Abstract
As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Evans et al., 2015), that described how we intended to replicate selected experiments from the paper 'Wnt activity defines colon cancer stem cells and is regulated by the microenvironment' (Vermeulen et al., 2010). Here, we report the results. Using three independent primary spheroidal colon cancer cultures that expressed a Wnt reporter construct we observed high Wnt activity was associated with the cell surface markers CD133, CD166, and CD29, but not CD24 and CD44, while the original study found all five markers were correlated with high Wnt activity (Figure 2F; Vermeulen et al., 2010). Clonogenicity was highest in cells with high Wnt activity and clonogenic potential of cells with low Wnt activity were increased by myofibroblast-secreted factors, including HGF. While the effects were in the same direction as the original study (Figure 6D; Vermeulen et al., 2010) whether statistical significance was reached among the different conditions varied. When tested in vivo, we did not find a difference in tumorigenicity between high and low Wnt activity, while the original study found cells with high Wnt activity were more effective in inducing tumors (Figure 7E; Vermeulen et al., 2010). Tumorigenicity, however, was increased with myofibroblast-secreted factors, which was in the same direction as the original study (Figure 7E; Vermeulen et al., 2010), but not statistically significant. Finally, we report meta-analyses for each results where possible.
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)
Factors secreted from cancer-associated fibroblasts (CAFs), which are an important component of the stroma, such as hepatocyte growth factor (HGF), were reported to play a role in the formation of the cancer stems cells (CSC) niche and tumorigenicity by activating the Wnt signaling pathway (Vermeulen et al, 2010)
Generation and characterization of primary spheroidal cultures of colon cancer cells To assess Wnt signaling activity in colon cancer stem cells (CSC), we transduced primary spheroidal cultures of colon cancer cells with the same Wnt reporter construct as the original study, which used a TCF/LEF-1 responsive promoter to drive expression of green fluorescent protein (GFP) (TOP-GFP; Reya et al, 2003)
Summary
The experimental approach to generate TOP-GFP expressing CSC cultures was described in Protocol 1 of the Registered Report (Evans et al, 2015). As stated in the Registered Report, we identified the clone to use as the one with the largest observed difference in clonogenicity between untreated TOP-GFPhigh and TOP-GFPlow cells, which, as described above, was the E450 culture.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
