Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Polyclonal B cell response wikipedia , lookup
Adaptive immune system wikipedia , lookup
DNA vaccination wikipedia , lookup
Monoclonal antibody wikipedia , lookup
Innate immune system wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Adoptive cell transfer wikipedia , lookup
Major urinary proteins wikipedia , lookup
X-linked severe combined immunodeficiency wikipedia , lookup
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.