Download Figures to be replicated

Reproducibility Project: Cancer Biology
Registered Report: Study 15
Study information
 Authors: Sugahara K.N., Teesalu T., Prakash Karmali P., Ramana Kotamraju
V., Agemy L., Greenwald D.R., Ruoslahti E.

Title: Coadministration of a Tumor-Penetrating Peptide Enhances the Efficacy
of Cancer Drugs

Journal: Science

DOI: 10.1126/science.1183057
Summary of Paper
 To be added upon finalization of protocol details
Key experimental results
 iRGD co-treatment with chemotherapies (ABX, DOX, trastuzumab) in
xenograph human prostate (22Rv1) tumors have increased drug penentrance
and accumulation, increased TUNEL staining, and decreases tumor
volume/weight without an effect on toxicity
Figures to be replicated
 Figure 2A: Representative images from mice bearing orthotopic human
prostate tumors (22Rv1) and injected with DOX with or without iRGD
o Protocol 2 (includes Protocol 1)
 Figure 2B: Graph of DOX accumulation from mice bearing orthotopic human
prostate tumor (22Rv1) and treated with or without iRGD
o Protocol 3 (includes Protocol 1)
 Figure 2C: Graph of tumor weight from mice bearing orthotopic human
prostate tumor (22Rv1) and treated with varying doses of DOX with or without
iRGD
o Protocol 4 (included Protocol 1)
 Figure 2D: Graph of TUNEL positive area from mice bearing orthotopic human
prostate tumor (22Rv1) and treated with varying doses of DOX with or without
iRGD
o Protocol 5 (follows from Protocol 4)
 Supp Figure 9A: Graph of body weight from mice bearing orthotopic human
prostate tumor (22Rv1) and treated with varying doses of DOX with or without
iRGD
o Protocol 5 (follows from Protocol 4)
General questions for the authors:
 We’ve decided to add an additional control to all of the replication
experiments; treating the mice with RDGfK in combination with other drugs.
We plan to use 2 µmol/kg, which is the amount you used in Figure 3. Is this a
reasonable concentration to extend to our replication experiments?
 Can you provide a cost estimate for having your chemists synthesize iRDG
and RDGfK for this replication project? Will you also be able to provide quality
control data (i.e. mass spec confirmation of peptide identity and sequence,
etc)?
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Reproducibility Project: Cancer Biology
Registered Report: Study 15
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Reproducibility Project: Cancer Biology
Registered Report: Study 15
Protocols
Protocol 1: Generation of mice bearing orthotopic prostate tumors
Number of replicates:
 This will be based on the total number of animals needed for further protocols.
Power calculations:
 Not applicable
Controls:
 Not applicable
Steps:
1. Culture RRv1 prostate cells (ATCC CRL-2505) in DMEM with 10% fetal bovine
serum and penicillin/streptomycin.
2. Resuspend 106 cells for injection.
a. What were cells resuspended in? What was the final volume for
injection?
3. Inject orthotopically into athymic BALB/c nude mice (Harlan Sprage Dawley,
Inc, Strain number ??).
a. Were mice anesthetized for injection? If so, how?
b. Can the authors please provide the catalog number or strain number for
the mice used?
Deliverables:
 Mice injected with RRv1 prostate cancer cells for further analysis
 Data to be collected:
o None
 Analysis:
o None
Known differences from original study:
 To be filled out after lab feedback
Protocol 2: Treatment of prostate orthotopic tumor bearing mice with Dox alone or
with Dox in combination with iRGD
Number of replicates:
Power calculations:
 How many mice are needed in each group?
o Treated with Dox and PBS
o Treated with Dox and RDGfK
o Treated with Dox and iRDG
Controls:
 Orthotopic tumor bearing mice treated with Dox alone
 Orthotopic tumor bearing mice treated with Dox in combination with RDGfK
o RDGfK is a version of iRDG that lacks the CendR motif that allows
tissue penetration
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Reproducibility Project: Cancer Biology
Registered Report: Study 15

o This control is not originally used in the paper, and has been added by
the RP:CB core team
Tumor immunostaining analysis:
o Sections treated with isotype control antibody
o Sections treated with secondary antibody only
Test conditions:
 Orthotopic tumor bearing mice treated with Dox in combination with iRDG
Steps:
1. Generate tumor bearing mice as per Protocol 1.
2. Allow tumors to grow for two weeks.
3. Inject mice with drugs in combination:
a. Negative control: Inject mice intravenously with 10 mg/kg Doxorubicin
(Dox) (Doxorubicin hydrochloride, Sigma-Aldrich Cat #????)
(suspended in 100 µL PBS) and with 100 µL PBS.
i. Can the authors please provide the catalog number of the Dox
purchased from Sigma?
b. Negative control: Inject mice intravenously with 10 mg/kg Dox
(suspended in 100 µL PBS) and (?) RDGfK (suspended in 100 µL
PBS).
c. Experimental: Inject mice intravenously with 10 mg/kg Dox (suspended
in 100 µL PBS) and 4 µmol/kg iRDG (suspended in 100 µL PBS).
4. One hour later, sacrifice mice and excise tumors:
a. Euthanize mice
i. How were mice euthanized? CO2 and cervical dislocation?
b. Perfuse with PBS + 1% BSA.
i. Was the PBS + 1% BSA cold? Perfusion was accomplished how
– through the left ventricle of the heart?
c. Excise prostate tumor tissue.
5. Process, embed and section tumor tissue.
a. Were tumors fixed? If so, in what fixative and for how long? Given that
Dox native fluorescence is used later in the protocol, fixation method
can affect the signal from the free Dox.
b. Embed tumor tissue in Optimal Cutting Temperature medium (OCT)
(purchased from where?) and freeze.
c. How thick were the sections cut from the tumors?
i. Materials and Methods section “Immunofluorescence” and
“Immunohistochemistry” state: “Tissue preparation and staining
of the cryosections were performed as described (3)”. There is
no reference 3 in the Supplemental Materials (only References
35-37), while Reference 3 in the main article is a listing of amino
acid abbreviations. Can the authors please provide the details of
tissue processing and staining?
6. Immunostain tumor tissue with rat anti-CD31 (BD Biosciences, ?). Use native
fluorescence to image Dox.
a. Can the authors please provide the Catalog number of the anti-CD31
antibody used?
b. At what wavelength does Dox autofluoresce?
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Reproducibility Project: Cancer Biology
Registered Report: Study 15
c. Negative control: Also stain tumor sections with an isotype control
antibody and only with secondary antibody.
7. Image stained tumor sections.
Deliverables:
 Data to be collected
o Figure 2A: Images of Dox-treated tumors and Dox+iRGD-treated
tumors
 Include all data images collected, including isotype control
staining and secondary only control staining
 Analysis
Known differences from original study:
 To be filled out after lab feedback
Protocol 3: Quantifying the amount of Dox present in tumor tissue and major organs
in mice treated with Dox with or without iRGD
Number of replicates:
Power calculations:
 How many mice are needed in each group?
o Treated with Dox and PBS
o Treated with Dox and RDGfK
o Treated with Dox and iRDG
 Do the OD490 readings need to be duplicated or triplicated?
Controls:
 Orthotopic tumor bearing mice treated with Dox alone
 Orthotopic tumor bearing mice treated with Dox in combination with RDGfK
o RDGfK is a version of iRDG that lacks the CendR motif that allows
tissue penetration
o This control is not originally used in the paper, and has been added by
the RP:CB core team
Test conditions:
 Orthotopic tumor bearing mice treated with Dox in combination with iRDG
Steps:
1. Generate tumor bearing mice as per Protocol 1.
2. Allow tumors to grow for two weeks.
3. Inject mice with drugs in combination:
a. Negative control: Inject mice intravenously with 10 mg/kg Dox
(suspended in 100 µL PBS) and with 100 µL PBS.
b. Negative control: Inject mice intravenously with 10 mg/kg Dox
(suspended in 100 µL PBS) and (?) RDGfK (suspended in 100 µL
PBS).
c. Experimental: Inject mice intravenously with 10 mg/kg Dox (suspended
in 100 µL PBS) and 4 µmol/kg iRDG (suspended in 100 µL PBS).
4. One hour later, sacrifice mice and excise tumors:
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Reproducibility Project: Cancer Biology
Registered Report: Study 15
5.
6.
7.
8.
9.
a. Euthanize mice
i. How were mice euthanized? CO2 and cervical dislocation?
b. Perfuse with BSA + 1% BSA
i. Was the PBS + 1% BSA cold? Perfusion was accomplished how
– through the left ventricle of the heart?
c. Excise prostate tumor tissue and the following organs; liver, spleen,
pancreas, heart, lung, kidneys, brain.
Homogenize each tissue separately in 1% sodium dodecyl sulfate and 1 mM
H2SO4 in water.
a. How much tissue is homogenized in what volume of vehicle?
Add 2 mLs of chloroform:isopropyl alcohol (1:1, v/v).
a. What volume of homogenate is the chloroform:isopropyl alcohol added
to?
Vortex and run through freeze/thaw cycles.
a. How long is the sample vortexed for? How many freeze/thaw cycles –
at what temperature and time per cycle?
Centrifuge samples at 14000xg for 15 minutes.
a. Is this spin done at room temperature or at 4°?
Measure the OD490 of the organic phase (the lowest phase).
a. How does the OD490 relate to the levels of Dox?
Deliverables:
 Figure 2B; raw readings of OD490 absorbance of each sample.
Analysis:
 Calculate the fold change in Dox level from mice treated with iRGD by dividing
by the absorbance reading of mice treated with Dox alone.
 Graph the fold change by tissue.
 Michael please add in the appropriate statistical test necessary to determine
significance of the changes in Dox level
Known differences from original study:
 To be filled out after lab feedback
Protocol 4: Effect of Dox alone or Dox in combination with iRGD on tumor growth and
total body weight
Number of replicates:

Power calculations:
 How many mice (or tumors) are needed in each group?
Controls:
 Orthotopic tumor bearing mice treated with PBS alone
 Orthotopic tumor bearing mice treated with Dox alone
 Orthotopic tumor bearing mice treated with Dox in combination with RDGfK
o RDGfK is a version of iRDG that lacks the CendR motif that allows
tissue penetration
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Reproducibility Project: Cancer Biology
Registered Report: Study 15
o This control is not originally used in the paper, and has been added by
the RP:CB core team
Test conditions:
 Orthotopic tumor bearing mice treated with:
o Group 1: PBS alone
o Group 2: 1 mg/kg Dox and PBS
o Group 3: 1 mg/kg Dox and iRDG
o Group 4: 3 mg/kg Dox and PBS
o Group 5: 3 mg/kg Dox with iRDG
o Group 6: iRDG and PBS
o Group 7: 1 mg/kg Dox and RDGfK
o Group 8: 3 mg/kg Dox and RDGfK
o Group 9: PBS and RDGfK
Steps:
1. Generate tumor bearing mice as per Protocol 1.
2. Allow tumors to grow for two weeks.
3. Inject mice with drugs according to their group; this is Day 0.
a. Repeat injection every other day for 24 days.
i. What is the vehicle for Dox, iRDG and RDGfK? What is the stock
concentration used?
4. Weigh mice every four days, starting on Day 0.
5. After 24 days of treatment, euthanize mice and harvest tumor tissue.
a. Does this mean that on day 24, the mice are injected and then
euthanized? Or are they injected on day 24, and euthanized the day
after or two days after the last injection?
b. Euthanize mice
i. How were mice euthanized? CO2 and cervical dislocation?
c. Perfuse with BSA + 1% BSA
i. Was the PBS + 1% BSA cold? Perfusion was accomplished how
– through the left ventricle of the heart?
d. Excise prostate tumor tissue and heart.
6. Weigh tumor tissue.
7. Process tumor tissue and hearts for further analysis (see Protocol 5); fix,
embed and section.
a. Were tissues fixed? If so, in what fixative and for how long? Given that
Dox native fluorescence is used later in the protocol, fixation method
can affect the signal from the free Dox.
b. Embed tumors in OCT and freeze.
c. How thick were the sections cut from the tumors?
i. Materials and Methods section “Immunofluorescence” and
“Immunohistochemistry” state: “Tissue preparation and staining
of the cryosections were performed as described (3)”. There is
no reference 3 in the Supplemental Materials (only References
35-37), while Reference 3 in the main article is a listing of amino
acid abbreviations. Can the authors please provide the details of
tissue processing and staining?
Deliverables:
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Reproducibility Project: Cancer Biology
Registered Report: Study 15



Figure 2C; Record of the drug treatment regimen and weight of each tumor.
Supp Figure 9A: Mouse body weight at time points during treatment
Tumor and heart tissue processed to sections for further analysis (see
Protocol 5)
Analysis:
 Graph of tumor weight by drug treatment in grams.
 Graph of change in body weight as a percentage of body weight on day 0.
 Michael please add in the appropriate statistical test necessary to assess
significance of weight changes in tumors and whole body
Known differences from original study:
 To be filled out after lab feedback
Protocol 5: Assessment of TUNEL staining of tumor and heart tissue after drug
treatment
Number of replicates:

Power calculations:
 How many mice (or tumors) are needed in each group?
Controls:
 See Protocol 4
Test conditions: (optional)
 See Protocol 4
Steps:
Note: This protocol uses tumor and heart tissue derived from Protocol 4
1. Perform TUNEL staining (In Situ Cell Death Detection Kit POD, Roche Applied
Science, 11684817910) of tumor and heart tissue sections.
a. What was the exact protocol used for TUNEL staining?
2. Scan the stained sections with a Scanscope CM-1 scanner and quantify areas
of TUNEL positive staining with ImageJ software.
a. How exactly was TUNEL positive area defined and measured?
b. How many areas and at what magnification were imaged per section?
(For example – 5 random fields at 40X.)
Deliverables:
 Figure 2D; area of TUNEL positive staining and the total area of image for
each image taken.
Analysis:
 Determine the ratio of TUNEL staining as fold change relative to staining in the
untreated (Group 1) tumors. Graph by treatment regimen.
 Michael please add in the appropriate statistical test necessary to assess
significance of changes in areas of TUNEL positivity
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Reproducibility Project: Cancer Biology
Registered Report: Study 15
Known differences from original study:
 To be filled out after lab feedback
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