• ■ Experiment has 2 cohorts:

  • ◯ Cohort 1: MMTV-PyMT^+/tg; Postn^+/+ female mice.

  • ◯ Cohort 2: MMTV-PyMT^+/tg; Postn^−/− female mice.

• ■ Experiment will use the following number of mice per cohort:

  • ◯ Cohort 1: 14 mice.

  • ◯ Cohort 2: 15 mice.

    • Note: Derive mice from consecutive litters and analyze development of tumors and metastases until the two cohorts reach the indicated numbers.

• ■ To account for outlier data, as presented in the original publication, 5% and 10% more mice were added to each cohort, respectively, to ensure at least 13 mice survive each cohort for a minimum power of 80%.

  • ◯ See ‘Power calculations’ section for details.

1. Breed MMTV-PyMT^+/tg; Postn^+/− or MMTV-PyMT^+/tg; Postn^−/− male mice with Postn^+/+ and Postn^−/− female mice to obtain MMTV-PyMT^+/tg; Postn^+/+ control and MMTV-PyMT^+/tg; Postn^−/− experimental female mice, respectively.

   • a. Do not use MMTV-PyMT female mice for breeding as they develop mammary tumors. Additionally, MMTV-PyMT^+/tg males cannot be crossed with MMTV-PyMT^+/tg females as the progeny will have a double dose of the oncogene and develop extremely aggressive tumors that cannot be used in this study.

   • b. For generation of the MMTV-PyMT^+/tg; Postn^−/− experimental female mice, male MMTV-PyMT^+/tg; Postn^−/− male mice will be crossed with Postn^−/− female mice.

   • c. For generation of the MMTV-PyMT^+/tg; Postn^+/+ control female mice, the MMTV-PyMT^+/tg; Postn^+/− male mouse will be crossed to Postn^+/+ female mice to obtain MMTV-PyMT^+/tg; Postn^+/+ male mice that will then be crossed with Postn^+/+ female mice.

   • d. As mice obtained were found to contain agents, cross-foster rederivation of mice will be performed based on the following procedure (Artwohl et al., 2008). Donor dam will be placed with male mice 5 days a week Monday afternoon to Friday morning and plugs will be checked daily. When dam is found plugged, she should be single housed and date of plug will be recorded as a sign of pregnancy. Once the pregnancy is confirmed, a timed pregnant mouse will be ordered to use as a foster recipient. Foster recipients will ideally have a different fur coat color than the donor dam, so the identification of the fostered pups will be easier. Consequently, the donor and recipient dams will be removed from their cages and placed in separate clean cages. The litter to be fostered will gently be picked up and cleaned with alcohol and passed to a clean tech to be mixed with dirty bedding, nestlet, and other pups from the recipient dam's cage. When mixing the pups, they will be gently arranged in the palm of the hand, in contact with nestlet and bedding from the recipient dam's cage to transfer the recipient dam's scent. All pups will be placed back in the nest and the recipient dam will be returned to the cage. The cage will be monitored visually every 15 min for the first hour; if there is evidence of rejection by the dam (agitation, carrying the pups around), the pups will be removed from the cage and humanely euthanized. The cages will be visually assessed at least twice daily and will not be disturbed for the first 72 hr after fostering in order to avoid any potential cannibalism. All experimental animals will not be treated with Ivermectin or Fenbendazole, as these could change a number of immune parameters affecting tumor growth and take rate.

2. Extract genomic DNA from mouse tail snips using DNA extraction kit following manufacturer instructions.

   • a. From manufacturer's instructions follow ‘Solid Tissue’ assay protocol.

3. Genotype mice by PCR with MMTV-PyMT and Postn primers.

   • a. MMTV-PYVT384 primer: GGA AGC AAG TAC TTC ACA ACG G.

     • i. This primer is one nucleotide different than what is listed on the Jackson Laboratory information for stock number 002374 (FVB/N-Tg(MMTV-PyVT)634Mul/J), but is used successfully by the original lab.

   • b. MMTV-PYVT385 primer: GGA AAG TCA CTA GGA GCA GGG.

   • c. Postn-5′ primer: GGT GCT TCT GTA AGG CCA TC.

   • d. Postn-3′ primer: GTG AGC CAG GAC CTT GTC ATA.

   • e. INT-as primer: AGC ACT GAC TGC GTT AGC AA.

   • f. Genotyping will be determined by examining both amplicon size and presence.

     • i. MMTV-PyMT conditions (oncogene = 556 bp band):

       10× PCR buffer	1.50 µl
       50 mM MgCl2	0.45 µl
       10 mM dNTPs	0.30 µl
       MMTV-PYVT384 primer	0.10 µl
       MMTV-PYVT385 primer	0.10 µl
       Taq-polymerase	0.20 µl
       H2O	Bring up to 13 µl
       DNA (1:20 dilution)	2 µl

       • Cycling parameters:

         1. 94°C pause.

         2. 94°C for 3 min.

         3. 12 cycles of:

            • i. 96°C for 20 s.

            • ii. 64°C for 30 s.

            • iii. 72°C for 65 s.

         4. 25 cycles of:

            • i. 94°C for 20 s.

            • ii. 58°C for 30 s.

            • iii. 72°C for 35 s.

         5. 72°C for 2 min.

         6. 20°C pause.

     • ii. Postn conditions (WT = 245 bp band; KO = 182 bp band):

       10× PCR buffer	1.50 µl
       50 mM MgCl2	0.45 µl
       10 mM dNTPs	0.30 µl
       Postn-5′ primer	0.10 µl
       Postn-3′ primer	0.10 µl
       INT-as primer	0.10 µl
       Taq-polymerase	0.20 µl
       H2O	Bring up to 13 µl
       DNA (1:20 dilution)	2 µl

       • Cycling parameters:

         1. 94°C pause.

         2. 94°C for 1 min.

         3. 45 cycles of:

            • i. 96°C for 6 s.

            • ii. 59°C for 20 s.

            • iii. 72°C for 30 s.

         4. 20°C pause.

4. Separate and image amplicons by agarose gel electrophoresis.

5. Monitor MMTV-PyMT^+/tg; Postn^+/+ and MMTV-PyMT^+/tg; Postn^−/− female mice for tumor development and keep until tumor disease is fully developed and the metastatic disease is estimated to occur.

   • a. Record age of mice when palpable tumors are detected.

     • i. Multiple tumors will form and grow until they reach a significant size.

   • b. Monitor health status of mice. If mice have to be euthanized prior to reaching fully developed primary tumors exclude mice from study and record reason for euthanasia.

   • c. Tumor disease is fully developed when primary tumors have developed in all mammary glands with an average weight around 1 g per tumor.

     • i. Both cohorts of mice will develop tumors approximately within 3–4 months of age and up to 6 months.

     • ii. Record mice with tumors that are large and form close to the neck as these may give metastasis more efficiently.

   • d. Record age of mice when sacrificed and determine time gap between detection and fully developed tumor.

     • i. Method of euthanasia is isoflurane overexposure (2–5% at 1 l/min) followed by cervical dislocation.

6. Dissect primary tumor and lung tissue from mice.

   • a. Weigh primary tumors.

     • i. Record total weight of all primary tumors together for each mouse.

     • ii. Record the number of primary tumors for each mouse.

     • iii. Divide total weight of all primary tumors by number of primary tumors to obtain a reference primary tumor weight for each mouse.

7. Dissect lungs and blindly count the number of macrometastatic nodules on every side of all separated lobes of the lung using a stereomicroscope.

   • a. Do not fix or stain tissues.

   • b. Quickly count large macrometastatic nodules (≥1 mm in diameter) on all sides.

8. Immediately after counting macrometastasis, use the first six lungs identified from MMTV-PyMT^+/tg;Postn^+/+ female mice that are positive with metastatic disease for further analysis (Protocol 2).

   • a. Should be approximately 5–6 months of age.

     • i. Exclude lungs from mice that are euthanized before this age.

   • b. Use mice with detectable metastatic disease and record number of macrometastases in each lung used and the total weight of the primary mammary tumors.

The original paper reported outliers in each cohort of mice. As these appear to have not been included in the original analysis, we will also remove any outliers from the analysis for comparison. But the analysis will also be performed with all data values. Outliers are determined as 1.5 times the interquartile range.

This replication attempt will perform the following statistical analysis listed below:

• ■ Statistical analysis:

  • ◯ Primary tumor weight in MMTV-PyMT^+/tg;Postn^+/+ mice relative to MMTV-PyMT^+/tg;Postn^−/− mice.

    • Unpaired two-tailed t-test.

  • ◯ Number of pulmonary macrometastases in MMTV-PyMT^+/tg;Postn^+/+ mice relative to MMTV-PyMT^+/tg;Postn^−/− mice.

    • Unpaired two-tailed t-test.

  • ◯ The replication attempt will also perform negative binomial regression analysis of the macrometastases count data.

• ■ Meta-analysis of effect sizes:

  • ◯ Compute the effect sizes of each comparison, compare them against the reported effect size in the original paper, and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot.

The mice in the original study were from an 8^th generation backcross to the FVB line, thus it was on a mixed background, while the mice used in the replication will be from a pure 10th generation backcross to the FVB line. This may make a difference in the effect size and will be included in the discussion of the results of the replication. Additionally, mice will undergo cross-foster rederivation to attempt to remove agents currently associated with the mice. All known differences of materials and reagents are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

Mice will undergo cross-foster rederivation to attempt to remove agents currently associated with the mice that could alter immune parameters affecting tumor growth and take rate. All data obtained from the experiment—raw data, data analysis, control data, and quality control data—will be made publicly available, either in the published manuscript or as an open access data set available on the Open Science Framework project page for this study (https://osf.io/vseix).

• ■ Experiment will use six lungs for a minimum power of 82%.

  • ◯ See appendix for detailed power calculations.

• ■ Each lung will be isolated into 3 cohorts:

  • ◯ Cohort 1: CD34⁺/CD31⁻ pulmonary fibroblasts from MMTV-PyMT^+/tg;Postn^+/+ mice.

  • ◯ Cohort 2: CD31⁺ endothelial cells from MMTV-PyMT^+/tg;Postn^+/+ mice.

  • ◯ Cohort 3: CD45⁺ immune cells from MMTV-PyMT^+/tg;Postn^+/+ mice.

    • Each cohort will be sorted using the following antibodies:

      • ◯ CD45.

      • ◯ CD31.

      • ◯ CD34.

      • ◯ Isotype controls.

      • ◯ Unstained control.

• ■ Each cohort will be analyzed for the following gene expression levels:

  • ◯ POSTN.

  • ◯ GAPDH.

Note:

• These metastatic positive lungs from MMTV-PyMT^+/tg; Postn^+/+ female mice come from Protocol 1.

1. Mince lungs with bended scissors to smooth paste without any clumps and transfer to tube.

   • a. Keep each set of lungs separate (do not pool).

2. Incubate tissue in 6× volume of digestion solution for 1 hr at 37°C with the tube horizontal and shaking at 100 rpm.

   • a. Digestion solution: HBSS supplemented with 0.4 U/ml liberase TM, 0.4 U/ml liberase TH, and 25 µg/ml DNase.

     • i. Liberase TM: stock solution = 26 U/ml = 5 mg/ml; store at −20°C; use at 1:66 dilution.

     • ii. Liberase TH: stock solution = 26 U/ml = 5 mg/ml; store at −20°C; use at 1:66 dilution.

     • iii. DNase: stock solution = 10 mg/ml in PBS; store at −20°C; use at 1:400 dilution.

3. Pellet cells at 180×g for 5 min at room temperature.

4. Resuspend cells in cold MACS buffer and filter through 100-µm cell strainer using a rubber tip of 2.5 ml syringe to smash remaining tissue pieces.

   • a. MACS buffer: 2 mM EDTA in PBS supplemented with 0.5% BSA.

5. Wash strainer extensively with MACS buffer to collect all cells and pellet cells at 180×g for 5 min.

6. Wash twice in MACS buffer, pelleting cells at 180×g for 5 min between washes.

7. Pellet cells at 180×g for 5 min, wash once in FACS buffer and pellet cells at 180×g for 5 min.

   • a. FACS buffer: 3% FBS in PBS.

8. Resuspend up to 5 × 10⁷ cells total in FACS buffer at 2 × 10⁷ cells/ml in polypropylene (opaque) FACS tubes.

9. Either add antibodies directly, or add antibody dilution mixes, and incubate on ice for 30 min in the dark (if staining high amount of cells put on roller at 4°C).

   • a. CD45-PE·Cy5.5 (use at 1:300 dilution).

   • b. CD31-Pac.Blue (use at 1:200 dilution).

   • c. CD34-PE (use at 1:50 dilution).

   • d. Include an unstained control for gating.

   • e. Include isotype control antibody stains.

     • i. Rat IgG2b, κ—PE-Cy5.5.

     • ii. Rat IgG2a, κ—Pac.Blue.

     • iii. Rat IgG2a, κ—PE.

10. Pellet cells at 180×g for 5 min, wash once with 4 ml FACS buffer, and pellet cells at 180×g for 5 min (if staining high amount of cells perform another wash).

11. Resuspend cells in 500 µl FACS buffer and filter through filter-membrane into polypropylene (opaque) FACS tubes protected from light.

12. Just before FACS add viability dye.

    • a. 7AAD (use at 1:1000 dilution).

13. Perform FACS analysis on cells.

    • a. Gate for viability (7-AAD⁻), then gate and collect the different populations to be analyzed:

      • i. CD34⁺/CD31⁻ cells.

      • ii. CD31⁺ cells.

      • iii. CD45⁺ cells.

      • iv. Use negative controls (unstained and isotype control antibodies) to determine gating of populations.

14. Isolate RNA from each collected cell population using TRI reagent following manufacturer's instructions.

    • a. Quantify RNA concentrations in each sample using a spectrometer.

      • i. Record sample purity (A_260/280 and A_260/230 ratios).

15. Reverse transcribe RNA:

    • a. cDNA synthesis:

      Total RNA	1 ng–5 µg
      oligodT(18)	40 pmol
      oligodT(24)	40 pmol
      10 mM dNTPs	1.0 µl
      H2O	Bring up to 12.5 µl

    • b. Heat to 70°C for 5 min, chill on ice for 2 min, then add:

      5× superscript II buffer	4.0 µl
      100 mM DTT	2.0 µl
      RNasin	0.5 µl

    • c. Incubate at 45°C for 2 min, then add:

      Superscript II

      1.0 µl

    • d. Incubate for 1 hr at 42°C.

    • e. Heat-inactivate at 70°C for 15 min.

16. Prepare samples in technical duplicates with two dilutions of cDNA (1:25 and 1:125) using POSTN and GAPDH primers and the Power SYBR green PCR Master Mix. Use GAPDH as control.

    • a. Primers:

      • i. POSTN 5′ primer: AAT GCT GCC CTG GCT ATA TG.

      • ii. POSTN 3′ primer: GTA TGA CCC TTT TCC TTC AA.

      • iii. GAPDH 5′ primer: CAA GCT CAT TTC CTG GTA TGA CAA T.

      • iv. GAPDH 3′ primer: GTT GGG ATA GGG CCT CTC TTG.

    • b. Set up SYBR mix (contains 1 mM MgCl₂): 10 µl of 1a into 1b (store on ice in the dark).

    • c. Set up PCR master mix (per reaction):

      Forward primer	1.0 µl of 5 µM (5 pmol/µl)
      Reverse primer	1.0 µl of 5 µM (5 pmol/µl)
      MgCl2	0.4 µl (for 2 mM)
      H2O	Bring up to 4.0 µl
      SYBR mix	1.0 µl

    • d. Add 5.0 µl of diluted cDNA and 5.0 µl of PCR master mix to Light cycler capillaries, spin down, and run quantitative PCR reaction following manufacturer's instructions.

      • i. Include negative control (no cDNA).

    • e. Analyze and compute ∆∆C_T values.

This replication attempt will perform the following statistical analysis listed below:

• ■ Statistical analysis:

Note: At the time of analysis, we will perform the Shapiro–Wilk test and generate a quantile–quantile (q–q) plot to assess the normality of the data and also perform Levene's test to assess homoscedasticity. If the data appear skewed, we will perform the appropriate transformation in order to proceed with the proposed statistical analysis. If this is not possible, we will perform the equivalent non-parametric test.

• • ◯ One-way ANOVA of POSTN RNA expression in CD34⁺/CD31⁻, CD31⁺, and CD45⁺ cells.

    • ◯ Planned comparisons with the Bonferroni correction:

      • CD34⁺/CD31⁻ vs CD45⁺.

      • CD31⁺ vs CD45⁺.

• ■ Meta-analysis of effect sizes:

  • ◯ Compute the effect sizes of each comparison, compare them against the reported effect size in the original paper and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot.

All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

Negative staining and isotype controls are included to assess antibody staining relative to background during FACS analysis. The sample purity (A_260/280 and A_260/230 ratios) of the isolated RNA from each sample will be reported. All data obtained from the experiment—raw data, data analysis, control data, and quality control data—will be made publicly available, either in the published manuscript or as an open access data set available on the Open Science Framework project page for this study (https://osf.io/vseix).

Test family

• ■ 2-tailed t-test, difference between two independent means, alpha error = 0.05.

Sensitivity calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007).

Test family

• ■ 2-tailed t-test, difference between two independent means, alpha error = 0.05.

Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007).

Test family

• ■ Negative binomial regression, alpha error = 0.05.

Analysis of original data: performed with R software, version 3.1.2 (R Core Development Team, 2014).

Chi-square goodness of fit test, p-value = 0.2535.

Regression coefficient, Genotype (Postn^−/−) = −1.742, incident rate ratio = 0.1752, p-value = 0.000923.

Predicted values from model:

Power Calculations performed with R software, version 3.1.2 (R Core Development Team, 2014).

• Reproducibility Project: Cancer Biology