Supplementary Figure 5: Clonal expansion of cells derived from Lgr5-expressing cells is not observed in primary SCCs in vivo.

Lineage tracing of Lgr5-expressing cells was performed in the same chemically induced primary (1°) SCC from Lgr5-EGFP-CreERT2 mice crossed with Rosa26-LSL-Tomato reporter mice by standard DMBA treatment followed by 20 weeks of tumor promotion using TPA. Once the SCC was readily visible, the mouse was treated with tamoxifen (TAM), and 2 d later initial labeling of Tomato⁺ cells derived from Lgr5-expressing cells was examined in the tumor tissue. (a–d) At 2 d after tamoxifen treatment, only rare Tomato⁺ cells derived from Lgr5-expressing cells (red; b) were readily observed in SCC epithelium, suggesting that Lgr5 is not a CSC marker in primary SCCs in vivo. The yellow dotted boxes demarcate the magnified regions in the insets. The white line indicates the epithelial–dermal border. DAPI (blue) staining was used to visualize cell nuclei (b,d). (a,c) Serial H&E sections of the images depicted in b and d to provide tumor histology. One SCC was used in this study to specifically trace progeny derived from Lgr5-expressing cells in the same tumor in vivo over time. Scale bar, 50 μm.