Figures and data in Replication Study: Wnt activity defines colon cancer stem cells and is regulated by the microenvironment

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Tables
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3 figures, 2 tables and 2 additional files

Figures

Figure 1 with 1 supplement

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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-GFP^low (10% lowest) and TOP-GFP^high (10% highest) populations indicated. (C) Single parameter histograms for the indicated cell surface markers for each of the indicated cultures. Gray denotes TOP-GFP^low (10% lowest) and green denotes TOP-GFP^high (10% highest) populations. (D) Density plots for CD29/CD24 and CD44/CD166 from TOP-GFP^low (gray) and TOP-GFP^high (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

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Flow cytometry gating strategy.

Representative density plots of gating strategy to assess cell surface markers from TOP-GFP^low and TOP-GFP^high 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-GFP^low (10% lowest) and TOP-GFP^high (10% highest) populations. TOP-GFP^low and TOP-GFP^high 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

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Clonogenicity assay of TOP-GFP cultures.

A limiting-dilution assay was performed on the TOP-GFP^low, TOP-GFP^high, or TOP-GFP^whole 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-GFP^low vs TOP-GFP^high: 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-GFP^low vs TOP-GFP^low + 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-GFP^low vs TOP-GFP^low + 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-GFP^low + HGF vs TOP-GFP^low + 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-GFP^low + MFCM vs TOP-GFP^low + 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-GFP^whole vs TOP-GFP^whole + 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

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Pilot of clonogenicity assay.

A limiting-dilution assay was performed on the TOP-GFP^low, TOP-GFP^high, or TOP-GFP^whole 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

Figure 3

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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-GFP^low vs TOP-GFP^high (meta-analysis p=0.094); TOP-GFP^low vs TOP-GFP^low + HGF (meta-analysis p=0.110); TOP-GFP^low vs TOP-GFP^low + MFCM (meta-analysis p=0.047); TOP-GFP^low + HGF vs TOP-GFP^low + HGF + PHA (meta-analysis p=0.218); TOP-GFP^low + MFCM vs TOP-GFP^low + MFCM + PHA (meta-analysis p=0.085); TOP-GFP^whole vs TOP-GFP^whole + 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-GFP^low vs TOP-GFP^high (meta-analysis p=0.330); TOP-GFP^low vs TOP-GFP^low + 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-GFP^low vs TOP-GFP^high (χ² = 0.084, uncorrected p=0.772, corrected p>0.99). Planned contrast between TOP-GFP^low vs TOP-GFP^low + 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

Line	Condition	10	100	1000	5000
E450	TOP-GFP Low	0/4	0/4	2/4	3/4
	TOP-GFP High	0/4	0/4	3/4	2/4
	TOP-GFP Low + MFCM	0/4	2/4	2/4	4/4

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Cell line (Homo sapiens)	Co100	doi:10.1038/ncb2048		shared by Medema lab, University of Amsterdam
Cell line (H. sapiens, female)	CSC1	ProMab Biotechnologies	cat# CC100103
Cell line (H. sapiens, female)	E450	this paper
Cell line (H. sapiens, female)	18Co	ATCC	cat# CRL-1459; RRID:CVCL_2379
Strain, strain background (Mus musculus, Athymic Nude, female)	athymic nude	Charles River	Strain code: 490; RRID:IMSR_CRL:490
Recombinant DNA reagent	TOP-GFP	doi:10.1038/nature01593	RRID:Addgene_14715	shared by Medema lab, University of Amsterdam
Chemical compound, drug	HGF	Sigma-Aldrich	cat# H5791	lot# MKBT3102V
Chemical compound, drug	PHA-665752	Sigma-Aldrich	cat# PZ0147
Other	Matrigel	Corning	cat# 356230
Antibody	PE-conjugated anti-CD133	Miltenyi Biotec	cat# 130-098-826; clone: AC133; RRID:AB_2660882	1:100 dilution
Antibody	PE-conjugated anti-CD24	BD Biosciences	cat# 560991; clone ML5; RRID:AB_10563074	1:100 dilution
Antibody	APC-conjugated anti-CD29	BD Biosciences	cat# 561794; clone: MAR4; RRID:AB_10898163	1:100 dilution
Antibody	PE-conjugated anti-CD166	R and D Systems	cat# FAB6561P; clone: 105902; RRID:AB_2223887	1:100 dilution
Antibody	APC-conjugated anti-CD44	BD Biosciences	cat# 560890; clone: G44-26; RRID:AB_2033959	1:100 dilution
Antibody	PE-conjugated mouse IgG1 isotype control	Miltenyi Biotec	cat# 130-098-106; clone: X-56; RRID:AB_2661463	1:100 dilution
Antibody	APC-conjugated mouse IgG2b, κ isotype control	BD Biosciences	cat# 555745; clone: 27–35; RRID:AB_398612	1:100 dilution
Antibody	PE-conjugated mouse IgG2a, κ isotype control	BD Biosciences	cat# 555574; clone: G155-178; RRID:AB_395953	1:100 dilution
Antibody	APC-conjugated mouse IgG1 isotype control	BD Biosciences	cat# 555751; clone: MOPC-21; RRID:AB_398613	1:100 dilution
Software, algorithm	FACS Sortware sorter	BD Biosciences	RRID:SCR_016722	version 1.2.0.142
Software, algorithm	HCS Studio Cell Analysis	Thermo Fisher Scientific	RRID:SCR_016787	version 6.6.0
Software, algorithm	FACSDiva	BD Biosciences	RRID:SCR_016722	version 6.1.3 or 8.0.1
Software, algorithm	FlowJo	Tree Star, Inc	RRID:SCR_008520	version 10
Software, algorithm	R Project for statistical computing	https://www.r-project.org	RRID:SCR_001905	version 3.5.1