1. Volume 19, Issue 5, Pages 981-994 (May 2017)
The Induction of Selected Wnt Target Genes by Tcf1 Mediates Generation of Tumorigenic Colon Stem Cells 
Daisuke Shiokawa, Ai Sato, Hirokazu Ohata, Michihiro Mutoh, Shigeki Sekine, Mamoru Kato, Tatsuhiro Shibata, Hitoshi Nakagama, Koji Okamoto 
Cell Reports 
Volume 19, Issue 5, Pages (May 2017)
DOI: /j.celrep
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2. Cell Reports 2017 19, 981-994DOI: (10.1016/j.celrep.2017.04.017)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Stratification of Colon Epithelial Cells into Functionally Distinct Cell Populations Using Single-Cell qPCR Analyses Revealed that Colon Carcinogenesis Is Associated with the Emergence of a Ceacam1-Negative Stem Cell Population
(A) Hierarchical clustering of the tumor and non-tumor colon epithelial cells into seven distinct cell populations. Expression data of single-cell qPCR for 43 genes (Table S1) are shown. The cellular identity of each cell population was assigned according to the expression of cell-type-specific genes and is shown above each cluster (Abs Diff, differentiated absorptive cells; Sec Diff, differentiated secretory cells; Sec Pro, secretory cell progenitors; Abs Pro, absorptive cell progenitors; stem T, tumor-specific stem cells; stem N, non-tumor stem cells). The color indicates the relative gene expression level from low (blue) to high (red). The black bars shown at the bottom indicate the original tissues from which the cells were prepared (N1–N3, non-tumor tissues; T1–T4, tumor tissues). Hierarchical clustering was performed using Ward’s method.
(B) Violin plots of the expression of the indicated genes among the seven cell populations shown in (A). Single-cell qPCR data from the seven examined tissues (three non-tumor and four tumor tissues) were used to produce the plots. The results are presented in the order of their p values, as determined by the Kruskal-Wallis test.
(C) Principal component analysis (PCA) of the distribution of the seven cell subpopulations in three non-tumor and four tumor epithelial tissues (far left column). The PCA map shown in Figure S1F is dissected into the original tissues (far left column), and the seven subpopulations in each of these original tissue are visualized in separate PCA maps (right columns). Each subpopulation is depicted by the indicated color.
(D) Stacked bar charts representing the proportions (%) of the seven cell subpopulations in each original tissue shown in (C).
(E) The expression levels of Ceacam1 and Lgr5 in individual cells from the stem T and stem N populations are shown on the PCA plots. The color indicates the relative expression level from low (blue) to high (red).
(F) Flow cytometry analyses of Ceacam1 expression in Lgr5-positive cells from the DSS-treated Lgr5-EGFP/ApcMin/+ mice. Epcam+/GFP+ epithelial cells from tumor and non-tumor tissues were analyzed for Ceacam1 expression.
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4. Figure 2
Single-Cell qPCR Analyses of Wnt Target Genes Reveal that Lgr5-Positive Colon Stem Cells Are Stratified into Four Distinct Cell Types
(A) Hierarchical clustering of Lgr5-positive epithelial cells into four distinct populations (shown here as stem A–D) according to Wnt target gene expression profiles. Tumor and non-tumor tissues of DSS-treated Lgr5-EGFP/ApcMin/+ mice were subjected to single-cell sorting to isolate Epcam+/CD24high cells. Following the verification of Lgr5 expression by standard PCR, cDNAs from each cell were used for multi-gene qPCR for 41 Wnt target genes. The black bars shown at the bottom indicate the original tissues from which the cells were prepared (N4, non-tumor tissue; T5, tumor tissue; T6, tumor tissue), as shown in Figure S2A. The color indicates the relative gene expression level from low (blue) to high (red).
(B) The four populations of Lgr5-positive stem cells (stem A, green; stem B, blue; stem C, purple; stem D, red) shown using a three-dimensional PCA.
(C) Hierarchical clustering of the Wnt target genes based on a correlation matrix: stem-A-associated genes (green), stem-B-associated genes (blue), stem-C-associated genes (purple), and stem-D-associated genes (red). Genes were clustered using Ward’s method.
(D) Principal component projection of each Wnt target gene on the three-dimensional PCA map.
(E) Identification of genes that were specifically expressed in each population (Wilcoxon test). The genes are ranked according to their p values. Genes that were expressed at higher or lower levels in a particular population than their global mean values are shown in pink or blue letters, respectively.
(F) Violin plots of the expression of the indicated cell-population-associated genes and Lgr5 expression in the four cell populations shown in (A).
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5. Figure 3
Ceacam1-Reduced Tumor-Specific Stem Cells Preferentially Express a Distinct Set of Wnt Target Genes
(A) Three-dimensional PCA analyses based on the expression of the Wnt target genes in Lgr5-positive epithelial cells derived from non-tumor (N4) and tumor (T5 and T6) epithelial tissues (total, far-left column) from DSS-treated ApcMin/+ mice. In each tissue, cells belonging to each cell type were segregated and are shown on the PCA map (right columns).
(B) Box-and-whisker plots of Ceacam1 expression in each of the four Lgr5-positive cell populations shown in (A); stem A (green), stem B (blue), stem C (purple), stem D (red). Outliers are shown as circles.
(C) Spearman’s correlation matrix between the expression of Ceacam1 and the Wnt target genes in stem A–D cells. Wnt target genes are grouped into four groups (stem A–D associated) according to the results of the hierarchical clustering shown in Figure 2C, and the correlations between the levels of each Wnt target gene and Ceacam1 were determined. The correlations with Lrig1 and Rgmb, which showed relatively low p values (see Figure S1H), are also shown.
(D) Violin plots of the expression of the indicated stem-D-associated genes in the seven cell populations shown in Figure S3B.
(E) Immunostaining of stem-D-associated Wnt targets (Lef1, Msi1, Stra6, and the long isoform of Tcf1) in the tumor and non-tumor tissues described in Figure S2A. Adjacent sections were stained with H&E. The bars represent 100 μm.
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6. Figure 4
Ceacam1-Reduced Tumor-Specific Stem Cells Show Enhanced Tumor-Initiating Capability
(A) In vitro organoid proliferation of tumor-derived Ceacam1–/low and Ceacam1high stem cells. Colon tumors were developed in DSS-treated Lgr5-EGFP/ApcMin/+ mice, and 5 × 103 GFP+/Ceacam1–/low and GFP+/Ceacam1high cells from the colon tumors were bulk-sorted by FACS and cultured as organoids in the absence of Wnt3a, Noggin, and R-spondin. The relative cell proliferation was evaluated on day 10. The normalized average values from four independent assays are shown (∗∗p < 0.01).
(B) Light microscopy of representative organoids from the GFP+/Ceacam1–/low and GFP+/Ceacam1high cells. The bar represents 100 μm.
(C) The numbers of formed organoids from the GFP+/Ceacam1–/low and GFP+/Ceacam1high cells from three independent experiments were counted (bottom panel) and shown as the means ± SD (upper panel) (∗∗p < 0.01).
(D) Representative xenograft tumors obtained 8 weeks after the subcutaneous injection into nude mice with the organoid cells (1 × 105 per injection) derived from GFP+/Ceacam1–/low cells described in (A) (top). H&E staining of a representative xenograft tumor (bottom). The bar represents 20 μm.
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7. Figure 5
The Expression of a Long Isoform of Tcf1 Is Required for Colon Tumorigenesis and the Unique Expression Profile of Wnt Target Genes in Tumorigenic Stem Cells
(A) Relative cell growth of organoids after CRISPR/Cas9-mediated knockout of the Wnt target genes belonging to the stem-D-associated group, relative to control-virus-infected organoids. The Ceacam1–/low tumor-derived organoids shown in Figure 4B were infected with the lentiviruses that expressed the indicated sgRNAs together with Cas9, and cell growth was quantified by using CellTiter-Glo assays (also see Figure S5A). Three sgRNAs were designed for each gene (Table S4). The knockout vectors whose expression caused a >2-fold increase or decrease in relative cell growth are shown in gray.
(B) Western blot of the tumor-derived organoids after infection with the control virus or the Tcf1 sgRNA-knockout viruses (Tcf1-#1, -#2, and -#3). The tumor-derived organoids (Tumor) were infected with the indicated Tcf1-knockout virus or the control virus and used for western blot analyses using the anti-Tcf1 antibody that detects only the long form (left panel) or one that detects both the long and short forms (right panel). Expression of Actin was used as a control. Non-tumor-derived organoids that were established from the wild-type mice (C57BL/6J) were also used as a reference (Non-T).
(C) Tumor volumes of the mouse xenografts after injection of the Tcf1 sgRNA-introduced organoid cells. The organoid cells infected with the indicated lentivirus were grown as organoids for 2 weeks, and 1 × 105 organoid cells were dissociated and used for subcutaneous injection into BALB/cnu/nu mice. The volumes of the formed tumors were measured 8 weeks after the injection. The experimental results from two independent experiments are shown.
(D) Immunohistological examination of the xenograft tumors described in (C). Tumors were immunostained with the indicated antibodies and co-stained with Hoechst H&E staining is shown in the far left column. The bar represents 100 μm.
(E) Western blot of the tumor-derived organoids after infection with the control viruses or the viruses expressing the HA-tagged short isoform of Tcf1.
(F) Relative cell proliferation of the organoids described in (E). The normalized average values from three independent assays are shown.
(G) Tumor volumes of the mouse xenografts after injection with the indicated organoid cells were shown as the means ± SD from three independent experiments.
(H) H&E staining of the xenograft tumors described in (G). The bar represents 100 μm.
(I) Waterfall plots (left panels) of the effect of Tcf1 knockout on the expression levels of the indicated stem-D-associated Wnt target genes in the tumor-derived organoids. Expression of the indicated genes in the organoids infected with the Tcf1 knockout sgRNAs (Tcf1-#1, Tcf1-#2) or controls were measured using qPCR. The relative effect of Tcf1 knockout on each gene was determined by comparing the expression levels in Tcf1 knockout cells with those in control organoids. Genes belonging to each group are depicted as follows: stem A associated (green), stem B associated (blue), stem C associated (purple), and stem D associated (red). Values are shown as the means ± SD from three independent experiments. The obtained results were categorized for each group and are shown as dot plots (right panel). The p values for comparisons of the indicated gene groups are shown.
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8. Figure 6
Expression of the Long Isoform of Tcf1 in Non-tumor Stem Cells Confers the Ability to Proliferate in a R-Spondin-Independent Manner
(A) Western blot of the non-tumor-derived organoids after infection with the control viruses or the viruses expressing the HA-tagged long form of Tcf1.
(B) Relative cell proliferation of the organoids described in (A).
(C) Light microscopy of representative organoids described in (A). The bar represents 100 μm.
(D) Relative cell proliferation of the organoids described in (A) in the presence of the indicated growth factors. The normalized average values from three independent assays are shown.
(E) Box-and-whisker plots of the Tcf1 RNA expression in human colon cancer at different clinical stages. The log2-transformed transcripts per million (TPM) values of the RNA sequencing (RNA-seq) data in human colon cancer were obtained from The Cancer Genome Atlas (TCGA) database and Tcf1 expression in the indicated stages of colon cancer (N, normal colon) are shown.
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