Download Figures to be replicated

Study information
Kang, TW; Yevsa, T; Woller, N; Hoenicke, L; Wuestefeld, T; Dauch, D; Hohmeyer, A;
Gereke, M; Rudalska, R; Potapova, A; Iken, M; Vucur, M; Weiss, S; Heikenwalder,
M; Khan, S; Gil, J; Bruder, D; Manns, M; Schirmacher, P; Tacke, F; Ott, M; Luedde,
T; Longerich, T; Kubicka, S; Zender, L
Title: Senescence surveillance of pre-malignant hepatocytes limits liver cancer
development
Journal: Nature
DOI: http://dx.doi.org/10.1038/nature10599
Key experimental results
Impaired senescence surveillance promotes liver cancer development
Figures to be replicated
2I: Nras-G12V-positive cells decrease over time compared to Nras-G12V/D38Apositive cells, which remain the same. Shows senescent hepatocytes might be
cleared by liver- infiltrating immune cells.
 Protocol 1
3B: C.B.-17 mice showed an accented reduction of Nras-positive cells over time
compared to no significant reduction in the number of Nras-positive cells in C.B17SCID mice. Shows that a defective adaptive immunity abrogates immune
surveillance of pre-malignant senescent hepatocytes.
 Protocol 1
3C: C.B.-17 mice showed an accented reduction of p21-positive cells over time
compared to a significantly less reduction in the number of p21-positive cells in C.B17SCID mice. Shows that a defective adaptive immunity abrogates immune
surveillance of pre-malignant senescent hepatocytes. Shows that some Nras
expressing hepatocytes at the later time points have escaped the senescence
program.
 Protocol 1
3D: C.B.-17 mice showed an accented reduction of p-Erk positive cells over time
compared to no significant reduction in the number of p-Erk-positive cells in C.B17SCID mice. Shows that a defective adaptive immunity abrogates immune
surveillance of pre-malignant senescent hepatocytes
 Protocol 1
3E: C.B.-17 mice showed an accented reduction of p16-positive cells over time
compared to a significantly less reduction in the number of p16-positive cells in C.B17SCID mice. Shows that a defective adaptive immunity abrogates immune
surveillance of pre-malignant senescent hepatocytes. Shows that some Nras
expressing hepatocytes at the later time points have escaped the senescence
program.
 Protocol 1
3F: C.B.-17 mice showed no tumor growth, compared to severe intrahepatic tumor
growth observed in Nras-G12V-transduced C.B.-17 SCID as well as C.B.-17
SCID/beige mice. Shows an impaired immune surveillance of pre-malignant
senescent hepatocytes results in HCC development.
 Protocol 1
S8A: Significantly less p21 and p16 positive than Nras-positive hepatocytes were
detected at later time points in C.B-17 SCID mice. Shows that some Nras expressing
hepatocytes at these time points have escaped the senescence program. Another
presentation of data from of Fig. 3B-3E
 Protocol 1
S8B: A diverse staining pattern of Nras positive cells in both Nras-G12V and NrasG12V/D38A. Shows that clonal expansion can be excluded. Representative image of
data collected in Fig. 3B-3E
 Protocol 1
S10: After 7 months there is no tumor growth in Nras-G12V/D38A-transduced mice.
Control for Fig. 3F
 Protocol 1
4A: WT (H-2b), CD8-/- and CD8-depleted WT mice displayed normal or nearly
normal senescence surveillance (decrease in Nras-positive cells in Nras-G12V
compared to Nras-G12V/D38A), compared to a completely blunted senescence
surveillance (no change in Nras-positive cells in Nras-G12V compared to NrasG12V/D38A) in CD4-/- or CD4-depleted WT mice. Shows CD4+ T-cell-dependent
surveillance of pre-malignant senescent hepatocytes.
 Protocol 2
4B: Stable delivery of oncogenic Nras-G12V results in massive intrahepatic tumor
growth in CD4-/- mice compared to WT. Shows CD4+ T-cell-dependent surveillance
of pre-malignant senescent hepatocytes.
 Protocol 2
S15: Stable delivery of oncogenic Nras-G12V resulted in massive intrahepatic tumor
growth in CD4-/- mice compared to normal mice. Shows CD4+ T-cell-dependent
surveillance of pre-malignant senescent hepatocytes. Related to Fig. 4B
 Protocol 2
Protocols
Protocol 1: Impaired senescence surveillance on liver cancer development
Number of replicates: X
Replication calculation needs to consider expected failure rate of tail vein injections.
 What is the expected failure rate of these injections?
Power calculations:
Data from Figure 2I
NrasG12V
Day 6
16.9 +/- 2.2
Day 12
6 +/- 1.4
Day 30
1.8 +/- 1.4
Day 60
0
Data from Figure 3B: Note these were estimated.
CB17
STDEV
CB17STDEV
SCID
Day 6
15.32
4.00
15.74
1.66
Day 12
7.09
2.21
13.30
1.38
Day 30
1.19
0.83
12.10
1.79
Day 60
0.00
0.00
13.52
3.03
Data from Figure 3C: Note these were estimated.
CB17
STDEV
CB17STDEV
SCID
Day 6
15.42
2.08
14.86
3.47
Day 12
6.81
2.50
13.89
2.22
Day 30
0.83
1.53
7.64
1.94
Day 60
0.00
0.00
0.00
0.00
Data from Figure 3D: Note these were estimated.
CB17
STDEV
CB17STDEV
SCID
Day 6
15.35
2.11
16.06
2.96
Day 12
8.45
2.68
14.37
2.68
Day 30
2.39
0.70
11.27
2.68
Day 60
0.00
0.00
10.42
1.13
Data from Figure 3E: Note these were estimated.
CB17
STDEV
CB17STDEV
SCID
Day 6
14.38
1.92
15.07
2.60
Day 12
5.48
2.60
12.88
1.64
Day 30
0.00
0.00
9.73
2.88
Day 60
0.00
0.00
5.62
3.97
Data from Figure S8A: Note these were estimated.
Nras
STDEV
p21
STDEV
Day 6
15.80
1.68
14.72
3.36
Day 12
13.26
1.45
14.32
2.29
Day 30
12.17
1.91
7.66
1.91
Day 60
13.37
3.05
0.00
0.00
NrasG12V/D38A
17.2 +/- 4.4
16.1 +/- 2.3
13.4 +/- 2.6
11.7+/- 3.4
Scidbeige
16.17
14.27
12.80
12.97
STDEV
SCIDbeige
15.14
13.89
8.75
0.00
STDEV
SCIDbeige
15.77
14.93
11.27
8.45
STDEV
SCIDbeige
15.62
13.56
10.55
3.84
STDEV
p16
15.10
12.94
9.56
5.57
3.03
3.86
2.90
3.86
2.08
1.94
3.89
0.00
3.80
2.68
1.69
0.99
2.05
1.78
3.97
1.10
STDEV
2.67
1.60
3.05
3.82
Can you share the data values shown in Figures 3B, 3C, 3D, 3E, and SF8A so our
power calculations are accurate?
Controls:
 NrasG12V/D38A
 C.B-17 WT mice injected with pPGK-empty vector [additional]
 secondary-only control
 isotype control
Outline of conditions:
X mice for each condition processed on 4 time points: 6, 12, 30, 60 days postinjection:
 C.B-17 WT mice
o injected with pPGK-empty vector + pT/CaggsNrasG12V
o injected with pPGK-empty vector + pT/CaggsNrasG12V/D38A
 C.B-17 WT mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
 C.B-17 SCID mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
 C.B-17 SCID/beige mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
X mice for each condition processed at 7 months post-injection:
 C.B-17 WT mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
 C.B-17 SCID mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
 C.B-17 SCID/beige mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
Steps:
1) Prepare all constructs using the EndoFreeMaxi Kit (Qiagen, 12362) following
manufacturer’s instructions.
a. Can you share the plasmids?
b. Did the authors specifically follow the Qiagen kit protocols?
c. Was there any endotoxin testing done on the output?
o pPGK-empty vector
o pPGK-SB13 transposase vector
o pT/CaggsNrasG12V transposon vector
o pT/CaggsNrasG12V/D38A transposon vector
2) Sequence plasmids to confirm identity and run on gel to confirm vector integrity.
a. Can you share the sequencing primers?
3) Inject C.B-17 (H-2d) WT (Charles River, 251), C.B-17 SCID mice (Charles River,
236), C.B-17 SCID/beige mice (Charles River, 250) mice with
transposon/transposase:
a. Mix at a 5:1 molar ratio of transposon (pT/CaggsNrasG12V or
pT/CaggsNrasG12V/D38A) to transposase-encoding plasmid (pPGK-empty
vector or pPGK-SB13) for a total injection mass of 30 µg.
b. Weigh mice.
c. Suspend 30 µg of plasmids in saline solution (?) at a final volume of 10% of
the animal's body weight.
d. Deliver DNA solution by hydrodynamic tail vein injection in <10 sec.
i. Can the authors supply more precedural detail on their
hydrodynamic injections?
ii. What gender and age were the mice?
iii. Can you please provide information about housing conditions?
4) At days 6, 12, 30, and 60 post-injection of DNA, euthanize mice and harvest liver
tissue.
5) Fix tissue and embed in paraffin.
a. What are the protocol details?
6) Section tissue
a. What thickness?
7) Stain different sections with primary and secondary antibodies:
Note: All antibodies will be validated as part of the antibody validation project.
a. Perform isotype control (?)
b. Perform secondary Ab only control.
c. What is the staining protocol? Was antigen retrieval used, if so, how was that
done?
d. What detection system was used (HRP, ABC, etc)?
o mouse-anti-Nras, clone F155 (Santa Cruz, sc-31) 1:100 dilution
o mouse-anti-p21, clone SXM30 (?) 1:50 dilution
i.
Original paper says Cell Signaling, but they don’t have this
clone, BD does:
http://www.bdbiosciences.com/ptProduct.jsp?ccn=556431.
Is this the same antibody?
o anti-pERK, Thr202/Tyr204 (Cell Signaling, ?) 1:100 dilution
i.
Which catalog number was used? #4370, #9106, #4377,
#4376, or #9101
o rabbit-anti-p16, clone M156 (Santa Cruz, sc-1207) 1:50 dilution
o anti-mouse IgG1-conjugate (?)
o anti-rabbit IgG- conjugate (?)
8) Blindly examine five 200x fields (Zeiss, Axio Imager M2) from two stained liver
sections from each mouse.
a. Count 200 total cells per field and record number of Nras-, p21-, pERK-, and
p16-positvely stained cells for each image.
9) At 7 months post-injection of DNA harvest livers from mice.
10) Photograph whole excised livers.
11) Measure detectable tumor nodules.
a. Record total number of detectable tumor nodules and nodules greater than 3
mm.
b. Is measurment greater than 3 mm in any dimension, or was there another way
of ensuring consistent measurement?
12) Fix liver tissue and embed in paraffin.
a. What are the protocol details?
13) Section tissue
a. What thickness?
14) Stain sections with H&E (?)
a. What is the staining protocol?
15) Blindly image and analyze images by a board certified pathologist for presence of
HCC.
Deliverables:
 Data to be collected:
o Mouse records (weight at time of injection, health records, etc)

o Images of all stained sections, including controls. (Compare to Figure
2I, and S8B)
o Raw total cell counts and Nras-, p21-, p16-, and pERK-positively
stained cells for each field examined.
o Graph of Nras-, p21-, p16-, and pERK-positively stained cells as a
percentage of total cells in field for each condition. (Compare to Figure
3B, 3C, 3D, 3E, and S8A)
o Images of each liver (Compare to Figure 3F and S10)
o Number and measurements of all tumor nodules detected in each liver.
o Number of tumor nodules greater than 3mm for each condition.
(Compare to Figure 3F and S10)
o Images of H&E stained liver sections and pathology report.
Analysis:
o Figure S8A: Percent of Nras-G12V-positive cells relative to p21-positive
cells in C.B-17 SCID mouse livers 30 days after stable delivery of Nras.
(unpaired two-tailed Student’s t-test). Also perform for 60 days after
stable delivery.
o Figure S8A: Percent of Nras-G12V-positive cells relative to p16-positive
cells in C.B-17 SCID mouse livers 30 days after stable delivery of Nras.
(unpaired two-tailed Student’s t-test)
o Percent of Nras-G12V-positive cells relative to Nras-G12V/D38A over
time in C.B-17 WT mouse livers after stable delivery of Nras. (test?)
Also perform for p21-, p16-, and pErk positive cells. Also perform for
C.B-17 SCID and C.B-17 SCID-beige mouse livers.
o Number of Nras-G12V tumor nodules greater than 3mm in C.B-17 WT
mice relative to C.B-17 SCID mice. (test?) Also perform for C.B-17
SCID/beige mice.
o Number of tumor nodules greater than 3mm in C.B-17 WT mice inject
with Nras-G12V relative to Nras-G12V/D38A. (test?) Also perform for
C.B-17 SCID and C.B-17 SCID/beige mice.
Protocol 2: T lymphocyte, B lymphocyte and NKT cells in senescence surveillance.
Number of replicates: X
Replication calculation needs to consider expected failure rate of tail vein injections.
 What is the expected failure rate of these injections?
Power calculations:
Data from Figure 4A
NrasG12V
BL/6 H-2b
5.96
CD8 -/8.30
CD8 depl
8.54
CD4-/18.48
CD4 depl
20.58
STDEV
1.87
1.40
2.46
3.63
3.16
NrasG12V/D38A
16.02
20.82
18.01
18.25
19.53
STDEV
2.46
3.86
2.11
4.91
2.69
Can you share the data values shown in Figure 4A so our power calculations are
accurate?
Controls:
 NrasG12V/D38A
 IgG2b injected C57/BL6 (H-2b) mice [additional]


secondary-only control
isotype control
Outline of conditions:
X mice for each condition processed at 12 days post-injection:
 C57/BL6 (H-2b) mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
 C57/BL6 CD4-/- mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
 C57/BL6 CD8-/- mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
 CD4 depleted C57/BL6 (H-2b) mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
 CD8 depleted C57/BL6 (H-2b) mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
 IgG2b injected C57/BL6 (H-2b) mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
X mice for each condition processed at 7 months post-injection:
 C57/BL6 (H-2b) mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
 C57/BL6 CD4-/- mice
o injected with pPGK-SB13 + pT/CaggsNrasG12V
o injected with pPGK-SB13 + pT/CaggsNrasG12V/D38A
Steps:
1) Prepare all constructs using the EndoFreeMaxi Kit (Qiagen, 12362) following
manufacturer’s instructions.
a. Can you share the plasmids?
b. Did the authors specifically follow the Qiagen kit protocols?
c. Was there any endotoxin testing done on the output?
o pPGK-SB13 transposase vector
o pT/CaggsNrasG12V transposon vector
o pT/CaggsNrasG12V/D38A transposon vector
2) Sequence plasmids to confirm identity and run on gel to confirm vector integrity.
a. Can you share the sequencing primers?
3) 7 days before injection of DNA vectors, administer anti-mouse CD4 clone GK1.5
(eBioscience, 16-0041) and rat-IgG2b (eBioscience, ?) to C57/BL6 (H-2b) mice
(Harlan, 044) at 7.5 µg/g of body weight intravenously.
a. Weigh mice
b. What gender and age were the mice?
c. What vehicle was used? What vein?
d. Can you please provide information about housing conditions?
4) 5 days before injection of DNA vectors, administer anti-mouse CD8a clone 53-6.7
(eBioscience, 16-0081) to C57/BL6 (H-2b) mice (Harlan, 044) at 7.5 µg/g of body
weight intravenously.
a. Weigh mice
b. What gender and age were the mice?
c. What vehicle was used? What vein?
d. Can you please provide information about housing conditions?
5) On the day of DNA injection, re-administer anti-mouse CD4 and rat-IgG2b
intravenously following step 1 above.
6) Inject CD4 depleted, CD8a depleted, C57/BL6 CD4-/- (B6.129S2-CD4tm1Mak/J)
(Jackson Laboratory, 002663), and C57/BL6 CD8-/- (B6.129S2-Cd8atm1Mak/J)
(Jackson Laboratory, 002665) mice with transposon/transposase:
a. Mix at a 5:1 molar ratio of transposon (pT/CaggsNrasG12V or
pT/CaggsNrasG12V/D38A) to transposase-encoding plasmid (pPGK-SB13)
for a total injection mass of 30 µg.
b. Weigh mice.
c. Suspend 30 µg of plasmids in saline solution (?) at a final volume of 10% of
the animal's body weight.
d. Deliver DNA solution by hydrodynamic tail vein injection in <10 sec.
i. Can the authors supply more precedural detail on their
hydrodynamic injections?
ii. What are the age and sex of the mice?
iii. Are the CD4-/- and CD8-/- stock # correct?
iv. Can you please provide information about housing conditions?
7) 5 days after injection of DNA, re-administer anti-mouse CD4 and rat-IgG2b
intravenously following step 1 above.
8) Confirm cell depletion in CD4 and CD8 depleted mice by flow cytometry:
a. Can you please provide protocols and gating strategies?
b. When was the depletion assessed?
o mouse-anti-CD4, clone GK1.5 (eBioscience, 16-0041)
o anti-CD4, clone RM4-5 (eBioscience, ?)
o mouse-anti-CD8a, clone 53-6.7 (eBioscience, 16-0081)
o anti-CD8b2, clone 53-5.8 (eBioscience, ?)
a. What is the catalog #, eBioscience does not have a clone 53-5.8?
9) At 12 days post-injection of DNA, harvest liver tissue.
10) Fix tissue and embed in paraffin.
a. What are the protocol details?
11) Section tissue
a. What thickness?
12) Stain sections with primary and secondary antibodies:
Note: All antibodies will be validated as part of the antibody validation project.
a. Perform isotype control (?)
b. Perform secondary Ab only control.
c. What is the staining protocol? Was antigen retrieval used, if so, how was that
done?
d. What detection system was used (HRP, ABC, etc)?
o mouse-anti-Nras, clone F155 (Santa Cruz, sc-31) 1:100 dilution
o anti-mouse IgG1-conjugate (?)
13) Blindly examine five 200X fields (Zeiss, Axio Imager M2) from two stained liver
sections from each mouse.
a. Count 200 total cells per field and record number of Nras-positvely stained
cells for each image.
14) At 7 months post-injection of DNA, harvest livers from mice.
15) Photograph whole excised livers.
16) Measure detectable tumor nodules.
a. Record total number of detectable tumor nodules and nodules greater than 3
mm.
b. Is measurment greater than 3 mm in any dimension, or was there another way
of ensuring consistent measurement?
17) Fix liver tissue and embed in paraffin.
a. What are the protocol details?
18) Section tissue
a. What thickness?
19) Stain sections with H&E (?)
a. What is the staining protocol?
20) Blindly image and analyze images by a board certified pathologist for presence of
HCC.
Deliverables:
 Data to be collected:
o Mouse records (weight at time of injections, health records, etc)
o Images of all stained sections, including controls.
o Raw total cell counts and Nras-positively stained cells for each field
examined.
o Graph of Nras-positively stained cells as a percentage of total cells in
field for each condition. (Compare to Figure 4A)
o Images of each liver (Compare to Figure 4B)
o Number and measurements of all tumor nodules detected in each liver.
o Number of tumor nodules greater than 3mm for each condition.
(Compare to Figure 4B)
o Images of H&E stained liver sections and pathology report. (Compare
to Figure S15)
 Analysis:
o Figure 4A: Percent of Nras-G12V-positive cells in livers of C57/BL6 (H2b) mice relative to C57/BL6 CD4-/- mice. (unpaired two-tailed
Student’s t-test). Also perform for C57/BL6 CD8-/- mice.
o Percent of Nras-G12V-positive cells in livers of IgG2b injected C57/BL6
(H-2b) mice relative to CD4 depleted C57/BL6 (H-2b) mice. (unpaired
two-tailed Student’s t-test). Also perform for CD8 depleted C57/BL6 (H2b) mice.
o Number of Nras-G12V tumor nodules greater than 3mm in C57/BL6 (H2b) mice relative to C57/BL6 CD4-/- mice. (test?)
o Number of tumor nodules greater than 3mm in C57/BL6 (H-2b) mice
inject with Nras-G12V relative to Nras-G12V/D38A. (test?) Also perform
for C57/BL6 CD4-/- mice.