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

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

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

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

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.

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

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).

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

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)

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

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).

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

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).

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

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.

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

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)