G) Mice (n=10 per group) were injected with the indicated 4T1 cells pre-
treated (2 days) with Doxycycline in order to induce the expression of the indicated shRNA.
Following injections, mice were treated with doxycycline during 2 days. Sixteen days later,
mice were sacrificed and lungs were harvested for metastatic nodule assessment (F) and
metastatic index calculation (G). H-J) Mice (n=10 per group) were injected with the indicated
4T1 cells. Two days after injection, mice were doxycycline-treated to induce the expression
of the indicated shRNA. Sixteen days later, mice were sacrificed and lungs were harvested
for metastatic nodule assessment (F) and metastatic index calculation (G). K-M) In mice
injected with 4T1 cells and treated with the indicated pharmacological inhibitors (see figure 6I
for the detailed procedure), the number of nodules normalized to the primary tumor volume
(K), the metastatic index (L), and the metastatic index normalized to the primary tumor
volume (M) were calculated. Paired samples were compared by 2-tailed Student’s t test,
while 1-way ANOVA and post-hoc Tukey’s tests were used for group comparisons. (*P <
0.05, **P < 0.01, ***P < 0.001)