UAB Barcelona · 927

κ

A B

2b 1

2a 3

4

4S

A B

A B

21 Gy   

95 days 4.5 months 6 months

Busulfan Melphalan

A B

-

-

-

-

-

α

-

-

-

®

®

1 3 5 7 9 11 13 15 17 190.0

4.0×1007

8.0×1007

1.2×1008

1.6×1008

0

156331256250125002500050000100000

200000_FLI_Fluc/mCherry only

780

13 min

200000

Time after D-luciferin Addition (min)

BLI

Inte

nsity

(ph/

s)

Cell number

1 3 5 7 9 11 13 15 17 190

1×1007

2×1007

3×1007

4×1007

5×1007

6×1007

0780156331256250125002500050000100000

13 min

200000

Time after D-luciferin Addition (min)

BLI

Inte

nsity

(ph/

s)

Cell number

A B

®

-

-

∼

1 3 4 4 MNA100

1000*

**

mR

NA

expr

essi

on (a

.u.)

PEA15

1 3 4 4 MNA0.1

1

10

100

***

FLIP

mR

NA

expr

essi

on (a

.u.)

1 3 4 4 MNA1

10

100

1000

******

*

LFG

mR

NA

expr

essi

on (a

.u.)

0 1000 2000 3000 4000 5000 60000

25

50

75

100

High (Above Median)Low (Below median)

n=125

n=126

p= 1.76·10-9

LFG

Days

Perc

ent s

urvi

val

0 1000 2000 3000 4000 5000 60000

25

50

75

100

High (Above Median)Low (Below median)

n=125

n=126

p=0.024

BAR

Days

Perc

ent s

urvi

val

0 1000 2000 3000 4000 5000 60000

25

50

75

100

High (Above Median)Low (Below median)

n=125

n=126

p= 5.6·10-4

IAP1

Days

Perc

ent s

urvi

val

1 3 4 4 MNA0.01

0.1

1

10

100

**

Rel

ativ

e LF

G e

xpre

ssio

n

LFG

A B

LFG

NB

MYCN non-amplified MYCN amplified

A4 A8 B8 C6 D3 D4 D6 D12

35

kDa

0 1000 2000 3000 4000 5000 60000

10

20

30

40

50

60

70

80

90

100

High (Above median)Low (Below median)

n=124

n=94

p<0.0001

Follow up in days

Per

cent

sur

viva

l

A Oberthuer GSE3446

High (Above median) Low (Below median)

n=51

n=51

p= 4.7 e-4 p= 0.232

n=36

n=36

High (Above median) Low (Below median)

GSE16476

B C

LFG

Favorable Unfavorable0.01

0.1

1

10

100

**

Rel

ativ

e m

RN

A ex

pres

sion

A8

B8

D4

D12

MY

CN

non

-am

plifi

ed, f

avor

able

his

tolo

gy

MY

CN

am

plifi

ed, u

nfav

orab

le h

isto

logy

Magnification

α

SK-N-BE(2)

RA (days) 0 1 3 5

LFG

α-tubulin

1.00 1.91 2.04 4.07

35

50

kDa

SH-SY5Y

RA (days) 0 1 3 5

LFG

α-tubulin

1.00 2.36 13.08 9.03

35

50

kDa

SH-SY5Y

0 1 2 3 4 5 60

1

2

3

4

0

50

100

150

200LFGTRKB

Retinoic Acid (days)

Rel

ativ

e LF

G e

xpre

ssio

n

Relative TR

KB expression

SK-N-BE(2)

0 1 2 3 4 5 60

1

2

3

0

20

40

60

80

100LFGTRKB

Retinoic Acid (days)

Rel

ativ

e LF

G e

xpre

ssio

n

Relative TR

KB expression

A

B

Adhere

nt

Sphere

Adhere

nt

Sphere

Adhere

nt

Sphere

0.0

0.5

1.0

1.5SK-N-BE(2)IMR-5IMR-32

***

LFG

Rel

ativ

e m

RN

A ex

pres

sion

Adherent Sphere

IMR-5 A B

α

SH-SY5Y +

Scr1 LFG1 Scr2 LFG2

LFG

α-tubulin

1 1 0.37 0.40

CHLA-90 + shRNA

Scr1 LFG1 Scr2 LFG2

LFG

α-tubulin

1 1 0.35 0.83

35

50

35

50

kDa kDa

A B

SH-SY5Y

0 1 2 3 4 5 6 70

1

2

3

4 shScrshLFGshScr 2shLFG 2

** ##,

Days

Cel

l Pro

lifer

atio

n (A

bs 5

90nm

)CHLA-90

0 1 2 3 4 5 6 70

1

2

3

4 shScrshLFGshScr 2shLFG 2

** #,

Days

Cel

l Pro

lifer

atio

n (A

bs 5

90nm

)

A B

CHLA-90

0 30 60 900.0

0.5

1.0

1.5

2.0 shScrshLFG

**

Time (min)

Cel

l Adh

esio

n (A

bs 5

90nm

)

SH-SY5Y

0 30 60 90 1200.0

0.1

0.2

0.3

0.4

0.5 shScrshLFG

*

Time (min)

Cel

l Adh

esio

n (A

bs 5

90nm

)

A B

shScr shLFG

LFG

α-tubulin

SK-N-BE(2)

1 0.31

35

50

kDa shScr shLFG

LFG

α-tubulin

IMR-32

1 0.63

35

50

kDa

A

B

C SK-N-BE(2)

0 20 40 60 80 1000.0

0.2

0.4

0.6 shScrshLFG

Time (min)

Cel

l Adh

esio

n (A

bs 5

90nm

)

IMR-32

0 20 40 60 80 1000.0

0.5

1.0

1.5

2.0 shScrshLFG

Time (min)

Cel

l Adh

esio

n (A

bs 5

90nm

)SK-N-BE(2)

0 1 2 3 40.0

0.5

1.0

1.5

2.0 shScrshLFG

Time (days)

Cel

l Pro

lifer

atio

n (A

bs 5

90nm

)

IMR-32

0 1 2 3 40

1

2

3

4

5 shLFGshScr

Time (days)

Cel

l Pro

lifer

atio

n (A

bs 5

90nm

)

shScr shLFG A B

shScr shLFG0

5

10

15 **

% o

f sph

ere-

form

ing

cells

ABCG2 CD133 KLF4 NANOG NESTIN NOTCH3 OCT4 SOX2

1

2 shScrshLFG

****Fo

ld c

hang

e vs

shS

cr

SNAI1 TCF3 ZEB1 ERBB3 KRT19 SNAI2 TWIST1

1

2 shScrshLFG ***** ***

Fold

cha

nge

vs s

hScr

TWIST1

High LFG Low LFG0

250

500

750p<0.0001

mR

NA

expr

essi

on (a

.u.)

FN1

High LFG Low LFG0

500

1000

1500

2000p=0.0041

mR

NA

expr

essi

on (a

.u.)

SNAI2

High LFG Low LFG0

25

50

75p=0.0060

mR

NA

expr

essi

on (a

.u.)

SPARC

High LFG Low LFG0

500

1000

1500

2000

2500p=0.0370

mR

NA

expr

essi

on (a

.u.)

TCF3

High LFG Low LFG0

50

100

150

200p<0.0001

mR

NA

expr

essi

on (a

.u.)

CALD1

High LFG Low LFG0

200

400

600p=0.0248

mR

NA

expr

essi

on (a

.u.)

ERBB3

High LFG Low LFG0

10

20

30

40p=0.0004

mR

NA

expr

essi

on (a

.u.)

KRT19

High LFG Low LFG0

200

400

600

800

1000

1200p=0.0163

mR

NA

expr

essi

on (a

.u.)

A

B

shScr shLFG0

2

4

6

*

Mig

ratio

n (fo

ld-in

duct

ion)

shScr shLFG0

50

100

150

Cel

l num

ber p

er fi

eld

A B

0 50000 100000 150000 2000000

2×1007

4×1007

6×1007

8×1007SH-SY5Y-shScrSH-SY5Y-shLFG

r2=0.99, p<0.0001

r2=0.99, p<0.0001

Number of Cells/Well

BLI I

nten

sity

(ph/

s)

8 11 15 170

20

40

60

80

100 shScrshLFG

Days post-injection

Tum

or in

cide

nce

(%)

0 5 10 15 20 250

500

1000

1500shScrshLFG

Days post-injection

Tum

or v

olum

e (m

m3 )

shScr shLFG0.0

0.5

1.0

1.5

Tum

or w

eigh

t (g)

A B

B

1.5

1.0

0.5

x106

Radiance (p/sec/cm2/sr)

shSc

r sh

LFG

A

1.5

1.0

0.5

x106

Radiance (p/sec/cm2/sr)

2.0

shSc

r sh

LFG

shScr shLFG105

106

107

108

109

Tota

l BLI

(ph/

sec)

shScr shLFG104

105

106

107

108

Tota

l BLI

( ph/

sec)

B

shLF

G

shSc

r

A

shLF

G

shSc

r

shScr shLFG0

5

10

15 *

Num

ber o

f Met

asta

ses

shScr shLFG0

20

40

60

Num

ber o

f Met

asta

ses

PC1 60.5%

PC2

29.6

%

0

010

5 10- 5- 10

515

-10

-15

-5

shLFG1

CTL1

CTL2

CTL3

shLFG2

shLFG3

PC1 60.5%

PC2

29.6

%

0

010

5 10- 5- 10

515

-10

-15

-5

shLFG1

CTL1

CTL2

CTL3

shLFG2

shLFG3

Colorec

tal ca

ncer

Cell cy

cle

Pancre

atic c

ance

r

Adhere

ns ju

nctio

n

Cell ad

hesio

n mole

cules

Osteoc

last d

ifferen

tiatio

n

Focal

adhe

sion

Pathway

s in c

ance

r

Metabo

lic pa

thway

s0

1

2

3

4

Odd

s R

atio

FGF14 STK36 BCL2 RAP1A TJP1 ITGA9 LFG-3

-2

-1

1

2

3 ArrayqPCR

Fold

cha

nge

vs s

hScr

Focal adhesion Cell adhesion

Adherent junctions

Cell cyclePathways in Cancer

Tet21N

Untreated Doxycycline

α

α

Tet21N + Dox (days)

0 1 3 5

LFG

α-tubulin

7

MYCN

1.00 0.55 1.76 3.66 4.53

Tet21N

0 1 2 3 4 5 6 70

10

20

30

0.0

0.5

1.0

1.5LFGMYCN

Dox (days)

Rel

ativ

e LF

G e

xpre

ssio

n

Relative M

YCN

expression

A B

35

50

64

kDa

IMR-32 shMYCN

0 1 2 30.0

0.5

1.0

1.5

2.0

2.5

0.0

0.5

1.0

1.5LFGMYCN

Dox (days)

Rel

ativ

e LF

G e

xpre

ssio

nR

elative MYC

N expression

1 2 30.0

0.5

1.0

1.5

2.0 VehicleDox

Days

LFG

mR

NA

expr

essi

on (a

.u.)

+ D

ox

- Dox

LFG

LAI-5S

0 1 2 30

5

10

15 5-azaDMSO

days

Rel

ativ

e LF

G e

xpre

ssio

n

SK-N-BE(2)

0 1 2 30

1

2

3

4

5 5-azaDMSO

days

Rel

ativ

e LF

G e

xpre

ssio

n

Tet21N

0 1 2 30

10

20

30 5-azaDMSO

days

Rel

ativ

e LF

G e

xpre

ssio

n

LAI-5S

0 1 20

2

4

6

8

10 NaBPBS

daysR

elat

ive

LFG

exp

ress

ion

SK-N-BE(2)

0 1 20

1

2

3 NaBPBS

days

Rel

ativ

e LF

G e

xpre

ssio

n

Tet21N

0 1 20

20

40

60

80

100 NaBPBS

days

Rel

ativ

e LF

G e

xpre

ssio

n

A

B

∼