Supplementary Figure 2: Lgr5+ cells are capable of giving rise to spindle carcinoma, but Lgr5 expression is not maintained in the resulting tumors.
From: Lgr6 is a stem cell marker in mouse skin squamous cell carcinoma
Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice developed papillomas when challenged with a back wound stimulus, and some of the papillomas eventually progressed to malignancy. (a) H&E-stained section of unwounded adult back skin from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice showing SG hyperplasia. (b) H&E-stained section of a typical papilloma harvested from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice. The inset is a high-power view showing the epithelial morphology of the component tumor cells. (c) H&E-stained section of a malignant tumor harvested from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice. The inset is a high-power view showing the spindle morphology of the component tumor cells. (d) Representative section showing anti-GFP immunofluorescence of normal adult skin from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice. (e) Representative section showing anti-GFP immunofluorescence of a spindle carcinoma from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice. (f,g) Three separate tumor cell lines were independently derived from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ spindle carcinomas. (f) TaqMan analysis showing Lgr4, Lgr5 and Lgr6 expression levels in the tumor cell lines and control normal back skin samples. Two different exon-spanning Lgr5 TaqMan probes that target different regions of the Lgr5 transcript were used. Data are presented as mean ± s.e. ***P < 0.001. n = 3. (g) Image analysis showing native GFP fluorescence in cells grown on chamber slides. Clockwise from top right: Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ tumor line 1, tumor line 2, tumor line 3 and positive-control spindle carcinoma cell line (D3) infected with the GFP-expressing F1HtUTG lentiviral vector89. Dotted lines demarcate the basement membrane and the outline of the hair follicles. Asterisks denote the autofluorescent hair shaft. White arrowheads point to specific GFP (Lgr5) expression in the outer root sheath of the hair follicles. Scale bar, 50 μm.
