## Replication Study: Wnt activity defines colon cancer stem cells and is regulated by the microenvironment <br> **Abstract:** As part of the [Reproducibility Project: Cancer Biology][1], we published a Registered Report ([Evans et al., 2015][2]), 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][3]). 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][3]). 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][3]) 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][3]). Tumorigenicity, however, was increased with myofibroblast-secreted factors, which was in the same direction as the original study (Figure 7E; [Vermeulen et al., 2010][3]), but not statistically significant. Finally, we report meta-analyses for each results where possible. ---------- ### Contents **Reports:** Read the [Replication Study][4], or view the [preprint versions][5]. To reproduce the Replication Study manuscript text run this in [R Studio][6] (note: downloads [Markdown script][7] directly from osf.io): library(httr) library(rmarkdown) GET("https://osf.io/d6qp8/?action=download",write_disk("Replication_Study_9.Rmd", overwrite = T)) render("Replication_Study_9.Rmd", output_format = "word_document") Also, explore the [Registered Report][2] and materials related to the Registered Report [here][8]. <br> **Data and Material Availability:** All associated data, protocols, analysis scripts, and other digital materials are available within this OSF project. Flow cytometry data for this study has also been deposited at Flow Repository, where it is directly accessible at https://flowrepository.org/id/FR-FCM-ZYUG. <br> **Experiments replicated:** Reproduce and explore the figures, analyses, data, and methods generated in this replication attempt. - [Protocols 1 & 2: Isolation of colon cancer stem cells and infection with TOP-GFP and flow cytometry analysis of CSC marker expression in TOP-GFP clones][9] - Protocol 1, as outlined in the [Registered Report][2], describes the isolation and culture of colon cancer stem cells. - Protocol 2 is similar to the experiment reported in figure 2F of [Vermeulen et al., 2010][3]. As outlined in the [Registered Report][2], This experiment will assess the association of TOP-GFP levels with CSC marker expression, specifically CD133, CD29, CD24, CD44, and CD166. - [Protocol 3: clonogenicity assay of TOP-GFP CSC clones][10] - Protocol 3 is a replication of the experiment reported in Figure 6D of [Vermeulen et al., 2010][3]. As outlined in the [Registered Report][2], this experiment will assess the effect of MFCM and recombinant HGF on the clonogenic potential of the TOP-GFP CSC clones and examine the ability of PHA-665752 to block MFCM- or HGF-triggered clonogenicity. - [Protocol 4: effect of MFCM on tumorigenicity in TOP-GFPlow CSC clone][11] - Protocol 4 assesses the effect of MFCM on the tumorigenicity potential of one of the TOP-GFP CSC clones, which is a replication of Figure 7E of [Vermeulen et al., 2010][3]. <br> **Meta-analyses:** As a measure of evaluating reproducibility a meta-analysis of each effect was performed. The forest plots, analyses, and data are available [here][12]. Questions about the project can be directed to [contact+rpcb@cos.io][13]. [1]: https://osf.io/e81xl/wiki/home/ [2]: https://elifesciences.org/articles/07301 [3]: https://www.nature.com/articles/ncb2048 [4]: https://doi.org/10.7554/eLife.45426 [5]: https://osf.io/gj63a/ [6]: https://www.rstudio.com [7]: https://osf.io/d6qp8/ [8]: https://osf.io/rfuj2/ [9]: https://osf.io/tfy28/wiki/home/ [10]: https://osf.io/k9vce/wiki/home/ [11]: https://osf.io/j73xu/wiki/home/ [12]: https://osf.io/g4ewk/wiki/home/ [13]:mailto:contact+rpcb@cos.io