Supplementary Figure 1. Immunohistochemical analysis of MYC induced liver cancer.
a. Immunohistochemical analysis of MYC transgene expression in the absence of MYC
expression in the liver of a doxycycline treated transgenic mouse liver (MYC OFF) and
in the liver of an untreated transgenic mouse liver tumor (MYC ON). Data shown is
representative of 1 of 5 different experiments. b. Tumor regression in a primary
transgenic tumor examined in situ, upon MYC inactivation. Liver tumor where MYC is
overexpressed (upper left) and histology (upper right). Regressed liver tumor after 2
weeks of MYC inactivation. (lower left) and histology of regressed tumor (lower right)
showing regressing tumor cells (r) and differentiated hepatocytes (h). c. Tumors restored
after MYC reactivation have similar phenotype to the primary tumors. Histology of
transplanted tumor overexpressing MYC (left) regressed tumor (middle) and tumor where
MYC is reactivated (right).
Supplementary Figure 2: MYC inactivation uncovers pluripotent differentiative
properties in hepatocellular cancer. Expression of the hepatocellular stem cell marker
CK19 in a. normal liver, hepatocellular cancer b. before and c. after MYC inactivation.
Supplementary Figure 3: Tumors that are restored upon MYC reactivation remained
dependent on MYC expression. Bioluminescence imaging (BLI) of luciferase labeled
liver tumors shows that transplanted tumors where MYC expression was inactivated and
then reactivated remain conditional to MYC expression and regress upon MYC
inactivation. Square symbol: MYC ON - MYC OFF - MYC ON - MYC OFF. Open
circle: MYC ON - MYC OFF. Representative pictures for mouse where MYC was on for
4 weeks (MYC ON), MYC on and then turned off for 6 weeks (MYC OFF), MYC ONOFF and then turned back on for 3 weeks (MYC Reactivated), and finally MYC ONOFF-ON and then turned back off (MYC OFF).
Supplementary Figure 4. Bioluminescence imaging can be used as a sensitive and
accurate strategy to measure tumor growth and regression. a. Sensitivity of in vivo
bioluminescence imaging. Luciferase expressing liver tumor cells at a dose range of
1x103 to 1x106 were transplanted subcutaneously into SCID mice and imaged
immediately. Using BLI, luciferase labeled cells could be detected with a minimum
sensitivity of at least 1x103 cells. Representative data from 1 of 4 experiments is shown.
b. Bioluminescence imaging correlated with measured tumor growth. 1x106 luciferase
positive tumor cells were transplanted into SCID mice and tumor number was followed
by BLI and caliper measurement. BLI images of representative mice are shown in the
lower panel. Experiment was performed 2 times with cohorts of 5 mice. c.
Bioluminescence imaging could be used to measure tumor regression. Tumor regression
was measured in the transplanted liver tumor cells after MYC inactivation by BLI and
tumor size was measured using calipers. Representative data is shown from 1 of 2
experiments with 5 mice in each group. Lower panels show representative
bioluminescence images.
Supplementary Figure 5: Normal liver cells do not engraft in SCID hosts.
Cell
suspensions of normal liver and/or liver tumor cells were transplanted subcutaneously in
SCID mice and then imaged by bioluminescence imaging.
a. Normal hepatocytes that
expressed luciferase and were induced for MYC expression (+) were transplanted
subcutaneously into SCID mice at a dose of 4 x 107 cells. b. Normal hepatocytes that
expressed luciferase and induced for MYC expression (+), were transplanted
subcutaneously into SCID mice at a dose of 4 x 107 together with 10 x106 tumor cells
thate luciferase negative (-). c. 10 x106 tumor cells that were luciferase positive (+) were
injected into SCID mice. Mice were imaged at various time points as indicated in the
figure. Representative data from one of four mice analyzed are shown.
Supplementary Figure 6: Relapsed tumor after MYC inactivation was found to exhibit
increased expression of endogenous N- and L- Myc. Western blot analysis of MYC in a
tumor (left) and relapsed tumor (right) for transgenic C-MYC and endogenous mouse cMyc and endogenous mouse N-Myc and L-Myc.
Supplementary Figure 7: Model for consequence of oncogene inactivation in
hepatocellular cancer. MYC induced liver cancers retain pluripotent differentiative cell
properties, which are revealed upon MYC inactivation. Upon MYC inactivation tumor
cells differentiated into both hepatocytes and bile duct cells. Some of the cells may
remain as a dormant cancer cell and upon MYC reactivation emerge from their dormant
state and regain their tumorigenic properties.