Replication study: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.

Amirali Afshari,Archana Gupta,Nicole Perfito,Timothy M Errington,Elizabeth Iorns,Rachel Tsui,Ranjita Sengupta,Vittorio Sebastiano,Young H Kim,Alexandria Denis,Reproducibility Project: Cancer Biology ,Jeewon Kim

doi:10.7554/elife.39944

Abstract

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Lesnik et al., 2016) that described how we intended to replicate selected experiments from the paper 'Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET' (Peinado et al., 2012). Here we report the results. We regenerated tumor cells stably expressing a short hairpin to reduce Met expression (shMet) using the same highly metastatic mouse melanoma cell line (B16-F10) as the original study, which efficiently downregulated Met in B16F10 cells similar to the original study (Supplementary Figure 5A; Peinado et al., 2012). Exosomes from control cells expressed Met, which was reduced in exosomes from shMet cells; however, we were unable to reliably detect phosphorylated Met in exosomes. We tested the effect of exosome-dependent Met signaling on primary tumor growth and metastasis. Similar to the results in the original study, we did not find a statistically significant change in primary tumor growth. Measuring lung and femur metastases, we found a small increase in metastatic burden with exosomes from control cells that was diminished when Met expression was reduced; however, while the effects were in the same direction as the original study (Figure 4E; Peinado et al., 2012), they were not statistically significant. Differences between the original study and this replication attempt, such as level of knockdown efficiency, cell line genetic drift, sample sizes, study endpoints, and variability of observed metastatic burden, are factors that might have influenced the outcomes. Finally, we report meta-analyses for each result.

Highlights

The Reproducibility Project: Cancer Biology (RP:CB) is a collaboration between the Center for Open Science and Science Exchange that seeks to address concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology (Errington et al, 2014)
To test the effect exosome-dependent Met signaling has on primary tumor growth and metastasis, we used the same highly metastatic mouse melanoma cell line (B16-F10) and the same lentiviral system as the original study to make B16-F10 cells stably expressing an shRNA targeting Met or a control shRNA using the same targeting sequences as the original study
We tested various multiplicity of infection (MOI) ratios, all of which displayed expression of the shRNA with corresponding decreased Met and Met levels in short hairpin to reduce Met expression (shMet) cells compared to shScr cells (Figure 1—figure supplement 1)

Summary

Introduction

The Reproducibility Project: Cancer Biology (RP:CB) is a collaboration between the Center for Open Science and Science Exchange that seeks to address concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology (Errington et al, 2014). For each of these papers a Registered Report detailing the proposed experimental designs and protocols for the replications was peer reviewed and published prior to data collection. The outcome measures reported in this Replication Study will be aggregated with those from the other Replication Studies to create a dataset that will be examined to provide evidence about reproducibility of cancer biology research, and to identify factors that influence reproducibility more generally

Results and discussion

Materials and methods

Funding Funder