1. Cancer Biology
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Replication Study: Wnt activity defines colon cancer stem cells and is regulated by the microenvironment

  1. Anthony Essex
  2. Javier Pineda
  3. Grishma Acharya
  4. Hong Xin
  5. James Evans
  6. Reproducibility Project: Cancer Biology  Is a corresponding author
  1. PhenoVista Biosciences, United States
  2. Explora BioLabs Inc, United States
Replication Study
Cite this article as: eLife 2019;8:e45426 doi: 10.7554/eLife.45426
3 figures, 2 tables and 2 additional files

Figures

Figure 1 with 1 supplement
Analysis of CSC marker expression in TOP-GFP cultures.

(A) Representative images of the three independent single-cell-cloned CSC cultures, lentivirally transduced with TOP-GFP. Phase contrast (top) and fluorescence microscopy (bottom) for each of the cultures indicated. Bar = 90 µm. (B) Single parameter histograms for GFP intensity for each of the TOP-GFP single-cell-cloned CSC cultures with the TOP-GFPlow (10% lowest) and TOP-GFPhigh (10% highest) populations indicated. (C) Single parameter histograms for the indicated cell surface markers for each of the indicated cultures. Gray denotes TOP-GFPlow (10% lowest) and green denotes TOP-GFPhigh (10% highest) populations. (D) Density plots for CD29/CD24 and CD44/CD166 from TOP-GFPlow (gray) and TOP-GFPhigh (green) populations of each culture. Additional details for this experiment can be found at https://osf.io/tfy28/.

https://doi.org/10.7554/eLife.45426.002
Figure 1—figure supplement 1
Flow cytometry gating strategy.

Representative density plots of gating strategy to assess cell surface markers from TOP-GFPlow and TOP-GFPhigh populations. Forward scatter area (FSC-A) and PerCP-Cy5.5 was used to gate on viable cells (PI negative cells), followed by forward verses side scatter area (FSC-A vs SSC-A) to identify cells of interest and exclude debris, which were then analyzed by FSC-A and forward scatter width (FSC-W), and then SSC-A and side scatter width (SSC-W) to exclude doublet cells. From the single-cell population, SSC and FITC were used to gate on the TOP-GFPlow (10% lowest) and TOP-GFPhigh (10% highest) populations. TOP-GFPlow and TOP-GFPhigh populations were then assessed for PE and APC to detect the fluorophores conjugated to antibodies against the cell surface markers analyzed in this study. Additional details for this experiment can be found at https://osf.io/tfy28/.

https://doi.org/10.7554/eLife.45426.003
Figure 2 with 1 supplement
Clonogenicity assay of TOP-GFP cultures.

A limiting-dilution assay was performed on the TOP-GFPlow, TOP-GFPhigh, or TOP-GFPwhole populations of the three indicated TOP-GFP cultures. Cells were left untreated, or treated with 25 ng/ml HGF, 1:2 dilution of MFCM, or 500 nM PHA-665752 (PHA), as indicated. The bar graphs present the clonogenic potential of each culture with error bars representing 95% confidence intervals (y-axis is log2 scale). This experiment was performed once for each culture. See Materials and methods and Registered Report (Evans et al., 2015) for details on limiting-dilution statistics and scheme. Planned contrast between TOP-GFPlow vs TOP-GFPhigh: E450 (χ2 = 39.8, uncorrected p=2.82×10−10, corrected p=1.69×10−9); CSC1 (χ2 = 4.82, uncorrected p=0.028, corrected p=0.169); Co100 (χ2 = 7.59, uncorrected p=0.0059, corrected p=0.035). Planned contrast between TOP-GFPlow vs TOP-GFPlow + HGF: E450 (χ2 = 1.49, uncorrected p=0.223, corrected p>0.99); CSC1 (χ2 = 0.337, uncorrected p=0.562, corrected p=0.99); Co100 (χ2 = 12.7, uncorrected p=3.70×10−4, corrected p=0.0022). Planned contrast between TOP-GFPlow vs TOP-GFPlow + MFCM: E450 (χ2 = 1.96, uncorrected p=0.162, corrected p=0.969); CSC1 (χ2 = 4.18, uncorrected p=0.041, corrected p=0.245); Co100 (χ2 = 28.7, uncorrected p=8.26×10−8, corrected p=4.96×10−7). Planned contrast between TOP-GFPlow + HGF vs TOP-GFPlow + HGF + PHA: E450 (χ2 = 0.376, uncorrected p=0.540, corrected p>0.99); CSC1 (χ2 = 34.0, uncorrected p=5.64×10−9, corrected p=3.39×10−8); Co100 (χ2 = 5.13, uncorrected p=0.024, corrected p=0.141). Planned contrast between TOP-GFPlow + MFCM vs TOP-GFPlow + MFCM + PHA: E450 (χ2 = 61.0, uncorrected p=5.71×10−15, corrected p=3.43×10−14); CSC1 (χ2 = 43.5, uncorrected p=4.14×10−11, corrected p=2.48×10−10); Co100 (χ2 = 17.6, uncorrected p=2.67×10−5, corrected p=1.60×10−4). Planned contrast between TOP-GFPwhole vs TOP-GFPwhole + PHA: E450 (χ2 = 68.3, uncorrected p=1.43×10−16, corrected p=8.56×10−16); CSC1 (χ2 = 72.2, uncorrected p=1.96×10−17, corrected p=1.17×10−16); Co100 (χ2 = 20.2, uncorrected p=6.91×10−6, corrected p=4.14×10−5). Additional details for this experiment can be found at https://osf.io/k9vce/.

https://doi.org/10.7554/eLife.45426.004
Figure 2—figure supplement 1
Pilot of clonogenicity assay.

A limiting-dilution assay was performed on the TOP-GFPlow, TOP-GFPhigh, or TOP-GFPwhole populations of the three indicated TOP-GFP cultures. Cells were left untreated. The bar graphs present the clonogenic potential of each culture with error bars representing 95% confidence intervals (y-axis is log2 scale). The pilot experiment was performed once for each culture. See Methods and Registered Report (Evans et al., 2015) for details on limiting-dilution scheme. Additional details for this experiment can be found at https://osf.io/k9vce/.

https://doi.org/10.7554/eLife.45426.005
Meta-analyses of each effect.

Effect size and 95% confidence interval are presented for Vermeulen et al. (2010), the results from this replication study (RP:CB), and a random effects meta-analysis of the effects. Cohen’s ω is a standardized measure of the association between the cells tested and clonogenic, or tumorigenic, frequency. The higher the value, the stronger the association, with an effect size of zero indicating there was no association. Sample sizes used in Vermeulen et al. (2010) and RP:CB are reported under the study name. (A) Comparison of clonogenic frequency between the indicated treated, or untreated, populations of TOP-GFP CSC cultures. TOP-GFPlow vs TOP-GFPhigh (meta-analysis p=0.094); TOP-GFPlow vs TOP-GFPlow + HGF (meta-analysis p=0.110); TOP-GFPlow vs TOP-GFPlow + MFCM (meta-analysis p=0.047); TOP-GFPlow + HGF vs TOP-GFPlow + HGF + PHA (meta-analysis p=0.218); TOP-GFPlow + MFCM vs TOP-GFPlow + MFCM + PHA (meta-analysis p=0.085); TOP-GFPwhole vs TOP-GFPwhole + PHA (meta-analysis p=0.498). (B) Comparison of frequency of tumorigenicity between the indicated treated, or untreated, populations of TOP-GFP CSC cultures injected into mice. TOP-GFPlow vs TOP-GFPhigh (meta-analysis p=0.330); TOP-GFPlow vs TOP-GFPlow + MFCM (meta-analysis p=0.033). Additional details for these meta-analyses can be found at https://osf.io/g4ewk/.

https://doi.org/10.7554/eLife.45426.007

Tables

Table 1
Tumorigenicity assay of TOP-GFP culture.

Cell numbers from the indicated populations were injected into female athymic nude mice. Cells were left untreated or treated with 1:2 dilution of MFCM for 2 hr before injection. The number of successful tumor initiations after nine weeks out of four injected mice for each condition is reported. Planned contrast between TOP-GFPlow vs TOP-GFPhigh (χ2 = 0.084, uncorrected p=0.772, corrected p>0.99). Planned contrast between TOP-GFPlow vs TOP-GFPlow + MFCM (χ2 = 3.32, uncorrected p=0.069, corrected p=0.137). Additional details for this experiment can be found at https://osf.io/j73xu/.

https://doi.org/10.7554/eLife.45426.006
LineCondition1010010005000
E450TOP-GFP Low0/40/42/43/4
TOP-GFP High0/40/43/42/4
TOP-GFP Low + MFCM0/42/42/44/4
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Cell line (Homo sapiens)Co100doi:10.1038/ncb2048shared by
Medema lab, University of
Amsterdam
Cell line (H. sapiens, female)CSC1ProMab Biotechnologiescat# CC100103
Cell line (H. sapiens, female)E450this paper
Cell line (H. sapiens, female)18CoATCCcat# CRL-1459; RRID:CVCL_2379
Strain, strain background (Mus musculus, Athymic Nude, female)athymic nudeCharles RiverStrain code: 490; RRID:IMSR_CRL:490
Recombinant DNA reagentTOP-GFPdoi:10.1038/nature01593RRID:Addgene_14715shared by Medema lab, University of Amsterdam
Chemical compound, drugHGFSigma-Aldrichcat# H5791lot# MKBT3102V
Chemical compound, drugPHA-665752Sigma-Aldrichcat# PZ0147
OtherMatrigelCorningcat# 356230
AntibodyPE-conjugated anti-CD133Miltenyi Bioteccat# 130-098-826; clone: AC133; RRID:AB_26608821:100 dilution
AntibodyPE-conjugated anti-CD24BD Biosciencescat# 560991; clone ML5; RRID:AB_105630741:100 dilution
AntibodyAPC-conjugated anti-CD29BD Biosciencescat# 561794; clone: MAR4; RRID:AB_108981631:100 dilution
AntibodyPE-conjugated anti-CD166R and D Systemscat# FAB6561P; clone: 105902; RRID:AB_22238871:100 dilution
AntibodyAPC-conjugated anti-CD44BD Biosciencescat# 560890; clone: G44-26; RRID:AB_20339591:100 dilution
AntibodyPE-conjugated mouse IgG1 isotype controlMiltenyi Bioteccat# 130-098-106; clone: X-56; RRID:AB_26614631:100 dilution
AntibodyAPC-conjugated mouse IgG2b, κ isotype controlBD Biosciencescat# 555745; clone: 27–35; RRID:AB_3986121:100 dilution
AntibodyPE-conjugated mouse IgG2a, κ isotype controlBD Biosciencescat# 555574; clone: G155-178; RRID:AB_3959531:100 dilution
AntibodyAPC-conjugated mouse IgG1 isotype controlBD Biosciencescat# 555751; clone: MOPC-21; RRID:AB_3986131:100 dilution
Software, algorithmFACS Sortware sorterBD BiosciencesRRID:SCR_016722version 1.2.0.142
Software, algorithmHCS Studio Cell AnalysisThermo Fisher ScientificRRID:SCR_016787version 6.6.0
Software, algorithmFACSDivaBD BiosciencesRRID:SCR_016722version 6.1.3 or 8.0.1
Software, algorithmFlowJoTree Star, IncRRID:SCR_008520version 10
Software, algorithmR Project for statistical computinghttps://www.r-project.orgRRID:SCR_001905version 3.5.1

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