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Experiment Overview

Repository ID: FR-FCM-ZYUG Experiment name: Replication Study: Wnt activity defines colon cancer stem cells and is regulated by the microenvironment MIFlowCyt score: 86.00%
Primary researcher: Timothy Errington PI/manager: Timothy Errington Uploaded by: Timothy Errington
Experiment dates: 2015-09-04 - 2018-06-20 Dataset uploaded: Jan 2019 Last updated: Jun 2019
Keywords: [cancer stem cells] [extreme limiting dilution analysis] [single cell sorting] [replication] [metascience] [Reproducibility Project: Cancer Biology] [Wnt signaling pathway] Manuscripts: [31215867] [PMC6584130] Elifelogo
Organizations: Center for Open Science, Charlottesville, VA (United States of America)
Science Exchange, Palo Alto, CA (United States of America)
PhenoVista Biosciences, n/a, San Diego, CA (United States)
Purpose: This experiment describes the isolation and culture of colon cancer stem cells (Protocol 1) and assess the association of TOP-GFP levels with CSC marker expression, specifically CD133, CD29, CD24, CD44, and CD166 (Protocol 2). These protocols are a replication of the experiment reported in Figure 2F of Vermeulen et al., 2010. Additionally, single cell sorting for extreme-limiting dilution analysis (in vitro and in vivo) was performed. This experiment will assess the effect of MFCM and recombinant HGF on the clonogenic potential of the TOP-GFP CSC clones and also examine the ability of the small molecular c-Met inhibitor, PHA-665752, to block MFCM- or HGF-triggered clonogenicity (Protocol 3). This is a replication of the experiment reported in Figure 6D of Vermeulen et al., 2010.. This experiment will assess the effect of MFCM on the tumorigenicity potential of one of the TOP-GFP CSC clones (Protocol 4), which is a replication of Figure 7E of Vermeulen et al., 2010.
Conclusion: We found the TOP-GFPhigh populations were more enriched for CD133+ or CD166+ cells compared to the TOP-GFPlow populations for each of the three cultures (Figure 1C,D). There were also more CD29+ cells in the TOP-GFPhigh populations for each of the three cultures, while the two TOP-GFP populations were mostly similar for CD24 expression. We also found the TOP-GFPlow populations from E450 and CSC1 cultures were more enriched for CD44+ cells, while both the populations displayed similar expression for Co100. We found the clonogenic potential of TOP-GFPhigh cells were greater than TOP-GFPlow cells for each of the three cultures (Figure 2). We also observed that the clonogenicity of TOP-GFPlow cells were increased in the presence of MCFM or HGF, which was reduced when PHA665752 was included, although to varying degrees across the different cultures. Interestingly, PHA665752 treatment on the whole population of TOP-GFP cells (TOP-GFPwhole) had varying effects on the clonogenicity among the different cultures tested. Both the Co100 and E450 cultures had decreased clonogenicity in the presence of PHA665752, while CSC1 cultures were increased. We found the frequency of tumorigenicity was similar when TOP-GFPhigh cells (1 in every 3332, 95% CI [9174, 1210]) or TOP-GFPlow cells (1 in every 2744, 95% CI [7377, 1020]) were injected (Table 1), which was not a statistically significant difference (
Comments: This is experiment is part of the Reproducibility Project: Cancer Biology (https://osf.io/e81xl/wiki/home/). 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.
Funding: The Reproducibility Project: Cancer Biology is funded by the Laura and John Arnold Foundation, provided to the Center for Open Science in collaboration with Science Exchange. The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Quality control: The cell lines used in this experiment underwent mycoplasma testing to ensure there is no contamination. Isotype control antibodies were used to confirm the specificity of the antibodies. A pilot assay for the in vitro extreme-limiting dilution assay was performed on untreated conditions to assess if the dilution was appropriate. We also determined the concentration of HGF in MFCM, which was determined by enzyme-linked immunosorbent assay (ELISA). Additional detailed experimental notes, data, and analysis are available on the Open Science Framework (OSF) (https://osf.io/tfy28/)


Experiment variables

Sample Type
· E450 Protocol_1_450_001.fcs · Protocol_2_450_45001 PE.fcs · Protocol_2_450_45002.fcs · Protocol_2_450_45005.fcs · Protocol_2_450_45006 APC.fcs · Protocol_2_450_45012 GFP.fcs · Protocol_2_450_45013 PI+.fcs · Protocol_2_Specimen_001_450.fcs · Protocol_3_Specimen_001_e450_003.fcs · Protocol_4_20180320_20180320.fcs
· CSC1 Protocol_1_CSC1.fcs · Protocol_2_CSC_CSC1.fcs · Protocol_2_CSC_CSC12.fcs · Protocol_2_CSC_CSC13 PI+.fcs · Protocol_2_CSC_CSC2.fcs · Protocol_2_CSC_CSC5.fcs · Protocol_2_CSC_CSC6APC.fcs · Protocol_2_CSC_CSC7 PE.fcs · Protocol_2_Specimen_001_CSC1.fcs · Protocol_3_Specimen_001_CSC1_002.fcs
· Co100 Protocol_1_C0100.fcs · Protocol_2_C0100_C01.fcs · Protocol_2_C0100_C010 PE.fcs · Protocol_2_C0100_C0100 13 PI.fcs · Protocol_2_C0100_C02.fcs · Protocol_2_C0100_C05.fcs · Protocol_2_C0100_c012 gfp.fcs · Protocol_2_C0100_c06 apc.fcs · Protocol_2_Specimen_001_neg.fcs · Protocol_3_Specimen_001_CO100_001.fcs · Protocol_3_pilot_co100_001.fcs · Protocol_3_pilot_neg_001.fcs

Conditions
· Protocol1 Protocol_1_450_001.fcs · Protocol_1_C0100.fcs · Protocol_1_CSC1.fcs
· Protocol2 Protocol_2_450_45001 PE.fcs · Protocol_2_450_45002.fcs · Protocol_2_450_45005.fcs · Protocol_2_450_45006 APC.fcs · Protocol_2_450_45012 GFP.fcs · Protocol_2_450_45013 PI+.fcs · Protocol_2_C0100_C01.fcs · Protocol_2_C0100_C010 PE.fcs · Protocol_2_C0100_C0100 13 PI.fcs · Protocol_2_C0100_C02.fcs · Protocol_2_C0100_C05.fcs · Protocol_2_C0100_c012 gfp.fcs · Protocol_2_C0100_c06 apc.fcs · Protocol_2_CSC_CSC1.fcs · Protocol_2_CSC_CSC12.fcs · Protocol_2_CSC_CSC13 PI+.fcs · Protocol_2_CSC_CSC2.fcs · Protocol_2_CSC_CSC5.fcs · Protocol_2_CSC_CSC6APC.fcs · Protocol_2_CSC_CSC7 PE.fcs · Protocol_2_Specimen_001_450.fcs · Protocol_2_Specimen_001_CSC1.fcs · Protocol_2_Specimen_001_neg.fcs
· Protocol3 Protocol_3_Specimen_001_CO100_001.fcs · Protocol_3_Specimen_001_CSC1_002.fcs · Protocol_3_Specimen_001_e450_003.fcs · Protocol_3_pilot_co100_001.fcs · Protocol_3_pilot_neg_001.fcs
· Protocol4 Protocol_4_20180320_20180320.fcs

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