Effects of declining sorafenib levels and dose on

acquired reversible resistance and toxicity in

hepatocellular carcinoma

Elizabeth A. Kuczynski, Christina R. Lee, Shan Man, Eric Chen,

Robert S. Kerbel

SUPPLEMENTARY FIGURES S1-5

C

Hep3B-hCG parental

Hep3B-hCG-R1

Hep3B-hCG-R2

D

0 10 20 30 40 50 60 70 800

100

200

300

400

500

600

700

800

900Vehicle controlSorafenib (30 mg/kg)Sorafenib treatment in-terval

day of therapy

urin

ary

hCG

/cre

atin

ine

(mIU

/mg)

A B

Parental R1 R20

50

100

150

200

250

300

mea

n #

cells

or

colo

nies

EGFSorafenib

P-ERK

ERK

Parental R1- + - + - + - +- - + + - - + +

E

0 20 40 60 80 100 120 1400

1000

2000

3000

4000

5000Vehicle controlSorafenib 30 mg/kg

day of therapy

Uri

nary

hC

G/c

reat

inin

e (m

IU/m

g)

0 10 20 300

1000

2000

3000

4000

5000Vehicle controlSorafenib 30 mg/kg

day of therapy

Uri

nary

hC

G/c

reat

inin

e (m

IU/m

g)

F G

Invasion

ProliferationTumor growth (hCG)

Clonogenic Survival

hCG: Orthotopic TransferhCG: Subcutaneous Transfer

-6.0 -5.8 -5.6 -5.4 -5.2 -5.00

25

50

75

100 Hep3B-hCG parental

Hep3B-hCG-R1

Hep3B-hCG-R2

Sorafenib concentration (M)

% c

ell v

iabi

lity

Supplementary Figure S1

IC50: 3.03µM (P), 2.92µM (R1), 2.36µM (R2)

Supplementary Figure S2

BhCG for Drug Level AssessmentA

Plasma Tumor

*

Sensit

ive

Early P

rogr

essio

n

Sensit

ive

Early P

rogr

essio

n0

2500

5000

7500

10000

sora

feni

b (n

g/m

l)

-10 0 10 20 30 40 50 600

500

1000

1500

2000

2500

3000

3500

4000Vehicle control

Sensitive (day 13)

Early progression

day of treatment

urin

ary

hCG

/cre

atin

ine

(mIU

/mg)

0 20 40 60 80 1000

1000

2000

3000

4000

5000

Vehicle to vehicleVehicle to sorafenibSorafenib to vehicle

day of treatment

urin

ary

hCG

/cre

atin

ine

(mIU

/mg)

hCG: 65 Day Sorafenib Pre-Conditioned Mice

A

B

Supplementary Figure S3

0 10 20 30 40 50 60 700

500

1000

1500

2000

2500

3000

Vehicle

Sorafenib (30 mg/kg)

Day treatment

urin

ary

hC

G/c

reat

inin

e (m

IU/m

g)

+2d implant, +5d treat

+2d implant, +5d treat

hCG: Indicator Group of SCID HCC mice

Sensitive Resistant0.0%

0.5%

1.0%

1.5%

2.0%

2.5%

3.0%Tissue Sorafenib N-oxide

Liver (tumor-free)Liver (HCC)Tumor (HCC)

% s

oraf

enib

N-o

xide

p=0.742

p=0.965

P=0.0659

C

Supplementary Figure S4

Weight Loss: HCC vs. Tumor-Free

F G

30 40 50 60 700

25

50

75

100

HCCTumor-free

day of treatment

% s

urvi

val p=0.266

Survival: HCC vs. Tumor-Free

D E

Control Sensitive Resistant Dose- escalated

0

1

2

3

mas

s li

ver

+ t

um

or

(g) ** **

Tumor + Liver Weight

30 40 50 60 700

25

50

75

100HCC(30 to 60mg/kg)

Tumor free(30 to 60mg/kg)

day of treatment

% s

urvi

val

p=0.600

Survival: Dose escalation

0 10 20 30 40 50 60 7070%

80%

90%

100%

HCC - 30 mg/kg sorafenib

Tumor-free - 30 mg/kg sorafenib

day of treatment

% w

eig

ht

loss

(30 mg/kg) (30 mg/kg)

p=0.165

p=0.285

-10 0 10 20 30 40 50 60 700

500

1000

1500

2000

Vehicle controlEarly progressor - 30 mg/kgEarly progressor - 30 to 60 mg/kgLate progressor - 30 mg/kg

day of treatmentur

inar

y hC

G/c

reat

inin

e (m

IU/m

g)

B hCG: Dose Escalation Early vs. Late Progressors

Tumor Liver Plasma0

20000

40000

60000

80000

100000Early ProgressorLate Progressor

Tot

al s

oraf

enib

(ng/

ml)

*p=0.0145

Sorafenib concentrations: day 14

Resistant Dose escalated0%

1%

2%

3%

4%

5%Tissue Sorafenib N-oxide

Liver (tumor-free)Liver (HCC)Tumor (HCC)

% s

oraf

enib

N-o

xide

C

p=0.202p=0.152

p=0.00554

A

A

C Liver Lysates - Tumor-Free

Control* Sensitive Resistant Escalated

Liver lysates - HCCSensitive Resistant EscalatedControl

P-STAT3

STAT3

P-STAT3

STAT3

Supplementary Figure S5

DControl Sensitive Resistant Escalated

CAIX

Actin

Control Sensitive Resistant Escalated

P-PDGFRβ

PDGFRβ

Tumor Lysates

Tumor Lysates

CoSen Res Esc

0

2

4

6

8

10

12 Tumor Mouse P-VEGFR2

P-T

yr /m

ouse

VE

GFR

2

B

P-ERK

ERK