1. Volume 21, Issue 5, Pages 650-664.e8 (November 2017)
TGF-β-Induced Quiescence Mediates Chemoresistance of Tumor-Propagating Cells in Squamous Cell Carcinoma 
Jessie A. Brown, Yoshiya Yonekubo, Nicole Hanson, Ana Sastre-Perona, Alice Basin, Julie A. Rytlewski, Igor Dolgalev, Shane Meehan, Aristotelis Tsirigos, Slobodan Beronja, Markus Schober 
Cell Stem Cell 
Volume 21, Issue 5, Pages e8 (November 2017)
DOI: /j.stem
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2. Cell Stem Cell 2017 21, 650-664.e8DOI: (10.1016/j.stem.2017.10.001)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
Label-Retaining Cells Reside at the Tumor-Stroma Interface in SCCs
(A) Schematic of proliferation reporter.
(B) Schematic of pulse-chase time course experiment.
(C) LR SCC cells (green, arrows) emerge at the tumor-stroma interface outlined by α6β4 (red). DAPI (blue) stains nuclei. These are composite images of four separate fields. Scale bars indicate 50 μm.
(D) Flow cytometric analyses of live RFP+α6hiβ1hi SCC cells.
(E) Plating efficiency of LR and non-LR cells. Bar graphs represent mean ± SEM (n = 7).
(F) Scatterplots show colony size distribution. Horizontal lines denote mean ± SEM.
(G) Tumor-initiating potential in limit dilution transplantation assays. Mean ± SEM (n = 6).
(H) Tumor growth curves of LR and non-LR SCC cells. Points represent mean ± SEM (n = 3).
See also Figures S1 and S2.
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4. Figure 2
Quiescent and Proliferative TPCs Maintain Long-Term Tumor Growth
(A) Scatterplot of CD71 and H2B-GFP expression.
(B) 8-hr EdU incorporation correlates with CD71. Bar graphs represent mean ± SEM. (n = 3).
(C) Cell-cycle analyses validate proliferation rates of SCC cell subpopulations.
(D) Colony formation on 3T3 feeders. Bar graphs represent mean ± SEM (n = 6, p < 0.05, Student’s t test).
(E) Scatterplots show colony size distribution. Horizontal lines denote mean ± SEM (p = Mann-Whitney non-parametric t test).
(F) Tumor-initiating potential in limit dilution transplantation assays. Mean ± SEM (n = 5).
(G) Tumor growth curves starting at the time of tumor detection. Points represent mean ± SEM (n = 5).
(H) Table summarizes limit dilution transplantation assays with 5 serial passages.
(I) Flow cytometry analyses of live RFP+α6hiβ1hi cells following serial transplantation. Scatterplots show similar composition of daughter tumors and their parent (A).
See also Figure S2 and Table S1.
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5. Figure 3 Quiescent TPCs Resist Cytotoxic Therapy
(A) Tumor growth curves of vehicle (gray) and 5-FU (red) treated SCCs. Points represent mean ± SEM (n = 3).
(B) Luciferase activity measurements indicate physiologically active tumor cells. Scale bar represents increasing signal intensity from blue to red.
(C) Scatterplots of vehicle and 5-FU-treated SCCs. H2B-GFP was chased for 10 days.
(D) Boxplots show relative changes in tumor composition after 5-FU (red). (n = 3).
(E) Cell-cycle profiles of TPC populations after vehicle (gray) or 5-FU (red).
(F) Stacked bar graphs represent relative increase of sub-G1 populations in GFPlo cohorts of 5-FU-treated SCCs. Bar graphs represent mean ± SEM (n = 3, p < 0.05, Student’s t test).
(G) Flow cytometric analyses of Casp3 activation on vehicle (gray) or 5-FU-treated (red) SCCs. Bar graphs represent mean ± SEM (n = 3, p < 0.05, Student’s t test).
(H) Representative image of γH2AX (red) on vehicle (left) or 5-FU-treated (right) SCCs. Asterisks denote γH2AX+ apoptotic cells. Arrows indicate LR H2B-GFPhi (green) cells. β4 (white) outlines the tumor-stroma interface, and DAPI (blue) stains the nuclei. These are composite images of four separate fields. Scale bar represents 50 μm.
(I) Dot plots show the percentage of γH2AX+ cells in LR and non-LR populations after 5-FU (red).
(J) Kaplan-Meier graphs show the tumor-initiating potential after transplantation of 100 purified TPCs, isolated from vehicle (left) or 5-FU-treated (right) SCCs. CD71hiGFPlo cells are in gray; CD71hiGFPhi cells are in light green; CD71loGFPlo cells are in teal; and CD71loGFPhi cells are in dark green. (n = 18, 3 independent experiments with 6 replicates each, p < 0.05, chi-square test.)
See also Figure S3.
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6. Figure 4
Differential Gene Expression Identifies Regulators of TPC Quiescence in SCCs
(A) Dendrogram and heatmap show differentially expressed transcripts. Blue indicates transcripts upregulated in proliferative (P)-TPCs, and red indicates transcripts upregulated in quiescent (Q)-TPCs.
(B) Venn diagram visualizes overlap in transcripts that are upregulated in Q-TPCs, LR-IFSCs, and Q-HFSCs.
(C) IPA prediction of upstream regulators. Z score denotes pathway activation (positive, red) or inhibition (negative, blue) in Q-TPCs.
(D–F) qPCR validation of Trp53 (D), Cdkn1a (E), and Tgfbr2 (F) knockdown. Bar graphs represent mean fold change. Error bars represent ± SEM (n = 3, p < 0.05, Student’s t test). Scatterplots show relative changes in tumor composition. Horizontal lines represent mean ± SEM (p = Mann-Whitney non-parametric t test).
(G) Scatterplot analyses of CD71 and H2B-GFP in live, RFP+ α6hiβ1hi Tgfbr2WT and Tgfbr2KO SCCs.
(H) Cell-cycle analyses of Tgfbr2WT and Tgfbr2KO SCC cells in vitro before and after 24 hr TGF-β1.
(I) Western blot analyses of SCC cells treated for 0, 1, and 8 hr with TGF-β1.
See also Figures S4 and S5 and Tables S2, S3, and S4.
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7. Figure 5
Chromatin-Accessible Regions Bound by Smad2/3 Are Enriched for Cell-Cycle Regulators in Quiescent TPCs
(A) Schematic representation of integrated ChIP, ATAC, and RNA-seq analyses.
(B) Global distribution of Smad2/3 bound regulatory elements.
(C) Venn diagram shows overlap of genes with Smad2/3 bound open chromatin in close proximity and transcripts that are differentially expressed between Q- and P-TPCs.
(D) Scatterplot visualizes GO data of Smad2/3-regulated genes with transcripts that are differentially expressed between Q- and P-TPCs. Color labels denote statistical significance, and circle sizes visualize GO term frequency (more general terms are larger).
(E) Bar graphs represent differential fold change of Cdc25b expression between Q- and P-TPCs in two independent SCCs.
(F) Histograms visualizing Smad2/3 binding and chromatin accessibility around the Cdc25b locus in SCC TPCs.
(G) ChIP-qPCR of Smad2/3 at the Cdc25b enhancer after 1hr TGF-β1 (blue). Bar graphs represent mean fold change. Error bars represent ± SEM (n = 3, p < 0.05, Student’s t test).
(H) Histograms show chromatin accessibility at Smad2/3 bound regulatory elements.
(I) Histograms show genome-wide chromatin accessibility at the TSS.
(J) Enrichment of transcription factor motifs at Smad2/3 bound enhancer sequences.
See also Table S5.
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8. Figure 6 Tgfbr2KO Tumors Are Highly Responsive to Chemotherapy
(A) Tumor growth curves of Tgfbr2KO SCCs treated daily with vehicle (gray) or 5-FU (red). Points represent mean ± SEM (n = 4).
(B) Luciferase activity measurements indicate physiologically active tumor cells. Arrows indicate decellularized plug. Scale bar represents increasing signal intensity from blue to red.
(C) Scatterplot analyses of CD71 and H2B-GFP in Tgfbr2KO SCCs after vehicle or 5-FU.
(D) Cell-cycle profiles after vehicle (gray) or 5-FU (red).
(E) Stacked bar graphs represent the mean percentage of cells in different stages of the cell cycle in vehicle (gray) and 5-FU-treated (red) Tgfbr2KO SCCs.
(F) Colony formation after vehicle (gray) or 5-FU (red) on 3T3 feeders. Bar graphs represent mean ± SEM (n = 3, p < 0.05, Student’s t test).
(G) Scatterplots show colony size distribution. Horizontal lines denote mean ± SEM (p = Mann-Whitney non-parametric t test).
(H) Kaplan-Meier curves show tumor-initiating potential after transplantation of 100 purified SCCs, isolated from vehicle (gray) or 5-FU-treated (red) tumors (n = 18, 3 experiments with 6 replicates each, p < 0.05, chi-square test).
(I) Growth analyses of Tgfbr2WT and Tgfbr2KO SCC cells treated with vehicle or 5-FU with or without TGF-β1. Bar graphs represent mean ± SEM (n = 3, p < 0.05, Student’s t test).
(J) Stacked bar graphs of cell-cycle phases of Tgfbr2WT and Tgfbr2KO SCC cells cultured with vehicle or 5-FU with or without TGF-β1 over time. Bars represent the mean percentage ± SEM (n = 3, p < 0.05, Student’s t test).
(K) Growth analyses of 5-FU-treated Tgfbr2WT SCC cells with (red) or without (light red) TGF-β1 for 5 days. Cells were released and regrown for 5 days. Bar graphs represent mean ± SEM (n = 3, p < 0.05, Student’s t test).
(L) Immunofluorescence microscopy images of γH2AX (red) on Tgfbr2WT SCC cells treated with vehicle or 5-FU with or without TGF-β1. DAPI (blue) stains nuclei. Boxplots of quantifications of γH2AX after 5-FU with or without TGF-β1. (n = 25, p < 0.05, Student’s t test).
(M) Western blot analyses of Tgfbr2WT and Tgfbr2KO SCC cells treated for 0, 24, and 48 hr with 5-FU with or without TGF-β1.
See also Figures S6 and S7.
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9. Figure 7
TGF-β Signaling Is Activated in HNSCC Patients with Progressive Disease
(A) Multi-dimensional scaling plot of HPV- HNSCC patients with complete response (blue) or progressive disease (red) after Cisplatin (P), Carboplatin (P), or Paclitaxel (T).
(B) Volcano plot of differentially expressed genes in responsive and progressive HNSCCs. Red denotes statistically significant transcript changes.
(C) Venn diagram visualizes overlap in transcripts upregulated in Q-TPCs (green), P-TPCs (gray), and patients with progressive HNSCCs (red) or responsive HNSCCs (blue).
(D) IPA prediction of upstream regulators. Z score indicates pathway activation (positive, red) or inhibition (negative, blue) in patients with progressive disease.
(E) Stacked bar graphs of cell-cycle phases of HNSCC25 cells cultured with vehicle or 5-FU with or without TGF-β1 in low-serum conditions. Bars represent the mean percentage ± SEM (n = 3, p < 0.05, Student’s t test).
(F) Dose-response curves of HNSCC25 cells pre-treated for 24 hr with DMSO or TGF-β1 before the addition of 5-FU for 72 hr. Fold change was measured by relative luminance units (RLUs). Points represent mean ± SEM (n = 3, nonlinear regression).
(G) Quantification of activity area fold change from dose-response assays of HNSCC25 cells in 10%, 5%, 0.5%, and 0.1% serum conditions with or without TGF-β1. Bar graphs represent mean ± SEM (n = 3, p < 0.05, Student’s t test).
See also Figure S7 and Table S6.
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