• Experiment will be conducted once.

  • This experiment will generate B16-F10 shMet and B16-F10 shScramble stable cells.

Note: Information that these are stable transfectants was communicated by authors.

Note:

• All cells will be sent for mycoplasma testing and short tandem repeat (STR) profiling.

• Cells maintained in DMEM supplemented with 10% exosome-depleted FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C in a humidified atmosphere at 5% CO₂.

1. Deplete exosomes from FBS by ultracentrifugation at 100,000xg for 70 min at 4˚C.

2. Transduce B16-F10 cells with pGIPZ mouse Met shRNA (shMet) or pGIPZ non-silencing shRNA (sh Scramble) lentiviral particles following provider standard protocol (see Guide to Lentiviral Packaging and Transduction, System Biosciences) with a multiplicty of infection (MOI) of 10:1 (lentivirus particles:cells) and incubate overnight (16 hr).

   1. Combine culture medium with TransDux to a 1X final concentration.

3. The next day (16 hr later) replace media.

4. Determine transduction efficiency by green fluorescent protein (GFP) expression:

   1. Three d after transduction (when efficiency is anticipated to be near 80–90%), use fluorescent microscopy to image cells to determine transduction efficiency based on percent of GFP-positive cells.

      1. Use untransduced B16-F10 cells as GFP-negative cell population.

5. After determining transduction efficiency, replace media supplemented with 1.5 µg/ml of puromycin to select for transduced cells.

   1. Use untransduced B16-F10 cells as control for puromycin selection treatment.

6. Continue culture and passage of B16-F10 shMet and B16-F10 shScramble stable cells in puromycin for at least 28 d before further analysis.

   1. After initial selection of 28 d, B16-F10 shMet and B16-F10 shScramble cells should be maintained in puromycin, however when cells are plated for experiments they do not need puromycin added.

   2. Record detailed notes about culturing and passaging of cells, paying particular attention to density.

• Data to be collected:

  • Detailed notes on cell culturing of both stable cell lines generated.

  • Transduction efficiency as a percentage of GFP-positive cells.

  • Fluorescent microscopy images of GFP-positive cells.

• Sample delivered for further analysis:

  • B16-F10 shMet and B16-F10 shScramble cells for exosome purification and Western blot analysis of METexpression for Protocol 2.

  • B16-F10 shMet and B16-F10 shScramble cells for exosome purification and mouse injection for Protocol 3.

No analysis performed.

Similar to the original study, the transduction efficiency, determined by GFP expression, and the knockdown efficiency, determined by Western blot, will be measured (Protocol 2). The original protocol for selecting GFP-positive cells included a step to perform fluorescence-activated cell sorting (FACS) of the population to achieve a 95–99% GFP-positive population prior to puromycin selection (information provided by authors). The replication attempt will not include the FACS sorting step and instead will select the stable cells for at least 28 d before further analysis and will maintain the cell lines when not used in experimental procedures under puromycin selection. 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.

The cell lines used in this experiment will undergo STR profiling to confirm their identity and will be sent for mycoplasma testing to ensure there is no contamination. Untransduced B16-F10 cells will be used to confirm the GFP-negative cell population and during puromycin selection to ensure efficient transduction occurs. Detailed cell culture notes will be recorded and made available to monitor growth rates of B16-F10 shScramble and B16-F10 shMet cells. All of the raw data will be uploaded to the project page on the Open Science Framework (OSF) (https://osf.io/ewqzf/) and made publically available.

• Experiment to be repeated a total of three times for a minimum power of 99%.

  • See Power calculations section for details.

• Each experiment has four cohorts:

  • Cohort 1: B16-F10 shScramble cells

  • Cohort 2: B16-F10 shMet cells

  • Cohort 3: Purified exosomes from B16-F10 shScramble cells

  • Cohort 4: Purified exosomes from B16-F10 shMet cells

• Each cohort is probed for:

  • HSC70 (exosome marker)

  • TSG101 (exosome marker)

  • CD63 (additional exosome marker)

  • MET

  • pMET Tyr1234/5

  • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; control)

Note:

• All cells will be sent for mycoplasma testing and STR profiling.

• B16-F10 shMet and B16-F10 shScramble stable cells were generated in Protocol 1.

• All cells maintained in DMEM supplemented with 10% exosome-depleted FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C in a humidified atmosphere at 5% CO₂.

• B16-F10 shMet and B16-F10 shScramble stable cells should have 1.5 µg/ml puromycin added to medium while maintaining the cell lines that are not used in the experimental procedure.

1. Deplete exosomes from FBS by ultracentrifugation at 100,000xg for 70 min at 4˚C.

2. Grow B16-F10 shScramble and B16-F10 shMet cells for exosome purification and direct lysis.

   1. For exosome purification plate ~5 x 10⁶ cells per 150 mm dish with 25 ml of media. Use two dishes per cell line.

   2. For direct lysis plate ~8–10 x 10⁶ cells per 100 mm dish with 10 ml of media. Use one dish per cell line.

   3. B16-F10 shMet and B16-F10 shScramble cells do not need puromycin added to plates used in the experimental procedure.

3. Grow exponentially until cells reach 80–90% confluence.

   1. Culture 100 mm dishes overnight.

   2. Culture 150 mm dishes for 48–72 hr.

4. Directly lyse in 100 mm dishes:

   1. Wash cells in PBS.

   2. Prepare RIPA buffer and harvest cells directly in lysis buffer.

      1. Add a complete protease inhibitor to RIPA buffer.

   3. Clear lysates by benchtop centrifugation at 14,000xg for 20 min at 4˚C.

   4. Measure protein concentration of supernatant with a BCA kit following manufacturer’s instructions.

   5. Prepare 30 µg of total protein per sample with 4X Laemmli buffer.

      1. Store at -20˚C until analysis.

5. Purify exosomes from 150 mm dishes:

   1. Collect supernatant from cell cultures in 50 ml centrifuge tubes.

   2. Pellet cells from supernatant in a benchtop centrifuge at 500xg for 10 min at 4˚C.

   3. Transfer supernatant to thick wall ultracentrifuge tubes.

   4. Remove cell debris by ultracentrifugation at 20,000xg for 20 min at 4˚C.

   5. Collect supernatant.

   6. Harvest exosomes by ultracentrifugation at 100,000xg for 70 min at 4˚C.

   7. Resuspend the exosome pellet in 20 ml of PBS.

   8. Pellet exosomes by ultracentrifugation at 100,000xg for 70 min at 4˚C.

   9. Resuspend exosome pellet in 100 µl PBS.

      1. Exosomes can be stored at -20˚C for 2–3 weeks.

   10. Measure protein concentration with a BCA kit following manufacturer’s instructions.

       1. Estimated yield of exosomes should be around 1–2 µg/1 x 10⁶ cells.

   11. Characterize exosome pellet using standard Nanosight NTA analysis.

       1. 10–20 µg of exosome protein is needed for analysis.

       2. Report on size distribution and concentration of exosomes.

   12. Prepare 30 µg of total protein per sample with 4X Laemmli buffer.

       1. Store at -20˚C until analysis.

6. Load 30 µg of total protein with sample buffer in each lane on an SDS-PAGE gel with a protein molecular weight marker.

7. Run electrophoresis at constant voltage (100 V) for ~1–2 hr in 1X electrophoresis buffer following manufacturer instructions.

8. Transfer gel to membrane using a wet transfer system at constant amperage (350 mA) for 45 min in 1X Tris-glycine buffer supplemented with 20% methanol.

   1. Pre-soak membrane with methanol and then 1X transfer buffer before assembly.

9. Block membranes in SuperBlock following manufacturer’s instructions.

10. Probe membranes with the following primary antibodies overnight at 4˚C diluted in SuperBlock.

    1. mouse anti-HSC70; use at 1:500; 70 kDa

    2. mouse anti-TSG101; use at 1:500; 45 kDa

    3. anti-CD63; use at 1:1000; 53 kDa

    4. mouse anti-MET; use at 1:1000; 145 kDa

    5. rabbit anti-pMET Tyr1234/5; use at 1:1000; 145 kDa

    6. rabbit anti-GAPDH; use at 1:1000; 37 kDa

11. Wash membranes three times 10 min in Ticket Tax Box Service (TTBS).

    1. TTBS = 1X TBS supplemented with 0.1% tween.

12. Detect primary antibodies with the following secondary antibodies diluted in SuperBlock for 1 hr.

    1. sheep anti-mouse-HRP; use at 1:4000–1:20,000

    2. donkey anti-rabbit-HRP; use at 1:4000–1:20,000

13. Wash membranes three times for 10 min each in TTBS.

14. Detect using ECL reagent following manufacturer’s instructions.

15. Quantify intensities of the immunoreactive bands by densitometry.

    1. Normalize total MET to GAPDH.

    2. Normalize pMET (Tyr1234/1235) to GAPDH.

16. Repeat independently two additional times.

• Data to be collected:

  • Exosome characterization data (including protein concentration using a BCA kit).

  • Images of probed membranes (full images with ladder) (compare to Figure S1C [right panel]).

  • Quantified levels of total MET and Phospho-MET normalized to GAPDH for all conditions (compare to Figure S5A).

This experiment assesses if knockdown of Met alters total MET and phospho-MET expression in cells and exosomes. This replication attempt will perform the following statistical analysis:

• Statistical analysis:

  • One-way multivariate analysis of variance (MANOVA) comparing the relative mean of photon signal for total MET and phospho-MET normalized to GAPDH from B16-F10 shScramble and B16-F10 shMet cells.

    • Planned comparisons with the Bonferroni correction:

      1. Total MET in B16-F10 shScramble cells compared with B16-F10 shMet cells

      2. Phospho-MET in B16-F10 shScramble cells compared with B16-F10 shMet cells

  • One-way MANOVA comparing the relative mean of photon signal for total MET and phospho-MET normalized to GAPDH from B16-F10 shScramble and B16-F10 shMet exosomes.

    • Planned comparisons with the Bonferroni correction:

      1. Total MET in B16-F10 shScramble exosomes compared to B16-F10 shMet exosomes

      2. Phospho-MET in B16-F10 shScramble exosomes compared to B16-F10 shMet exosomes

• 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 cell lines when not used in experimental procedures will be maintained under puromycin selection. The replication attempt will include an additional exosome marker, CD63, not included in the original study. 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.

The cell lines used in this experiment will undergo STR profiling to confirm their identity and will be sent for mycoplasma testing to ensure there is no contamination. This protocol analyzes the knockdown efficiency of c-Met in B16-F10 cells. Isolated exosomes are characterized by NanoSight and are analyzed for typical exosome markers by Western blot, including CD63, an additional marker not included in the original study. All of the raw data, including the image files and quantified bands from the Western blot, will be uploaded to the project page on the OSF (https://osf.io/ewqzf/) and made publically available.

• Experiment will use seven mice per cohort for a minimum power of 82%.

  • See Appendix for detailed power calculations

• Each experiment has three cohorts:

  • Cohort 1: Synthetic unilamellar liposomes injected into C57BL/6 mice

  • Cohort 2: B16-F10 shScramble exosomes injected into C57BL/6 mice

  • Cohort 3: B16-F10 shMet exosomes injected into C57BL/6 mice

Note:

• All cells will be sent for mycoplasma testing and STR profiling.

• B16-F10 shMet and B16-F10 shScramble stable cells were generated in Protocol 1.

• All cells maintained in DMEM supplemented with 10% exosome-depleted FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C in a humidified atmosphere at 5% CO₂.

• B16-F10 shMet and B16-F10 shScramble stable cells should have 1.5 µg/ml puromycin added to medium while maintaining the cell lines that are not used in the experimental procedure.

• The original study indicated mice were 8–10 weeks old, however the authors clarified that the mice were 6 weeks old.

1. Deplete exosomes from FBS by ultracentrifugation at 100,000xg for 70 min at 4˚C.

2. Two–three days before needing exosomes, plate ~5 x 10⁶ B16-F10 shScramble and B16-F10 shMet cells per 150 mm dish with 25 ml of media for exosome purification.

   1. Use three to six dishes per cell line to obtain needed amount of exosomes.

   2. B16-F10 shMet and B16-F10 shScramble cells do not need puromycin added to plates used in the experimental procedure.

3. Culture cells exponentially for 48–72 hr until cells reach 80–90% confluence.

4. Purify exosomes from 150 mm dishes:

   1. Collect supernatant from cell cultures in 50 ml centrifuge tubes.

   2. Pellet cells from supernatant in a benchtop centrifuge at 500xg for 10 min at 4˚C.

   3. Transfer supernatant to thick wall ultracentrifuge tubes.

   4. Remove cell debris by ultracentrifugation at 20,000xg for 20 min at 4˚C.

   5. Collect supernatant.

   6. Harvest exosomes by ultracentrifugation at 100,000xg for 70 min at 4˚C.

   7. Resuspend the exosome pellet in 20 ml of PBS.

   8. Pellet exosomes by ultracentrifugation at 100,000xg for 70 min at 4˚C.

   9. Resuspend exosome pellet in 100 µl PBS.

      1. An aliquot of the exosome preparation can be stored at -20˚C until Nanosight analysis.

   10. Measure protein concentration with a BCA kit following manufacturer’s instructions.

       1. Estimated yield of exosomes should be around 1–2 µg/1x10⁶ cells.

   11. Characterize exosome pellets using standard Nanosight NTA analysis.

       1. An aliquot of each exosome preparation will be stored at -20˚C and analyzed all at once following the final preparation.

       2. 10–20 µg of exosome protein is needed for analysis.

       3. Report on size distribution and concentration of exosomes.

       4. Report the number of exosomes per µg protein (as measured by BCA) for each sample.

   12. Prepare samples at a concentration of 50 ng/µl to achieve 5 µg of total protein diluted in 100 µl of PBS.

5. Following protein quantification of each preparation, inject intravenously, via retro-orbital injection, freshly isolated B16-F10 shScramble or B16-F10 shMet exosomes, or synthetic unilamellar 100 nm liposomes into 6-week-old C57BL/6 female mice three times a week for a total of 28 d.

   1. Sample injection schedule:

      1. Each cohort will be injected on Monday, Wednesday, and Friday with a fresh exosome preparation (>35 µg total) each week for a total of 4 weeks.

      2. Step 4 will be performed for each injection day.

   2. It is crucial to inject fresh exosomes every time, do not freeze down, and inject right after purification and quantification following steps 2–4 above.

   3. Volume of injection is 100 µl.

   4. Amount of synthetic liposomes injected, 1.25 µg of L-α-phosphatidylcholine (PC) will mimic 5 µg of exosome protein, which is based on a theoretical 4:1 protein:PC ratio (communicated by authors).

      1. Dilute 1.92 µl of a 1:20 dilution of the stock concentration of synthetic liposomes into 100 µl PBS for each injection.

6. After 28 d, inject 1 x 10⁶ B16-F10-luciferase cells diluted in 100 µl of PBS subcutaneously in the flank of mice.

7. Measure primary tumor volume three times a week for a total of 21 d.

   1. Use calipers to measure width and height with volume determined as (length x width²)/2. (additional recorded information)

      1. Note: tumor volume detection will be limited to <1000 mm³.

   2. Record latency. (additional recorded information)

      1. Note: Perform Steps 8 through 10 (luciferin injection, euthanasia, dissection, and imaging) from mice from different cohorts in parallel (i.e., one from each of the three cohorts) so variation during the procedure is equal across cohorts. (additional detail)

8. After 21 d anesthetize mice and inject 50 mg/kg of D-luciferin via retro-orbital injection in 100–200 µl PBS (volume depending on the body weight).

   1. Weigh mice.

   2. Use isoflurane and O₂ to anesthetize mice.

9. Five min later euthanize mice by cervical dislocation under anesthesia and dissect tissues (lungs and femurs).

   1. Dissect out primary tumors and record weight (additional parameter).

10. Image dissected primary lungs and bones (femurs) for luciferase expression in IVIS Imaging system

    1. Record the time from euthanasia to imaging for each mouse.

    2. Record photon flux.

       1. Take two–three exposure times for each sample.

          1. Use same exposure time for each tissue from all mice during analysis.

• Data to be collected:

  • Mouse health records (injection schedule, time from tumor cell injection to detectable tumors [latency], weight of mice at end of experiment, mortality report)

  • Exosome characterization data (including protein concentration using a BCA kit).

  • Raw numbers and calculated tumor volume for all mice, and graph of tumor volume versus time for all conditions during course of treatment.

  • Time of euthanasia to imaging for each mouse.

  • Images of lungs and bones for luciferase expression (compare to Figure 4E).

  • Raw photon flux values of all analyzed images of each tissue for all conditions using the same exposure time (compare to Figure 4E).

  • Final weight of tumors.

This experiment assesses if knockdown of Met alters primary tumor growth and metastasis. This replication attempt will perform the following statistical analysis:

• Statistical Analysis:

  • Bonferroni corrected one-way ANOVAs comparing lung photon flux of mice treated with synthetic unilamellar liposomes, or exosomes from B16-F10 shScramble or B16-F10 shMet cells and the following planned comparisons with the Fisher’s least significant difference (LSD) test:

    1. Synthetic unilamellar liposomes compared to B16-F10 shScramble exosomes

    2. B16-F10 shMet exosomes compared to B16-F10 shScramble exosomes

  • Bonferroni corrected Kruskal–Wallis test comparing bone photon flux of mice treated with synthetic unilamellar liposomes, or exosomes from B16-F10 shScramble or B16-F10 shMet cells and the following planned comparisons (Wilcoxon–Mann–Whitney test) with the Fisher’s LSD test:

    1. Synthetic unilamellar liposomes compared to B16-F10 shScramble exosomes

    2. B16-F10 shMet exosomes compared to B16-F10 shScramble exosomes

• 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.

• Exploratory analysis:

  •  Comparison of primary tumor growth rates.

    • This is exploratory analysis. We will measure tumor growth rates across all mouse cohorts over the length of the study. These data were collected, but not reported in the original study, and found to not be different. We will plot growth curves and calculate area under the curve for each mouse. We will then perform a one-way analysis of variance (ANOVA) analysis, with the following planned comparisons with the Fisher’s LSD test:

      1. Synthetic unilamellar liposomes compared to B16-F10 shScramble exosomes

      2. B16-F10 shMet exosomes compared to B16-F10 shScramble exosomes

  • Comparison of final primary tumor weights.

    • This is exploratory analysis. We will measure tumor weights across all mouse cohorts at the end of the study. These data were not reported in the original study. We will perform a one-way ANOVA analysis, with the following planned comparisons with the Fisher’s LSD test:

      1. Synthetic unilamellar liposomes compared to B16-F10 shScramble exosomes

      2. B16-F10 shMet exosomes compared to B16-F10 shScramble exosomes

The cell lines when not used in experimental procedures will be maintained under puromycin selection. The number of exosomes injected (based on protein content) will be reported for each preparation from each cohort. The original data on primary tumor growth were not shown and final primary tumor weights were not recorded. The replication attempt will record and present these data as well as tumor latency. 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.

The cell lines used in this experiment will undergo STR profiling to confirm their identity and will be sent for mycoplasma testing to ensure there is no contamination. Isolated exosomes will be injected immediately after protein quantification and will be characterized by NanoSight following the final preparation to ensure the integrity of the samples. Exosomes and luciferin will be injected intravenously, via retro-orbital injection, similar to the original study. While it will be attempted to be the same for all animals, the time from euthanasia to imaging for each mouse will be recorded as an additional quality control parameter. All of the raw data, including the image files and photo flux values, will be uploaded to the project page on the OSF (https://osf.io/ewqzf/) and made publically available.

Not applicable

Summary of original data reported in Figure S5A (calculated from data shared by original authors):

The replication will also include analysis of total MET and phospho-MET in exosomes from B16-F10 shScramble and B16-F10 shMet cells. We will use the same calculated sample size as determined for the cells with the assumption that the cells and exosomes have similar values, which Peinado and colleagues (2012) present in Figure 4A.

Test family

• two-tailed t test, difference between two independent means, Bonferroni’s correction: alpha error = 0.025

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

Total Met:

Phospho-Met:

Summary of original data reported in Figure 4E (calculated from data shared by original authors):

Lung photo flux:

Test family

• ANOVA: Fixed effects, omnibus, one-way, Bonferroni’s correction: alpha error = 0.025.

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

ANOVA F-test statistic performed with R software, version 3.1.2 (Team, 2014).

Partial η² calculated from (Lakens, 2013).

Test family

• 2 tailed t test, difference between two independent means, Fisher’s LSD correction: alpha error = 0.025.

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

Bones photon flux:

Test family

• ANOVA: Fixed effects, omnibus, one-way, Bonferroni’s correction: alpha error = 0.025.

Power calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F-test statistic performed with R software, version 3.1.2 (Team, 2014). Partial η² calculated from (Lakens, 2013).

Test family

• 2 tailed t test, Wilcoxon–Mann–Whitney test (two groups), Fisher’s LSD correction: alpha error = 0.025.

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

• Reproducibility Project: Cancer Biology